Posts Tagged ‘IL-2’

Gluten-free Diets

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



Clinical, Serologic, and Histologic Features of Gluten Sensitivity in Children

Ruggiero Francavilla, Fernanda Cristofori, Stefania Castellaneta, et al.
J Pediatr 2014; 164:463-7.

Until a few years ago, the spectrum of gluten-related disorders included only celiac disease (CD) and wheat allergy (WA).  Recent data, however, suggest the existence of another form of gluten intolerance, known as nonceliac gluten sensitivity, or simply gluten sensitivity (GS). Some individuals experience distress after eating gluten-containing products and show improvement after institution of a gluten-free diet (GFD). Although the gastrointestinal symptoms may resemble those seen in CD, patients with CD do not have positive celiac-related antibodies or intestinal damage. This entity was described more than 30 years ago in 8 adult females suffering from abdominal pain and chronic diarrhea who experienced relief from a GFD and a return of symptoms on a gluten challenge.

GS is diagnosed in patients with symptoms that respond to removal of gluten from the diet, after CD and WA are excluded. Currently, it is a clinical diagnosis based on response to the GFD and relapse after gluten challenge; no specific blood test is available for GS. Sapone et al, aiming to elucidate the underlying pathophysiological mechanisms of GS, found that GS, as opposed to CD, is a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.

Recently, the existence of GS was confirmed by Biesiekierski et al in a double-blind, randomized, placebo-controlled challenge trial performed in a selected group of patients with irritable bowel syndrome who were symptomatically controlled on a GFD. Patients with irritable bowel syndrome-GS frequently demonstrate serum IgG class native anti-gliadin antibodies (AGA) as a possible marker of immune activation to gluten.

Objective To describe the clinical, serologic, and histologic characteristics of children with gluten sensitivity (GS). Study design We studied 15 children (10 males and 5 females; mean age, 9.6 + 3.9 years) with GS who were diagnosed based on a clear-cut relationship between wheat consumption and development of symptoms, after excluding celiac disease (CD) and wheat allergy, along with 15 children with active CD (5 males and 10 females; mean age, 9.1 + 3.1 years) and 15 controls with a functional gastrointestinal disorder (6males and 9 females; mean age, 8.6 + 2.7 years). Method: All children underwent CD panel testing (native anti-gliadin antibodies IgG and IgA, anti-tissue transglutaminase antibody IgA and IgG, and anti-endomysial antibody IgA), hematologic assessment (hemoglobin, iron, ferritin, aspartate aminotransferase, erythrocyte sedimentation rate), HLA typing, and small intestinal biopsy (on a voluntary basis in the children with GS).
Results Abdominal pain was the most prevalent symptomin the children with GS (80%), followed by chronic diarrhea in (73%), tiredness (33%), bloating (26%), limb pain, vomiting, constipation, headache (20%), and failure to thrive (13%). Native antigliadin antibodies IgG was positive in 66% of the children with GS. No differences in nutritional, biochemical, or inflammatory markers were found between the children with GS and controls. HLA-DQ2 was found in 7 children with GS. Histology revealed normal to mildly inflamed mucosa (Marsh stage 0-1) in the children with GS. Conclusion Our findings support the existence of GS in children across all ages with clinical, serologic, genetic, and histologic features similar to those of adults. (J Pediatr 2014;164:463-7).


Coeliac disease

C Leivers, G Martin, M Gasparetto, H Shelley, M Valente
Paediatrics and Child Health  2014; 24(11):481-84

Celiac disease is an immune-mediated systemic disorder, which is triggered by dietary gluten in genetically susceptible individuals. It is characterised by the presence of HLA-DQ2 or HLADQ8 genetic haplotypes, gluten-dependent signs and symptoms, celiac-specific antibodies and enteropathy.

