Archive for the ‘Umbilical cord cells’ Category

Treatment of Acute Leukemias

Author and Curator: Larry H. Bernstein, MD, FCAP

2.4.4 Treatment of Acute Leukemias

Treatment of Acute Lymphoblastic Leukemia

Ching-Hon Pu, and William E. Evans
N Engl J Med Jan 12, 2006; 354:166-178

Although the overall cure rate of acute lymphoblastic leukemia (ALL) in children is about 80 percent, affected adults fare less well. This review considers recent advances in the treatment of ALL, emphasizing issues that need to be addressed if treatment outcome is to improve further.

Acute Lymphoblastic Leukemia

Ching-Hon Pui, Mary V. Relling, and James R. Downing
N Engl J Med Apr 8, 2004; 350:1535-1548

This comprehensive survey emphasizes how recent advances in the knowledge of molecular mechanisms involved in acute lymphoblastic leukemia have influenced diagnosis, prognosis, and treatment.

Gene-Expression Patterns in Drug-Resistant Acute Lymphoblastic Leukemia Cells and Response to Treatment

Amy Holleman, Meyling H. Cheok, Monique L. den Boer, et al.
N Engl J Med 2004; 351:533-42

Childhood acute lymphoblastic leukemia (ALL) is curable with chemotherapy in approximately 80 percent of patients. However, the cause of treatment failure in the remaining 20 percent of patients is largely unknown.

Methods We tested leukemia cells from 173 children for sensitivity in vitro to prednisolone, vincristine, asparaginase, and daunorubicin. The cells were then subjected to an assessment of gene expression with the use of 14,500 probe sets to identify differentially expressed genes in drug-sensitive and drug-resistant ALL. Gene-expression patterns that differed according to sensitivity or resistance to the four drugs were compared with treatment outcome in the original 173 patients and an independent cohort of 98 children treated with the same drugs at another institution.

Results We identified sets of differentially expressed genes in B-lineage ALL that were sensitive or resistant to prednisolone (33 genes), vincristine (40 genes), asparaginase (35 genes), or daunorubicin (20 genes). A combined gene-expression score of resistance to the four drugs, as compared with sensitivity to the four, was significantly and independently related to treatment outcome in a multivariate analysis (hazard ratio for relapse, 3.0; P=0.027). Results were confirmed in an independent population of patients treated with the same medications (hazard ratio for relapse, 11.85; P=0.019). Of the 124 genes identified, 121 have not previously been associated with resistance to the four drugs we tested.

Conclusions  Differential expression of a relatively small number of genes is associated with drug resistance and treatment outcome in childhood ALL.

Leukemias Treatment & Management

Author: Lihteh Wu, MD; Chief Editor: Hampton Roy Sr

The treatment of leukemia is in constant flux, evolving and changing rapidly over the past few years. Most treatment protocols use systemic chemotherapy with or without radiotherapy. The basic strategy is to eliminate all detectable disease by using cytotoxic agents. To attain this goal, 3 phases are typically used, as follows: remission induction phase, consolidation phase, and maintenance therapy phase.

Chemotherapeutic agents are chosen that interfere with cell division. Tumor cells usually divide more rapidly than host cells, making them more vulnerable to the effects of chemotherapy. Primary treatment will be under the direction of a medical oncologist, radiation oncologist, and primary care physician. Although a general treatment plan will be outlined, the ophthalmologist does not prescribe or manage such treatment.

  • The initial treatment of ALL uses various combinations of vincristine, prednisone, and L-asparaginase until a complete remission is obtained.
  • Maintenance therapy with mercaptopurine is continued for 2-3 years following remission.
  • Use of intrathecal methotrexate with or without cranial irradiation to cover the CNS varies from facility to facility.
  • Daunorubicin, cytarabine, and thioguanine currently are used to obtain induction and remission of AML.
  • Maintenance therapy for 8 months may lengthen remission. Once relapse has occurred, AML generally is curable only by bone marrow transplantation.
  • Presently, treatment of CLL is palliative.
  • CML is characterized by a leukocytosis greater than 100,000 cells. Emergent treatment with leukopheresis sometimes is necessary when leukostastic complications are present. Otherwise, busulfan or hydroxyurea may control WBC counts. During the chronic phase, treatment is palliative.
  • When CML converts to the blastic phase, approximately one third of cases behave as ALL and respond to treatment with vincristine and prednisone. The remaining two thirds resemble AML but respond poorly to AML therapy.
  • Allogeneic bone marrow transplant is the only curative therapy for CML. However, it carries a high early mortality rate.
  • Leukemic retinopathy usually is not treated directly. As the hematological parameters normalize with systemic treatment, many of the ophthalmic signs resolve. There are reports that leukopheresis for hyperviscosity also may alleviate intraocular manifestations.
  • When definite intraocular leukemic infiltrates fail to respond to systemic chemotherapy, direct radiation therapy is recommended.
  • Relapse, manifested by anterior segment involvement, should be treated by radiation. In certain cases, subconjunctival chemotherapeutic agents have been injected.
  • Optic nerve head infiltration in patients with ALL is an emergency and requires prompt radiation therapy to try to salvage some vision.

Treatments and drugs

Common treatments used to fight leukemia include:

  • Chemotherapy. Chemotherapy is the major form of treatment for leukemia. This drug treatment uses chemicals to kill leukemia cells.

Depending on the type of leukemia you have, you may receive a single drug or a combination of drugs. These drugs may come in a pill form, or they may be injected directly into a vein.

  • Biological therapy. Biological therapy works by using treatments that help your immune system recognize and attack leukemia cells.
  • Targeted therapy. Targeted therapy uses drugs that attack specific vulnerabilities within your cancer cells.

For example, the drug imatinib (Gleevec) stops the action of a protein within the leukemia cells of people with chronic myelogenous leukemia. This can help control the disease.

  • Radiation therapy. Radiation therapy uses X-rays or other high-energy beams to damage leukemia cells and stop their growth. During radiation therapy, you lie on a table while a large machine moves around you, directing the radiation to precise points on your body.

You may receive radiation in one specific area of your body where there is a collection of leukemia cells, or you may receive radiation over your whole body. Radiation therapy may be used to prepare for a stem cell transplant.

  • Stem cell transplant. A stem cell transplant is a procedure to replace your diseased bone marrow with healthy bone marrow.

Before a stem cell transplant, you receive high doses of chemotherapy or radiation therapy to destroy your diseased bone marrow. Then you receive an infusion of blood-forming stem cells that help to rebuild your bone marrow.

You may receive stem cells from a donor, or in some cases you may be able to use your own stem cells. A stem cell transplant is very similar to a bone marrow transplant. Acute Myeloid Leukemia

New treatment approaches in acute myeloid leukemia: review of recent clinical studies.

Norsworthy K1Luznik LGojo I.
Rev Recent Clin Trials. 2012 Aug; 7(3):224-37.

Standard chemotherapy can cure only a fraction (30-40%) of younger and very few older patients with acute myeloid leukemia (AML). While conventional allografting can extend the cure rates, its application remains limited mostly to younger patients and those in remission. Limited efficacy of current therapies and improved understanding of the disease biology provided a spur for clinical trials examining novel agents and therapeutic strategies in AML. Clinical studies with novel chemotherapeutics, antibodies, different signal transduction inhibitors, and epigenetic modulators demonstrated their clinical activity; however, it remains unclear how to successfully integrate novel agents either alone or in combination with chemotherapy into the overall therapeutic schema for AML. Further studies are needed to examine their role in relation to standard chemotherapy and their applicability to select patient populations based on recognition of unique disease and patient characteristics, including the development of predictive biomarkers of response. With increasing use of nonmyeloablative or reduced intensity conditioning and alternative graft sources such as haploidentical donors and cord blood transplants, the benefits of allografting may extend to a broader patient population, including older AML patients and those lacking a HLA-matched donor. We will review here recent clinical studies that examined novel pharmacologic and immunologic approaches to AML therapy.

Novel approaches to the treatment of acute myeloid leukemia.

Roboz GJ1
Hematology Am Soc Hematol Educ Program. 2011:43-50.

Approximately 12 000 adults are diagnosed with acute myeloid leukemia (AML) in the United States annually, the majority of whom die from their disease. The mainstay of initial treatment, cytosine arabinoside (ara-C) combined with an anthracycline, was developed nearly 40 years ago and remains the worldwide standard of care. Advances in genomics technologies have identified AML as a genetically heterogeneous disease, and many patients can now be categorized into clinicopathologic subgroups on the basis of their underlying molecular genetic defects. It is hoped that enhanced specificity of diagnostic classification will result in more effective application of targeted agents and the ability to create individualized treatment strategies. This review describes the current treatment standards for induction, consolidation, and stem cell transplantation; special considerations in the management of older AML patients; novel agents; emerging data on the detection and management of minimal residual disease (MRD); and strategies to improve the design and implementation of AML clinical trials.

Age ≥ 60 years has consistently been identified as an independent adverse prognostic factor in AML, and there are very few long-term survivors in this age group.5 Poor outcomes in elderly AML patients have been attributed to both host- and disease-related factors, including medical comorbidities, physical frailty, increased incidence of antecedent myelodysplastic syndrome and myeloproliferative disorders, and higher frequency of adverse cytogenetics.28 Older patients with multiple poor-risk factors have a high probability of early death and little chance of long-term disease-free survival with standard chemotherapy. In a retrospective analysis of 998 older patients treated with intensive induction at the M.D. Anderson Cancer Center, multivariate analysis identified age ≥ 75 years, unfavorable karyotype, poor performance status, creatinine > 1.3 mg/dL, duration of antecedent hematologic disorder > 6 months, and treatment outside a laminar airflow room as adverse prognostic indicators.29 Patients with 3 or more of these factors had expected complete remission rates of < 20%, 8-week mortality > 50%, and 1-year survival < 10%. The Medical Research Council (MRC) identified cytogenetics, WBC count at diagnosis, age, and de novo versus secondary disease as critical factors influencing survival in > 2000 older patients with AML, but cautioned in their conclusions that less objective factors, such as clinical assessment of “fitness” for chemotherapy, may be equally important in making treatment decisions in this patient population.30 It is hoped that data from comprehensive geriatric assessments of functional status, cognition, mood, quality of life, and other measures obtained during ongoing cooperative group trials will improve our ability to predict how older patients will tolerate treatment.

Current treatment of acute myeloid leukemia.

Roboz GJ1.
Curr Opin Oncol. 2012 Nov; 24(6):711-9.

The objectives of this review are to discuss standard and investigational nontransplant treatment strategies for acute myeloid leukemia (AML), excluding acute promyelocytic leukemia.

RECENT FINDINGS: Most adults with AML die from their disease. The standard treatment paradigm for AML is remission induction chemotherapy with an anthracycline/cytarabine combination, followed by either consolidation chemotherapy or allogeneic stem cell transplantation, depending on the patient’s ability to tolerate intensive treatment and the likelihood of cure with chemotherapy alone. Although this approach has changed little in the last three decades, increased understanding of the pathogenesis of AML and improvements in molecular genomic technologies are leading to novel drug targets and the development of personalized, risk-adapted treatment strategies. Recent findings related to prognostically relevant and potentially ‘druggable’ molecular targets are reviewed.

SUMMARY: At the present time, AML remains a devastating and mostly incurable disease, but the combination of optimized chemotherapeutics and molecularly targeted agents holds significant promise for the future.

Adult Acute Myeloid Leukemia Treatment (PDQ®)

About This PDQ Summary

This summary is reviewed regularly and updated as necessary by the PDQ Adult Treatment Editorial Board, which is editorially independent of the National Cancer Institute (NCI). The summary reflects an independent review of the literature and does not represent a policy statement of NCI or the National Institutes of Health (NIH).

Board members review recently published articles each month to determine whether an article should:

  • be discussed at a meeting,
  • be cited with text, or
  • replace or update an existing article that is already cited.

Treatment Option Overview for AML

Successful treatment of acute myeloid leukemia (AML) requires the control of bone marrow and systemic disease and specific treatment of central nervous system (CNS) disease, if present. The cornerstone of this strategy includes systemically administered combination chemotherapy. Because only 5% of patients with AML develop CNS disease, prophylactic treatment is not indicated.[13]

Treatment is divided into two phases: remission induction (to attain remission) and postremission (to maintain remission). Maintenance therapy for AML was previously administered for several years but is not included in most current treatment clinical trials in the United States, other than for acute promyelocytic leukemia. (Refer to the Adult Acute Myeloid Leukemia in Remission section of this summary for more information.) Other studies have used more intensive postremission therapy administered for a shorter duration of time after which treatment is discontinued.[4] Postremission therapy appears to be effective when given immediately after remission is achieved.[4]

Since myelosuppression is an anticipated consequence of both the leukemia and its treatment with chemotherapy, patients must be closely monitored during therapy. Facilities must be available for hematologic support with multiple blood fractions including platelet transfusions and for the treatment of related infectious complications.[5] Randomized trials have shown similar outcomes for patients who received prophylactic platelet transfusions at a level of 10,000/mm3 rather than 20,000/mm3.[6] The incidence of platelet alloimmunization was similar among groups randomly assigned to receive pooled platelet concentrates from random donors; filtered, pooled platelet concentrates from random donors; ultraviolet B-irradiated, pooled platelet concentrates from random donors; or filtered platelets obtained by apheresis from single random donors.[7] Colony-stimulating factors, for example, granulocyte colony–stimulating factor (G-CSF) and granulocyte-macrophage colony–stimulating factor (GM-CSF), have been studied in an effort to shorten the period of granulocytopenia associated with leukemia treatment.[8] If used, these agents are administered after completion of induction therapy. GM-CSF was shown to improve survival in a randomized trial of AML in patients aged 55 to 70 years (median survival was 10.6 months vs. 4.8 months). In this Eastern Cooperative Oncology Group (ECOG) (EST-1490) trial, patients were randomly assigned to receive GM-CSF or placebo following demonstration of leukemic clearance of the bone marrow;[9] however, GM-CSF did not show benefit in a separate similar randomized trial in patients older than 60 years.[10] In the latter study, clearance of the marrow was not required before initiating cytokine therapy. In a Southwest Oncology Group (NCT00023777) randomized trial of G-CSF given following induction therapy to patients older than 65 years, complete response was higher in patients who received G-CSF because of a decreased incidence of primary leukemic resistance. Growth factor administration did not impact on mortality or on survival.[11,12] Because the majority of randomized clinical trials have not shown an impact of growth factors on survival, their use is not routinely recommended in the remission induction setting.

The administration of GM-CSF or other myeloid growth factors before and during induction therapy, to augment the effects of cytotoxic therapy through the recruitment of leukemic blasts into cell cycle (growth factor priming), has been an area of active clinical research. Evidence from randomized studies of GM-CSF priming have come to opposite conclusions. A randomized study of GM-CSF priming during conventional induction and postremission therapy showed no difference in outcomes between patients who received GM-CSF and those who did not receive growth factor priming.[13,14][Level of evidence: 1iiA] In contrast, a similar randomized placebo-controlled study of GM-CSF priming in patients with AML aged 55 to 75 years showed improved disease-free survival (DFS) in the group receiving GM-CSF (median DFS for patients who achieved complete remission was 23 months vs. 11 months; 2-year DFS was 48% vs. 21%), with a trend towards improvement in overall survival (2-year survival was 39% vs. 27%, = .082) for patients aged 55 to 64 years.[15][Level of evidence: 1iiDii]


  1. Kebriaei P, Champlin R, deLima M, et al.: Management of acute leukemias. In: DeVita VT Jr, Lawrence TS, Rosenberg SA: Cancer: Principles and Practice of Oncology. 9th ed. Philadelphia, Pa: Lippincott Williams & Wilkins, 2011, pp 1928-54.
  2. Wiernik PH: Diagnosis and treatment of acute nonlymphocytic leukemia. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 283-302.
  3. Morrison FS, Kopecky KJ, Head DR, et al.: Late intensification with POMP chemotherapy prolongs survival in acute myelogenous leukemia–results of a Southwest Oncology Group study of rubidazone versus adriamycin for remission induction, prophylactic intrathecal therapy, late intensification, and levamisole maintenance. Leukemia 6 (7): 708-14, 1992. [PUBMED Abstract]
  4. Cassileth PA, Lynch E, Hines JD, et al.: Varying intensity of postremission therapy in acute myeloid leukemia. Blood 79 (8): 1924-30, 1992. [PUBMED Abstract]
  5. Supportive Care. In: Wiernik PH, Canellos GP, Dutcher JP, et al., eds.: Neoplastic Diseases of the Blood. 3rd ed. New York, NY: Churchill Livingstone, 1996, pp 779-967.
  6. Rebulla P, Finazzi G, Marangoni F, et al.: The threshold for prophylactic platelet transfusions in adults with acute myeloid leukemia. Gruppo Italiano Malattie Ematologiche Maligne dell’Adulto. N Engl J Med 337 (26): 1870-5, 1997. [PUBMED Abstract]
  7. Leukocyte reduction and ultraviolet B irradiation of platelets to prevent alloimmunization and refractoriness to platelet transfusions. The Trial to Reduce Alloimmunization to Platelets Study Group. N Engl J Med 337 (26): 1861-9, 1997. [PUBMED Abstract]
  8. Geller RB: Use of cytokines in the treatment of acute myelocytic leukemia: a critical review. J Clin Oncol 14 (4): 1371-82, 1996. [PUBMED Abstract]
  9. Rowe JM, Andersen JW, Mazza JJ, et al.: A randomized placebo-controlled phase III study of granulocyte-macrophage colony-stimulating factor in adult patients (> 55 to 70 years of age) with acute myelogenous leukemia: a study of the Eastern Cooperative Oncology Group (E1490). Blood 86 (2): 457-62, 1995. [PUBMED Abstract]
  10. Stone RM, Berg DT, George SL, et al.: Granulocyte-macrophage colony-stimulating factor after initial chemotherapy for elderly patients with primary acute myelogenous leukemia. Cancer and Leukemia Group B. N Engl J Med 332 (25): 1671-7, 1995. [PUBMED Abstract]
  11. Dombret H, Chastang C, Fenaux P, et al.: A controlled study of recombinant human granulocyte colony-stimulating factor in elderly patients after treatment for acute myelogenous leukemia. AML Cooperative Study Group. N Engl J Med 332 (25): 1678-83, 1995. [PUBMED Abstract]
  12. Godwin JE, Kopecky KJ, Head DR, et al.: A double-blind placebo-controlled trial of granulocyte colony-stimulating factor in elderly patients with previously untreated acute myeloid leukemia: a Southwest oncology group study (9031). Blood 91 (10): 3607-15, 1998. [PUBMED Abstract]
  13. Buchner T, Hiddemann W, Wormann B, et al.: GM-CSF multiple course priming and long-term administration in newly diagnosed AML: hematologic and therapeutic effects. [Abstract] Blood 84 (10 Suppl 1): A-95, 27a, 1994.
  14. Löwenberg B, Boogaerts MA, Daenen SM, et al.: Value of different modalities of granulocyte-macrophage colony-stimulating factor applied during or after induction therapy of acute myeloid leukemia. J Clin Oncol 15 (12): 3496-506, 1997. [PUBMED Abstract]
  15. Witz F, Sadoun A, Perrin MC, et al.: A placebo-controlled study of recombinant human granulocyte-macrophage colony-stimulating factor administered during and after induction treatment for de novo acute myelogenous leukemia in elderly patients. Groupe Ouest Est Leucémies Aiguës Myéloblastiques (GOELAM). Blood 91 (8): 2722-30, 1998. [PUBMED Abstract]

Read Full Post »

Treatments other than Chemotherapy for Leukemias and Lymphomas

Author, Curator, Editor: Larry H. Bernstein, MD, FCAP

2.5.1 Radiation Therapy

Radiation therapy, also called radiotherapy or irradiation, can be used to treat leukemia, lymphoma, myeloma and myelodysplastic syndromes. The type of radiation used for radiotherapy (ionizing radiation) is the same that’s used for diagnostic x-rays. Radiotherapy, however, is given in higher doses.

Radiotherapy works by damaging the genetic material (DNA) within cells, which prevents them from growing and reproducing. Although the radiotherapy is directed at cancer cells, it can also damage nearby healthy cells. However, current methods of radiotherapy have been improved upon, minimizing “scatter” to nearby tissues. Therefore its benefit (destroying the cancer cells) outweighs its risk (harming healthy cells).

When radiotherapy is used for blood cancer treatment, it’s usually part of a treatment plan that includes drug therapy. Radiotherapy can also be used to relieve pain or discomfort caused by an enlarged liver, lymph node(s) or spleen.

Radiotherapy, either alone or with chemotherapy, is sometimes given as conditioning treatment to prepare a patient for a blood or marrow stem cell transplant. The most common types used to treat blood cancer are external beam radiation (see below) and radioimmunotherapy.
External Beam Radiation

External beam radiation is the type of radiotherapy used most often for people with blood cancers. A focused radiation beam is delivered outside the body by a machine called a linear accelerator, or linac for short. The linear accelerator moves around the body to deliver radiation from various angles. Linear accelerators make it possible to decrease or avoid skin reactions and deliver targeted radiation to lessen “scatter” of radiation to nearby tissues.

The dose (total amount) of radiation used during treatment depends on various factors regarding the patient, disease and reason for treatment, and is established by a radiation oncologist. You may receive radiotherapy during a series of visits, spread over several weeks (from two to 10 weeks, on average). This approach, called dose fractionation, lessens side effects. External beam radiation does not make you radioactive.

2.5.2  Bone marrow (BM) transplantation

There are three kinds of bone marrow transplants:

Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.

Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.

Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.

Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

A stem cell transplant is usually done after chemotherapy and radiation is complete. The stem cells are delivered into your bloodstream usually through a tube called a central venous catheter. The process is similar to getting a blood transfusion. The stem cells travel through the blood into the bone marrow. Most times, no surgery is needed.

Donor stem cells can be collected in two ways:

  • Bone marrow harvest. This minor surgery is done under general anesthesia. This means the donor will be asleep and pain-free during the procedure. The bone marrow is removed from the back of both hip bones. The amount of marrow removed depends on the weight of the person who is receiving it.
  • Leukapheresis. First, the donor is given 5 days of shots to help stem cells move from the bone marrow into the blood. During leukapheresis, blood is removed from the donor through an IV line in a vein. The part of white blood cells that contains stem cells is then separated in a machine and removed to be later given to the recipient. The red blood cells are returned to the donor.

Why the Procedure is Performed

A bone marrow transplant replaces bone marrow that either is not working properly or has been destroyed (ablated) by chemotherapy or radiation. Doctors believe that for many cancers, the donor’s white blood cells can attach to any remaining cancer cells, similar to when white cells attach to bacteria or viruses when fighting an infection.

Your doctor may recommend a bone marrow transplant if you have:

Certain cancers, such as leukemia, lymphoma, and multiple myeloma

A disease that affects the production of bone marrow cells, such as aplastic anemia, congenital neutropenia, severe immunodeficiency syndromes, sickle cell anemia, thalassemia

Had chemotherapy that destroyed your bone

2.5.3 Autologous stem cell transplantation

Phase II trial of 131I-B1 (anti-CD20) antibody therapy with autologous stem cell transplantation for relapsed B cell lymphomas

O.W Press,  F Appelbaum,  P.J Martin, et al.

25 patients with relapsed B-cell lymphomas were evaluated with trace-labelled doses (2·5 mg/kg, 185-370 MBq [5-10 mCi]) of 131I-labelled anti-CD20 (B1) antibody in a phase II trial. 22 patients achieved 131I-B1 biodistributions delivering higher doses of radiation to tumor sites than to normal organs and 21 of these were treated with therapeutic infusions of 131I-B1 (12·765-29·045 GBq) followed by autologous hemopoietic stem cell reinfusion. 18 of the 21 treated patients had objective responses, including 16 complete remissions. One patient died of progressive lymphoma and one died of sepsis. Analysis of our phase I and II trials with 131I-labelled B1 reveal a progression-free survival of 62% and an overall survival of 93% with a median follow-up of 2 years. 131I-anti-CD20 (B1) antibody therapy produces complete responses of long duration in most patients with relapsed B-cell lymphomas when given at maximally tolerated doses with autologous stem cell rescue.

Autologous (Self) Transplants

An autologous transplant (or rescue) is a type of transplant that uses the person’s own stem cells. These cells are collected in advance and returned at a later stage. They are used to replace stem cells that have been damaged by high doses of chemotherapy, used to treat the person’s underlying disease.

In most cases, stem cells are collected directly from the bloodstream. While stem cells normally live in your marrow, a combination of chemotherapy and a growth factor (a drug that stimulates stem cells) called Granulocyte Colony Stimulating Factor (G-CSF) is used to expand the number of stem cells in the marrow and cause them to spill out into the circulating blood. From here they can be collected from a vein by passing the blood through a special machine called a cell separator, in a process similar to dialysis.

Most of the side effects of an autologous transplant are caused by the conditioning therapy used. Although they can be very unpleasant at times it is important to remember that most of them are temporary and reversible.

Procedure of Hematopoietic Stem Cell Transplantation

Hematopoietic stem cell transplantation (HSCT) is the transplantation of multipotent hematopoietic stem cells, usually derived from bone marrow, peripheral blood, or umbilical cord blood. It may be autologous (the patient’s own stem cells are used) or allogeneic (the stem cells come from a donor).

Hematopoietic Stem Cell Transplantation

Author: Ajay Perumbeti, MD, FAAP; Chief Editor: Emmanuel C Besa, MD

Hematopoietic stem cell transplantation (HSCT) involves the intravenous (IV) infusion of autologous or allogeneic stem cells to reestablish hematopoietic function in patients whose bone marrow or immune system is damaged or defective.

The image below illustrates an algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy.

An algorithm for typically preferred hematopoietic stem cell transplantation cell source for treatment of malignancy: If a matched sibling donor is not available, then a MUD is selected; if a MUD is not available, then choices include a mismatched unrelated donor, umbilical cord donor(s), and a haploidentical donor.

Supportive Therapies

2.5.4  Blood transfusions – risks and complications of a blood transfusion

  • Allogeneic transfusion reaction (acute or delayed hemolytic reaction)
  • Allergic reaction
  • Viruses Infectious Diseases

The risk of catching a virus from a blood transfusion is very low.

HIV. Your risk of getting HIV from a blood transfusion is lower than your risk of getting killed by lightning. Only about 1 in 2 million donations might carry HIV and transmit HIV if given to a patient.

Hepatitis B and C. The risk of having a donation that carries hepatitis B is about 1 in 205,000. The risk for hepatitis C is 1 in 2 million. If you receive blood during a transfusion that contains hepatitis, you’ll likely develop the virus.

Variant Creutzfeldt-Jakob disease (vCJD). This disease is the human version of Mad Cow Disease. It’s a very rare, yet fatal brain disorder. There is a possible risk of getting vCJD from a blood transfusion, although the risk is very low. Because of this, people who may have been exposed to vCJD aren’t eligible blood donors.

  • Fever
  • Iron Overload
  • Lung Injury
  • Graft-Versus-Host Disease

Graft-versus-host disease (GVHD) is a condition in which white blood cells in the new blood attack your tissues.