The pathogenesis of celiac disease is complex and involves both genetic and environmental factors. Genetics is important: there is a high concordance in monozygotic twins (between 70 and 86%) and the HLA haplotype of DQ2/DQ8 is the principal genetic factor described. In the Caucasian population, between 30 and 35% will be carriers of these markers, but only 2-5% will go on to develop celiac disease. In patients with coeliac disease, 95% have HLA-DQ2 and 5-10% will carry HLA-DQ8.

Non-celiac gluten sensitivity

This occurs in those who have had a diagnosis of celiac disease excluded, but whereby there is a clear adverse response associated with gluten ingestion. Non-IgE mediated food allergy is a potential cause.

Differential diagnoses/causes of villous atrophy

  • Coeliac disease
  • Food protein hypersensitivity (particularly cow’s milk and/or soya proteins)
  • Eosinophilic gastroenteritis
  • Hypogammaglobulinemia
  • Whipple diseases
  • Abetalipoproteinaemia (Bassen-Kornzweig syndrome)
  • Intestinal lymphoma
  • Crohn’s disease
  • Infectious diseases (e.g. tuberculosis, giardiasis, parasitic infestations, infectious enteritis)
  • Small bowel bacterial overgrowth
  • Severe malnutrition
  • Small bowel ischemia
  • Radiotherapy
  • Autoimmune enteropathy
  • Cytotoxic drugs

Gluten challenge

Routine gluten challenge is not recommended. However, the process is advised when the initial diagnosis was not secure. Challenges should be undertaken at age 6-7 years or when pubertal growth is complete. Prior to a challenge HLA-DQ2/8 haplotype should be determined; if absent, celiac disease is unlikely. At least 4-6 weeks (ideally three months) of a normal gluten-containing diet (2-3 meals per day containing at least 5 g of gluten) is recommended prior to testing. Celiac serology and symptoms should be closely monitored to decide on the timing of biopsies. A pediatric dietitian is useful to support families and clinicians through this process.

Serological testing

Blood tests including tTG-IgA and full blood count are undertaken and growth is assessed. tTG-IgA levels are used to assess recovery and dietary adherence, particularly in asymptomatic patients. The high sensitivity and specificity of tTG-IgA in the diagnosis of celiac disease, has been extensively validated for diagnostic and follow up purposes.

Dietetic assessment and interview

A dietary assessment includes a review of the gluten free diet; the child and family’s level of adherence and its overall nutritional adequacy. In particular, the child’s intake of calcium and iron is assessed and if required, the family is advised on how to increase the intake of these nutrients up to the Reference Nutrient Intake (RNI).

Practice points

  1. The incidence of coeliac disease remains high, currently estimated to be around 1% of the UK population, although only 10-20% of these are diagnosed
  2. Prior to confirming a diagnosis, it is important to ensure the child is on a gluten-containing diet
  3. The diagnostic process in children has changed and depends on whether the child is symptomatic or asymptomatic, and on the level of their tTG
  4. Duodenal biopsy may be avoided in symptomatic children who meet strict criteria upon further laboratory testing
  5. A lifelong gluten free diet is currently the only treatment for celiac disease
  6. The management of coeliac disease involves examination, repeat serology and dietetic interview and support
  7. Prolonged, untreated coeliac disease has associated morbidity and mortality


Controlled Trial of Gluten-Free Diet in Patients with Irritable Bowel Syndrome-Diarrhea: Effects on Bowel Frequency and Intestinal Function

Maria I. Vazquez–Roque, Michael Camilleri, Thomas Smyrk, et al.
Gastroenterology 2013;144:903–911