2.5.5 Erythropoietin

Erythropoietin, (/ɨˌrɪθrɵˈpɔɪ.ɨtɨn/UK /ɛˌrɪθr.pˈtɪn/) also known as EPO, is a glycoprotein hormone that controls erythropoiesis, or red blood cell production. It is a cytokine (protein signaling molecule) for erythrocyte (red blood cell) precursors in the bone marrow. Human EPO has a molecular weight of 34 kDa.

Also called hematopoietin or hemopoietin, it is produced by interstitial fibroblasts in the kidney in close association with peritubular capillary and proximal convoluted tubule. It is also produced in perisinusoidal cells in the liver. While liver production predominates in the fetal and perinatal period, renal production is predominant during adulthood. In addition to erythropoiesis, erythropoietin also has other known biological functions. For example, it plays an important role in the brain’s response to neuronal injury.[1] EPO is also involved in the wound healing process.[2]

Exogenous erythropoietin is produced by recombinant DNA technology in cell culture. Several different pharmaceutical agents are available with a variety ofglycosylation patterns, and are collectively called erythropoiesis-stimulating agents (ESA). The specific details for labelled use vary between the package inserts, but ESAs have been used in the treatment of anemia in chronic kidney disease, anemia in myelodysplasia, and in anemia from cancer chemotherapy. Boxed warnings include a risk of death, myocardial infarction, stroke, venous thromboembolism, and tumor recurrence.[3]

2.5.6  G-CSF (granulocyte-colony stimulating factor)

Granulocyte-colony stimulating factor (G-CSF or GCSF), also known as colony-stimulating factor 3 (CSF 3), is a glycoprotein that stimulates the bone marrow to produce granulocytes and stem cells and release them into the bloodstream.

There are different types, including

  • Lenograstim (Granocyte)
  • Filgrastim (Neupogen, Zarzio, Nivestim, Ratiograstim)
  • Long acting (pegylated) filgrastim (pegfilgrastim, Neulasta) and lipegfilgrastim (Longquex)

Pegylated G-CSF stays in the body for longer so you have treatment less often than with the other types of G-CSF.

2.5.7  Plasma Exchange (plasmapheresis)

Plasmapheresis is a term used to refer to a broad range of procedures in which extracorporeal separation of blood components results in a filtered plasma product.[1, 2] The filtering of plasma from whole blood can be accomplished via centrifugation or semipermeable membranes.[3] Centrifugation takes advantage of the different specific gravities inherent to various blood products such as red cells, white cells, platelets, and plasma.[4] Membrane plasma separation uses differences in particle size to filter plasma from the cellular components of blood.[3]

Traditionally, in the United States, most plasmapheresis takes place using automated centrifuge-based technology.[5] In certain instances, in particular in patients already undergoing hemodialysis, plasmapheresis can be carried out using semipermeable membranes to filter plasma.[4]

In therapeutic plasma exchange, using an automated centrifuge, filtered plasma is discarded and red blood cells along with replacement colloid such as donor plasma or albumin is returned to the patient. In membrane plasma filtration, secondary membrane plasma fractionation can selectively remove undesired macromolecules, which then allows for return of the processed plasma to the patient instead of donor plasma or albumin. Examples of secondary membrane plasma fractionation include cascade filtration,[6] thermofiltration, cryofiltration,[7] and low-density lipoprotein pheresis.

The Apheresis Applications Committee of the American Society for Apheresis periodically evaluates potential indications for apheresis and categorizes them from I to IV based on the available medical literature. The following are some of the indications, and their categorization, from the society’s 2010 guidelines.[2]

  • The only Category I indication for hemopoietic malignancy is Hyperviscosity in monoclonal gammopathies

2.5.8  Platelet Transfusions

Indications for platelet transfusion in children with acute leukemia

Scott Murphy, Samuel Litwin, Leonard M. Herring, Penelope Koch, et al.
Am J Hematol Jun 1982; 12(4): 347–356;jsessionid=A6001D9D865EA1EBC667EF98382EF20C.f03t01

In an attempt to determine the indications for platelet transfusion in thrombocytopenic patients, we randomized 56 children with acute leukemia to one of two regimens of platelet transfusion. The prophylactic group received platelets when the platelet count fell below 20,000 per mm3 irrespective of clinical events. The therapeutic group was transfused only when significant bleeding occurred and not for thrombocytopenia alone. The time to first bleeding episode was significantly longer and the number of bleeding episodes were significantly reduced in the prophylactic group. The survival curves of the two groups could not be distinguished from each other. Prior to the last month of life, the total number of days on which bleeding was present was significantly reduced by prophylactic therapy. However, in the terminal phase (last month of life), the duration of bleeding episodes was significantly longer in the prophylactic group. This may have been due to a higher incidence of immunologic refractoriness to platelet transfusion. Because of this terminal bleeding, comparison of the two groups for total number of days on which bleeding was present did not show a significant difference over the entire study period.

Clinical and Laboratory Aspects of Platelet Transfusion Therapy
Yuan S, Goldfinger D

INTRODUCTION — Hemostasis depends on an adequate number of functional platelets, together with an intact coagulation (clotting factor) system. This topic covers the logistics of platelet use and the indications for platelet transfusion in adults. The approach to the bleeding patient, refractoriness to platelet transfusion, and platelet transfusion in neonates are discussed elsewhere.

Pooled Platelets – A single unit of platelets can be isolated from every unit of donated blood, by centrifuging the blood within the closed collection system to separate the platelets from the red blood cells (RBC). The number of platelets per unit varies according to the platelet count of the donor; a yield of 7 x 1010 platelets is typical [1]. Since this number is inadequate to raise the platelet count in an adult recipient, four to six units are pooled to allow transfusion of 3 to 4 x 1011 platelets per transfusion [2]. These are called whole blood-derived or random donor pooled platelets.

Advantages of pooled platelets include lower cost and ease of collection and processing (a separate donation procedure and pheresis equipment are not required). The major disadvantage is recipient exposure to multiple donors in a single transfusion and logistic issues related to bacterial testing.

Apheresis (single donor) Platelets – Platelets can also be collected from volunteer donors in the blood bank, in a one- to two-hour pheresis procedure. Platelets and some white blood cells are removed, and red blood cells and plasma are returned to the donor. A typical apheresis platelet unit provides the equivalent of six or more units of platelets from whole blood (ie, 3 to 6 x 1011 platelets) [2]. In larger donors with high platelet counts, up to three units can be collected in one session. These are called apheresis or single donor platelets.

Advantages of single donor platelets are exposure of the recipient to a single donor rather than multiple donors, and the ability to match donor and recipient characteristics such as HLA type, cytomegalovirus (CMV) status, and blood type for certain recipients.

Both pooled and apheresis platelets contain some white blood cells (WBC) that were collected along with the platelets. These WBC can cause febrile non-hemolytic transfusion reactions (FNHTR), alloimmunization, and transfusion-associated graft-versus-host disease (ta-GVHD) in some patients.

Platelet products also contain plasma, which can be implicated in adverse reactions including transfusion-related acute lung injury (TRALI) and anaphylaxis. (See ‘Complications of platelet transfusion’ .)

Read Full Post »

Allogeneic Stem Cell Transplantation

Writer and Curator: Larry H. Bernstein, MD, FCAP

This article has the following structure:

9.3.1  Cell based immunotherapy

9.3.2  Photodynamic therapy (PDT)

9.3.3  Small molecules targeted therapy drugs; Tyrosine kinase inhibitors; imatinib (Gleevec/Glivec) and gefitinib (Iressa).

9.3.4 Graft versus Host Disease

9.3.5 Aspergillus Complicating Allogeneic Transplantation


9.3.1 Allogeneic Stem Cell Treatment

Allogeneic stem cell transplantation involves transferring the stem cells from a healthy person (the donor) to your body after high-intensity chemotherapy or radiation.

Allogeneic stem cell transplantation is used to cure some patients who:

  • Are at high risk of relapse
  • Don’t respond fully to treatment
  • Relapse after prior successful treatment

Allogeneic stem cell transplantation can be a high-risk procedure. The high-conditioning regimens are meant to severely or completely impair your ability to make stem cells and you will likely experience side effects during the days you receive high-dose conditioning radiation or chemotherapy. The goals of high-conditioning therapy are to:

treat the remaining cancer cells intensively, thereby making a cancer recurrence less likely
inactivate the immune system to reduce the chance of stem cell graft rejection
enable donor cells to travel to the marrow (engraftment), produce blood cells and bring about graft versus tumor effect

Possible Adverse Effects

The immune system and the blood system are closely linked and can’t be separated from each other. Because of this, allogeneic transplantation means that not only the donor’s blood system but also his or her immune system is transferred. As a result, these adverse effects are possible:

  • Immune rejection of the donated stem cells by the recipient (host-versus-graft effect)
  • Immune reaction by the donor cells against the recipient’s tissues (graft-versus-host disease [GVHD])

The immune reaction, or GVHD, is treated by administering drugs to the patient after the transplant that reduce the ability of the donated immune cells to attack and injure the patient’s tissues. See Graft Versus Host Disease.

Allogeneic stem cell transplants for patients who are older or have overall poor health are relatively uncommon. This is because the pre-transplant conditioning therapy is generally not well tolerated by such patients, especially those with poorly functioning internal organs. However, reduced intensity allogeneic stem cell transplants may be an appropriate treatment for some older or sicker patients.

T-Lymphocyte Depletion

One goal of allogeneic stem cell transplant is to cause the T lymphocytes in the donor’s blood or marrow to take hold (engraft) and grow in the patient’s marrow. Sometimes the T lymphocytes attack the cancer cells. When this happens, it’s called graft versus tumor (GVT) effect (also called graft versus cancer effect). The attack makes it less likely that the disease will return. This effect is more common in myeloid leukemias than it is in other blood cancers.

Unfortunately, T lymphocytes are the same cells that cause graft versus host disease (GVHD). Because of this serious and sometimes life-threatening side effect, doctors in certain cases want to decrease the number of T lymphocytes to be infused with the stem cells. This procedure, called T-lymphocyte depletion, is currently being studied by researchers. The technique involves treating the stem cells collected for transplant with agents that reduce the number of T lymphocytes.

The aim of T-lymphocyte depletion is to lessen GVHD’s incidence and severity. However, it can also cause increased rates of graft rejection, a decreased GVT effect and a slower immune recovery. Doctors must be careful about the number of T lymphocytes removed when using this technique.

Stem Cell Selection

Stem cell selection is another technique being studied in clinical trials that can reduce the number of T lymphocytes that a patient receives. Because of specific features on the outer coat of stem cells, doctors can selectively remove stem cells from a cell mixture. This technique produces a large number of stem cells and fewer other cells, including T lymphocytes.

9.3.2 Defining Characteristics of  Stem Cells

Stem cells have the remarkable potential to develop into many different cell types in the body during early life and growth. In addition, in many tissues they serve as a sort of internal repair system, dividing essentially without limit to replenish other cells as long as the person or animal is still alive. When a stem cell divides, each new cell has the potential either to remain a stem cell or become another type of cell with a more specialized function, such as a muscle cell, a red blood cell, or a brain cell.

Stem cells are distinguished from other cell types by two important characteristics. First, they are unspecialized cells capable of renewing themselves through cell division, sometimes after long periods of inactivity. Second, under certain physiologic or experimental conditions, they can be induced to become tissue- or organ-specific cells with special functions. In some organs, such as the gut and bone marrow, stem cells regularly divide to repair and replace worn out or damaged tissues. In other organs, however, such as the pancreas and the heart, stem cells only divide under special conditions.

Until recently, scientists primarily worked with two kinds of stem cells from animals and humans: embryonic stem cells and non-embryonic “somatic” or “adult” stem cells. The functions and characteristics of these cells will be explained in this document. Scientists discovered ways to derive embryonic stem cells from early mouse embryos more than 30 years ago, in 1981. The detailed study of the biology of mouse stem cells led to the discovery, in 1998, of a method to derive stem cells from human embryos and grow the cells in the laboratory. These cells are called human embryonic stem cells. The embryos used in these studies were created for reproductive purposes through in vitro fertilization procedures.

When they were no longer needed for that purpose, they were donated for research with the informed consent of the donor. In 2006, researchers made another breakthrough by identifying conditions that would allow some specialized adult cells to be “reprogrammed” genetically to assume a stem cell-like state. This new type of stem cell is called induced pluripotent stem cells (iPSCs).

Stem cells differ from other kinds of cells in the body. All stem cells—regardless of their source—have three general properties: they are capable of dividing and renewing themselves for long periods; they are unspecialized; and they can give rise to specialized cell types.

Stem cells are capable of dividing and renewing themselves for long periods. Unlike muscle cells, blood cells, or nerve cells—which do not normally replicate themselves—stem cells may replicate many times, or proliferate. A starting population of stem cells that proliferates for many months in the laboratory can yield millions of cells. If the resulting cells continue to be unspecialized, like the parent stem cells, the cells are said to be capable of long-term self-renewal.

Scientists are trying to understand two fundamental properties of stem cells that relate to their long-term self-renewal:

  1. Why can embryonic stem cells proliferate for a year or more in the laboratory without differentiating, but most adult stem cells cannot; and
  2. What are the factors in living organisms that normally regulate stem cell proliferation and self-renewal?

Discovering the answers to these questions may make it possible to understand how cell proliferation is regulated during normal embryonic development or during the abnormal cell division that leads to cancer.

Stem cells are unspecialized. One of the fundamental properties of a stem cell is that it does not have any tissue-specific structures that allow it to perform specialized functions. For example, a stem cell cannot work with its neighbors to pump blood through the body (like a heart muscle cell), and it cannot carry oxygen molecules through the bloodstream (like a red blood cell). However, unspecialized stem cells can give rise to specialized cells, including heart muscle cells, blood cells, or nerve cells.

Stem cells can give rise to specialized cells. When unspecialized stem cells give rise to specialized cells, the process is called differentiation. While differentiating, the cell usually goes through several stages, becoming more specialized at each step. Scientists are just beginning to understand the signals inside and outside cells that trigger each step of the differentiation process. The internal signals are controlled by a cell’s genes, which are interspersed across long strands of DNA and carry coded instructions for all cellular structures and functions. The external signals for cell differentiation include chemicals secreted by other cells, physical contact with neighboring cells, and certain molecules in the microenvironment. The interaction of signals during differentiation causes the cell’s DNA to acquire epigenetic marks that restrict DNA expression in the cell and can be passed on through cell division.

Adult stem cells typically generate the cell types of the tissue in which they reside. For example, a blood-forming adult stem cell in the bone marrow normally gives rise to the many types of blood cells. It is generally accepted that a blood-forming cell in the bone marrow—which is called a hematopoietic stem cell—cannot give rise to the cells of a very different tissue, such as nerve cells in the brain.

Through years of experimentation, scientists have established some basic protocols or “recipes” for the directed differentiation of embryonic stem cells into some specific cell types (Figure 1). (For additional examples of directed differentiation of embryonic stem cells, refer to the NIH stem cell report available at

stem cell differentiation figure1_sm

stem cell differentiation figure1_sm

9.3.3 Types of Stem Cell Transplants for Treating Cancer

In a typical stem cell transplant for cancer very high doses of chemo are used, often along with radiation therapy, to try to destroy all the cancer cells. This treatment also kills the stem cells in the bone marrow. Soon after treatment, stem cells are given to replace those that were destroyed. These stem cells are given into a vein, much like a blood transfusion. Over time they settle in the bone marrow and begin to grow and make healthy blood cells. This process is called engraftment.

There are 3 basic types of transplants. They are named based on who gives the stem cells.

  • Autologous (aw-tahl-uh-gus)—the cells come from you
  • Allogeneic (al-o-jen-NEE-ick or al-o-jen-NAY-ick)—the cells come from a matched related or unrelated donor
  • Syngeneic (sin-jen-NEE-ick or sin-jen-NAY-ick)—the cells come from your identical twin or triplet
hematopoietic stem cell transplant

hematopoietic stem cell transplant

Autologous stem cell transplants

These stem cells come from you alone. In this type of transplant, your stem cells are taken before you get cancer treatment that destroys them. Your stem cells are removed, or harvested, from either your bone marrow or your blood and then frozen. To find out more about that process, please see the section “What’s it like to donate stem cells?” After you get high doses of chemo and/or radiation the stem cells are thawed and given back to you.

One advantage of autologous stem cell transplant is that you are getting your own cells back. When you donate your own stem cells you don’t have to worry about the graft attacking your body (graft-versus-host disease) or about getting a new infection from another person. But there can still be graft failure, and autologous transplants can’t produce the “graft-versus-cancer” effect.

This kind of transplant is mainly used to treat certain leukemias, lymphomas, and multiple myeloma. It’s sometimes used for other cancers, like testicular cancer and neuroblastoma, and certain cancers in children.

Getting rid of cancer cells in autologous transplants

A possible disadvantage of an autologous transplant is that cancer cells may be picked up along with the stem cells and then put back into your body later. Another disadvantage is that your immune system is still the same as before when your stem cells engraft. The cancer cells were able to grow despite your immune cells before, and may be able to do so again. The need to remove cancer cells from transplants or transplant patients and the best way to do it is being researched.

Doing 2 autologous transplants in a row is known as a tandem transplant or a double autologous transplant. In this type of transplant, the patient gets 2 courses of high-dose chemo, each followed by a transplant of their own stem cells. All of the stem cells needed are collected before the first high-dose chemo treatment, and half of them are used for each transplant. Most often both courses of chemo are given within 6 months, with the second one given after the patient recovers from the first one.

Allogeneic stem cell transplants

In the most common type of allogeneic transplant, the stem cells come from a donor whose tissue type closely matches the patient’s. (This is discussed later under “HLA matching” in the section called “ Donor matching for allogeneic transplant.”) The best donor is a close family member, usually a brother or sister. If you do not have a good match in your family, a donor might be found in the general public through a national registry. This is sometimes called a MUD (matched unrelated donortransplant. Transplants with a MUD are usually riskier than those with a relative who is a good match.

Blood taken from the placenta and umbilical cord of newborns is a newer source of stem cells for allogeneic transplant. Called cord blood, this small volume of blood has a high number of stem cells that tend to multiply quickly. But the number of stem cells in a unit of cord blood is often too low for large adults, so this source of stem cells is limited to small adults and children. Doctors are now looking at different ways to use cord blood for transplant in larger adults, such as using cord blood from 2 donors.

Pros of allogeneic stem cell transplant: The donor stem cells make their own immune cells, which could help destroy any cancer cells that remain after high-dose treatment. This is called the graft-versus-cancer effect. Other advantages are that the donor can often be asked to donate more stem cells or even white blood cells if needed, and stem cells from healthy donors are free of cancer cells.

Cons to allogeneic stem cell transplants: The transplant, also known as the graft, might not take — that is, the donor cells could die or be destroyed by the patient’s body before settling in the bone marrow. Another risk is that the immune cells from the donor may not just attack the cancer cells – they could attack healthy cells in the patient’s body. This is called graft-versus-host disease (described in the section called “Problems that may come up shortly after transplant”). There is also a very small risk of certain infections from the donor cells, even though donors are tested before they donate. A higher risk comes from infections you have had, and which your immune system has under control. These infections often surface after allogeneic transplant because your immune system is held in check (suppressed) by medicines called immunosuppressive drugs. These infections can cause serious problems and even death.

Allogeneic transplant is most often used to treat certain types of leukemia, lymphomas, multiple myeloma,myelodysplastic syndrome, and other bone marrow disorders such as aplastic anemia.

Mini transplants (non-myeloablative transplants)

For some people, age or certain health conditions make it more risky to wipe out all of their bone marrow before a transplant. For those people, doctors can use a type of allogeneic transplant that’s sometimes called a mini-transplant. Compared with a standard allogeneic transplant, this one uses less chemo and/or radiation to get the patient ready for the transplant. Your doctor might refer to it as a non-myeloablative transplant or mention reduced-intensity conditioning (RIC). The idea here is to kill some of the cancer cells along with some of the bone marrow, and suppress the immune system just enough to allow donor stem cells to settle in the bone marrow.

Unlike the standard allogeneic transplant, cells from both the donor and the patient exist together in the patient’s body for some time after a mini-transplant. But slowly, over the course of months, the donor cells take over the bone marrow and replace the patient’s own bone marrow cells. These new cells can then develop an immune response to the cancer and help kill off the patient’s cancer cells — the graft-versus-cancer effect.

Syngeneic stem cell transplants – for those with an identical sibling

This is a special kind of allogeneic transplant that can only be used when the recipient has an identical sibling (twin or triplet) who can donate — someone who will have the same tissue type. An advantage of syngeneic stem cell transplant is that graft-versus-host disease will not be a problem. There are no cancer cells in the transplant, either, as there would be in an autologous transplant.

A disadvantage is that because the new immune system is so much like the recipient’s immune system, there is no graft-versus-cancer effect, either. Every effort must be made to destroy all the cancer cells before the transplant is done to help keep the cancer from relapsing (coming back).

9.3.4 Graft versus Host Disease–GVHD-/

Graft-versus-host disease(GVHD) occurs because of differences between the cells of your body and the donated cells and is a common side effect of an allogeneic bone marrow transplant.

An allogeneic transplant uses blood cells from a family member, unrelated donor or cord blood unit. GVHD can affect many different parts of the body including the skin, eyes, mouth, stomach, and intestines.

There are two types of GVHD:

  • Acute GVHD: Develops in the first 100 days or so after transplant but can occur later. This primarily affects the skin, stomach, intestines, and liver.
  • Chronic GVHD: Usually develops 3-6 months after transplant, but signs can appear earlier or later. If you have had or currently have acute GVHD, you are more likely to have chronic GVHD.

The severity of acute and chronic GVHD can range from mild to life-threatening.

Doctors often see mild GVHD as a good thing after an allogeneic transplant when the transplant was done for a blood cancer. It is a sign that the donor’s immune system is working to destroy any remaining cancer cells. Patients who experience some GVHD have a lower risk of the cancer returning after transplant than patients who do not develop GVHD. If the transplant was to treat a disease other than cancer disease, like aplastic anemia, then the doctor may want to treat even mild GVHD.

Graft-versus-Host Disease

JLM FerraraJE LevineP Reddy, and E Holler
Lancet. 2009 May 2; 373(9674): 1550–1561.

The number of allogeneic hematopoietic cell transplantations (HCT) continues to increase with more than 25,000 allogeneic transplantations performed annually. The graft-versus-leukemia / tumor (GVL) effect during allogeneic HCT effectively eradicates many hematological malignancies.1 The development of novel strategies that use donor leukocyte infusions, non-myeloablative conditioning and umbilical cord blood (UCB) transplantation have helped expand the indications for allogeneic HCT over the last several years, especially among older patients.2 Improvements in infectious prophylaxis, immunosuppressive medications, supportive care and DNA-based tissue typing have also contributed to improved outcomes after allogeneic HCT.1 Yet the major complication of allogeneic HCT, graft-versus-host disease (GVHD), remains lethal and limits the use of this important therapy.2 Given current trends, the number of transplants from unrelated donors is expected to double within the next five years, significantly increasing the population of patients with GVHD. In this seminar we review advances made in identifying the genetic risk factors and pathophysiology of this major HCT complication, as well as its prevention, diagnosis and treatment.

Etiology and Clinical Features

Fifty years ago Billingham formulated three requirements for the development of GVHD: the graft must contain immunologically competent cells; the recipient must express tissue antigens that are not present in the transplant donor; and the recipient must be incapable of mounting an effective response to eliminate the transplanted cells.3 We know now that the immunologically competent cells are T cells, and that GVHD can develop in various clinical settings when tissues containing T cells (blood products, bone marrow, and solid organs) are transferred from one person to another who is not able to eliminate those cells.45 Patients, whose immune systems are suppressed, and who receive white blood cells from another individual, are at particularly high risk for GVHD.

GVHD occurs when donor T cells respond to genetically defined proteins on host cells. The most important proteins are Human Leukocyte Antigens (HLA)267, which are highly polymorphic and are encoded by the major histocompatibility complex (MHC). Class I HLA (A, B, and C) proteins are expressed on almost all nucleated cells of the body at varying densities. Class II proteins (DR, DQ, and DP) are primarily expressed on hematopoietic cells (B cells, dendritic cells, monocytes), but their expression can be induced on many other cell types following inflammation or injury. High-resolution DNA typing of HLA genes with polymerase chain reaction (PCR)-based techniques have now largely replaced earlier methods. The incidence of acute GVHD is directly related to the degree of mismatch between HLA proteins89 and thus ideally, donors and recipients are matched at HLA-A, -B, -C, and -DRB1, (“8/8 matches”), but mismatches may be tolerated for UCB grafts (see below).1012

Non-HLA Genetics

Despite HLA identity between a patient and donor, approximately 40% of patients receiving HLA-identical grafts develop acute GVHD due to genetic differences that lie outside the HLA loci, or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY and HA-3, are expressed on all tissues and are targets for both GVHD and GVL.13 Other minor HAs, such as HA-1 and HA-2, are expressed most abundantly on hematopoietic cells (including leukemic cells) and may therefore induce a greater GVL effect with less GVHD.1314

Polymorphisms in both donors and recipients for cytokines that are involved in the classical `cytokine storm’ of GVHD (discussed below) have been implicated as risk factors for GVHD.15 Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10), Interferon-γ (IFNγ) variants have correlated with GVHD in some, but not all, studies.1618 Genetic polymorphisms of proteins involved in innate immunity, such as nucleotide oligomerization domain 2 and Keratin 18 receptors, have also been associated with GVHD.1922 Future strategies to identify the best possible transplant donor will probably incorporate both HLA and non-HLA genetic factors.

Clinical Features of Acute GVHD

Based on an early Seattle experience, acute GVHD was defined to occur prior to day 100, whereas chronic GVHD occurred after that time.2325 This definition is far from satisfactory, and a recent National Institutes of Health classification includes late-onset acute GVHD (after day 100) and an overlap syndrome with features of both acute and chronic GVHD.26 Late-onset acute GVHD and the overlap syndrome occur with greater frequency after reduced-intensity conditioning (RIC), an increasingly widespread technique (see below). As shown in Table 1, the clinical manifestations of acute GVHD occur in the skin, gastrointestinal tract and liver.27 In a comprehensive review, Martin et al found that at the onset of acute GVHD, 81% of patients had skin involvement, 54% had GI involvement, and 50% had liver involvement.23 Recent data suggest that lungs might also be targets of experimental GVHD.28

Acute GVHD Symptoms

Table 1

Pathophysiology of Acute GVHD

Two important principles are important to consider regarding the pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated but normal inflammatory mechanisms mediated by donor lymphocytes infused into the recipient where they function appropriately, given the foreign environment they encounter. Second, the recipient tissues that stimulate donor lymphocytes have usually been damaged by underlying disease, prior infections, and the transplant conditioning regimen.29 As a result, these tissues produce molecules (sometimes referred to as “danger” signals) that promote the activation and proliferation of donor immune cells.4245 Mouse models havebeen central to our identification and understanding of the pathophysiologic mechanisms of GVHD, and canine models have been critical to the development of clinically useful strategies for GVHD prophylaxis and treatment and to the development of donor leukocyte infusions.364647 Based largely on these experimental models, the development of acute GVHD can be conceptualized in three sequential steps or phases: (1) activation of the APCs; (2) donor T cell activation, proliferation, differentiation and migration; and (3) target tissue destruction (Figure 3).

Figure 3

GVHD Pathophysiology

In Phase I, the recipient conditioning regimen damages host tissues and causes release of inflammatory cytokines such as TNFα, IL-1 and IL-6. Increased levels of these cytokines leads to activation of host antigen presenting cells (APCs). In Phase II, host APCs activate mature donor cells. The subsequent proliferation and differentiation of these activated T cells produces additional effectors that mediate the tissue damage, including Cytotoxic T Lymphocytes, Natural Killer (NK) cells, TNFα and IL-1. Lipopolysaccharide (LPS) that has leaked through the damaged intestinal mucosa triggers additional TNFα production. TNFα can damage tissue directly by inducing necrosis and apoptosis in the skin and GI tract through either TNF receptors or the Fas pathway. TNFα plays a direct role in intestinal GVHD damage which further amplifies damage in the skin, liver and lung in a “cytokine storm.”

GVHD pathophysiology nihms-115970-f0003

GVHD pathophysiology nihms-115970-f0003

Phase I: Activation of Antigen Presenting Cells (APCs)

The first step involves the activation of APCs by the underlying disease and the HCT conditioning regimen. Damaged host tissues respond by producing “danger” signals, including proinflammatory cytokines (e.g., TNF-α), chemokines, and increased expression of adhesion molecules, MHC antigens and costimulatory molecules on host APCs.424850 A recent report demonstrated that at one week after HCT, increased levels of TNF-α receptor I, a surrogate marker for TNF-α, strongly correlated with the later development of GVHD.51 Damage to the GI tract from the conditioning is particularly important because it allows for systemic translocation of additional inflammatory stimuli such as microbial products including lipopolysaccaride (LPS) or other pathogen-associated molecular patterns that further enhance the activation of host APCs.49 The secondary lymphoid tissue in the GI tract is likely the initial site of interaction between activated APCs and donor T cells.52 These observations have led an important clinical strategy to reduce acute GVHD by reducing the intensity of the conditioning regimen. Experimental GVHD can also be reduced by manipulating distinct subsets of APCs.53,54 In addition, non-hematopoietic stem cells, such as mesenchymal stem cells or stromal cells, can reduce allogeneic T cell responses, although the mechanism for such inhibition remains unclear.2

The concept that enhanced activation of host APCs increases the risk for acute GVHD unifies a number of seemingly disparate clinical associations with that risk, such as advanced stages of malignancy, more intense transplant conditioning regimens and histories of viral infections. APCs detect infections by recognizing conserved molecular patterns that are unique to microbes, called pathogen-associated molecular patterns (PAMPs). Among the classes of receptors that recognize such patterns, the Toll-like receptors (TLR) are the best characterized.55 For example, TLR4 recognizes LPS55 and mice with mutant TLR4 receptors that do not respond to LPS cause less GVHD when used as donors.56 Other TLRs that recognize viral DNA or RNA also activate APCs and may enhance GVHD, providing a potential mechanistic basis for increased GVHD associated with viral infections such as cytomegalovirus (CMV).57

Phase II: Donor T Cell Activation

The core of the GVH reaction is Step 2, where donor T cells proliferate and differentiate in response to host APCs. The “danger” signals generated in Phase I augment this activation at least in part by increasing the expression of costimulatory molecules.58 Blockade of co-stimulatory pathways to prevent GVHD is successful in animal models, but this approach has not yet been tested in large clinical trials.2

In mouse models, where genetic differences between donor and recipient strains can be tightly controlled, CD4+ cells induce acute GVHD to MHC class II differences, and CD8+ cells induce acute GVHD to MHC class I differences.5961 In the majority of HLA-identical HCTs, both CD4+ and CD8+ subsets respond to minor histocompatibility antigens and can cause GVHD in HLA-identical HCT.

Regulatory T cells can suppress the proliferation of conventional T cells and prevent GVHD in animal models when added to donor grafts containing conventional T cells.62 In mice, the Foxp3 protein functions as a master switch in the development of regulatory T cells, which normally constitute 5% of the CD4+ T cell population.62 Regulatory T cells secrete anti-inflammatory cytokines IL-10 and Transforming Growth Factor(TGF)-β and can also act through contact-dependent inhibition of APCs.62 It is likely that the use of regulatory T cells in clinical acute GVHD will require improved techniques to identify and expand them.

Natural Killer T cell (NKT) 1.1+ subsets of both the host and donors that have been shown to modulate acute GVHD.63 Host NKT cells have been shown to suppress acute GVHD in an IL-4 dependent manner.64 A recent clinical trial of total lymphoid irradiation used as conditioning significantly reduced GVHD and enhanced host NKT cell function.65 By contrast, donor NKT cells can reduce GVHD and enhance perforin mediated GVL in an experimental model.66

Activation of immune cells results in rapid intracellular biochemical cascades that induce transcription of genes for many proteins including cytokines and their receptors. Th1 cytokines (IFN-γ, IL-2 and TNF-α) are produced in large amounts during acute GVHD. IL-2 production by donor T cells remains the principal target of many current clinical therapeutic and prophylactic approaches to GVHD, such as cyclosporine, tacrolimus and monoclonal antibodies (mAbs) directed against IL-2 and its receptor.9 But emerging data indicate an important role for IL-2 in the generation and maintenance of CD4+ CD25+ T regs, suggesting that prolonged interference with IL-2 may have an unintended consequence of preventing the development of long term tolerance after allogeneic HCT.67 IFN-γ has multiple functions and can either amplify or reduce GVHD.68,69 IFN-γ may amplify GVHD by increasing the expression of molecules such as chemokines receptors, MHC proteins, and adhesion molecules; it also increases the sensitivity of monocytes and macrophages to stimuli such as LPS and accelerates intracellular cascades in response to these stimuli.70Early polarization of donor T cells so that they secrete less IFN-γ and more IL-4 can also attenuate experimental acute GVHD.71 IFN-γ may amplify GVHD by directly damaging epithelium in the GI tract and skin and inducing immnosuppression through the induction of nitric oxide.72 By contrast, IFN-γ may suppress GVHD by hastening the apoptosis of activated donor T cells.6973. This complexity means the manipulation of IFN-γ may have diverse effects in vivo, making it a challenging target with respect to therapeutic intervention. IL-10 plays a key role in suppression of immune responses, and clinical data suggest it may regulate acute GVHD.17 TGF-β, another suppressive cytokine can suppress acute GVHD but exacerbate chronic GVHD.74 Thus the timing and duration of the secretion of any given cytokine may determine the specific effects of that cytokine on GVHD severity.

Phase III: Cellular and Inflammatory Effector Phase

The effector phase of this process is a complex cascade of both cellular mediators such as cytotoxic T lymphocytes(CTLs) and NK cells and soluble inflammatory mediators such as TNF-α, IFN-γ, IL-1 and nitric oxide.229 These soluble and cellular mediators synergize to amplify local tissue injury and further promote inflammation and target tissue destruction.

Cellular Effectors

The cellular effectors of acute GVHD are primarily CTLs and NK cells.49 CTLs that preferentially use the Fas/FasL pathway of target lysis and appear to predominate in GVHD liver damage (hepatocytes express large amounts of Fas) whereas GVHD CTLs that use the perforin /granzyme pathways are more important in the GI tract and skin.275 Chemokines direct the migration of donor T cells from lymphoid tissues to the target organs where they cause damage. Macrophage inflammatory protein-1alpha (MIP-1α) and other chemokines such as CCL2-5, CXCL2, CXCL9-11, CCL17 and CCL27 are over-expressed and enhance the homing of cellular effectors to target organs during experimental GVHD.76Expression of integrins, such as α4β7 and its ligand MadCAM-1, are also important for homing of donor T cells to Peyer’s patches during intestinal GVHD.527778

Prevention of GVHD

Based on the evidence from animal models regarding the central role of T cells in initiating GVHD, numerous clinical studies evaluating T cell depletion (TCD) as prophylaxis for GVHD were performed in the 1980’s and 1990’s. There were three principal TCD strategies: (1) negative selection of T cells ex vivo, (2) positive selection of CD34+ stem cells ex vivo; and (3) anti-T cell antibodies in vivo.83Most strategies showed a significant limitation in both acute and chronic GVHD.8488 Unfortunately, the lower incidence of severe GVHD was offset by high rates of graft failure, relapse of malignancy, infections, and Epstein-Barr virus-associated lymphoproliferative disorders. Negative selection purging strategies using various anti-T cell antibodies achieved similar long-term results regardless of the breadth of antibody specificity.8993 One large registry study demonstrated that purging strategies using antibodies with broad specificities produced inferior leukemia-free survival than standard immunosuppression in patients receiving unrelated donor transplants.94 Several studies have investigated partial T cell depletion, either by eliminating specific T cell subsets (e.g., CD8+) or by titrating the dose of T cells present in the inoculum.9597 None of these approaches, however, has convincingly demonstrated an optimal strategy that improves long-term survival.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein expressed on a broad spectrum of leukocytes including lymphocytes, monocytes, and dendritic cells. Its use in GVHD prophylaxis in a Phase II trial decreased the incidence of acute and chronic GVHD following reduced intensity transplant.98 In two prospective studies, patients who received alemtuzumab rather than methotrexate showed significantly lower rates of acute and chronic GVHD,99 but experienced more infectious complications and higher rates of relapse, so that there was no overall survival benefit. Alemtuzumab may also contribute to graft failure when used with minimal intensity conditioning regimens.100

An alternative strategy to TCD attempted to induce anergy in donor T cells by ex vivo antibody blockade of co-stimulatory pathways prior to transplantation. A small study using this approach in haploidentical HCT recipients was quite encouraging, but has not yet been replicated.101 Thus the focus of most prevention strategies remains pharmacological manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis has also been extensively tested. The best studied drugs are anti-thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations. These sera, which have high titers of polyclonal antibodies, are made by immunizing animals (horses or rabbits) to thymocytes or lymphocytes, respectively. A complicating factor in determining the role of these polyclonal sera in transplantation is the observation that even different brands of the same class of sera exert different biologic effects.102 However, the side effects of ATG/ALG infusions are common across different preparations and include fever, chills, headache, thrombocytopenia (from cross-reactivity to platelets), and, infrequently, anaphylaxis. In retrospective studies, rabbit ATG reduced the incidence of GVHD in related donor HSCT recipients without appearing to improve survival.103104 In recipients of unrelated donor HSCT, addition of ALG to standard GVHD prophylaxis effectively prevented severe GVHD, but did not result in improved survival because of increased infections.105 In a long term follow-up study, however, pretransplant ATG provided significant protection against extensive chronic GVHD and chronic lung dysfunction.106

The primary pharmacologic strategy to prevent GVHD is the inhibition of the cytoplasmic enzyme, calcineurin, that is critical for in the activation of T cells. The calcineurin inhibitors, cyclosporine and tacrolimus, have similar mechanisms of action, clinical effectiveness and toxicity profiles, including hypomagnesemia, hyperkalemia, hypertension, and nephrotoxicity.9107 Serious side effects include transplant-associated thrombotic microangiopathy (TAM) and neurotoxicity that can lead to premature discontinuation. Although clinically similar to thrombotic thrombocytopenic purpura, TAM does not reliably respond to therapeutic plasmapheresis, carries a high mortality rate, and removal of the offending agent does not always result in improvement.108 Posterior reversible encephalopathy syndrome includes mental status changes, seizures, neurological deficits and characteristic magnetic resonance imaging findings; this syndrome has been seen in 1-2% of HCT recipients receiving and calcineurin inhibitors.109 Side effects of these drugs decrease as the dose is tapered, usually two to four months after HCT.

Calcineurin inhibitors are often administered in combination with other immunosuppressants, such as methotrexate, which is given at low doses in the early post-transplant period.9107 The toxicities of methotrexate (neutropenia and mucositis) have led some investigators to replace it with mycophenolate mofetil (MMF). In one prospective randomized trial, patients who received MMF as part of GVHD prophylaxis experienced significantly less severe mucositis and more rapid neutrophil engraftment than those who received methotrexate.110 The incidence and severity of acute GVHD was similar between the two groups, but the study closed early due to superiority of the MMF arm with respect to reduced mucositis and the speed of hematopoietic engraftment. A desire for faster neutrophil engraftment has led to the use of MMF in UCB blood transplants where graft failure is a major concern.111 MMF is also often used after RIC regimens for similar reasons.112113

Sirolimus is an immunosuppressant that is structurally similar to tacrolimus but does not inhibit calcineurin. In a small Phase II trial, it showed excellent efficacy in combination with tacrolimus;114 the drug damages endothelial cells, however, and it may enhance TAM that is associated with calcineurin inhibitors.115 The combination of tacrolimus and sirolimus is currently being compared in a large randomized multi-center trial.

RIC regimens attempt to suppress the host immune system sufficiently so that donor T cells can engraft and then ablate the lympho-hematopoietic compartment of the recipient. The term “non-myeloablative” is therefore somewhat misleading. RIC regimens produce less tissue damage and lower levels of the inflammatory cytokines that are important in the initiation of GVHD pathophysiology; this effect may explain the reduced incidence of severe GVHD following RIC compared to the full intensity conditioning used in historical controls.98116 The onset of acute GVHD may be delayed after RIC until after day 100, however, and it may present simultaneously with elements of chronic GVHD (“overlap syndrome”).116120

Treatment of Acute GVHD

GVHD generally first develops in the second month after HCT, during continued treatment with calcineurin-based prophylaxis.23121 Steroids, with their potent antilymphocyte and anti-inflammatory activity, are the gold standard for treatment of GVHD. Many centers treat mild GVHD of the skin (Grade I) with topical steroids alone, but for more severe skin GVHD and any degree of visceral GVHD involvement, high-dose systemic steroids are usually initiated. Steroid therapy results in complete remission in less than half of the patients,122 and more severe GVHD is less likely to respond to treatment.123124 In a prospective randomized study, the addition of ATG to steroids as primary therapy did not increase the response rate.124 In a retrospective study, the use of ATG in patients who showed early signs of steroid-resistance was beneficial,122 but not all studies show such benefit and ATG is not standardly used because of increased infection risks.106125126.

An increasingly common treatment for GVHD is extracorporeal photopheresis (ECP). During ECP, the patient’s white blood cells are collected by apheresis, incubated with the DNA-intercalating agent, 8-methoxypsoralen, exposed to ultraviolet light (UVA), and returned to the patient. ECP is known to induce cellular apoptosis, which has strong anti-inflammatory effects in a number of systems, including prevention of rejection of solid organ grafts.127 Animal studies show that ECP reverses acute GVHD by increasing the number of regulatory T cells.128 A Phase II clinical study of steroid-dependent or steroid refractory GVHD showed resolution of GVHD in a large majority of patients, with 50% long-term survival in this very high risk group.129 Randomized multi-center studies of this approach are needed to determine its place in the management of acute GVHD.

Another interesting strategy to treat GVHD is the blockade of the inflammatory cytokine TNF-α. TNF-α can activate APCs, recruit effector cells and cause direct tissue damage.130 In animal models, TNF-α plays a central role in GVHD of the GI tract, which is central to the “cytokine storm” and plasma levels of TNFR I (a surrogate marker for TNF-α) rise in patients before the clinical manifestations of GVHD appear. 51 A recent Phase II trial of etanercept, a solubilized TNFR II, showed significant efficacy when added to systemic steroids as primary therapy for acute GVHD. Seventy percent of patients had complete resolution of all GVHD symptoms within one month, with 80% complete responses in the GI tract and the skin. The authors also showed that plasma levels of TNFR I were a significant biomarker for clinical GVHD.131

Treatment of Chronic GVHD

In contrast to acute GVHD, the pathophysiology of chronic GVHD remains poorly understood, and it is treated with a variety of immunosuppressive agents. The response of chronic GVHD to treatment is unpredictable, and mixed responses in different organs can occur in the same patient. Confounding variables such as infection and co-morbidities also make responses hard to measure. The use of corticosteroids (with or without a calcineurin inhibitor) is the standard of care, but a randomized trial of more than 300 patients with chronic GVHD found no difference between cyclosporine plus prednisone versus prednisone alone.132 Chronic immunosuppressants, especially those containing steroids, are highly toxic and result in infectious deaths. Many second line therapies have been studied, but none has achieved widespread acceptance. As mentioned above, ECP shows some promise, with significant response rates in high-risk patients. The best responses were observed in skin, liver, oral mucosa, eye, and lung.133 This observation is particularly relevant because lung GVHD has the potential to be a particularly devastating complication necessitating lung transplant as the only therapeutic option.134135

Essential Supportive Care in GVHD Patients

Meticulous supportive care is critical for patients with both acute and chronic GVHD because of the extended duration of immunosuppressive treatments and because the multiple medications required may have synergistic toxicities. Such care includes extensive infectious prophylaxis, early interventions in cases of suspected infections, and prophylaxis against non-infectious side effects of medications (See Table 3). These complications often require rapid responses to prevent serious or irreversible damage, and are best handled in close collaboration between the primary physician and the transplant specialist.

Table 3

Recommendations for Supportive Care

All patients should receive at least fluconazole as prophylaxis against fungal infections. Invasive molds, especially aspergillus, are common in patients with prolonged steroid use.136 Prophylaxis with voriconazole or posaconazole should be considered for these patients. Usual sites of infection are the lungs, sinuses, brain, skin,137 and serial galactomannan assays may aid in the early detection.138 Candida can cause lesions in the lung and spleen, which may need screening with ultrasonography. Pneumocystis is another opportunistic infection that should receive cotrimoxazol (bactrim) prophylaxis.139

Viral infections are frequent in these patients with GVHD. Cytomegalovirus causes interstitial pneumonia and gastritis. Patients who are at risk should have their blood monitored several times monthly. Techniques that directly detect virus should be performed, such as CMV PCR or pp65 antigen, and evidence of increased viral load should prompt preemptive treatment with ganciclovir or foscarnet prior to clinical manifestations of disease. Shingles is not uncommon and acyclovir prophylaxis may be beneficial.140 Patients and caregivers should receive vaccinations against influenza, and treatment with neuraminidase inhibitors is recommended in the event of influenza infection.141142

Patients with GVHD often have IgG2 and IgG4 subclass deficiencies despite normal lgG levels, making them susceptible to infections with encapsulated organisms. Treatment of severe hypogammaglobulinemia with intravenous immunoglobulin is standard in many centers,143 but the level that triggers replacement varies considerably among transplant specialists. There is little supporting evidence for the routine use of intravenous immunoglobulin as prophylaxis144 but patients should receive routine prophylaxis (penicillin or its equivalent) due to the increased risk of streptococcal sepsis.145 Pneumococcal conjugate and hemophilus influenza vaccine may provide additional protection and are also recommended for all patients, including those with chronic GVHD.139146147 The sites of any indwelling catheters should be assessed regularly and early treatment of a suspected infection initiated. Early signs or symptoms of septic shock such as shaking chills or low blood pressure requires prompt evaluation with chest X-ray and/or CT scan, blood culture and broad spectrum antibiotics because shock may progress rapidly in these patients.

9.3.5 Aspergillus Complicating Allogeneic Transplantation

Aspergillus infections in allogeneic stem cell transplant recipients: have we made any progress?

E Jantunen, V-J Anttila and T Ruutu
BMT 2002; 30(12):925-929

Invasive aspergillosis (IA) is common in allogeneic SCT recipients, with an incidence of 4-10%. The majority of these infections are diagnosed several months after SCT and they are frequently associated with GVHD. The diagnosis is difficult and often delayed. Established IA is notoriously difficult to treat with a death rate of 80-90%. This review summarises recent data on this problem to assess whether there has been any progress. Effective prophylactic measures are still lacking. Severe immunosuppression is the main obstacle to the success of therapy. Recent and ongoing developments in diagnostic measures and new antifungal agents may improve treatment results to some extent, but Aspergillus infections still remain a formidable problem in allogeneic transplantation. Further studies in this field will focus on the role of various cytokines and combinations of antifungal agents.


Read Full Post »

Graft-versus-Host Disease

Writer and Curator: Larry H. Bernstein, MD, FCAP 



This piece is a follow up to the article on allogeneic transfusion reactions, which extends into transplantation and transplantation outcomes for hematological diseases, both malignant and nonmalignant. The safety of transfusions in Western countries has improved substantially, and the causes for transfusion mishaps has been reduced to unexpected infectious sources, uncommon immune incompatibilities, and errors in processing the blood products.  The greatest risk is incurred in platelet transfusions because of the short shelf-life of the product, and the time needed for testing prior to release.  This portion of the review is concerned with Graft-versus-Host Disease, which is unique to transfusion and transplanting of blood. In other transplantation, there is graft failure because of host versus graft incompatibility or complications.  The reverse order applies to blood.  In this case, on the contrary, the transfused or grafted donor tissue becomes a pursuer after the recipients hematopoietic cells.

Peter Brian Medawar: Father of Transplantation

Thomas E. Starzl, M.D., PH.D., F.A.C.S.
J Am Coll Surg. 1995 Mar; 180(3): 332–336

Most of the surgical specialities can be tracked to the creative vision of a surgeon. Transplantation is an exception. Here, the father of the field is succinctly defined in the dictionary as: “Peter Brian Medawar: a Brazilian born British Zoologist who at the age of 45 shared a 1960 Nobel Prize for his work on acquired immunologic tolerance”. Medawar was mysteriously overwhelming to many colleagues and observers, even when he was young. He was the son of a Lebanese father and an English mother—tall, athletic, abnormally handsome, hypnotically articulate in public, and politely cordial in his personal relations. In September 1969, at the age of 54, he had the first of a series of strokes. These crippled him physically but not in spirit. Although I saw Medawar often professionally and privately over a 22 year period, before and after he was disabled, this sporadic exposure was not enough to understand him. My sense is that no one did, except perhaps Jean, his wife for nearly 50 years.

Medawar’s dazzling personality before and great courage after his strokes was inspirational, but his fame was based on the unique achievement in 1953 captured by the terse dictionary mention of “acquired immunologic tolerance.” The roots leading to this accomplishment had fed on the blood of war. More than 12 years earlier, the recently wed zoologist Medawar—24 years of age and fresh from graduate studies at Oxford University—was assigned to
the service of the British surgeon, Dr. Thomas Gibson, to determine if skin allografts could be used to treat casualties from the Battle of Britain. First,
in human studies with Gibson, and then with simple and logical rabbit experiments, Medawar showed that rejection of the skin was an immunologic phenomenon. This later was shown  to be analogous to the cell-mediated delayed hypersensitivity that confers immunity to diseases such as tuberculosis. The principal evidence in the early studies was that repetitive grafts from the same donor were rejected more rapidly with each successive attempt —the sensitization and donor specificity confirming an earlier clinical observations by Emil Holman of Stanford in skin-grafted burn victims. Once it was established that rejection was an immune reaction, strategies began to evolve to weaken the recipient immune system. By 1953, total body irradiation and adrenal cortical steroids had been shown to delay skin rejection. However, this immunosuppressive effect was either minor if the animals survived, or lethal to the recipient if the grafts were spared.


In the resulting atmosphere of nihilism about clinical applications, a three and one-half page article by Billingham, Brent, and Medawar in the October 3, 1953 issue of Nature describing acquired tolerance, came as a blinding beacon of hope. The three men had learned that donor splenocytes could be engrafted by their intravenous infusion into immunologically immature mice in utero or perinatally. When these inoculated recipients matured, they could accept skin and other tissues from the donor (but from no other) mouse strain. The immune system of the recipients had been populated by the immunocytes of the donor, meaning that they were now chimeras. The race was on to convert this principle to humans. However, the dark side of their accomplishment soon was revealed by the two younger members of Medawar’s team, Billingham and Brent and by the Dane, Simonsen. The engrafted donor cells could turn the tables and reject the defenseless recipient unless the tissue match was a good one. This was the dreaded graft versus host disease (GVHD) in which transplanted donor cells attacked the recipient skin, gastrointestinal tract, lungs, liver, and the bone marrow itself. Medawar’s dream of 1953 was suddenly a nightmare. Or was it?

On the contrary, the work took a straight line to clinical application, after the demonstration by Prehn and Main that similar tolerance could be induced in adult mice rendered immunologically defenseless by total body irradiation before splenocyte (or later bone marrow) infusion. The recipient conditioning, known as cytoablation, also could be accomplished with myelotoxic drugs. However, as Billingham, Brent, and Medawar had predicted, donor specific tolerance could be induced in humans without GVHD only if there was a good tissue (HLA) match. In 1968, 15 years after the epic Billingham, Brent and Medawar publication, Robert Good and Fritz Bach reported the first two successful human bone marrow transplants. Both recipients of well matched bone marrow from blood relatives are still alive. This was a triumph in which the principal clinicians were internists, as summarized 25 years later in the acceptance speech by the 1990 Nobel Laureate Donnall Thomas.

The growth of bone marrow and whole organ transplantation

The growth of bone marrow and whole organ transplantation

The growth of bone marrow (right) and whole organ transplantation (left) from the seed planted by Peter Medawar during World War II. GVHD, Graft versus host disease.

Immunological Tolerance: Medawar Nobel Acceptance Lecture

“Immunological tolerance” may be described as a state of indifference or non-reactivity towards a substance that would normally be expected to excite an immunological response. The term first came to be used in the context of tissue transplantation immunity, i.e. of the form of immunity that usually prohibits the grafting of tissues between individuals of different genetic make-up; and it was used to refer only to a non-reactivity caused by exposing animals to antigenic stimuli before they were old enough to undertake an immunological response. For example, if living cells from a mouse of strain CBA are injected into an adult mouse of strain A, the CBA cells will be destroyed by an immunological process, and the A-line mouse that received them will destroy any later graft of the same origin with the speed to be expected of an animal immunologically forearmed. But if the CBA cells are injected into a foetal or newborn A-line mouse, they are accepted; more than that, the A-line mouse, when it grows up, will accept any later graft from a CBA donor as if it were its own. I shall begin by using the term “immunological tolerance” in the rather restricted sense that is illustrated by this experiment, and shall discuss its more general usage later on.

The experiment I have just described can be thought of as an artificial reproduction of an astonishing natural curiosity, the phenomenon of red-cell chimerism in certain dizygotic twins. The blood systems of twin cattle before birth are not sharply distinct from each other, as they are in most other twins; instead, the blood systems make anastomoses with each other, with the effect that the twins can indulge in a prolonged exchange of blood before birth. In 1945, R.D. Owen2 made the remarkable discovery that most twin cattle are born with, and may retain throughout life, a stable mixture – not necessarily a fifty-fifty mixture – of each other’s red cells; it followed, then, that the twin cattle must have exchanged red-cell precursors and not merely red cells in their mutual transfusion before birth. This is the first example of the phenomenon we came to call immunological tolerance; the red cells could not have “adapted” themselves to their strange environment, because they were in fact identified as native or foreign by those very antigenie properties which, had an adaptation occurred, must necessarily have been transformed. A few years later R.E. Billingham and I3, with the help of three members of the scientific staff of the Agricultural Research Council, showed that most dizygotic cattle twins would accept skin grafts from each other, and that this mutual tolerance was specific, for skin transplanted from third parties was cast off in the expected fashion.