Background & Aims: Patients with diarrhea-predominant irritable bowel syndrome (IBS-D) could benefit from a gluten-free diet (GFD).
Methods: We performed a randomized controlled 4-week trial of a gluten-containing  diet (GCD) or GFD in 45 patients with IBS-D; genotype analysis was performed for HLA-DQ2 and HLA-DQ8. Twenty-two patients were placed on the GCD (11 HLA-DQ2/8 negative and 11 HLA-DQ2/8 positive) and 23 patients were placed on the GFD (12 HLA-DQ2/8 negative and 11 HLADQ2/8 positive). We measured bowel function daily, small bowel (SB) and colonic transit, mucosal permeability (by lactulose and mannitol excretion), and cytokine production by peripheral blood mononuclear cells after exposure to gluten and rice. We collected rectosigmoid biopsy specimens from 28 patients, analyzed levels of messenger RNAs encoding tight junction proteins, and performed H&E staining and immune-histochemical analyses. Analysis of covariance models was used to compare data from the GCD and GFD groups.
Results: Subjects on the GCD had more bowel movements per day (P < .04); the GCD had a greater effect on bowel movements per day of HLA-DQ2/8–positive than HLA-DQ2/8–negative patients (P < .019). The GCD was associated with higher SB permeability (based on 0-2 h levels of mannitol and the lactulose/
mannitol ratio); SB permeability was greater in HLA-DQ2/8–positive than HLADQ2/8–negative patients (P < .018). No significant differences in colonic permeability were observed. Patients on the GCD had a small decrease in expression of zonula occludens 1 in SB mucosa and significant decreases in expression of zonula occludens 1, claudin-1, and occludin in rectosigmoid mucosa; the effects of the GCD on expression were significantly greater in HLA-DQ2/8–positive patients. The GCD vs the GFD had no significant effects on transit or histology. Peripheral blood mononuclear cells produced higher levels of interleukin-10, granulocyte colony-stimulating factor, and transforming growth factor-α in response to gluten than rice (unrelated to HLA genotype). Conclusions: Gluten alters bowel barrier functions in patients with IBS-D, particularly in HLA-DQ2/8–positive patients. These findings reveal a reversible mechanism for the disorder. Clinical trials.gov NCT01094041.

Our data convincingly showed effects of gluten on the increased mRNA expression of all the measured TJ proteins in colonic tissue relative to GFD. One limitation of the study was the inability to document alterations in colonic permeability using the 2-sugar excretion profile from 8 to 24 hours. We perceive that this may represent a lack of sensitivity of the lactulose and mannitol excretion test, for example, because of the metabolism of both sugars by colonic bacteria. There are advantages to measuring both tissue and in vivo markers of barrier function. Another limitation was that the mechanism for improvement in stool frequency on a GFD in the absence of changes in colonic transit was not elucidated by our studies. Because it is unclear whether gluten or its metabolic products induce specific secretory mechanisms, the current hypothesis is that change in stool frequency may reflect change in fluid secretion from increased mucosal permeability. Our current studies did not evaluate effects of gluten on the microbiome, afferent functions, or cytokine expression in the mucosal biopsy specimens from patients before and after the interventions. These would be interesting parameters to include in future studies. Finally, our study did not specifically address the effects of gluten protein per se, and it is possible that other proteins in wheat flour may be responsible for the changes observed.

Overall, our data provide mechanistic explanations for the observation that gluten withdrawal may improve patient symptoms in IBS. The data also explain, in part, the observation of the relationship of HLA genotype to beneficial effects of gluten withdrawal in view of our results showing that biological effects of gluten were associated with HLA-DQ2 or HLA-DQ8 genotype. The data suggest that the relationship of dietary factors, innate and adaptive immune responses, and mucosal interactions in IBS-D deserve further study, and they support the need for further clinical intervention studies to evaluate the clinical effects of gluten withdrawal in patients with IBS-D.