Some properties of the tolerant state

The main points that emerged from our analysis of the tolerant state were these. In the first place, tolerance must be due to an alteration of the host, not to an antigenic adaptation of the grafted cells, for grafts newly transplanted in adult life have no opportunity to adapt themselves, and the descendants of the cells injected into foetal or newborn animals can be shown by N.A. Mitcbison’s methods to retain their antigenic power10. Once established, the state of tolerance is systemic; if one part of the body will tolerate a foreign graft, so will another; we found no evidence that a tolerated graft builds up a privileged position for itself within its own lymphatic territory. The stimulus that is responsible for instating tolerance is an antigenic stimulus – one which, had it been applied to older animals, would have caused them to become sensitive or immune. A plural stimulus can induce plural tolerance; the donor will usually contain several important antigens that are lacking in the recipient, and long-lasting tolerance must imply tolerance of them all. The state of tolerance is specific in the sense that it will discriminate between one individual and another, for an animal made tolerant of grafts from one individual will not accept grafts from a second individual unrelated to the first; but it will not discriminate between one tissue and another from the same donor.

Tolerance and auto-immunity: 50 years after Burnet.

Martini A1, Burgio GR
Eur J Pediatr. 1999 Oct;158(10):769-75.

Fifty years ago Sir F. Macfarlane Burnet published his first fundamental contribution to the theory of immune tolerance he perfected 10 years later. Since then an impressive amount of new information on the function of the immune system has been gathered. As any original meaningful theory, Burnet’s hypothesis on the development of immune tolerance has undergone extensive modifications to take into account all these new findings. An improved understanding of the mechanisms of tolerance has led to new possibilities for the treatment of auto-immune diseases.

Clonal Selection

Clonal selection theory is a scientific theory in immunology that explains the functions of cells (lymphocytes) of the immune system in response to specific antigens invading the body. The concept was introduced by an Australian doctor Frank Macfarlane Burnet in 1957 in an attempt to explain the formation of a diversity of antibodies during initiation of the immune response. The theory has become a widely accepted model for how the immune system responds to infection and how certain types of B and T lymphocytes are selected for destruction of specific antigens.

The theory states that in a pre-existing group of lymphocytes (specifically B cells), a specific antigen only activates (i.e. selection) its counter-specific cell so that particular cell is induced to multiply (producing its clones) for antibody production. In short the theory is an explanation of the mechanism for the generation of diversity of antibody specificity. The first experimental evidence came in 1958, when Gustav Nossal and Joshua Lederberg showed that one B cell always produces only one antibody. The idea turned out to be the foundation of molecular immunology, especially in adaptive immunity.

The fundamental contribution of Robert A. Good to the discovery of the crucial role of thymus in mammalian immunity

Domenico Ribatti
Immunology. 2006 Nov; 119(3): 291–295.

Robert Alan Good was a pioneer in the field of immunodeficiency diseases. He and his colleagues defined the cellular basis and functional consequences of many of the inherited immunodeficiency diseases. His was one of the groups that discovered the pivotal role of the thymus in the immune system development and defined the separate development of the thymus-dependent and bursa-dependent lymphoid cell lineages and their responsibilities in cell-mediated and humoral immunity.  Keywords: bursa of Fabricius, history of medicine, immunology, thymus

Robert Alan Good (May 21, 1922 – June 13, 2003) was an American physician who performed the first successful human bone marrow transplant

Robert A. Good began his intellectual and experimental queries related to the thymus in 1952 at the University of Minnesota, initially with pediatric patients. However, his interest in the plasma cell, antibodies and the immune response began in 1944, while still in Medical School at the University of Minnesota in Minneapolis, with his first publication appearing in 1945.

Idiopathic Acquired Agammaglobulinemia Associated with Thymoma (1953)

  • a markedly deficient ability to produce antibodies and significant deficits of all or most of the cell-mediated immunities
  • in no instance did removal of the thymic tumour restore immunological function or correct the protein deficit

Good syndrome: thymoma with immunodeficiency

  • increased susceptibility to bacterial infections by encapsulated organisms and opportunistic viral and fungal infections
  • immunodeficiencies, leukopenia, lymphopenia and eosinophylopenia
  • severely hypogammaglobulinemic rather than agammaglobulinemic

Good and others found that the patients lacked all of the subsequently described immunoglobulins. These patients were found not to have plasma cells or germinal centers in their hematopoietic and lymphoid tissues. They possessed circulating lymphocytes in normal numbers.

Speculation on the reason for immunological failure following neonatal thymectomy has centered on the thymus as a source of cells or humoral factors essential to normal lymphoid development and immunological maturation.

The bursa of Fabricius and the thymus are ‘central lymphoid organs’ in the chicken, essential to the ontogenetic development of adaptive immunity in that species. Studies by Papermaster and co-workers in Good’s laboratory34,35 indicated that bursectomy in the newly hatched chicks did not completely abolish immunological potential in the adult animal but rather produced a striking quantitative reduction insufficient to eliminate the homograft reaction. The failure of thymectomy in newly hatched chicks to alter the immunological potential of the maturing animal probably only reflected the participation of the bursa of Fabricius in the development of full immunological capacity.

Bursectomized and irradiated birds were completely devoid of germinal centers, plasma cells and the capacity to make antibodies yet they had perfectly normal development of thymocytes and lymphocytes elsewhere in the body that mediated cellular immune reactions. On the other hand, thymectomized and irradiated animals were deficient in lymphocytes that mediated cellular immunity as assessed by skin graft rejection, delayed-type hypersensitivity and graft versus host assays, but they still produced germinal centers, plasma cells and circulating immunoglobulins.


Graft vs Host Disease

Graft-versus-host disease (GVHD) is a complication that can occur after a stem cell or bone marrow transplant. With GVHD, the newly transplanted donor cells attack the transplant recipient’s body.

Graft-versus-host disease (GVHD) is a common complication following an allogeneic tissue transplant. It is commonly associated with stem cell or bone marrow transplant but the term also applies to other forms of tissue graft. Immune cells (white blood cells) in the tissue (the graft) recognize the recipient (the host) as “foreign“. The transplanted immune cells then attack the host’s body cells. GVHD can also occur after a blood transfusion if the blood products used have not been irradiated or treated with an approved pathogen reduction system.


GVHD may occur after a bone marrow or stem cell transplant in which someone receives bone marrow tissue or cells from a donor. This type of transplant is called allogeneic. The new, transplanted cells regard the recipient’s body as foreign. When this happens, the newly transplanted cells attack the recipient’s body.

GVHD does not occur when someone receives his or her own cells during a transplant. This type of transplant is called autologous.

Before a transplant, tissue and cells from possible donors are checked to see how closely they match the person having the transplant. GVHD is less likely to occur, or symptoms will be milder, when the match is close. The chance of GVHD is:

  • Around 30 – 40% when the donor and recipient are related
  • Around 60 – 80% when the donor and recipient are not related

There are two types of GVHD: acute and chronic. Symptoms in both acute and chronic GVHD range from mild to severe.

  • Acute GVHD usually happens within the first 6 months after a transplant.
  • Chronic GVHD usually starts more than 3 months after a transplant, and can last a lifetime.

Bone marrow transplant

A bone marrow transplant is a procedure to replace damaged or destroyed bone marrow with healthy bone marrow stem cells.  Stem cells are immature cells in the bone marrow that give rise to all of your blood cells.

There are three kinds of bone marrow transplants:

  • Autologous bone marrow transplant: The term auto means self. Stem cells are removed from you before you receive high-dose chemotherapy or radiation treatment. The stem cells are stored in a freezer (cryopreservation). After high-dose chemotherapy or radiation treatments, your stems cells are put back in your body to make (regenerate) normal blood cells. This is called a rescue transplant.
  • Allogeneic bone marrow transplant: The term allo means other. Stem cells are removed from another person, called a donor. Most times, the donor’s genes must at least partly match your genes. Special blood tests are done to see if a donor is a good match for you. A brother or sister is most likely to be a good match. Sometimes parents, children, and other relatives are good matches. Donors who are not related to you may be found through national bone marrow registries.
  • Umbilical cord blood transplant: This is a type of allogeneic transplant. Stem cells are removed from a newborn baby’s umbilical cord right after birth. The stem cells are frozen and stored until they are needed for a transplant. Umbilical cord blood cells are very immature so there is less of a need for matching. But blood counts take much longer to recover.

Before the transplant, chemotherapy, radiation, or both may be given. This may be done in two ways:

  • Ablative (myeloablative) treatment: High-dose chemotherapy, radiation, or both are given to kill any cancer cells. This also kills all healthy bone marrow that remains, and allows new stem cells to grow in the bone marrow.
  • Reduced intensity treatment, also called a mini transplant: Patients receive lower doses of chemotherapy and radiation before a transplant. This allows older patients, and those with other health problems to have a transplant.

Histocompatibility antigen:

  • A histocompatibility antigen blood test looks at proteins called human leukocyte antigens (HLAs). These are found on the surface of almost all cells in the human body. HLAs are found in large amounts on the surface of white blood cells. They help the immune system tell the difference between body tissue and substances that are not from your own body.

Induction of transplantation tolerance in haploidenical transplantation under reduced intensity conditioning: The role of ex-vivo generated donor CD8+ T cells with central memory phenotype

Eran Ophir, Y Eidelstein, E Bachar-Lustig, D Hagin, N Or-Geva, A Lask, , Y Reisner
Best Practice & Research Clinical Haematology 24 (2011) 393–401

Haploidentical hematopoietic stem cell transplantation (HSCT) offers the advantage of readily available family member donors for nearly all patients. A ‘megadose’ of purified CD34þ hematopoietic stem cells is used to overcome the host’s residual immunity surviving the myeloablative conditioning, while avoiding severe GVHD. However, the number of CD34+ cells that can be harvested is insufficient for overcoming the large numbers of host T cells remaining after reduced intensity conditioning (RIC). Therefore, combining a ‘megadose’ of CD34+ HSCT with other tolerizing cells could potentially support and promote successful engraftment of haploidentical purified stem cell transplantation under a safer RIC. One approach to address this challenge
could be afforded by using Donor CD8 T cells directed against 3rd-party stimulators, bearing an ex-vivo induced central memory phenotype (Tcm). These Tcm cells, depleted of GVH reactivity, were shown to be highly
efficient in overcoming host T cells mediated rejection and in promoting
fully mismatched bone-marrow (BM) engraftment, in HSCT murine models.
This is likely due to the marked lymph node homing of the Tcm, their strong proliferative capacity and prolonged persistence in BM transplant recipients. Thus, combining anti 3rd-party Tcm cell therapy with a ‘megadose’ of purified CD34+ stem cells, could offer a safer RIC protocol for attaining hematopoietic chimerism in patients with hematological diseases and as a platform for organ transplantation or cell therapy in cancer patients.

Induction of tolerance in organ recipients by hematopoietic stem cell transplantation

Eran Ophir, Yair Reisner
International Immunopharmacology 9 (2009) 694–700

The use of hematopoietic stem cell transplantation (HSCT) for the establishment of mixed chimerism represents a viable and attractive approach for generating tolerance in transplantation biology, as it generally leads to durable immune tolerance, enabling the subsequent engraftment of organ transplants without the need for a deleterious continuous immunosuppressive therapy. However, in order to apply HSCT to patients in a manner that enables long term survival, transplant-related mortality must be minimized by eliminating the risk for graft-versus-host-disease (GVHD) and by reducing the toxicity of the conditioning protocol. T-cell depleted bone marrow transplants (TDBMT) have been shown to adequately eliminate GVHD. However, even in leukemia patients undergoing supralethal conditioning, mismatched TDBMT are vigorously rejected. This barrier can be overcome through the modulatory activity of CD34 cells, which are endowed with veto activity, by the use of megadose stem cell transplants. In mice, megadoses of Sca+linhematopoietic stem cells can induce mixed chimerism following sub-lethal conditioning. Nevertheless, the number of human CD34 cells that can be harvested is not likely to be sufficient to overcome rejection under reduced intensity conditioning (RIC), which might be acceptable in recipients of organ transplantation. To address this challenge, we investigated a novel source of veto cells, namely anti 3rd-party cytotoxic T cells (CTLs) which are depleted of GVH reactivity, combined with megadoses of purified stem cells and a RIC protocol. This approach might provide a safer modality for the induction of durable chimerism.

Intrinsic unresponsiveness of Mertk/B cells to chronic graft-versus-host disease is associated with unmodulated CD1d expression

Wen-Hai Shao, Y Zhen, FD Finkelman, RA Eisenberg, PL Cohen
Journal of Autoimmunity 39 (2012) 412e419

Activation and migration of marginal zone B (MZB) cells into follicular (FO) regions of the spleen has been proposed as one of the mechanisms that regulate the development of autoreactive B cells. The mer receptor tyrosine kinase (Mertk) mediates apoptotic cell clearance and regulates activation and cytokine secretion. In the well-studied class II chronic GVH model of bm12 cells into B6 hosts, we observed that Mertk deficient B6 mice did not generate autoantibodies in response to this allogeneic stimulus. We posited that Mertk is important in MHC-II-mediated B cell signaling. In the present study, we show that B cells from Mertk-/- mice but not WT B6 mice exhibited decreased calcium mobilization and tyrosine phosphorylation when stimulated by MHC-II cross-linking. The finding that Mertk was important for class II signaling in B cells was further supported by the preponderance of a-allotype autoantibodies in cGVH in RAG-KO mice reconstituted with a mixture of bone marrow from Mertk-/-mice (b-allotype) and C20 mice (a-allotype). MZB cells from Mertk-/-  mice were unable to down regulate surface CD1d expression and subsequent inclusion in the MZ, associated with significantly lower germinal center responses compared to MZB cells from WT. Moreover, Mertk-/- mice treated with an anti-CD1d down regulating antibody responded significantly to bm12 cells, while no response was observed in Mertk-/- mice treated with control antibodies. Taken together, these findings extend the role of Mertk to include CD1d down regulation on MZB cells, a potential mechanism limiting B cell activation in cGVH.

Galectin-9 ameliorates acute GVH disease through the induction of T-cell apoptosis

Kazuki Sakai, Eri Kawata, Eishi Ashihara, Yoko Nakagawa, et al.
Eur. J. Immunol. 2011. 41: 67–75

Galectins comprise a family of animal lectins that differ in their affinity for β-galactosides. Galectin-9 (Gal-9) is a tandem-repeat-type galectin that was recently shown to function as a ligand for T-cell immunoglobin domain and mucin domain-3 (Tim-3) expressed on terminally differentiated CD41 Th1 cells. Gal-9 modulates immune reactions, including the induction of apoptosis in Th1 cells. In this study, we investigated the effects of Gal-9 in murine models of acute GVH disease (aGVHD). First, we demonstrated that recombinant human Gal-9 inhibit MLR in a dose-dependent manner, involving both Ca21 influx and apoptosis in T cells. Next, we revealed that recombinant human Gal-9 significantly inhibit the progression of aGVHD in murine BM transplantation models. In conclusion, Gal-9 ameliorates aGVHD, possibly by inducing T-cell apoptosis, suggesting that gal-9 may be an attractive candidate for the treatment of aGVHD.


GVHD Prevention: An Ounce Is Better Than a Pound

Pavan Reddy, Gerard Socie, Corey Cutler, Daniel Weisdorf
Biol Blood Marrow Transplant 18:S17-S26, 2012

The pathophysiology of acute graft-versus-host disease (aGVHD) is known to involve donor T cells responding to host histoincompatible allo-antigens presented by the host antigen presenting cells (APCs) and the subsequent induction of pro-inflammatory cytokines and cellular effectors that cause target organ damage. In a more general sense, GVHD can be considered as an immune response against foreign antigens that has gone awry. Similar to all immune responses, GVHD, can be understood as a process that consists of (A) triggers, (B) sensors, (C) mediators, and (D) effectors of GVHD.

Like all immune responses, certain triggers are critical for induction of acute graft-versus-host disease (aGVHD). These include: (1) Disparities between histocompatibility antigens: antigen disparity can be at the level of major histocompatibility complex (MHC), that is, MHC mismatched or at the level of minor histocompatibility antigens (miHA), that is, MHC matched but miHA mismatched. The severity of aGVHD is directly related to the degree of M HC mismatch. In bone marrow transplants (BMT) that are MHC matched but miHA disparate, donor T cells still recognize MHC-peptide derived from the products of recipient polymorphic genes, the miHAs.

Damage induced by conditioning regimens and underlying diseases: under most circumstances, the initiation of an adaptive immune response is triggered by the innate immune response. The innate immune system is triggered by certain exogenous and endogenous molecules. This is likely the case in the induction of aGVHD. Pattern recognition receptors such as Toll-like receptors (TLR), nucleotide-binding oligomerization domain containing 2 (NOD2) play an essential role in innate immunity and in initiating the cellular signaling pathways that activate cytokine secretion, such as NF-kB. Some of their ligands, such as lipopolysaccharide, CpG, and MDP2, which is recognized by TLR-4, TLR-9, and NOD2, respectively, are released by the preparative regimens and contribute to the induction and enhancement of allo-T cell responses. In this way, the conditioning regimens amplify the secretion of proinflammatory cytokines like interleukin (IL)-1, tumor necrosis factor (TNF)-α,  IL-6, and other interferon family members in a process described as a ‘‘cytokine storm.’’

The triggers that initiate an immune response have to be sensed and presented. APCs might be considered the sensors for aGVHD. The APCs sense the DAMPs, present the MHC disparate or miHA disparate protein, and provide the critical secondary (costimulatory) and tertiary (cytokine) signals for activation of the alloreactive T cells, the mediators of aGVHD. APCs sense allo-disparity through MHC and peptide complexes. Dendritic cells (DCs) are the most potent APCs and the primary sensors of allo-disparity.

APCs provide the critical costimulation signals for turning on the aGVHD process. The interaction between the MHC/allopeptide complex on APCs and the T cell receptor of donor T cells along with the signal via T cell costimulatory molecules and their ligands on APCs is required to achieve T cell activation, proliferation, differentiation, and survival and the in vivo blockade of positive costimulatory molecules (such as CD28, ICOS, CD40, CD30, etc.), or inhibitory signals (such as PD-1 and CTLA-4) mitigate or exacerbate aGVHD, respectively.

Evidence suggests that alloreactive donor T cells consist of several subsets with different stimuli responsiveness, activation thresholds, and effector functions.

The allo-antigen composition of the host determines which donor T cells subsets differentiate and proliferate. As mentioned previously, in the majority of HLA-matched HCT, aGVHD may be induced by either or both CD41 and CD81 subsets responses to miHAs. The repertoire and immunodominance of the GVHD-associated peptides presented by MHC class I and class II molecules has not been defined. Donor naive CD62L1 T cells are the primary alloreactive T cells that drive the GVHD reaction while the donor effector memory CD62L2 T cells do not. Interestingly, donor regulatory T cells (Tregs) expressing CD62L are also critical to the regulation of GVHD. We now know that it is possible to modulate the alloreactivity of na€ıve T cells by inducing anergy with costimulation blockade, deletion via cytokine modulation, or mixed chimerism. Donor effector memory T cells that are nonalloreactive do not induce GVHD, yet are able to transfer functional memory. In contrast, memory T cells that are alloreactive can cause severe GVHD.

The effector phase that leads to GVHD target organ damage is a complex cascade that involves cytolytic cellular effectors such as CD8 cytotoxic T lymphocytes (CTLs), CD4 T cells, natural killer cells, and inflammatory molecules such as IL-1β, TNF-α, IFN-ϒ, IL-6, and reactive oxygen species. The cellular effectors require cell-cell contact to kill the cells of the target tissues via activation of perforin granzyme, Fas-FasL (CD95-CD95L), or TNFR TRAIL pathways. Other CTLs killing mechanisms such as TWEAK, and LTβ/LIGHT pathways have also been implicated in GVHD. It is important to note that
CTL pathways are essential for GVL effects as well.

All of the above aspects of the biology of aGVHD have been summarized in the mold of a normal immune response. Although this allows for accessing the biology of GVHD, it is important to note that GVHD is a complicated systemic process with as yet still many unknowns and is not a simplified, linear, or cyclical process.

Kinetics of CD4+ and CD8+ T-cell subsets in graft-versus-host reaction (GVHR) in ginbuna crucian carp Carassius auratus langsdorfii

Yasuhiro Shibasakia, H Todaa, Isao kobayashib, T Moritomoa, T Nakanishia
Developmental and Comparative Immunology 34 (2010) 1075–1081

We have previously demonstrated the presence of graft-versus-host reaction (GVHR) in fish employing a model system of clonal triploid ginbuna and tetraploid ginbuna-goldfish hybrids. To elucidate the role of CD8+ T cells in the induction of GVHR, we investigate the kinetics of CD4+ and CD8+ T-cell subsets in GVHR along with the pathological changes associated with GVH disease (GVHD) in ginbuna. GVHR was not induced with a leukocyte fraction lacking CD8+ T cells separated by magnetic cell sorting. Ploidy and immunofluorescence analysis revealed that CD4+ and CD8+  T cells from sensitized donors greatly

increased in the host trunk kidney, constituting more than 80% of total cells 1–2 weeks after donor cell injection, while those from non-sensitized donors constituted less than 50% of cells present. The increase of CD4+ T cells was greater and more rapid than that of CD8+ T cells. The number of donor CD4+ and CD8+ T cells was highest in trunk kidney followed by spleen. Increases in donor CD4+ and CD8+ T cells were also found in liver and PBL, although the percentages were not as high. Pathologic changes similar to those in human and murine acute GVHD were observed in the lymphoid organs as well as target organs such as skin, liver and intestine, including the destruction of cells and tissues and massive leukocyte infiltration. The pathologic changes became more severe with the increase of CD8+ T cells. These results suggest that donor-derived CD8+ T cells play essential roles for the induction of acute GVHR/D in teleosts as in mammals.

Fludarabine and Exposure-Targeted Busulfan Compares Favorably with Busulfan/Cyclophosphamide-Based Regimens in Pediatric Hematopoietic
Cell Transplantation: Maintaining Efficacy with Less Toxicity

I.H. Bartelink, E.M.L. van Reij, C.E. Gerhardt, E.M. van Maarseveen, et al
Biol Blood Marrow Transplant 20 (2014) 345e353

Busulfan (Bu) is used as a myeloablative agent in conditioning regimens before allogeneic hematopoietic cell transplantation (allo-HCT). In line with strategies explored in adults, patient outcomes may be optimized by replacing cyclophosphamide (Cy) with or without melphalan (Mel) with fludarabine (Flu). We compared outcomes in 2 consecutive cohorts of HCT recipients with a nonmalignant HCT indication, a myeloid malignancy, or a lymphoid malignancy with a contraindication for total body irradiation (TBI). Between 2009 and 2012, 64 children received Flu + Bu at a target dose of 80-95 mg-h/L, and between 2005 and 2008, 50 children received Bu targeted to 74-80 mg-h/L þ Cy. In the latter group, Mel was added for patients with myeloid malignancy (n = 12). Possible confounding effects of calendar time were studied in 69 patients receiving a myeloablative dose of TBI between 2005 and 2012. Estimated 2-year survival and event-free survival were 82% and 78%, respectively, in the FluBu arm and 78% and 72%, respectively, in the BuCy (Mel) arm (P,  not significant). Compared with the BuCy (Mel) arm, less toxicity was noted in the FluBu arm, with lower rates of acute (noninfectious) lung injury (16% versus 36%; P < .007), veno-occlusive disease (3% versus 28%; P < .003), chronic graft-versus-host disease (9% versus 26%; P < .047), adenovirus infection (3% versus 32%; P < .001), and human herpesvirus 6 infection reactivation (21% versus 44%; P < .005). Furthermore, the median duration of neutropenia was shorter in the FluBu arm (11 days versus 22 days; P < .001), and the patients in this arm required fewer transfusions. Our data indicate that Flu (160 mg/m2) with targeted myeloablative Bu (90 mg-h/L) is less toxic than and equally effective
as BuCy (Mel) in patients with similar indications for allo-HCT.

Fibrotic and Sclerotic Manifestations of Chronic Graft-versus-Host Disease

Carrie L. Kitko, Eric S. White, Kristin Baird
Biol Blood Marrow Transplant 18:S46-S52, 2012

Chronic graft-versus-host disease (cGVHD) is a common cause of morbidity
and mortality following allogeneic stem cell transplantation (HCT), with approximately 50% to 60% of long-term HCT survivors developing one or more manifestations of the disorder. Although acute GVHD is typically limited to skin, liver, and gastrointestinal involvement, virtually every organ is at risk for the development of cGVHD. Although the pathophysiology of cGVHD remains poorly understood, some of the most severe organ manifestations are linked by end-organ fibrosis. In particular, fibrotic cutaneous and bronchiolar changes, resulting in scleroderma-like changes and bronchiolitis obliterans syndrome (BOS), respectively, are two of the most devastating outcomes for these patients. Both sclerotic GVHD (ScGVHD) and BOS have been reported in 5% to 15% of patients with cGVHD.

Many of the manifestations of cGVHD share clinical characteristics seen in nontransplant conditions, including systemic sclerosis or pulmonary fibrosis. Thus, understanding the pathophysiology underlying these related conditions may help identify potential mechanisms and ultimately new therapeutic options for patients with cGVHD.

Tyrosine kinase inhibitors (TKIs) have been shown to inhibit two different profibrotic pathways (transforming growth factor β [TGF-β] and platelet-derived growth factor [PDGF]) in various mouse models of fibrotic disease and offer a possible novel treatment approach for cGVHD patients suffering from severe sclerosis. Likewise, overexpression of TNF-α has been shown to induce fibrogenesis in experimental hepatocellular disease and has been linked with human scleroderma-associated interstitial pulmonary fibrosis and profibrotic responses in human osteoarthritic hip joint fibroblasts. The use of TNF antagonists has been examined in some clinical situations associated with fibrosis, suggesting they may also be of some benefit to patients with cGVHD; however, this must first be prospectively tested.

Table. Proposed Modifications to NIH BOS Clinical Definition

  • Absence of infection (no change)
  • Another cGVHD manifestation in another organ (no change)
  • FEV1 <75% predicted (no change) or >10% decline from pre-HCT value (modification)
  • Signs of Obstruction
  • FEV1/SVC ratio <0.7 (modification), or
  • RV >120% predicted (no change), or
  • RV/TLC >120% (modification), and
  • HRCT with evidence of air trapping (no change)

SVC indicates slow vital capacity; RV, residual volume; TLC, total lung capacity; HRCT, high-resolution computed tomography

Figure (not shown)
Effect of etanercept on survival in post-HCT patients with subacute lung injury. (A) Overall 5-year survival by pulmonary function testing defect. Patients with an obstructive defect (solid line) had a 5-year survival of 67% compared with 44% in those with a restrictive lung defect (dashed line) (P 5 .19). (B) Overall 5-year survival by response to therapy. Patients who responded to etanercept therapy (solid line) had a 5-year survival of 90% compared with 55% in patients who failed to respond (dashed line) (P 5.07). (Figures reprinted with permission, Biol Blood and Marrow Trans).

Extensive, sclerotic skin changes with superficial or deep subcutaneous or fascial involvement are seen in approximately 4% to 13% of patients with cGVHD and can be a life-threatening manifestation. ScGVHD of the skin includes several cutaneous presentations characterized by inflammation and progressive fibrosis of the dermis and subcutaneous tissues. These changes can resemble morphea, systemic sclerosis, or eosinophilic fasciitis and may or may not occur in the setting of concurrent overlying epidermal GVHD. When severe, ScGVHD can result in contractures, severe wasting, and chest wall restriction.

Development of clinical trials for patients with cGVHD is difficult because of the complexity and heterogeneity of disease, variable approaches to treatment, and the lack of standardized assessments of disease. In particular, the study of ScGVHD lacks universally accepted measures of disease burden and response. Investigators have used several measures to assess ScGVHD involvement including body surface area, magnetic resonance imaging, ultrasound, and range-of-motion measurements. Additionally, investigators have tried to apply the Rodnan score, the standardmeasure for skin involvement in scleroderma. Thus far, none of these measures has proven
to be completely reliable in the setting of ScGVHD, and it is likely that multiple measures will need to be integrated into the assessment of ScGVHD.

Imatinib mesylate (Gleevec in the US; Glivec in Europe, Australia, and Latin America, marketed by Novartis) is a TKI that has biological activity against both PDGF and TGF-β signaling pathways. Both cytokines have been implicated in the pathogenesis of several fibrosing diseases, including hepatic, renal, and lung, as well as in scleroderma, a disease that closely resembles ScGVHD. In addition, stimulatory antibodies specific for the PDGF receptor (PDGFR) were identified in a series of 39 patients with extensive cGVHD with higher levels detected in those patients with skin involvement. Similar stimulatory antibodies targeting PDGFR have been reported in patients with scleroderma, suggesting an important therapeutic target for these fibrosing conditions. Imatinib mesylate has particularly potent activity against PDGF and is FDA approved in the United States for the treatment of several disorders associated with aberrant PDGFR signaling. The side effect profile of the drug is well established in non-HCT patients, which is helpful in the setting of a therapy for allogenic HCT patients, many of whom have multiorgan system symptoms and possible dysfunction and who will require ongoing immunosuppressive therapy.

Through the efforts of the Chronic GVHD Consortium, led by Stephanie Lee at the Fred Hutchinson Cancer Research Center, there is a multicenter, ongoing prospective evaluation of the NIH diagnostic and assessment tools. This effort has already resulted in several publications that have further refined essential criteria for cGVHD evaluation, including organ-specific manifestations such as BOS and ScGVHD. Currently, the Consortium is conducting a multicenter prospective clinical trial of fluticasone propionate, azithromycin, and montelukast for the treatment of BOS ( NCT01307462); a separate trial of imatinib versus rituximab for treatment of ScGVHD is also enrolling subjects ( NCT01309997).

Although cGVHD remains a significant problem for many long-term survivors of HCT, critical advances in cGVHD research and treatment can be achieved by cooperative group efforts such as those put forth by the Chronic GVHD Consortium and the Clinical Trials Network.

Hematopoietic stem cell transplantation (HSCT): An approach to autoimmunity

Carmen Alaez, Mariana Loyola, Andrea Murguıa, Hilario Flores, et al.
Autoimmunity Reviews 5 (2006) 167– 179

HSCT provides the opportunity to replace a damaged tissue. It is the most important treatment for high risk hematologic malignant and nonmalignant disorders. An important challenge in the identification of matched donors/patients is the HLA diversity. The Mexican Bone Marrow Registry (DONORMO) has nowadays N5000 donors. The prevalent alleles are Amerindian, Mediterranean (Semitic and Spanish genes) and African. In theory, it is possible to find 11% of 6/6 A–B–DR low resolution matches for 70% of patients with Mexican ancestry. We contributed with 39 unrelated, cord blood and autologous HSCT for patients with malignant, genetic and autoimmune disorders. Overall disease survival was 50% (2–7 years) depending on the initial diagnosis, conditioning, disease evolution or other factors. Clinical studies using autologous and unrelated HSC are performed on patients with refractory autoimmune diseases producing mixed results: mainly, T1D, RA, MS, SLE. Improvement has been observed in skin damage and quality of life in SLE and systemic sclerosis. Disease stabilization in 2/3 of MS patients. However, in RA and T1D, initial benefits have been followed by eventual relapse. With growing clinical experience and protocol improvement, treatment-related mortality is decreasing. Proof efficacy will be achieved by comparing HSCT with standard therapy in autoimmunity.

Monoclonal Antibody-Mediated Targeting of CD123, IL-3 Receptor α Chain, Eliminates Human Acute Myeloid Leukemic Stem Cells

Liqing Jin, Erwin M. Lee, Hayley S. Ramshaw, Samantha J. Busfield, et al.
Cell: Stem Cell 5, 31–42, July 2, 2009

Leukemia stem cells (LSCs) initiate and sustain the acute myeloid leukemia (AML) clonal hierarchy and possess biological properties rendering them resistant to conventional chemotherapy. The poor survival of AML patients raises expectations that LSC-targeted therapies might achieve durable remissions. We report that an anti-interleukin-3 (IL-3) receptor α chain (CD123)-neutralizing antibody (7G3) targeted AML-LSCs, impairing homing
to bone marrow (BM) and activating innate immunity of nonobese diabetic/ severe-combined immunodeficient (NOD/SCID) mice. 7G3 treatment profoundly reduced AML-LSC engraftment and improved mouse survival.
Mice with preestablished disease showed reduced AML burden in the BM
and periphery and impaired secondary transplantation upon treatment, establishing that AMLLSCs were directly targeted. 7G3 inhibited IL-3-mediated intracellular signaling of isolated AML CD34+ CD38[1] cells in vitro and reduced their survival. These results provide clear validation for therapeutic monoclonal antibody (mAb) targeting of AML-LSCs and for translation of in vivo preclinical research findings toward a clinical application.

Many Days at Home during Neutropenia after Allogeneic Hematopoietic Stem Cell Transplantation Correlates with Low Incidence of Acute Graft-versus-Host Disease

Olle Ringdén, Mats Remberger, Katarina Holmberg, Charlotta Edeskog, et al.
Biol Blood Marrow Transplant 19 (2013) 314e320

Patients are isolated in the hospital during the neutropenic phase after allogeneic hematopoietic stem cell transplantation. We challenged this by allowing patients to be treated at home. A nurse from the unit visited and checked the patient. One hundred forty-six patients treated at home were compared with matched hospital control subjects. Oral intake was intensified from September 2006 and improved (P < .002). We compared 4 groups: home care and control subjects before and after September 2006. The cumulative incidence of acute graft-versus-host disease (GVHD) of grades II to IV was 15% in the “old” home care group, which was significantly lower than that of 32% to 44% in the other groups (P <.03). Transplantation-related mortality, chronic GVHD, and relapse were similar in the groups. The “new” home care patients spent fewer days at home (P < .002). In multivariate analysis, GVHD of grades 0 to I was associated with home care (hazard ratio [HR], 2.46; P <.02) and with days spent at home (HR, .92; P < .005) but not with oral nutrition (HR, .98; P = .13). Five year survival was 61% in the home care group as compared with 49% in the control subjects (P < .07). Home care is safe. Home care and many days spent at home were correlated with a low risk of acute GVHD.

Impact on Outcomes of Human Leukocyte Antigen Matching by Allele-Level Typing in Adults with Acute Myeloid Leukemia Undergoing Umbilical Cord Blood Transplantation

Jaime Sanz, Francisco J. Jaramillo, Dolores Planelles, Pau Montesinos, et al.
Biol Blood Marrow Transplant 20 (2014) 106e110

This retrospective study analyzed the impact of directional donor-recipient human leukocyte antigen (HLA) disparity using allele-level typing at HLA-A, -B, -C, and -DRB1 in 79 adults with acute myeloid leukemia (AML) who received single-unit umbilical cord blood (UCB) transplant at a single institution. With extended high resolution HLA typing, the donor-recipient compatibility ranged from 2/8 to 8/8. HLA disparity showed no negative impact on nonrelapse mortality (NRM), graft-versus-host (GVH) disease or engraftment. Considering disparities in the GVH direction, the 5-year cumulative incidence of relapse was 44% and 22% for patients receiving an UCB unit matched > 6/8 and < 6/8, respectively (P <.04). In multivariable analysis, a higher HLA disparity in the GVH direction using extended high-resolution typing (Risk ratio [RR] 2.8; 95% confidence interval [CI], 1.5 to 5.1; P ¼.0009) and first complete remission at time of transplantation (RR 2.1; 95% CI, 1.2 to 3.8; P < .01) were the only variables significantly associated with an improved disease-free survival. In conclusion, we found that in adults with AML undergoing single-unit UCBT, an increased number of HLA disparities at allele-level typing improved disease-free survival by decreasing the relapse rate without a negative effect on NRM.

HLA mismatch direction in cord blood transplantation: impact on outcome and implications for cord blood unit selection
Cladd E. Stevens, C Carrier, C Carpenter, D Sung, and A Scaradavou

Blood. 2011; 118(14):3969-3978

Donor-recipient human leukocyte antigen mismatch level affects the outcome of unrelated cord blood (CB) transplantation. To identify possible “permissive” mismatches, we examined the relationship between  direction of human leukocyte antigen mismatch (“vector”) and transplantation outcomes in 1202 recipients of single CB units from the New York Blood Center National Cord Blood Program treated in United States Centers from 1993-2006. Altogether, 98 donor/patient pairs had only unidirectional mismatches: 58 in the graft-versus-host (GVH) direction only (GVH-O) and 40 in the host-versus-graft or rejection direction only (R-O). Engraftment was faster in patients with GVH-O mismatches compared with those with 1 bidirectional mismatch (hazard ratio [HR] = 1.6, P < .003). In addition, patients with hematologic malignancies given GVH-O grafts had lower transplantation-related mortality (HR = 0.5, P < .062), overall mortality (HR = 0.5, P < .019), and treatment failure (HR = 0.5, P < .016), resulting in outcomes similar to those of matched CB grafts. In contrast, R-O mismatches had slower engraftment, higher graft failure, and higher relapse rates (HR = 2.4, P < .010). Based on our findings, CB search algorithms should be modified to identify unidirectional mismatches. We recommend that transplant centers give priority to GVH-O-mismatched units over other mismatches and avoid selecting R-O mismatches, if possible.

Mutation of the NPM1 gene contributes to the development of donor cell–derived acute myeloid leukemia after unrelated cord blood transplantation
for acute lymphoblastic leukemia

G Rodríguez-Macías, C Martínez-Laperche, J Gayoso, V Noriega, .., Ismael Buño
Human Pathology (2013) 44, 1696–1699

Donor cell leukemia (DCL) is a rare but severe complication after allogeneic stem cell transplantation. Its true incidence is unknown because of a lack of correct recognition and reporting, although improvements in molecular analysis of donor-host chimerism are contributing to a better diagnosis of this complication. The mechanisms of leukemogenesis are unclear, and multiple factors can contribute to the development of DCL. In recent years, cord blood has emerged as an alternative source of hematopoietic progenitor cells, and at least 12 cases of DCL have been reported after unrelated cord blood transplantation. We report a new case of DCL after unrelated cord blood transplantation in a 44-year-old woman diagnosed as having acute lymphoblastic leukemia with t(1;19) that developed acute myeloid leukemia with normal karyotype and nucleophosmin (NPM1) mutation in donor cells. To our knowledge, this is the first report of NPM1 mutation contributing to DCL development.

Graft-versus-leukemia in the bone marrow
Blood, 23 JAN 2014; 123(4)

63-year-old female with relapsed acute myeloid leukemia (AML) after allogeneic stem cell transplantation reached CR2 after re-induction therapy followed by consolidation with donor lymphocyte infusions: 3 x 107/kg and 3 x 108/kg after 1 and 2.5 months, respectively. No signs of graft-versus-host disease were observed at this time. At 5 months follow-up, her blood count deteriorated: hemoglobin: 6.9 mmol/L, thrombocytes: 58 x 109/L and leukocytes: 1.37 x 109/L. Bone marrow aspirate was not evaluable. Bone marrow trephine biopsy showed relapse AML with hypercellularity in the H&E staining (340 objective lens, panel A) and 20% CD341 blast cells without any signs of maturation (panel B). Also, a high number of CD3 positive T cells (panel C) was noted, intermingling with the CD34 positive blasts, both staining positively with CD43 (panel D). Only supportive care was given. However, normalization of the blood count was observed in the following months and she developed graft-versus-host disease of the lung, which was treated with ciclosporin and prednisone. A bone marrow aspirate performed 3 months after relapse showed a third remission with 0.8% myeloid blasts. In retrospect, one could therefore consider the picture of the bone marrow trephine biopsy at the second relapse as graft-versus-leukemia in the bone marrow.

GVL- panel A

GVL- panel A

GVL - panel B

GVL – panel B

GVL - panel C

GVL – panel C

GVL - panel D

Long-Term Outcomes of Alemtuzumab-Based Reduced-Intensity Conditioned Hematopoietic Stem Cell Transplantation for Myelodysplastic Syndrome and Acute Myelogenous Leukemia Secondary to Myelodysplastic Syndrome

Victoria T. Potter, Pramila Krishnamurthy, Linda D. Barber, ZiYi Lim, et al.
Biol Blood Marrow Transplant 20 (2014) 111e117

Allogeneic hematopoietic stem cell transplantation (HSCT) with reduced-intensity conditioning (RIC) offers a potential cure for patients with myelodysplastic syndrome (MDS) who are ineligible for standard-intensity regimens. Previously published data from our institution suggest excellent outcomes at 1 yr using a uniform fludarabine, busulfan, and alemtuzumab-based regimen. Here we report long-term follow-up of 192 patients with MDS and acute myelogenous leukemia (AML) secondary to MDS (MDS-AML) transplanted with this protocol, using sibling (n = 45) or matched unrelated (n = 147) donors. The median age of the cohort was 57 yr (range, 21 to 72 yr), and median follow-up was 4.5 yr (range, 0.1 to 10.6 yr). The 5-yr overall survival (OS), event-free survival, and nonrelapse mortality were 44%, 33%, and 26% respectively. The incidence of de novo chronic graft-versus-host disease (GVHD) was low at 19%, illustrating the efficacy of alemtuzumab for GVHD prophylaxis. Conversely, the 5-yr relapse rate was 51%. For younger patients (age <50 yr), the 5-yr OS and relapse rates were 58% and 39%, respectively. On multivariate analysis, advanced age predicted significantly worse outcomes, with patients age >60 yr having a 5-yr OS of 15% and relapse rate of 66%. Patients receiving preemptive donor lymphocyte infusions had an impressive 5-yr OS of 67%, suggesting that this protocol may lend itself to the incorporation of immunotherapeutic strategies. Overall, these data demonstrate good 5-yr OS for patients with MDS and MDS-AML undergoing alemtuzumab-based RIC-HSCT. The low rate of chronic GVHD is encouraging, and comparative studies with other RIC protocols are warranted.

Natural killer cell activity influences outcome after T cell depleted stem cell transplantation from matched unrelated and haploidentical donors

Peter Lang, Matthias Pfeiffer,  Heiko-Manuel Teltschik, Patrick Schlegel, et al.
Best Practice & Research Clinical Haematology 24 (2011) 403–411

Lytic activity and recovery of natural killer (NK) cells was monitored in pediatric patients with leukemias (ALL, AML, CML, JMML) and myelodysplastic syndromes after transplantation of T cell depleted stem cells from matched unrelated (n = 18) and mismatched related (haploidentical, n = 29) donors. CD34+ selection with magnetic microbeads resulted in 8 x 103/kg residual T cells. No post-transplant immune suppression was given. NK cells recovered rapidly after transplantation (300 CD56+/mL at day 30, median), whereas T cell recovery was delayed (median: 12 CD3+/mL at day 90). NK activity was measured as specific lysis of K 562 targets several times (mean: 3 assays per patient). Four temporal patterns of lytic activity could be differentiated: consistently low, consistently high, decreasing and increasing activity. Patients with consistently high or increasing activity had significantly lower relapse probability than patients with consistently low or decreasing levels (0.18 vs 0.73 at 2 years, p < 0.05). The subgroup of patients with ALL showed similar results (0.75 vs 0.14 at 2 years, p < 0.05). Speed of T cell recovery had no influence. These data suggest that both achieving and maintaining a high level of NK activity may contribute to prevent relapse. Since NK activity could be markedly increased by in vitro stimulation with Interleukin 2 (IL-2), in vivo administration should be considered.

Graft-versus-host disease: Pathogenesis and clinical manifestations of graft-versus-host disease

Sharon R. Hymes, Amin M. Alousi,  and Edward W. Cowen
J Am Acad Dermatol  2012; 66: 515.e1-18.

  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality in patients who undergo allogeneic hematopoietic cell transplantation.
  • Acute graft-versus-host disease manifests as a skin exanthem, liver dysfunction, and gastrointestinal involvement.
  • Chronic graft-versus-host disease of the skin is remarkably variable in its clinical presentation.
  • Chronic graft-versus-host disease is a multisystem disorder that may affect nearly any organ; the most common sites are the skin, oral mucosa, and eyes.

Key points

  • Allogeneic transplantation is in widespread use for hematologic malignancies, but is also increasingly used for marrow failure syndromes, immunodeficiencies, and other life-threatening conditions
  • Graft-versus-host disease is the primary cause of morbidity and nonerelapse related mortality after allogeneic hematopoietic cell transplantation
  • Minimizing graft-versus-host disease without losing the graft-versus-tumor effect is an area of active research
  • The skin is the most common organ affected in patients with graft-versus-host disease

Outcomes of Thalassemia Patients Undergoing Hematopoietic Stem Cell Transplantation by Using a Standard Myeloablative versus a Novel Reduced-Toxicity Conditioning Regimen According to a New Risk Stratification

Usanarat Anurathapan, S Pakakasama, P Mekjaruskul, N Sirachainan, et al.
Biol Blood Marrow Transplant 20 (2014) 2056e2075

Improving outcomes among class 3 thalassemia patients receiving allogeneic hematopoietic stem cell transplantations (HSCT) remains a challenge. Before HSCT, patients who were > 7 years old and had a liver size > 5 cm constitute what the Center for International Blood and Marrow Transplant Research defined as a very high risk subset of a conventional high-risk class 3 group (here referred to as class 3 HR). We performed HSCT in 98 patients with related and unrelated donor stem cells. Seventy-six of the patients with age < 10 years received the more conventional myeloablative conditioning (MAC) regimen (cyclophos-phamide, busulfan,  + fludarabine); the remaining 22 patients with age > 10 years and hepatomegaly (class 3 HR), and in several instances additional comorbidity problems, underwent HSCT with a novel reduced-toxicity conditioning (RTC) regimen (fludarabine and busulfan). We then compared the outcomes between these 2 groups (MAC versus RTC). Event-free survival (86% versus 90%) and overall survival (95% versus 90%) were not significantly different between the respective groups; however, there was a higher incidence of serious treatment-related complications in the MAC group, and although we experienced 6 graft failures in the MAC group (8%), there were none in the RTC group. Based on these results, we suggest that (1) class 3HRthalassemia patients can safely receive HSCT with our novel RTC regimen and achieve the same excellent outcome as low/standard-risk thalassemia patients who received the standard MAC regimen, and further, (2) that this novel RTC approach should be tested in the low/standard-risk patient population.

Pharmacological Immunosuppression Reduces But Does Not Eliminate the Need for Total-Body Irradiation in Nonmyeloablative Conditioning Regimens for Hematopoietic Cell Transplantation

Marco Mielcarek, Beverly Torok-Storb, Rainer Storb
Biol Blood Marrow Transplant 17: 1255-1260 (2011)

In the dog leukocyte antigen (DLA)-identical hematopoietic cell transplantation (HCT) model, stable marrow engraftment can be achieved with total-body irradiation (TBI) of 200 cGy when used in combination with postgrafting immunosuppression. The TBI dose can be reduced to 100 cGy without compromising engraftment rates if granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMC) are infused with the marrow. T cell-depleting the G-PBMC product abrogates this effect. These results were interpreted to suggest that the additional T cells provided with G-PBMC facilitated engraftment by overcoming host resistance.We therefore hypothesized that the TBI dose may be further reduced to 50 cGy by augmenting immunosupression either by (1) tolerizing or killing recipient T cells, or (2) enhancing the graft-versus-host (GVH) activity of donor T cells. To test the first hypothesis, recipient T cells were activated before HCT by repetitive donor-specific PBMC infusions followed by administration of methotrexate (MTX) (n 5 5), CTLA4-Ig (n = 4), denileukin diftitox (Ontak; n = 4), CTLA4-Ig 1 MTX (n = 8), or 5c8 antibody (anti-CD154) 1 MTX (n = 3). To test the second hypothesis, recipient dendritic cells were expanded in vivo by infusion of Flt3 ligand given either pre-HCT (n = 4) or pre- and post-HCT (n = 5) to augment GVH reactions. Although all dogs showed initial allogeneic engraftment, sustained engraftment was seen in only 6 of 42 dogs (14% of all dogs treated in 9 experimental groups). Hence, unless more innovative pharmacotherapy can be developed that more forcefully shifts the immunologic balance in favor of the donor, noncytotoxic immunosuppressive drug therapy as the sole component of HCT preparative regimens may not suffice to ensure sustained engraftment.

Pretransplant Immunosuppression followed by Reduced-Toxicity Conditioning and Stem Cell Transplantation in High-Risk Thalassemia: A Safe Approach to Disease Control

Usanarat Anurathapan, S Pakakasama, P Rujkijyanont, N Sirachainan, et al.
Biol Blood Marrow Transplant 19 (2013) 1254e1270

Patients with class 3 thalassemia with high-risk features for adverse events after high-dose chemotherapy with hematopoietic stem cell transplantation (HSCT) are difficult to treat, tending to either suffer serious toxicity or fail to establish stable graft function. We performed HSCT in 18 such patients age 7 years and hepatomegaly using a novel approach with pretransplant immunosuppression followed by a myeloablative reduced-toxicity conditioning regimen (fludarabine and i.v. busulfan [Flu-IV Bu]) and then HSCT. The median patient age was 14 years (range, 10 to 18 years). Before the Flu-IV Bu þ antithymocyte globulin conditioning regimen, all patients received 1 to 2 cycles of pretransplant immunosuppression with fludarabine and dexamethasone. Thirteen patients received a related donor graft, and 5 received an unrelated donor graft. An initial prompt engraftment of donor cells with full donor chimerism was observed in all 18 patients, but 2 patients developed secondary mixed chimerism that necessitated withdrawal of immunosuppression to achieve full donor chimerism. Two patients (11%) had acute grade III-IV graft-versus-host disease, and 5 patients had limited chronic graft-versus-host disease. The only treatment-related mortality was from infection, and with a median follow-up of 42 months (range, 4 to 75), the 5-year overall survival and thalassemia-free survival were 89%. We conclude that this novel sequential immunoablative pretransplant-ation conditioning program is safe and effective for patients with high-risk class 3 thalassemia exhibiting additional comorbidities.

Profiling antibodies to class II HLA in transplant patient sera

Curtis McMurtrey, D Lowe, R Buchli, S Daga, D Royer, A Humphrey, et al.
Human Immunology 75 (2014) 261–270

Immunizing events including pregnancy, transfusions, and transplantation promote strong alloantibody responses to HLA. Such alloantibodies to HLA preclude organ transplantation, foster hyperacute rejection, and contribute to chronic transplant failure. Diagnostic antibody-screening assays detect alloreactive antibodies, yet key attributes including antibody concentration and isotype remain largely unexplored. The goal here was to provide a detailed profile of allogeneic antibodies to class II HLA. Methodologically, alloantibodies were purified from sensitized patient sera using an HLA-DR11 immunoaffinity column and subsequently categorized. Antibodies to DR11 were found to fix complement, exist at a median serum concentration of 2.3 lg/mL, consist of all isotypes, and isotypes IgG2, IgM, and IgE were elevated. Because multimeric isotypes can confound diagnostic determinations of antibody concentration, IgM and IgA isotypes were removed and DR11-IgG tested alone. Despite removal of multimeric isotypes, patient-to patient antibody concentra-tions did not correlate with MFI values. In conclusion, allogeneic antibody responses to DR11 are comprised of all antibody isotypes at differing proportions, these combined isotypes fix complement at nominal serum concentrations, and enhancements other than the removal of IgM and IgA multimeric isotypes may be required if MFI is to be used as a means of determining anti-HLA serum antibody concentrations in diagnostic clinical assays.

Reduced-intensity conditioning and HLA-matched hemopoietic stem-cell transplantation in patients with chronic granulomatous disease: a prospective multicenter study

Tayfun Güngör, P Teira, M Slatter, G Stussi, P Stepensky, D Moshous, et al.
Lancet 2014; 383: 436–48

Background In chronic granulomatous disease allogeneic hemopoietic stem-cell transplantation (HSCT) in adolescents and young adults and patients with high-risk disease is complicated by graft-failure, graft-versus-host disease (GVHD), and transplant-related mortality. We examined the effect of a reduced-intensity conditioning regimen designed to enhance myeloid engraftment and reduce organ toxicity in these patients.       Methods This prospective study was done at 16 centers in ten countries worldwide. Patients aged 0–40 years with chronic granulomatous disease were assessed and enrolled at the discretion of individual centers. Reduced-intensity conditioning consisted of high-dose fludarabine (30 mg/m² [infants <9 kg 1∙2 mg/kg]; one dose per day on days –8 to –3), serotherapy (anti-thymocyte globulin [10 mg/kg, one dose per day on days –4 to –1; or thymoglobulin 2·5 mg/kg, one dose per day on days –5 to –3]; or low-dose alemtuzumab [<1 mg/kg on days –8 to –6]), and low-dose (50–72% of myeloablative dose) or targeted busulfan administration (recommended cumulative area under the curve: 45–65 mg/L × h). Busulfan was administered mainly intravenously and exceptionally orally from days –5 to –3. Intravenous busulfan was dosed according to weight-based recommendations and was administered in most centers (ten) twice daily over 4 h. Unmanipulated bone marrow or peripheral blood stem cells from HLA-matched related donors or HLA-9/10 or HLA-10/10 matched unrelated-donors were infused. The primary endpoints were overall survival and event-free survival (EFS), probabilities of overall survival and EFS at 2 years, incidence of acute and chronic GVHD, achievement of at least 90% myeloid donor chimerism, and incidence of graft failure after at least 6 months of follow-up. Findings 56 patients (median age 12∙7 years; IQR 6·8–17·3) with chronic granulomatous disease were enrolled from June 15, 2003, to Dec 15, 2012. 42 patients (75%) had high-risk features (ie, intractable infections and autoinflammation), 25 (45%) were adolescents and young adults (age 14–39 years). 21 HLA-matched related-donor and 35 HLA-matched unrelated-donor transplants were done. Median time to engraftment was 19 days (IQR 16–22) for neutrophils and 21 days (IQR 16–25) for platelets. At median follow-up of 21 months (IQR 13–35) overall survival was 93% (52 of 56) and EFS was 89% (50 of 56). The 2-year probability of overall survival was 96% (95% CI 86∙46–99∙09) and of EFS was 91% (79∙78–96∙17). Graft-failure occurred in 5% (three of 56) of patients. The cumulative incidence of acute GVHD of grade III–IV was 4% (two of 56) and of chronic graft-versus-host disease was 7% (four of 56). Stable (≥90%) myeloid donor chimerism was documented in 52 (93%) surviving patients. Interpretation This reduced-intensity conditioning regimen is safe and efficacious in high-risk patients with chronic granulomatous disease.