Ingestion of oats and barley in patients with celiac disease mobilizes cross-reactive T cells activated by avenin peptides and immuno-dominant hordein peptides

Melinda Y. Hardy, Jason A. Tye-Din, Jessica A. Stewart, et al.
Journal of Autoimmunity 56 (2015) 56-65

Celiac disease (CD) is a common CD4+ T cell mediated enteropathy driven by gluten in wheat, rye, and barley. Whilst clinical feeding studies generally support the safety of oats ingestion in CD, the avenin protein from oats can stimulate intestinal gluten-reactive T cells isolated from some CD patients in vitro. Our objective was to establish whether ingestion of oats or other grains toxic in CD stimulate an avenin specific T cell response in vivo. We fed participants a meal of oats (100 g/day over 3 days) to measure the in vivo polyclonal avenin-specific T cell responses to peptides contained within comprehensive avenin peptide libraries in 73 HLADQ2.5+ CD patients. Grain cross-reactivity was investigated using oral challenge with wheat, barley, and rye. Avenin-specific responses were observed in 6/73 HLA-DQ2.5+ CD patients (8%), against four closely related peptides. Oral barley challenge efficiently induced cross-reactive avenin/hordein-specific T cells in most CD patients, whereas wheat or rye challenge did not. In vitro, immunogenic avenin peptides were susceptible to digestive endopeptidases and showed weak HLA-DQ2.5 binding stability. Our findings indicate that CD patients possess T cells capable of responding to immuno-dominant hordein epitopes and homologous avenin peptides ex vivo, but the frequency and consistency of these T cells in blood is substantially higher after oral challenge with barley compared to oats. The low rates of T cell activation after a substantial oats challenge (100 g/d) suggests that doses of oats commonly consumed are insufficient to cause clinical relapse, and supports the safety of oats demonstrated in long-term feeding studies.


Diagnosis and classification of celiac disease and gluten sensitivity

Elio Tonutti, Nicola Bizzaro
Autoimmunity Reviews 13 (2014) 472–476
Celiac disease is a complex disorder, the development of which is controlled by a combination of genetic (HLA alleles) and environmental (gluten ingestion) factors. New diagnostic guidelines developed by ESPGHAN emphasize the crucial role of serological tests in the diagnostic process of symptomatic subjects, and of the detection of HLA DQ2/DQ8 alleles in defining a diagnosis in asymptomatic subjects belonging to at-risk groups. The serological diagnosis of CD is based on the detection of class IgA anti-tissue transglutaminase (anti-tTG) and anti-endomysial antibodies. In patients with IgA deficiency, anti-tTG or anti-deamidated gliadin peptide antibody assays of the IgG class are used. When anti-tTG antibody levels are very high, antibody specificity is absolute and CD can be diagnosed without performing a duodenum biopsy. Non-celiac gluten sensitivity is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out. Diagnostic criteria include the presence of symptoms similar to those of celiac or allergic patients; negative allergological tests and absence of anti-tTG and EMA antibodies; normal duodenal histology; evidence of disappearance of the symptoms with a gluten-free diet; relapse of the symptoms when gluten is reintroduced.

Celiac disease (CD) is a chronic, immune-mediated, gluten-induced gut disorder that manifests itself with a range of clinical symptoms in genetically susceptible subjects. Immune reaction to wheat, barley and rye gliadin fractions and glutenins triggers an inflammatory state of the duodenal mucosa: the result is reduced intestinal villus height and hyperplastic cryptae that may lead to complete villus atrophy. The critical role played by gluten is demonstrated by the fact that in CD patients on a gluten free diet (GFD) clinical symptoms disappear, anti-transglutaminase 2 antibodies (anti-tTG2, the serological markers of the disorder) normalize, and villus atrophy recedes. As to the role
of genetic factors, CD development has been demonstrated to be closely associated with MHC class II HLA-DQ2 and HLA-DQ8 molecules; in fact, virtually all CD patients express at least one of these HLA molecules compared to the general population in which about 30–35% have either DQ2 or DQ8.

A new gluten-associated clinical condition, named ‘non-celiac gluten sensitivity’ (NCGS) [4], also described in literature as gluten hypersensitivity or gluten intolerance, has been recently identified. NCGS is characterized by gastrointestinal or extra-intestinal symptoms comparable, in many cases, to those of CD patients; however, to date no specific immunological mechanisms or serological markers have been identified for this disorder. The diagnosis is made by exclusion of CD or IgE-mediated allergy to wheat, and is based on the direct association between gluten ingestion and symptom onset.