Refinement of the Definition of Permissible HLA-DPB1 Mismatches with Predicted Indirectly ReCognizable HLA-DPB1 Epitopes

Kirsten A. Thus, MTA Ruizendaal, TA de Hoop, Eric Borst, et al.
Biol Blood Marrow Transplant 20 (2014) 1705e1710

Hematopoietic stem cell transplantation with HLA-DPB1emismatched donors leads to an increased risk of acute graft-versus-host disease (GVHD). Studies have indicated a prognostic value for classifying HLA-DPB1 mismatches based on T cell epitope (TCE) groups. The aim of this study was to determine the contribution of indirect recognition of HLA-DPe derived epitopes, as determined with the Predicted Indirectly ReCognizable HLA Epitopes (PIRCHE) method. We therefore conducted a retrospective single-center analysis on 80 patients transplanted with a 10/10 matched unrelated donor that was HLA-DPB1 mismatched. HLADPB1 mismatches that were classified as GVH nonpermissive by the TCE algorithm correlated to higher numbers of HLA class I as well as HLA class II presented PIRCHE (PIRCHE-I and -II) compared with permissive or host-versus-graft nonpermissive mismatches. Patients with acute GVHD grades II to IV presented significantly higher numbers of PIRCHE-I compared with patients without acute GVHD (P < .05). Patients were divided into 2 groups based on the presence or absence of PIRCHE. Patients with PIRCHE-I or -II have an increased hazard of acute GVHD when compared with patients without PIRCHE-I or -II (hazard ratio [HR], 3.19; 95% confidence interval [CI], 1.10 to 9.19; P <.05; and HR, 4.07; 95% CI, .97 to 17.19; P < .06, respectively). Patients classified as having an HLA-DPB1 permissive mismatch by the TCE model had an increased risk of acute GVHD when comparing presence of PIRCHE-I with absence of PIRCHE-I (HR, 2.96; 95% CI, .84 to 10.39; P < .09). We therefore conclude that the data presented in this study describe an attractive and feasible possibility to better select permissible HLA-DPB1 mismatches by including both a direct and an indirect recognition model.

Treosulfan-Thiotepa-FludarabineeBased Conditioning Regimen for
Allogeneic Transplantation in Patients with Thalassemia Major: A
Single-Center Experience from North India

Dharma Choudhary, SK Sharma, N Gupta,…, Satyendra Katewa
Biol Blood Marrow Transplant 19 (2013) 492e503

Hematopoietic stem cell transplantation (HSCT) is the definite treatment
for patients with thalassemia major. A busulfan (Bu) and cyclophosphamide
(Cy)ebased regimen has been the standard myeloablative chemotherapy,
but it is associated with higher treatment-related toxicity, particularly in
patients classified as high risk by the Pesaro criteria. Treosulfan-based
conditioning regimens have been found to be equally effective and less
toxic. Consequently, we analyzed the safety and efficacy of treosulfan/
thiotepa/fludarabine (treo/thio/flu)-based conditioning regimens for
allogeneic HSCT in patients with thalassemia major between February
2010 and September 2012. We compared those results retrospectively
with results in patients who underwent previous HSCT with a Bu/Cy/
antithymocyte globulin (ATG)ebased conditioning regimen. A treo/thio/
flu-based conditioning regimen was used in 28 consecutive patients with
thalassemia major. The median patient age was 9.7 years (range, 2-18
years), and the mean CD34+ stem cell dose was 6.18 x 106/kg. Neutrophil
and platelet engraftment occurred at a median of 15 days (range, 12-23
days) and 21 days (range, 14-34 days), respectively. Three patients
developed veno-occlusive disease, 4 patients developed acute graft
versus-host disease (GVHD), and 2 patients had chronic GVHD. Treatment-
related mortality (TRM) was 21.4%. Two patients experienced secondary
graft rejection. We compared these results with results in patients who
underwent previous HSCT using a Bu/Cy/ATG-based conditioning regimen.
Twelve patients were treated with this protocol, at a median age of 7.2
years (range, 2-11 years). One patient had moderate veno-occlusive disease,
2 patients developed acute GVHD, 2 patients had chronic GVHD, and 2
patients experienced graft rejection. There was no TRM in this group. We
found no significant differences between the 2 groups (treo/thio/flu vs Bu/
Cy/ATG) in terms of the incidence of acute GVHD, chronic GVHD, TRM,
and graft failure, although a trend toward higher TRM was seen with the
treo/thio/flu regimen.

Graft-versus-Host Disease
James L.M. Ferrara, John E. Levine, Pavan Reddy, and Ernst Holler
Lancet. 2009 May 2; 373(9674): 1550–1561

The number of allogeneic hematopoietic cell transplantations (HCT)
continues to increase with more than 25,000 allogeneic transplantations
performed annually. The graft-versus leukemia/ tumor (GVL) effect during
allogeneic HCT effectively eradicates many hematological malignancies.
The development of novel strategies that use donor leukocyte infusions,
non-myeloablative conditioning and umbilical cord blood (UCB)
transplantation have helped expand the indications for allogeneic HCT
over the last several years, especially among older patients. Improvements
in infectious prophylaxis, immunosuppressive medications, supportive care
and DNA-based tissue typing have also contributed to improved outcomes
after allogeneic HCT. Yet the major complication of allogeneic HCT, graft-
versus-host disease (GVHD), remains lethal and limits the use of this
important therapy. Given current trends, the number of transplants from
unrelated donors is expected to double within the next five years,
significantly increasing the population of patients with GVHD. In this
seminar we review advances made in identifying the genetic risk
factors and pathophysiology of this major HCT complication, as well
as its prevention, diagnosis and treatment.

Non-HLA Genetics—Despite HLA identity between a patient and donor,
approximately 40% of patients receiving HLA-identical grafts develop
acute GVHD due to genetic differences that lie outside the HLA loci,
or “minor” histocompatibility antigens (HA). Some minor HAs, such as HY
and HA-3, are expressed on all tissues and are targets for both GVHD
and GVL. Other minor HAs, such as HA-1 and HA-2, are expressed most
abundantly on hematopoietic cells (including leukemic cells) and may
therefore induce a greater GVL effect with less GVHD. Polymorphisms
in both donors and recipients for cytokines that are involved in the
classical `cytokine storm’ of GVHD have been implicated as risk factors
for GVHD. Tumor Necrosis Factor (TNF)-α, Interleukin 10 (IL-10),
Interferon-γ (IFNγ) variants have correlated with GVHD in some, but
not all, studies. Genetic polymorphisms of proteins involved in innate
immunity, such as nucleotide oligomerization domain 2 and Keratin 18
receptors, have also been associated with GVHD.

Future strategies to identify the best possible transplant donor will
probably incorporate both HLA and non-HLA genetic factors. Skin is most
commonly affected and is usually the first organ involved, often coinciding
with engraftment of donor cells. The characteristic maculopapular rash is
pruritic and can spread throughout the body, sparing the scalp. In severe
cases the skin may blister and ulcerate. Apoptosis at the base of epidermal
rete pegs is a characteristic pathologic finding. Other features include
dyskeratosis, exocytosis of lymphocytes, satellite lymphocytes adjacent
to dyskeratotic epidermal keratinocytes, and a perivascular lymphocytic
infiltration in the dermis.

Gastrointestinal tract involvement of acute GVHD usually presents as
diarrhea but may also include vomiting, anorexia, and/or abdominal pain
when severe. The diarrhea of GVHD is secretory and often voluminous
(greater than two liters per day). Bleeding, which carries a poor prognosis,
occurs as a result of mucosal ulceration but patchy involvement of the
mucosa often leads to a normal appearance on endoscopy.

The incidence of the severity of acute GVHD is determined by the extent
of involvement of  three principal target organs. The overall grades are
classified as I (mild), II (moderate), III (severe) and IV (very severe). Severe
GVHD carries a poor prognosis, with 25% long term survival for grade III and
5% for grade IV. The incidence of acute GVHD is directly related to the
degree of mismatch between HLA proteins and ranges from 35-45% in
recipients of full matched sibling donor grafts to 60-80% in recipients of
one-antigen HLA mismatched unrelated donor grafts. The same degree
of mismatch causes less GVHD using UCB grafts and incidence of acute
GVHD is lower following the transplant of partially matched UCB units
and ranges from 35-65%.

Two important principles are important to consider regarding the
pathophysiology of acute GVHD. First, acute GVHD reflects exaggerated
but normal inflammatory mechanisms mediated by donor lymphocytes infused
into the recipient where they function appropriately, given the foreign
environment they encounter. Second, the recipient tissues that stimulate
donor lymphocytes have usually been damaged by underlying disease,
prior infections, and the transplant conditioning regimen. As
a result, these tissues produce molecules (sometimes referred to as
“danger” signals) that promote the activation and proliferation of donor
immune cells.  Based largely on experimental models, the development
of acute GVHD can be conceptualized in three sequential steps or phases:
(1) activation of the APCs; (2) donor T cell activation, proliferation,
differentiation and migration; and (3) target tissue destruction.

Alemtuzumab is a monoclonal antibody that binds CD52, a protein
expressed on a broad spectrum of leukocytes including lymphocytes,
monocytes, and dendritic cells. Its use in GVHD prophylaxis in a
Phase II trial decreased the incidence of acute and chronic GVHD
following reduced intensity transplant.98 In two prospective studies,
patients who received alemtuzumab rather than methotrexate showed
significantly lower rates of acute and chronic GVHD, but experienced
more infectious complications and higher rates of relapse, so that there
was no overall survival benefit. Alemtuzumab may also contribute to
graft failure when used with minimal intensity conditioning regimens.

An alternative strategy to TCD attempted to induce anergy in donor
T cells by ex vivo antibody blockade of co-stimulatory pathways prior
to transplantation. A small study using this approach in haploidentical
HCT recipients was quite encouraging, but has not yet been replicated.
Thus the focus of most prevention strategies remains  pharmacological
manipulation of T cells after transplant.

Administration of anti-T cell antibodies in vivo as GVHD prophylaxis
has also been extensively tested. The best studied drugs are anti-
thymocyte globulin (ATG) or antilymphocyte globulin (ALG) preparations.
These sera, which have high titers of polyclonal antibodies, are made
by immunizing animals (horses or rabbits) to thymocytes or lymphocytes,
respectively. A complicating factor in determining the role of these
polyclonal sera in transplantation is the observation that even different
brands of the same class of sera exert different biologic effects. However,
the side effects of ATG/ALG infusions are common across different
preparations and include fever, chills, headache, thrombocytopenia
(from cross-reactivity to platelets), and, infrequently, anaphylaxis. In
retrospective studies, rabbit ATG reduced the incidence of GVHD in
related donor HSCT recipients without appearing to improve survival.
In recipients of unrelated donor HSCT, addition of ALG to standard
GVHD prophylaxis effectively prevented severe GVHD, but did not
result in improved survival because of increased infections. In a long
term follow-up study, however, pretransplant ATG provided significant
protection against extensive chronic GVHD and chronic lung dysfunction.

As allogeneic transplantation becomes an increasingly attractive therapeutic
option, the need for novel approaches to GVHD has accelerated. The
number of patients receiving transplants from unrelated donors is
expected to double in the next five years, significantly increasing
the population of patients with GVHD. The advent of RIC regimens
has reduced transplant-related mortality and lengthened the period
during which acute GVHD may develop (many new cases present up
to day 200) and the need for close monitoring of patients in this period
has increased. Patients have often returned to the care of their primary
hematologists by this time, increasing the need for these physicians to
collaborate with transplant specialists in the management of GVHD and
its complications.

Identification of biomarkers for GVHD with diagnostic (and possibly
prognostic) significance may eventually make the treatment of GVHD
preemptive rather than prophylactic. The use of cellular component therapy,
such as regulatory T cells that have been expanded ex vivo. will also
enter clinical trials in the near future, but the extensive infrastructure
required for such cellular approaches will likely limit their use initially.

Immunomodulatory Effects of Palifermin (Recombinant Human
Keratinocyte Growth Factor) in 
an SLE-Like Model of Chronic
Graft-Versus-Host Disease

C. A. Ellison, Y. V. Lissitsyn, I. Gheorghiu & J. G. Gartner
Scandinavian Journal of Immunology 2011; 75, 69–76

Keratinocyte growth factor (KGF) promotes epithelial cell proliferation
and survival. Recombinant human KGF, also known as palifermin, protects
epithelial cells from injury induced by chemicals, irradiation and acute murine
graft versus-host disease (GVHD). Findings from our studies and others
have shown that palifermin also has immunomodulatory properties. In a
model of acute GVHD, we showed that it shifts the immune response
from one in which Th1 cytokines dominate to mixed Th1 and Th2 cytokine
profile. Using the DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-hybrid model of chronic,
systemic lupus erythematosus-like GVHD, we showed that palifermin
treatment is associated with higher levels of Th2 cytokines, the production
of anti-nuclear antibodies, cryoglobulinemia and the development of more
severe pathological changes in the kidney. The aim of our current study
was to gain a better understanding of the immunobiology of KGF by
further characterizing the palifermin-mediated effects in this model of
chronic GVHD. Because the pathological changes we observed resemble
those seen in thymic stromal lymphopoietin (TSLP) transgenic mice, we
had originally hypothesized that palifermin might augment TSLP levels.
Surprisingly, we did not observe an increase in thymic

TSLP mRNA expression in palifermin-treated recipients. We did, however,
observe some differences in the percentages of CD4+CD25+Foxp3+
regulatory T cells in the spleen at some time points in palifermin-treated
recipients. Most importantly, we found that TGFβ levels were higher in
palifermin-treated recipients early in the GVH reaction, raising the
possibility that KGF might indirectly induce the development of fibrosis
and glomerulonephritis through a pathway involving TGFβ.

Keratinocyte growth factor (KGF) is an epithelial cell growth factor that is
produced by both mesenchymal cells and intraepithelial cdT cells. It is
also known as fibroblast growth factor 7. Its receptor, (KGFR⁄FGF7R), an
alternatively spliced form of FGFR2 ⁄ bek, is found on epithelial cells in
the intestine, mammary glands, ovaries and urinary tract, and on
hepatocytes, keratinocytes and alveolar type II cells. Previously, it
was shown that recombinant human KGF, also known as palifermin,
can protect the lung, bladder or intestine from chemical- or irradiation-
induced injury. This has been attributed to the ability of KGF to reduce
oxidative damage and enhance DNA repair.

Our own studies have provided a better understanding of the immuno-
biological properties of KGF in pathologically distinct models of systemic
disease driven by intense immunological and inflammatory responses.
The acute GVHD that develops in the C57BL ⁄ 6 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model is characterized by the activation of alloreactive donor T cells,
the production of Th1 cytokines and tissue injury in the skin, gastrointestinal
tract, liver, thymus and lung, where epithelia are present. Injury to the
intestinal mucosa permits the translocation of endotoxin into the system,
which, if untreated, leads to the development of endotoxemic shock. We
showed that palifermin treatment protects recipients from epithelial
cell injury, endotoxemia and morbidity in GVH mice. Palifermin also
shifts the immune response away from one that is predominated by Th1
cytokines towards a profile of mixed Th1 and Th2 cytokines, with a
preponderance of Th2 cytokines. The DBA⁄ 2 fi (C57BL ⁄ 6 · DBA⁄ 2)F1-
hybrid model of chronic GVHD is characterized by pathological changes
resembling those seen in systemic lupus erythematosus (SLE). Using this
model, we showed that palifermin treatment augments the production of Th2
cytokines such as IL-4, IL-5 and IL-13 and obviates IFN-c production. Both
untreated and palifermin-treated recipients developed pathological changes
in the kidney, but these changes were more severe in palifermin-treated
recipients. Some of the changes that developed in the palifermin-treated
recipients resemble those seen in thymic stromal lymphopoietin (TSLP)
transgenic mice. These similarities include the presence of ANA in the
sera, the development of cryoglobulinemia and the development of
glomerulonephritis featuring the deposition of immune complexes
consisting of IgG, IgA, IgM and C3 in the mesangium and the glomerular
capillaries. This led us to hypothesize that treating the recipient mice with
palifermin might induce TSLP expression in this model.

In this study, we were interested in determining whether palifermin
treatment was indeed associated with increased TSLP expression.
We were also interested in knowing whether palifermin treatment
changes the percentage of CD4+CD25+FoxP3+ cells in the spleen,
because palifermin treatment has been associated with increased
percentages of CD4+CD25+FoxP3+ cells in other studies including
our own. Lastly, we wished to study the effect of palifermin treatment
on TGFb levels, because this cytokine is known to play a pivotal role
in the development of glomerulonephritis.

We studied the histopathological changes to confirm that the pathological
changes seen in the kidney in this study were the same as those reported
by us previously.We examined kidney sections from both untreated and
palifermin-treated recipients. In these experiments, we were able to
reproduce findings from an earlier study that showed that palifermin-
treated recipients mice in this model of chronic GVHD develop a severe,
extracapillary proliferative glomerular nephritis characterized by epithelial
crescents and hyaline thrombi. These changes were associated with higher
levels of protein in the urine and the development of ascites, presumably
related to the development of nephrotic syndrome, as a consequence
of glomerular injury.

Pathological changes in the kidney

Pathological changes in the kidney. (A) shows a section from a BDF1-hybrid control
mouse that did not receive a graft. (B) shows increased epithelial cellularity within a
glomerulus from an untreated recipient with chronic graft-versus-host disease, on
day 50. No crescents were observed in sections from this group of recipients.
(C and D) show examples of pathological changes observed in kidneys from
palifermin-treated recipients on day 50. Arrows indicate examples of crescentic
glomerulonephritis and the development of protein casts within tubular lumena.
(E and F) show examples of the hyaline thrombi (arrows) seen in the glomeruli
in kidney sections from palifermin-treated recipients on day 50. All sections
were stained with haematoxylin and eosin except for that shown in (F), which
was stained with Masson Trichrome. The concentration of protein measured in
the urine is shown in the lower left corner of each photomicrograph. Original
magnification: ·200 (B–E) and ·400 (A and F).

TGFβ is a highly pleiotropic cytokine with three isoforms, TGFβ1, TGFβ2 and
TGFβ3 . Nearly, all cells have receptors for at least one of these isoforms,
but cells of the immune system primarily express TGFβ1. This cytokine
was implicated in the development of experimental glomerulonephritis in
experiments in which rats were treated with antiserum directed against
TGFβ1. The ability of palifermin to induce TGFβ release and reverse
limited airflow was demonstrated in a mouse model of emphysema. The
authors further showed that palifermin induced the release of TGFβ1
from primary cultures of mouse alveolar type 2 cells. Our results show
that palifermin treatment is associated with a rise in splenic TGFβ levels
during the first month of the GVH reaction. It is possible that by inducing
TGFβ production shortly after transplantation, palifermin treatment is able
to promote the development of the severe, crescentic glomerulonephritis
that we observed at later time points. As such, our findings raise the
possibility that endogenous KGF might play a role in the development
of glomerulonephritis and ⁄ or other autoimmune phenomena associated
with chronic GVHD and ⁄ or SLE.

T cells, murine chronic graft-versus-host disease and autoimmunity

Robert A. Eisenberg, Charles S. Via
Journal of Autoimmunity 39 (2012) 240e247

The chronic graft-versus-host disease (cGVHD) in mice is characterized by
the production of autoantibodies and immunopathology characteristic of
systemic lupus erythematosus (lupus). The basic pathogenesis involves
the cognate recognition of foreign MHC class II of host B cells by alloreactive
CD4 T cells from the donor. CD4 T cells of the host are also necessary for
the full maturation of host B cells before the transfer of donor T cells.
CD8 T cells play critical roles as well. Donor CD8 T cells that are highly
cytotoxic can ablate or prevent the lupus syndrome, in part by killing
recipient B cells. Host CD8 T cells can reciprocally downregulate donor
CD8 T cells, and thus prevent them from suppressing the autoimmune
process. Thus, when the donor inoculum contains both CD4 T cells and
CD8 T cells, the resultant syndrome depends on the balance of activities
of these various cell populations. For example, in one cGVHD model
(DBA/2 (C57BL/6xDBA/2)F1, the disease is more severe in females, as
it is in several of the spontaneous mouse models of lupus, as well as in
human disease. The mechanism of this female skewing of disease
appears to depend on the relative inability of CD8 cells of the female host
to downregulate the donor CD4 T cells that drive the autoantibody response.
In general, then, the abnormal CD4 T cell help and the modulating roles
of CD8 T cells seen in cGVHD parallel the participation of T cells in
genetic lupus in mice and human lupus, although these spontaneous
syndromes are presumably not driven by overt alloreactivity.

Systemic lupus erythematosus (SLE) is characterized by a spectrum of auto-
antibodies that targets multiple normal cellular components, particularly
nucleic acids or proteins that are physiologically bound to nucleic acids.
Although SLE is highly diverse in its manifestations, a common theme
is the loss of B cell tolerance to these cellular autoantigens. More than
for any other human condition, several spontaneously arising mouse
models for SLE have been described, beginning with the New Zealand
strains in 1959. These models are largely genetic. In some cases, an
individual gene such as fas or Yaa plays a major role in driving the loss
of tolerance. However, in general the genetic contribution is complex and
involves multiple loci, which are not yet fully defined.

Despite extensive investigations, the failures in immunoregulation that
underlie the genetic SLE models remain poorly understood. It is not known
for sure which B cell tolerance checkpoints are breached in a given model,
and why. The autoantibody response to DNA, Sm, and other autoantigens
resembles the normal response to exogenous antigens: it involves clonal
expansion, somatic mutation, and a pattern of isotype use characteristic of
a T-cell dependent immunization. Thus the cellular dynamics of the response
may be basically normal. Yet the B-cell repertoire is abnormally autoreactive.

In this review we wish to focus more on the role of the T cell in SLE. As
stated above, the loss of B cell tolerance in SLE does appear in general
to require the participation of T cells. Multiple T cells abnormalities have
been described in human and in murine SLE, although in most cases it is
not clear if these are primary or secondary manifestations. Nevertheless,
it is striking how difficult it has been to demonstrate definitively the specificity
of the T cells that provide help for autoantibody production.

The key cellular mechanism in the cGVHD that results in the loss of B cell
tolerance and the production of the autoantibodies typical of SLE is the
cognate interaction of CD4 T cells with an MHC class II determinant on
the B cell surface. A variety of protocols have achieved this interaction.
In general, either the donor/recipient strains are paired in such away
that they only differ at the MHC class II loci, or the CD4 cells are isolated
free of CD8 cells that would recognize MHC class I. If the allorecognition
involves both CD4 T cell interaction with MHC II and CD8 interaction with
MHC I, an acute GVHD occurs, which is immunosuppressive, rather than
immunostimulatory. The DBA/2 (C57BL/6 DBA/2)F1 (B6D2F1) and the
BALB/c (BALB/c A/J)F1 models are exceptions to this rule. The former
has been investigated extensively for a deficiency in CD8 cytotoxic

The MHC class II recognition may be at either the I-A or the I-E locus.
However, the autoantibody specificities seen and the degree of immuno-
pathology differ depending on the locus targeted. In one set of experiments,
F1 mice were bred between either B6 or coisogenic bm12 mice and
B10.A(2R) or B10.A(4R) MHC recombinant congenics. The MHC class II
of B6 is I-Ab, while that of bm12 is I-Abm12. These two alleles differ by
only three amino acids, which is sufficient for a full strength MLR (mixed
lymphocyte reaction) between the two strains. Otherwise B6 and bm12
are identical. B10.A(2R) and B10.A(4R) differ only by the expression of
I-E in the former strain, but not in the latter strain. Thus, donor/recipient
combinations could be employed that provided for allogeneic differs only
at I-A, only at I-E, or at both loci.

Results from Busser et al. delineate requirements for this MHC class II
recognition. Utilizing several transgenic mouse strains that express a
more or less constricted CD4 autoreactive repertoire, they showed that
a diverse repertoire was essential to the production of SLE autoantibodies
by MHC II recognition. On the other hand, the non-specific, early polyclonal
B cell activation phase of cGVHD occurred even with a limited CD4 repertoire.

Figure not shown. Chronic GVHD in bm12 C57BL/6 mice. The MHC of the
bm12 donor differs from the MHC of the C57BL/6 recipient just in three
amino acids in the I-A class II molecule. Thus donor CD4 T cells recognize
MHC IIþ B cells as foreign. Donor CD8 T cells see only self MHC I. All T
cells do not express MHC II. Polyclonal activation and specific lupus
autoantibody responses ensue..

Lupus can result from unchecked CD4 T cell cognate help to a polyclonal
population of B cells. CD8 T cells can downregulate this CD4 driven B-cell
hyperactivity through CD8 CTL effectors and can maintain remission,
possibly through memory CD8 T cells. Whether CD8 CTL actually prevent
lupus in normals and fail in lupus prone individuals is not known; however,
data from the P F1 model suggest that therapeutic induction of CD8 CTL
and possibly long term memory cells may be beneficial in preventing or
limiting disease expression. The potential major role played by either
IFNa and IL-21 in both lupus expression and CD8 CTL function remains
to be further defined, but already these cytokines are being targeted in
human or murine lupus.

It is not surprising that the T cells have been shown to have diverse roles in
the autoimmune cGVHD in mice. Donor CD4 T cells drive the host B cell
activation, while host CD4 T cells are required to mature these B cells prior
to their encounter with donor T cells. Donor CD4 T cells also help activate
donor CD8 T cells, which in turn can downregulate or even ablate the
autoimmune response. Donor CD4 T cells license host DC cells, which in
turn can interact with donor CD8 T cells. Host CD8 T cells can suppress
the activity of donor CD8 T cells, and thereby favor the development of
the lupus syndrome. Although the precise mechanisms of T cell participation
in spontaneous lupus are still being defined, it seems reasonable to probe
these syndromes in humans and in mice for T cell mechanism that have
been shown to participate in cGVHD, CD4-B cell interactions almost
certainly are central to the pathogenesis of spontaneous lupus, and they
have been a target of investigation for several decades. If we understood
the peptide specificity of the alloreactive CD4 T cells that drive the formation
of the characteristic lupus autoantibodies, we would have a much clearer
idea where to look for such epitopes in spontaneous disease. Much less
is known about the other T cell activities defined in cGVHD, particularly
those that involve CD8 T cells. This area should invite further detailed
investigation. For example, the striking role of CD8 T cells in the stronger
female disease in the DBA BDF1 model clearly demands that similar
mechanisms be sought for in spontaneous disease.

Understanding Chronic GVHD from Different Angles

Bruce Blazar, Eric S. White, Daniel Couriel
Biol Blood Marrow Transplant 18:S184-S188, 2012

Whereas acute graft-versus-host disease (aGVHD) rates have decreased
with more intensive GVHD preventive agents and use of single and double
umbilical cord blood units as a source of donor cells in adult recipients,
significant chronic GVHD (cGVHD) rates unexpectedly have remained high.
Moreover, granulocyte colony stimulating factor mobilized peripheral blood
stem cell grafts have been associated with an increased overall risk of
cGVHD. As such, cGVHD has emerged as a primary cause of morbidity
and mortality following allogeneic hematopoietic stem cell transplantation.
Progress in developing cGVHD interventional strategies has been hampered
by variable onset and clinical and pathological manifestations of cGVHD, now
better defined by the National Institutes of Health (NIH) consensus conference,
and a dearth of preclinical models that closely mimic the conditions in which
cGVHD is generated and manifested. Although the exact causes of cGVHD
remain unknown, higher antibody levels have been associated with auto-
immunity and implicated in cGVHD. Newly diagnosed patients with
extensive cGVHD had elevated soluble B cell activating factor levels and
anti-double-strand DNA antibodies were found, which was associated with
higher circulating levels of pregerminal center (GC) B cells and post-GC
plasmablasts. B cells from cGVHD patients were hyperresponsive to Toll-like
receptor-9 signaling and have up-regulated CD86 levels.

By using a Cy and low doses of donor T cells, aGVHD was avoided and
cGVHD with BO favored. Histologic changes were similar to the findings in
human cGVHD with peribronchiolar and perivascular cuffing and infiltration
of the airway epithelium. The liver had inflammation and lymphocytic
infiltration, along with collagen deposition. The parotid and submandibular
salivary glands displayed lymphocytic infiltrates in both the bone marrow
and cGVHD groups, likely because of transplantation conditioning.

Treatment of steroid refractory cGVHD patients with rituximab, a B cell–
depleting anti-CD20 monoclonal antibody, has shown a beneficial role in
resolution of the autoimmune disorders such as systemic lupus erythmatosus
and rheumatoid arthritis, andcGVHD, with overall response rates of 29%
to 36% for oral, hepatic, gastrointestinal, and lung cGVHD, and 60% for
cutaneous cGVHD in aggregate data from multiple trials. Thus, we recently
undertook studies to identify the presence of CD41 T helper cells and B2201
B cells in the airways of mice that had BO, tissue-specific antibodies from sera,
and alloantibody deposition in the lung and liver of cGVHD recipients. cGVHD
development was associated with IgG2c deposition in the lung and liver,
abrogated if the donor bone marrow was deficient in mature B cells or
incapable of producing antihost reactive IgG. Robust GC formation was
seen in mice with cGVHD. Alleviation of symptoms in mice that received
B cell–deficient bone marrow confirms the requirement of B cells for lung
dysfunction and inflammation and fibrosis in the lung and liver.

Given a role for IgG antibodies, allo- or auto-Ab binding to the cGVHD organs
could enable tissue destruction or the pathology could be defined by the
specific function of these secreted antibodies. Pathogenic antibody production
therefore is likely to be an important inducer of cGVHD, and targeting this
specific function of the B cells is an attractive strategy for cGVHD. Because
GC B cells display lower susceptibility to rituximab-mediated clearance, probably
because they reside in a nonoptimal environment for antibody-based depletion,
our observation that GC B cells are critical to the development of cGVHD
suggests that agents that are more effective at disrupting the GC might be
more clinically useful. Treatment with LTbR-Ig, a fusion protein that blocks
interactions between LTbR and its ligands, had a direct effect on the
symptoms of cGVHD, at least in part by blocking GC formation and suggest
that LTbR-Ig could be a potential clinical interventional strategy for prevention
and therapy of cGVHD.

Fibrosis is the end result of a number of inflammatory and other injurious events,
resulting in replacement of normal tissue with a dense extracellular matrix (ECM)
scar composed primarily of collagens. While some degree of tissue fibrosis is
considered protective (e.g. in the setting of cutaneous wound healing),
exaggerated or unrelenting ECM deposition with replacement of the normal
tissue architecture is considered pathologic. Fibroproliferative disorders as
a class involving multiple organs (e.g. cGVHD following hematopoietic stem
cell transplant [affecting up to 30% of recipients surviving more than 100 days,
scleroderma [estimated to affect 70,000 in the US], idiopathic pulmonary fibrosis
[estimated to affect 200,000 in the US], hepatic cirrhosis [estimated to affect
up to 400,000 in the US], and renal fibrosis due to diabetic nephropathy and
other causes [estimated to affect over 400,000 in the US]) are a major cause
of morbidity and mortality. Combined, these disorders alone are conservatively
estimated to affect approximately 1 in 300 persons in the United States. When
coupled with a host of other disorders in which tissue fibrosis contributes to
morbidity (e.g. fibroproliferative acute respiratory distress syndrome,
hypersensitivity pneumonitis, solid organ transplant rejection), that estimate
is likely to be much greater.

Wound healing occurs by a highly orchestrated, complex process that has
been well defined. In general, wound repair occurs in 4 stages which overlap
considerably: clotting/coagulation, inflammation, fibroproliferation, and tissue
remodeling. The initial injury leads to a local disruption of epithelial and
endothelial barriers resulting in the elaboration of inflammatory mediators and
extravasation of cells and plasma proteins that serve to achieve hemostasis
and provide a provisional fibrin-rich matrix for the influx of inflammatory and
other reparative cells. Simultaneously, platelet degranulation provides a local
“boost” of vasodilators, growth factors, and ECM proteins that aid in the wound
healing response. Inflammatory cell influx occurs next, with polymorphonuclear
leukocytes (PMNs) arriving first. Following PMN degranulation, mononuclear
cells (macrophages and lymphocytes) arrive next and, along with PMN derived
products, sterilize and remove foreign materials from the wound. This process
also results in the elaboration of cytokines and chemokines designed to
augment the inflammatory response, to promote angiogenesis (allowing for
enhanced nutrient and oxygen delivery to the wound bed), and to recruit
fibroblasts to the wound bed. Fibroblast recruitment and transdifferentiation to
myofibroblasts (or recruitment of already-differentiated myofibroblasts or
fibroblast precursors; this point is still controversial) marks the fibroproliferative
stage, with the result being the elaboration of ECM proteins (collagens,
fibronectins) to repair the tissue defect.

Vorinostat plus tacrolimus and mycophenolate to prevent graft-versus-host
disease after related-donor reduced-intensity conditioning allogeneic
stem-cell transplantation: a phase 1/2 trial

Sung Won Choi, T Braun, L Chang, JLM Ferrara, A Pawarode, et al.
Lancet Oncol 2014; 15: 87–95

Background Acute graft-versus-host disease (GVHD) remains a barrier to more
widespread application of allogeneic hemopoietic stem-cell transplantation.
Vorinostat is an inhibitor of histone deacetylases and was shown to attenuate
GVHD in preclinical models. We aimed to study the safety and activity of
vorinostat, in combination with standard immunoprophylaxis, for prevention of
GVHD in patients undergoing related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation. Methods Between March 31, 2009,
and Feb 8, 2013, we did a prospective, single-arm, phase 1/2 study at two
centers in the USA. We recruited adults (aged ≥18 years) with high-risk
hematological malignant diseases who were candidates for reduced-intensity
conditioning hemopoietic stem-cell transplantation and had an available 8/8
or 7/8 HLA matched related donor. All patients received a conditioning regimen
of fl udarabine (40 mg/m² daily for 4 days) and busulfan (3·2 mg/kg daily for
2 days) and GVHD immunoprophylaxis of mycophenolate mofetil (1 g three
times a day, days 0–28) and tacrolimus (0·03 mg/kg a day, titrated to a goal
level of 8–12 ng/mL, starting day –3 until day 180). Vorinostat (either 100 mg
or 200 mg, twice a day) was initiated 10 days before haemopoietic stem-cell
transplantation until day 100. The primary endpoint was the cumulative
incidence of grade 2–4 acute GVHD by day 100. This trial is registered with, number NCT00810602.
Findings 50 patients were assessable for both toxic effects and response;
eight additional patients were included in the analysis of toxic effects. All
patients engrafted neutrophils and platelets at expected times after
hemopoietic stem-cell transplantation. The cumulative incidence of grade
2–4 acute GVHD by day 100 was 22% (95% CI 13–36). The most common
non-hematological adverse events included electrolyte disturbances (n=15),
hyperglycemia (11), infections (six), mucositis (four), and increased activity
of liver enzymes (three). Non-symptomatic thrombocytopenia after
engraftment was the most common hematological grade 3–4 adverse
event (nine) but was transient and all cases resolved swiftly.
Interpretation Administration of vorinostat in combination with standard
GVHD prophylaxis after related-donor reduced-intensity conditioning
hemopoietic stem-cell transplantation is safe and is associated with a
lower than expected incidence of severe acute GVHD. Future studies
are needed to assess the effect of vorinostat for prevention of GVHD in
broader settings of hemopoietic stem-cell transplantation.

Read Full Post »

Summary – Volume 4, Part 2: Translational Medicine in Cardiovascular Diseases

Summary – Volume 4, Part 2:  Translational Medicine in Cardiovascular Diseases

Author and Curator: Larry H Bernstein, MD, FCAP


We have covered a large amount of material that involves

  • the development,
  • application, and
  • validation of outcomes of medical and surgical procedures

that are based on translation of science from the laboratory to the bedside, improving the standards of medical practice at an accelerated pace in the last quarter century, and in the last decade.  Encouraging enabling developments have been:

1. The establishment of national and international outcomes databases for procedures by specialist medical societies

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

On Devices and On Algorithms: Prediction of Arrhythmia after Cardiac Surgery and ECG Prediction of an Onset of Paroxysmal Atrial Fibrillation
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions
Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Survivals Comparison of Coronary Artery Bypass Graft (CABG) and Percutaneous Coronary Intervention (PCI) /Coronary Angioplasty
Larry H. Bernstein, MD, Writer And Aviva Lev-Ari, PhD, RN, Curator

Revascularization: PCI, Prior History of PCI vs CABG
Curator: Aviva Lev-Ari, PhD, RN

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Endovascular Lower-extremity Revascularization Effectiveness: Vascular Surgeons (VSs), Interventional Cardiologists (ICs) and Interventional Radiologists (IRs)
Curator: Aviva Lev-Ari, PhD, RN

and more

2. The identification of problem areas, particularly in activation of the prothrombotic pathways, infection control to an extent, and targeting of pathways leading to progression or to arrythmogenic complications.

Cardiovascular Complications: Death from Reoperative Sternotomy after prior CABG, MVR, AVR, or Radiation; Complications of PCI; Sepsis from Cardiovascular Interventions Author, Introduction and Summary: Justin D Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN

Anticoagulation genotype guided dosing
Larry H. Bernstein, MD, FCAP, Author and Curator

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

The Effects of Aprotinin on Endothelial Cell Coagulant Biology
Co-Author (Kamran Baig, MBBS, James Jaggers, MD, Jeffrey H. Lawson, MD, PhD) and Curator

Outcomes in High Cardiovascular Risk Patients: Prasugrel (Effient) vs. Clopidogrel (Plavix); Aliskiren (Tekturna) added to ACE or added to ARB
Reporter and Curator: Aviva Lev-Ari, PhD, RN

Pharmacogenomics – A New Method for Druggability  Author and Curator: Demet Sag, PhD

Advanced Topics in Sepsis and the Cardiovascular System at its End Stage    Author: Larry H Bernstein, MD, FCAP

3. Development of procedures that use a safer materials in vascular management.

Stent Design and Thrombosis: Bifurcation Intervention, Drug Eluting Stents (DES) and Biodegrable Stents
Curator: Aviva Lev-Ari, PhD, RN

Biomaterials Technology: Models of Tissue Engineering for Reperfusion and Implantable Devices for Revascularization
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

Vascular Repair: Stents and Biologically Active Implants
Author and Curator: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, RN, PhD

Drug Eluting Stents: On MIT’s Edelman Lab’s Contributions to Vascular Biology and its Pioneering Research on DES
Author: Larry H Bernstein, MD, FACP and Curator: Aviva Lev-Ari, PhD, RN

MedTech & Medical Devices for Cardiovascular Repair – Curations by Aviva Lev-Ari, PhD, RN

4. Discrimination of cases presenting for treatment based on qualifications for medical versus surgical intervention.

Treatment Options for Left Ventricular Failure – Temporary Circulatory Support: Intra-aortic balloon pump (IABP) – Impella Recover LD/LP 5.0 and 2.5, Pump Catheters (Non-surgical) vs Bridge Therapy: Percutaneous Left Ventricular Assist Devices (pLVADs) and LVADs (Surgical)
Author: Larry H Bernstein, MD, FCAP And Curator: Justin D Pearlman, MD, PhD, FACC

Coronary Reperfusion Therapies: CABG vs PCI – Mayo Clinic preprocedure Risk Score (MCRS) for Prediction of in-Hospital Mortality after CABG or PCI
Writer and Curator: Larry H. Bernstein, MD, FCAP and Curator: Aviva Lev-Ari, PhD, RN

ACC/AHA Guidelines for Coronary Artery Bypass Graft Surgery Reporter: Aviva Lev-Ari, PhD, RN

Mitral Valve Repair: Who is a Patient Candidate for a Non-Ablative Fully Non-Invasive Procedure?
Author, and Content Consultant to e-SERIES A: Cardiovascular Diseases: Justin Pearlman, MD, PhD, FACC and Article Curator: Aviva Lev-Ari, PhD, RN 

5.  This has become possible because of the advances in our knowledge of key related pathogenetic mechanisms involving gene expression and cellular regulation of complex mechanisms.

What is the key method to harness Inflammation to close the doors for many complex diseases?
Author and Curator: Larry H Bernstein, MD, FCAP

CVD Prevention and Evaluation of Cardiovascular Imaging Modalities: Coronary Calcium Score by CT Scan Screening to justify or not the Use of Statin
Curator: Aviva Lev-Ari, PhD, RN

Richard Lifton, MD, PhD of Yale University and Howard Hughes Medical Institute: Recipient of 2014 Breakthrough Prizes Awarded in Life Sciences for the Discovery of Genes and Biochemical Mechanisms that cause Hypertension
Curator: Aviva Lev-Ari, PhD, RN

Pathophysiological Effects of Diabetes on Ischemic-Cardiovascular Disease and on Chronic Obstructive Pulmonary Disease (COPD)
Curator:  Larry H. Bernstein, MD, FCAP

Atherosclerosis Independence: Genetic Polymorphisms of Ion Channels Role in the Pathogenesis of Coronary Microvascular Dysfunction and Myocardial Ischemia (Coronary Artery Disease (CAD))
Reviewer and Co-Curator: Larry H Bernstein, MD, CAP and Curator: Aviva Lev-Ari, PhD, RN

Notable Contributions to Regenerative Cardiology  Author and Curator: Larry H Bernstein, MD, FCAP and Article Commissioner: Aviva Lev-Ari, PhD, RD

As noted in the introduction, any of the material can be found and reviewed by content, and the eTOC is identified in attached:


This completes what has been presented in Part 2, Vol 4 , and supporting references for the main points that are found in the Leaders in Pharmaceutical Intelligence Cardiovascular book.  Part 1 was concerned with Posttranslational Modification of Proteins, vital for understanding cellular regulation and dysregulation.  Part 2 was concerned with Translational Medical Therapeutics, the efficacy of medical and surgical decisions based on bringing the knowledge gained from the laboratory, and from clinical trials into the realm opf best practice.  The time for this to occur in practice in the past has been through roughly a generation of physicians.  That was in part related to the busy workload of physicians, and inability to easily access specialty literature as the volume and complexity increased.  This had an effect of making access of a family to a primary care provider through a lifetime less likely than the period post WWII into the 1980s.

However, the growth of knowledge has accelerated in the specialties since the 1980’s so that the use of physician referral in time became a concern about the cost of medical care.  This is not the place for or a matter for discussion here.  It is also true that the scientific advances and improvements in available technology have had a great impact on medical outcomes.  The only unrelated issue is that of healthcare delivery, which is not up to the standard set by serial advances in therapeutics, accompanied by high cost due to development costs, marketing costs, and development of drug resistance.

I shall identify continuing developments in cardiovascular diagnostics, therapeutics, and bioengineering that is and has been emerging.

1. Mechanisms of disease

REPORT: Mapping the Cellular Response to Small Molecules Using Chemogenomic Fitness Signatures 

Science 11 April 2014:
Vol. 344 no. 6180 pp. 208-211

Abstract: Genome-wide characterization of the in vivo cellular response to perturbation is fundamental to understanding how cells survive stress. Identifying the proteins and pathways perturbed by small molecules affects biology and medicine by revealing the mechanisms of drug action. We used a yeast chemogenomics platform that quantifies the requirement for each gene for resistance to a compound in vivo to profile 3250 small molecules in a systematic and unbiased manner. We identified 317 compounds that specifically perturb the function of 121 genes and characterized the mechanism of specific compounds. Global analysis revealed that the cellular response to small molecules is limited and described by a network of 45 major chemogenomic signatures. Our results provide a resource for the discovery of functional interactions among genes, chemicals, and biological processes.


Laura Zahn
Sci. Signal. 15 April 2014; 7(321): ec103.

In order to identify how chemical compounds target genes and affect the physiology of the cell, tests of the perturbations that occur when treated with a range of pharmacological chemicals are required. By examining the haploinsufficiency profiling (HIP) and homozygous profiling (HOP) chemogenomic platforms, Lee et al.(p. 208) analyzed the response of yeast to thousands of different small molecules, with genetic, proteomic, and bioinformatic analyses. Over 300 compounds were identified that targeted 121 genes within 45 cellular response signature networks. These networks were used to extrapolate the likely effects of related chemicals, their impact upon genetic pathways, and to identify putative gene functions

Key Heart Failure Culprit Discovered

A team of cardiovascular researchers from the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai, Sanford-Burnham Medical Research Institute, and University of California, San Diego have identified a small, but powerful, new player in thIe onset and progression of heart failure. Their findings, published in the journal Nature  on March 12, also show how they successfully blocked the newly discovered culprit.
Investigators identified a tiny piece of RNA called miR-25 that blocks a gene known as SERCA2a, which regulates the flow of calcium within heart muscle cells. Decreased SERCA2a activity is one of the main causes of poor contraction of the heart and enlargement of heart muscle cells leading to heart failure.

Using a functional screening system developed by researchers at Sanford-Burnham, the research team discovered miR-25 acts pathologically in patients suffering from heart failure, delaying proper calcium uptake in heart muscle cells. According to co-lead study authors Christine Wahlquist and Dr. Agustin Rojas Muñoz, developers of the approach and researchers in Mercola’s lab at Sanford-Burnham, they used high-throughput robotics to sift through the entire genome for microRNAs involved in heart muscle dysfunction.

Subsequently, the researchers at the Cardiovascular Research Center at Icahn School of Medicine at Mount Sinai found that injecting a small piece of RNA to inhibit the effects of miR-25 dramatically halted heart failure progression in mice. In addition, it also improved their cardiac function and survival.

“In this study, we have not only identified one of the key cellular processes leading to heart failure, but have also demonstrated the therapeutic potential of blocking this process,” says co-lead study author Dr. Dongtak Jeong, a post-doctoral fellow at the Cardiovascular Research Center at Icahn School of  Medicine at Mount Sinai in the laboratory of the study’s co-senior author Dr. Roger J. Hajjar.

Publication: Inhibition of miR-25 improves cardiac contractility in the failing heart.Christine Wahlquist, Dongtak Jeong, Agustin Rojas-Muñoz, Changwon Kho, Ahyoung Lee, Shinichi Mitsuyama, Alain Van Mil, Woo Jin Park, Joost P. G. Sluijter, Pieter A. F. Doevendans, Roger J. :  Hajjar & Mark Mercola.     Nature (March 2014)


“Junk” DNA Tied to Heart Failure

Deep RNA Sequencing Reveals Dynamic Regulation of Myocardial Noncoding RNAs in Failing Human Heart and Remodeling With Mechanical Circulatory Support

Yang KC, Yamada KA, Patel AY, Topkara VK, George I, et al.
Circulation 2014;  129(9):1009-21.    …/CIRCULATIONAHA.113.003863.full

The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support. These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.

Junk DNA was long thought to have no important role in heredity or disease because it doesn’t code for proteins. But emerging research in recent years has revealed that many of these sections of the genome produce noncoding RNA molecules that still have important functions in the body. They come in a variety of forms, some more widely studied than others. Of these, about 90% are called long noncoding RNAs (lncRNAs), and exploration of their roles in health and disease is just beginning.

The Washington University group performed a comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

In their study, the researchers found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“The myocardial transcriptome is dynamically regulated in advanced heart failure and after LVAD support. The expression profiles of lncRNAs, but not mRNAs or miRNAs, can discriminate failing hearts of different pathologies and are markedly altered in response to LVAD support,” wrote the researchers. “These results suggest an important role for lncRNAs in the pathogenesis of heart failure and in reverse remodeling observed with mechanical support.”

‘Junk’ Genome Regions Linked to Heart Failure

In a recent issue of the journal Circulation, Washington University investigators report results from the first comprehensive analysis of all RNA molecules expressed in the human heart. The researchers studied nonfailing hearts and failing hearts before and after patients received pump support from left ventricular assist devices (LVAD). The LVADs increased each heart’s pumping capacity while patients waited for heart transplants.

“We took an unbiased approach to investigating which types of RNA might be linked to heart failure,” said senior author Jeanne Nerbonne, the Alumni Endowed Professor of Molecular Biology and Pharmacology. “We were surprised to find that long noncoding RNAs stood out.

In the new study, the investigators found that unlike other RNA molecules, expression patterns of long noncoding RNAs could distinguish between two major types of heart failure and between failing hearts before and after they received LVAD support.

“We don’t know whether these changes in long noncoding RNAs are a cause or an effect of heart failure,” Nerbonne said. “But it seems likely they play some role in coordinating the regulation of multiple genes involved in heart function.”

Nerbonne pointed out that all types of RNA molecules they examined could make the obvious distinction: telling the difference between failing and nonfailing hearts. But only expression of the long noncoding RNAs was measurably different between heart failure associated with a heart attack (ischemic) and heart failure without the obvious trigger of blocked arteries (nonischemic). Similarly, only long noncoding RNAs significantly changed expression patterns after implantation of left ventricular assist devices.


Decoding the noncoding transcripts in human heart failure

Xiao XG, Touma M, Wang Y
Circulation. 2014; 129(9): 958960, 

Heart failure is a complex disease with a broad spectrum of pathological features. Despite significant advancement in clinical diagnosis through improved imaging modalities and hemodynamic approaches, reliable molecular signatures for better differential diagnosis and better monitoring of heart failure progression remain elusive. The few known clinical biomarkers for heart failure, such as plasma brain natriuretic peptide and troponin, have been shown to have limited use in defining the cause or prognosis of the disease.1,2 Consequently, current clinical identification and classification of heart failure remain descriptive, mostly based on functional and morphological parameters. Therefore, defining the pathogenic mechanisms for hypertrophic versus dilated or ischemic versus nonischemic cardiomyopathies in the failing heart remain a major challenge to both basic science and clinic researchers. In recent years, mechanical circulatory support using left ventricular assist devices (LVADs) has assumed a growing role in the care of patients with end-stage heart failure.3 During the earlier years of LVAD application as a bridge to transplant, it became evident that some patients exhibit substantial recovery of ventricular function, structure, and electric properties.4 This led to the recognition that reverse remodeling is potentially an achievable therapeutic goal using LVADs. However, the underlying mechanism for the reverse remodeling in the LVAD-treated hearts is unclear, and its discovery would likely hold great promise to halt or even reverse the progression of heart failure.


Efficacy and Safety of Dabigatran Compared With Warfarin in Relation to Baseline Renal Function in Patients With Atrial Fibrillation: A RE-LY (Randomized Evaluation of Long-term Anticoagulation Therapy) Trial Analysis

Circulation. 2014; 129: 951-952​CIR.0000000000000022

In patients with atrial fibrillation, impaired renal function is associated with a higher risk of thromboembolic events and major bleeding. Oral anticoagulation with vitamin K antagonists reduces thromboembolic events but raises the risk of bleeding. The new oral anticoagulant dabigatran has 80% renal elimination, and its efficacy and safety might, therefore, be related to renal function. In this prespecified analysis from the Randomized Evaluation of Long-Term Anticoagulant Therapy (RELY) trial, outcomes with dabigatran versus warfarin were evaluated in relation to 4 estimates of renal function, that is, equations based on creatinine levels (Cockcroft-Gault, Modification of Diet in Renal Disease (MDRD), Chronic Kidney Disease Epidemiology Collaboration [CKD-EPI]) and cystatin C. The rates of stroke or systemic embolism were lower with dabigatran 150 mg and similar with 110 mg twice daily irrespective of renal function. Rates of major bleeding were lower with dabigatran 110 mg and similar with 150 mg twice daily across the entire range of renal function. However, when the CKD-EPI or MDRD equations were used, there was a significantly greater relative reduction in major bleeding with both doses of dabigatran than with warfarin in patients with estimated glomerular filtration rate ≥80 mL/min. These findings show that dabigatran can be used with the same efficacy and adequate safety in patients with a wide range of renal function and that a more accurate estimate of renal function might be useful for improved tailoring of anticoagulant treatment in patients with atrial fibrillation and an increased risk of stroke.

Aldosterone Regulates MicroRNAs in the Cortical Collecting Duct to Alter Sodium Transport.

Robert S Edinger, Claudia Coronnello, Andrew J Bodnar, William A Laframboise, Panayiotis V Benos, Jacqueline Ho, John P Johnson, Michael B Butterworth

Journal of the American Society of Nephrology (Impact Factor: 8.99). 04/2014;     http://dx.

Source: PubMed

ABSTRACT A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells.

Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport.

Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein.

A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3′ untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.


2. Diagnostic Biomarker Status

A prospective study of the impact of serial troponin measurements on the diagnosis of myocardial infarction and hospital and 6-month mortality in patients admitted to ICU with non-cardiac diagnoses.

Marlies Ostermann, Jessica Lo, Michael Toolan, Emma Tuddenham, Barnaby Sanderson, Katie Lei, John Smith, Anna Griffiths, Ian Webb, James Coutts, John hambers, Paul Collinson, Janet Peacock, David Bennett, David Treacher

Critical care (London, England) (Impact Factor: 4.72). 04/2014; 18(2):R62.

Source: PubMed

ABSTRACT Troponin T (cTnT) elevation is common in patients in the Intensive Care Unit (ICU) and associated with morbidity and mortality. Our aim was to determine the epidemiology of raised cTnT levels and contemporaneous electrocardiogram (ECG) changes suggesting myocardial infarction (MI) in ICU patients admitted for non-cardiac reasons.
cTnT and ECGs were recorded daily during week 1 and on alternate days during week 2 until discharge from ICU or death. ECGs were interpreted independently for the presence of ischaemic changes. Patients were classified into 4 groups: (i) definite MI (cTnT >=15 ng/L and contemporaneous changes of MI on ECG), (ii) possible MI (cTnT >=15 ng/L and contemporaneous ischaemic changes on ECG), (iii) troponin rise alone (cTnT >=15 ng/L), or (iv) normal. Medical notes were screened independently by two ICU clinicians for evidence that the clinical teams had considered a cardiac event.
Data from 144 patients were analysed [42% female; mean age 61.9 (SD 16.9)]. 121 patients (84%) had at least one cTnT level >=15 ng/L. A total of 20 patients (14%) had a definite MI, 27% had a possible MI, 43% had a cTNT rise without contemporaneous ECG changes, and 16% had no cTNT rise. ICU, hospital and 180 day mortality were significantly higher in patients with a definite or possible MI.Only 20% of definite MIs were recognised by the clinical team. There was no significant difference in mortality between recognised and non-recognised events.At time of cTNT rise, 100 patients (70%) were septic and 58% were on vasopressors. Patients who were septic when cTNT was elevated had an ICU mortality of 28% compared to 9% in patients without sepsis. ICU mortality of patients who were on vasopressors at time of cTNT elevation was 37% compared to 1.7% in patients not on vasopressors.
The majority of critically ill patients (84%) had a cTnT rise and 41% met criteria for a possible or definite MI of whom only 20% were recognised clinically. Mortality up to 180 days was higher in patients with a cTnT rise.


Prognostic performance of high-sensitivity cardiac troponin T kinetic changes adjusted for elevated admission values and the GRACE score in an unselected emergency department population.

Moritz BienerMatthias MuellerMehrshad VafaieAllan S JaffeHugo A Katus,Evangelos Giannitsis

Clinica chimica acta; international journal of clinical chemistry (Impact Factor: 2.54). 04/2014;

Source: PubMed

ABSTRACT To test the prognostic performance of rising and falling kinetic changes of high-sensitivity cardiac troponin T (hs-cTnT) and the GRACE score.
Rising and falling hs-cTnT changes in an unselected emergency department population were compared.
635 patients with a hs-cTnT >99th percentile admission value were enrolled. Of these, 572 patients qualified for evaluation with rising patterns (n=254, 44.4%), falling patterns (n=224, 39.2%), or falling patterns following an initial rise (n=94, 16.4%). During 407days of follow-up, we observed 74 deaths, 17 recurrent AMI, and 79 subjects with a composite of death/AMI. Admission values >14ng/L were associated with a higher rate of adverse outcomes (OR, 95%CI:death:12.6, 1.8-92.1, p=0.01, death/AMI:6.7, 1.6-27.9, p=0.01). Neither rising nor falling changes increased the AUC of baseline values (AUC: rising 0.562 vs 0.561, p=ns, falling: 0.533 vs 0.575, p=ns). A GRACE score ≥140 points indicated a higher risk of death (OR, 95%CI: 3.14, 1.84-5.36), AMI (OR,95%CI: 1.56, 0.59-4.17), or death/AMI (OR, 95%CI: 2.49, 1.51-4.11). Hs-cTnT changes did not improve prognostic performance of a GRACE score ≥140 points (AUC, 95%CI: death: 0.635, 0.570-0.701 vs. 0.560, 0.470-0.649 p=ns, AMI: 0.555, 0.418-0.693 vs. 0.603, 0.424-0.782, p=ns, death/AMI: 0.610, 0.545-0.676 vs. 0.538, 0.454-0.622, p=ns). Coronary angiography was performed earlier in patients with rising than with falling kinetics (median, IQR [hours]:13.7, 5.5-28.0 vs. 20.8, 6.3-59.0, p=0.01).
Neither rising nor falling hs-cTnT changes improve prognostic performance of elevated hs-cTnT admission values or the GRACE score. However, rising values are more likely associated with the decision for earlier invasive strategy.


Troponin assays for the diagnosis of myocardial infarction and acute coronary syndrome: where do we stand?

Arie Eisenman

ABSTRACT: Under normal circumstances, most intracellular troponin is part of the muscle contractile apparatus, and only a small percentage (< 2-8%) is free in the cytoplasm. The presence of a cardiac-specific troponin in the circulation at levels above normal is good evidence of damage to cardiac muscle cells, such as myocardial infarction, myocarditis, trauma, unstable angina, cardiac surgery or other cardiac procedures. Troponins are released as complexes leading to various cut-off values depending on the assay used. This makes them very sensitive and specific indicators of cardiac injury. As with other cardiac markers, observation of a rise and fall in troponin levels in the appropriate time-frame increases the diagnostic specificity for acute myocardial infarction. They start to rise approximately 4-6 h after the onset of acute myocardial infarction and peak at approximately 24 h, as is the case with creatine kinase-MB. They remain elevated for 7-10 days giving a longer diagnostic window than creatine kinase. Although the diagnosis of various types of acute coronary syndrome remains a clinical-based diagnosis, the use of troponin levels contributes to their classification. This Editorial elaborates on the nature of troponin, its classification, clinical use and importance, as well as comparing it with other currently available cardiac markers.

Expert Review of Cardiovascular Therapy 07/2006; 4(4):509-14. 


Impact of redefining acute myocardial infarction on incidence, management and reimbursement rate of acute coronary syndromes.

Carísi A Polanczyk, Samir Schneid, Betina V Imhof, Mariana Furtado, Carolina Pithan, Luis E Rohde, Jorge P Ribeiro

ABSTRACT: Although redefinition for acute myocardial infarction (AMI) has been proposed few years ago, to date it has not been universally adopted by many institutions. The purpose of this study is to evaluate the diagnostic, prognostic and economical impact of the new diagnostic criteria for AMI. Patients consecutively admitted to the emergency department with suspected acute coronary syndromes were enrolled in this study. Troponin T (cTnT) was measured in samples collected for routine CK-MB analyses and results were not available to physicians. Patients without AMI by traditional criteria and cTnT > or = 0.035 ng/mL were coded as redefined AMI. Clinical outcomes were hospital death, major cardiac events and revascularization procedures. In-hospital management and reimbursement rates were also analyzed. Among 363 patients, 59 (16%) patients had AMI by conventional criteria, whereas additional 75 (21%) had redefined AMI, an increase of 127% in the incidence. Patients with redefined AMI were significantly older, more frequently male, with atypical chest pain and more risk factors. In multivariate analysis, redefined AMI was associated with 3.1 fold higher hospital death (95% CI: 0.6-14) and a 5.6 fold more cardiac events (95% CI: 2.1-15) compared to those without AMI. From hospital perspective, based on DRGs payment system, adoption of AMI redefinition would increase 12% the reimbursement rate [3552 Int dollars per 100 patients evaluated]. The redefined criteria result in a substantial increase in AMI cases, and allow identification of high-risk patients. Efforts should be made to reinforce the adoption of AMI redefinition, which may result in more qualified and efficient management of ACS.

International Journal of Cardiology 03/2006; 107(2):180-7. · 5.51 Impact Factor


3. Biomedical Engineerin3g

Safety and Efficacy of an Injectable Extracellular Matrix Hydrogel for Treating Myocardial Infarction 

Sonya B. Seif-Naraghi, Jennifer M. Singelyn, Michael A. Salvatore,  et al.
Sci Transl Med 20 February 2013 5:173ra25

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of application with substantial intrinsic hurdles, but where human translation is now occurring.

 Acellular Biomaterials: An Evolving Alternative to Cell-Based Therapies

J. A. Burdick, R. L. Mauck, J. H. Gorman, R. C. Gorman,
Sci. Transl. Med. 2013; 5, (176): 176 ps4

Acellular biomaterials can stimulate the local environment to repair tissues without the regulatory and scientific challenges of cell-based therapies. A greater understanding of the mechanisms of such endogenous tissue repair is furthering the design and application of these biomaterials. We discuss recent progress in acellular materials for tissue repair, using cartilage and cardiac tissues as examples of applications with substantial intrinsic hurdles, but where human translation is now occurring.

Instructive Nanofiber Scaffolds with VEGF Create a Microenvironment for Arteriogenesis and Cardiac Repair

Yi-Dong Lin, Chwan-Yau Luo, Yu-Ning Hu, Ming-Long Yeh, Ying-Chang Hsueh, Min-Yao Chang, et al.
Sci Transl Med 8 August 2012; 4(146):ra109. 10.1126/scitranslmed.3003841

Angiogenic therapy is a promising approach for tissue repair and regeneration. However, recent clinical trials with protein delivery or gene therapy to promote angiogenesis have failed to provide therapeutic effects. A key factor for achieving effective revascularization is the durability of the microvasculature and the formation of new arterial vessels. Accordingly, we carried out experiments to test whether intramyocardial injection of self-assembling peptide nanofibers (NFs) combined with vascular endothelial growth factor (VEGF) could create an intramyocardial microenvironment with prolonged VEGF release to improve post-infarct neovascularization in rats. Our data showed that when injected with NF, VEGF delivery was sustained within the myocardium for up to 14 days, and the side effects of systemic edema and proteinuria were significantly reduced to the same level as that of control. NF/VEGF injection significantly improved angiogenesis, arteriogenesis, and cardiac performance 28 days after myocardial infarction. NF/VEGF injection not only allowed controlled local delivery but also transformed the injected site into a favorable microenvironment that recruited endogenous myofibroblasts and helped achieve effective revascularization. The engineered vascular niche further attracted a new population of cardiomyocyte-like cells to home to the injected sites, suggesting cardiomyocyte regeneration. Follow-up studies in pigs also revealed healing benefits consistent with observations in rats. In summary, this study demonstrates a new strategy for cardiovascular repair with potential for future clinical translation.

Manufacturing Challenges in Regenerative Medicine

I. Martin, P. J. Simmons, D. F. Williams.
Sci. Transl. Med. 2014; 6(232): fs16.

Along with scientific and regulatory issues, the translation of cell and tissue therapies in the routine clinical practice needs to address standardization and cost-effectiveness through the definition of suitable manufacturing paradigms.




Read Full Post »

Circulating Endothelial Progenitors Cells (cEPCs) as Biomarkers

Article Curator: Larry H. Bernstein, MD, FCAP


Topic Curator: Aviva Lev-Ari, PhD, RN

Circulating progenitor cells have gained much interest rapidly in the past year primarily in identification of damaged tissue that has turnover of cells that are identifiable in the circulation.  This has to require a sensitivity for identification at one or two logs lower than circulating hematopoietic cells.  I mention this untested view only because cells of the circulation are detected routinely by automated hematology instruments like those of Beckman-Coulter and Siemens, with graphical presentation of results.  The Sysmex also reports immature granulocytes that are a small percent of the neutrophil count.  In the evaluation of leukemias, flow cytometry has been used for years, but require a preparative step.  Cell types have been identified by acidic and basic dye stains to identify basophilic, acidophilic and neutrophilic granulocyte series, and by size of the cell population, and nuclear features, differentiating mature and nucleated red cells, the granulocyte series, monocytes and lymphocytes, as well as platelets (aggregation gives an underestimate of platelet count).  But to detect cancer cells or damaged endothelial cells, the number of cells in the circulation requires and antibody to the surface with a visualizable ligand attached to an antibody for identification.  Visualization could be by a fluorophor, or perhaps a luciferase reaction.  Here are two articles that identify circulating endothelial cells, making them suitable for biomarkers of cardiovascular injury.  Whether they can detect early predictive ischemia, or frank AMI needs investigation.  The concept of piecemeal necrosis in the heart may be applicable to cardiomyocyte injury that is found unexpectedly at autopsy as “silent infarct”.

Circulating endothelial progenitors–cells as biomarkers

Rosenzweig, Anthony
N Engl J Med. 2005 Sep 8;353(10):1055-7

Comment on

Circulating endothelial progenitor cells and cardiovascular outcomes

[N Engl J Med. 2005]  PMID: 16148292 [PubMed – indexed for MEDLINE]

Endothelial injury and dysfunction are thought to be critical events in the  pathogenesis of atherosclerosis. Thus,

  • understanding the mechanisms that  maintain and restore endothelial function
    • may have important clinical  implications.

A series of clinical and basic studies prompted by the discovery 

  • of bone marrow derived endothelial progenitor cells1 have
  • provided insights into these processes and
    • opened a door to the development of new therapeutic approaches.

Growing evidence suggests that bone marrow derived endothelial progenitor cells circulate in the blood and

  • play an important role in the formation of new blood vessels as well as
  • contribute to vascular homeostasis in the adult.

Circulating endothelial progenitor cells were initially identified

  • through their expression of CD34
    (a surface marker common to hematopoietic stem cells and mature endothelial cells)
  • and vascular endothelial cell growth-factor receptor 2
    (VEGFR2 or kinase-domain related [KDR] receptor),

but not of other markers seen on fully differentiated endothelial cells.1

Subsequent studies have also used other identifiers, such as

  • the stem-cell marker CD133, and
  • functional assays, including
    • the ability to form endothelial colonies.

Endothelial progenitor cells defined in these ways probably represent

  • a heterogeneous population, which,
  • in combination with the lack of a consensual definition,

complicates the interpretation of work in this field.

Nevertheless, numerous studies in animals have shown that endothelial  progenitor cells can integrate into new and existing blood vessels.2,3,4
Intravenous injection of cytokine-mobilized human endothelial progenitor cells

  • improved myocardial neoangiogenesis and
  • the recovery of functioning in a rat model of infarction.3

Repeated injection of bone marrow derived cells in a mouse model of atherosclerosis

  • reduced the rate of plaque formation without altering serum lipids levels, and
  • donor endothelial progenitor cells could subsequently be identified in the recipient’s blood vessels.4

Previous clinical studies have shown that

  • traditional risk factors for coronary atherosclerosis
  • are associated with low levels of circulating endothelial progenitor cells,5 whereas
  • protective interventions, including statin therapy6 and exercise,7
    • appear to increase the supply of these cells.

Hill et al. found that even in healthy volunteers,

  • levels of endothelial progenitor cells were inversely correlated with the Framingham risk score and
  • actually appeared to predict vascular function better than the Framingham risk score.5

Together, these data suggest that circulating endothelial progenitor cells may participate

  • not only in forming new blood vessels
  • but also in maintaining the integrity and function of vascular  endothelium,

thereby mitigating disease processes such as atherosclerosis.

In this issue of the Journal, Werner and colleagues have further advanced our understanding of the clinical implications of endothelial progenitor cells.8 Endothelial progenitor cells were quantitated in 519 patients with coronary artery disease who

  • were followed for one year after undergoing catheterization.

Patients with higher levels of endothelial progenitor cells had

  • a reduced risk of death from cardiovascular causes and of
  • the composite end point of major cardiovascular events.

These relationships were preserved even

  • after adjustment for traditional risk factors and prognostic variables.

A similar relationship was seen

  • whether endothelial progenitor cells were  identified by virtue of expression
    either of CD34 and KDR or of CD133 or
  • because of their ability to form endothelial colonies,

further strengthening the authors’ conclusions. Repeated catheterization was not performed in this  cohort, so

  • we do not know whether the reduction in clinical events reflected a slowed progression of atherosclerosis or some other clinical effect.

A  dissociation between anatomical measures of atherosclerosis and clinical events has been well documented in other settings.

Although this study is consistent with prior work suggesting that circulating endothelial progenitor cells may play a protective role in vascular homeostasis, other explanations

  • for the association between endothelial progenitor number and outcome remain possible.

Changes in the number of endothelial progenitor cells and

  • in clinical events might reflect a common underlying etiology,
      • rather than a causal relation.

For example, a defect in the production of nitric oxide, which plays an important role

  • in both the mobilization of endothelial progenitor cells9 and blood-vessel function, might account for both observations.

Similarly, the number of endothelial progenitor cells

  • may mirror a person’s regenerative capacity more broadly and
  • predict clinical events on that basis.

Even if endothelial progenitor cells are mechanistically linked to clinical cardiovascular events,

  • such clinical studies do not distinguish between the possibility
  • that the protection is mediated through the integration of endothelial  progenitor cells into blood vessels and

its possible mediation by other  mechanisms, such as the

  • paracrine benefits of endothelial progenitor  cell secreted products.

Although such questions will undoubtedly continue to provide fertile ground  for fundamental investigation,

  • the report by Werner and colleagues has more  immediate clinical implications.

First, it suggests that circulating cell  populations may represent a new class of biomarkers

  • that naturally integrate  diverse genetic and environmental effects,
  • thereby providing robust  physiological and prognostic insights.

Second, in the context of coronary  disease, the study shows that

  • the number of endothelial progenitor cells is an independent predictor of hard clinical outcomes.

As with other biomarkers, a demonstration of clinical usefulness will ultimately require

  • the examination of other patient populations, as well as
  • a demonstration that clinical therapy can be guided and enhanced by this information.

Finally, the increased risk associated with reduced levels of endothelial progenitor cells

  • supports the growing interest in the therapeutic potential of enhancing the level of these cells.

The most dramatic extension of this line of reasoning involves

transferring  bone marrow or peripheral blood cells that are likely to include endothelial  progenitor cells to patients with coronary artery disease. Although it would be premature to judge the clinical success of these strategies, early trials, including one randomized (though incompletely blinded) trial, have suggested

  • at least short-term functional benefits of intracoronary infusion of bone marrow cells after acute infarction.10

Trials are planned to address more definitively the potential benefits of such cells

  • in the settings of acute infarction and chronic ischemic cardiomyopathy.

Such efforts would be aided substantially by the identification of specific markers as well as

  • an improved understanding of the role of subtypes of endothelial progenitor cells and
  • of the mechanisms by which they work.

Ironically, the data presented by Werner and colleagues in combination with work showing

  • the impaired functioning of endothelial progenitor cells in high-risk patients5 suggest
  • that the patients most in need of endothelial progenitor cells may be
      • those who are least able to donate them for autologous transplantation.

Whether these limitations can be overcome through

  • ex vivo expansion or  genetic modification of endothelial progenitor cells is unclear.

In addition to possible cell-based therapies, work on endothelial progenitor cells provides yet another rationale

  • for redoubling efforts to comply with established therapeutic guidelines,
  • including lifestyle modifications and the use of statin therapy,
      • both of which appear to enhance the number of circulating endothelial progenitor cells.

Whether there will be a downside to enhancing the number and function of  endothelial progenitor cells remains unclear,

  • although obvious concerns  include exacerbating conditions that are characterized by adverse vessel  formation,
    • such as diabetic retinopathy and tumor angiogenesis.

Small studies have suggested an association between high levels of circulating endothelial progenitor cells and the risk of certain cancers, such as multiple myeloma.11 Moreover, studies in animals show that

  • bone marrow derived endothelial progenitors participate in tumor angiogenesis, thereby
      • enhancing tumor growth.12

In the study by Werner and colleagues,

  • the number of deaths from cardiovascular causes among patients with high levels of endothelial progenitor cells
  • was substantially lower than that among patients with lower levels of these cells,
  • without a reduction in the risk of death overall.8

Although this finding could raise the specter of a counterbalancing adverse effect of endothelial progenitor cells,

  • there was no apparent pattern in the deaths due to other causes,
  • and no deaths from cancer were noted in this population.

It is possible that as we learn more about the biology of endothelial progenitor cells, there may be opportunities

  • to target vessel formation more specifically.

In addition, therapeutic strategies

  • tailored to individualized risk will undoubtedly help in practice.

For example, in the study by Werner et al.,

  • patients in the group with the lowest baseline levels of endothelial progenitor cells
  • had a risk of death from cardiovascular causes of 8.3 percent during one year of follow-up,
  • suggesting that the benefits of enhancing the function and number of endothelial progenitor cells
      • may well outweigh the risks in such high-risk populations.

Additional studies will be necessary to address these questions definitively. Larger studies

  • of longer duration performed in different cohorts will be required to determine fully
    • the clinical usefulness of endothelial progenitor cells as a biomarker.

Rigorous interventional studies will indicate

  • whether levels of endothelial progenitor cells can be used to guide therapy and
  • whether cell transfer has a role in augmenting the levels of these cells.

Basic-science studies should help guide these clinical efforts by

  • further defining the desirable subpopulations of endothelial progenitor cells and
  • the mechanisms by which they mediate their effects.

By establishing a connection between circulating endothelial progenitor cells and hard clinical end points, Werner and colleagues

  • provide a potent stimulus for clinical and basic studies to address these important issues.

Source Information

From the Program in Cardiovascular Gene Therapy, Massachusetts General  Hospital, and Harvard Medical School ― both in Boston.


Asahara T, Murohara T, Sullivan A, et al. Isolation of putative progenitor  endothelial cells for angiogenesis. Science 1997;275:964-967.

Takahashi T, Kalka C, Masuda H, et al. Ischemia- and cytokine-induced  mobilization of bone marrow-derived endothelial progenitor cells for  neovascularization. Nat Med 1999;5:434-438.

Kocher AA, Schuster MD, Szabolcs MJ, et al. Neovascularization of ischemic myocardium by human bone-marrow-derived angioblasts prevents cardiomyocyte apoptosis, reduces remodeling and improves cardiac function. Nat Med 2001; 7: 430-436.

Rauscher FM, Goldschmidt-Clermont PJ, Davis BH, et al. Aging, progenitor cell exhaustion, and atherosclerosis. Circulation 2003; 108: 457-463.

Hill JM, Zalos G, Halcox JPJ, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med 2003;348:593-600.

Vasa M, Fichtlscherer S, Adler K, et al. Increase in circulating endothelial  progenitor cells by statin therapy in patients with stable coronary artery  disease. Circulation 2001; 103: 2885-2890.

Laufs U, Werner N, Link A, et al. Physical training increases endothelial  progenitor cells, inhibits neointima formation, and enhances angiogenesis.  Circulation 2004; 109: 220-226.

Werner N, Kosiol S, Schiegl T, et al. Circulating endothelial progenitor cells and cardiovascular outcomes. N Engl J Med 2005; 353: 999-1007.

Aicher A, Heeschen C, Mildner-Rihm C, et al. Essential role of endothelial nitric oxide synthase for mobilization of stem and progenitor cells. Nat Med  2003; 9: 1370-1376.

Wollert KC, Meyer GP, Lotz J, et al. Intracoronary autologous bone-marrow  cell transfer after myocardial infarction: the BOOST randomised controlled  clinical trial. Lancet 2004; 364: 141-148.

Zhang H, Vakil V, Braunstein M, et al. Circulating endothelial progenitor cells in multiple myeloma: implications and significance. Blood 2005; 105: 3286-3294.

Lyden D, Hattori K, Dias S, et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat Med 2001;7:1194-1201.

Fluid phase biopsy for detection and characterization of circulating endothelial cells in myocardial infarction.

Kelly Bethel, Madelyn S Luttgen, Samir Damani, Anand Kolatkar, Rachelle Lamy, Mohsen Sabouri-Ghomi, Sarah Topol, Eric J Topol, Peter Kuhn

Physical Biology (Impact Factor: 2.62). 01/2014; 11(1):016002.
Source: PubMed

Elevated levels of circulating endothelial cells (CECs) occur in response to various pathological conditions including myocardial infarction (MI). Here, we adapted

  • a fluid phase biopsy technology platform that successfully detects circulating tumor cells in the blood of cancer patients (HD-CTC assay),
  • to create a high-definition circulating endothelial cell (HD-CEC) assay for the detection and characterization of CECs.

Peripheral blood samples were collected from 79 MI patients, 25 healthy controls and six patients undergoing vascular surgery (VS). CECs were defined

  • by positive staining for DAPI, CD146 and von Willebrand Factor
  • and negative staining for CD45.

In addition, CECs exhibited distinct morphological features that

  • enable differentiation from surrounding white blood cells.
  1. CECs were found both as individual cells and as aggregates.
  2. CEC numbers were higher in MI patients compared with healthy controls.
  3. VS patients had lower CEC counts when compared with MI patients

but were not different from healthy controls.

Both HD-CEC and CellSearch® assays could discriminate

  • MI patients from healthy controls with comparable accuracy

but the HD-CEC assay exhibited

  • higher specificity while maintaining high sensitivity.

Our HD-CEC assay may be used as a robust diagnostic biomarker in MI patients.

MicroRNA function in endothelial cells

Solving the mystery of an unknown target gene using microRNA Target Site Blockers
Dr. Virginie Mattot
Dr. Virgine Mattot works in the team “Angiogenesis, endothelium activation and Cancer” directed by Dr. Fabrice Soncin at the Institut de Biologie de Lille in France where she studies the roles played by microRNAs in endothelial cells during physiological and pathological processes such as angiogenesis or endothelium activation. She has been using Target Site Blockers to investigate the role of microRNAs on putative targets which functions are yet unknown.
What is the main focus of the research conducted in your lab?
We are studying endothelial cell functions with a particular interest
  • in angiogenesis and endothelium activation during physiological and tumoral vascular development.
How did your research lead to the study of microRNAs?
A few years ago, we identified in my team
  • a new endothelial cell-specific gene which harbors a microRNA in its intronic sequence.

We have since been working on understanding

  • the functions of both this new gene and
  • its intronic microRNA in endothelial cells

What is the aim of your current project?

While we were searching for the functions of the intronic microRNA,
  • we identified an unknown gene as a putative target.
The aim of my project was to investigate if this unknown gene was actually a genuine target and
  • if regulation of this gene by the microRNA was involved in endothelial cell function.
We had already characterized the endothelial cell phenotype associated with the inhibition of our intronic microRNA.
We then used miRCURY LNA™ Target Site Blockers to demonstrate
  • that the expression of this unknown gene is actually controlled by this microRNA.
Further, we also demonstrated that the microRNA regulates
  • specific endothelial cell properties through regulation of this unknown gene.
How did you perform the experiments and analyze the results?
LNA™ enhanced target site blockers (TSB) for our microRNA were designed by Exiqon.
We transfected the TSBs into endothelial cells using our standard procedure and
  • analysed the induced phenotype.
As a control for these experiments, a mutated version of the TSB was designed by Exiqon and
  • transfected into endothelial cells.
We first verified that this TSB was functional by
  • analyzing the expression of the miRNA target
      • against which the TSB was directed in transfected cells.
Finally, we showed that the TSB induced similar phenotypes as those found when we inhibited the microRNA in the same cells. 
What were some specific challenges in your experiments and how did you overcome them?
The fact that the target gene for our microRNA was unknown was a major challenge. Without specific available tools, like antibodies,
  • it becomes difficult to demonstrate the effect of the microRNA on the gene in question and
  • to show that the unknown gene is indeed responsible for the functions of the microRNA.
However through the use of specific target site blockers, we were able to demonstrate
  • that this unknown gene was associated with the phenotype observed
    • when the microRNA was inhibited in endothelial cells.
How do you feel about your results so far?
We are very pleased with the results of the TSB experiments and
  • altogether these results demonstrate that our miRNA of interest
  • is functional in endothelial cells
    • through the regulation of a target gene with a previously unknown role.
What do you find to be the main benefits/advantage of the LNA™ microRNA target site blockers from Exiqon?
Target Site Blockers are efficient tools to demonstrate the

  • specific involvement of putative microRNA targets
  • in the function played by this microRNA.
The use of LNA™ allows the design of short oligonucleotides that are very specific and easy to work with. 
What would be your advice to colleagues about getting started with microRNA functional analysis?
In order to address the role played by a microRNA,
  • it is essential to perform both gain and loss of functions experiments.
What are the next steps in the current project and how do you plan to perform them?
We plan to use microRNA inhibitor libraries to identify
  • more microRNAs specifically involved in the processes that we currently study.
When and where will be hear /read more about your studies?
We are currently in the process of submitting a manuscript regarding the function of my microRNA of interest.

Read Full Post »