The development of highly sensitive immunological methods for identifying diagnostic antibodies (e.g. anti-tTG autoantibodies and anti-DGP antibodies) has enabled an increasing number of CD patients with vague or asymptomatic clinical presentations to be identified. Population-based studies now indicate that approximately 0.5–1% of the Western European and Northern American populations suffer from CD. In a recent paper, Abadie and coworkers correlate gluten consumption with HLA DQ2 and DQ8 haplotype frequency in the populations of the different world countries. The authors found a significant correlation between CD prevalence and wheat consumption, and between CD prevalence and DQ2–DQ8 frequency in most countries. However, outlier countries have been observed: Finland and Russia, for example, have similar wheat consumption levels and comparable HLA haplotype frequencies, but the prevalence of CD in Finland is 1–2.4% whereas in the adjacent Russian republic of Karelia the prevalence of CD is considerably lower (0.2%). In the Maghreb area, wheat and barley are the major staple foods. Despite similar frequencies of the DR3–DQ2 and DR4–DQ8 haplotypes, the prevalence of CD in Algeria (5.6%) is by far the highest reported worldwide, whereas CD prevalence in Tunisia (0.28%) remains one of the lowest. These observations suggest that similar levels of wheat consumption and predisposing HLA expression can be associated with strikingly different levels of CD prevalence.

CD is characterized by multiple clinical expressions. An ESPGHAN (European Society for Paediatric Gastroenterology, Hepatology and Nutrition) working group has recently developed new guidelines for the diagnosis of CD based on scientific and technical developments using an evidence-based approach. The ESPGHAN working group decided to revise the classification, also taking into consideration signs and symptoms that had not been considered in the previous classification. In particular, it was deemed advisable to eliminate the distinction between classic and atypical CD based on symptoms, as atypical signs and symptoms (e.g. anemia, neuropathy, reduced bone density) may be considerably more common than classic symptoms (e.g. chronic diarrhea).

Patients suffering from certain disorders (especially Hashimoto’s thyroiditis, type I diabetes, IgA deficiency and Down’s syndrome) have a higher risk of developing CD than the normal population. In these patients it is advisable to perform HLA DQ2/DQ8 and serological tests for CD even in the absence of symptoms.

CD and NGCS cannot be distinguished clinically, since the symptoms experienced by NGCS patients are often seen in CD. The definition of NGCS is a gluten reaction in which both allergic and autoimmune mechanisms have been ruled out (diagnosis by exclusion criteria).

Specifically: symptoms similar to those of celiac or allergic patients must be present; in vivo and in vitro wheat allergy tests (prick test and specific IgE), as well as anti-tTG and EMA antibodies must be negative; duodenal histology must be normal; the patients must also experience a disappearance of the symptoms when on a GFD and their reappearance after the reintroduction of gluten. The most frequent symptoms in NGCS patients are abdominal pain, eczema or rash, headache, blurred vision, fatigue, diarrhea, depression, anemia, numbness in the legs, arms or fingers, and joint pain.

Signs and symptoms of patients with non-celiac gluten sensitivity (NCGS)

Abdominal pain
Abdominal distension/bloating
Foggy mind
Numbness in the legs, arms
Joint pain

An important aspect, confirmed by numerous studies, is the correlation between anti-tTG count and histological damage. One of the latest studies assessed retrospectively 412 consecutive anti-tTG and EMA patients who received a biopsy for suspected CD: the subjects whose levels of anti-tTG were greater than 7-fold the cut-off value had a 99.7% positive predictive value for histological damage (with Marsh score N2). To date, there are no specific laboratory markers for NCGS; a recent study by Volta found that 78 patients with NCGS were AGA IgG positive in 56.4% of the cases and AGA IgA positive in 7.7% of the cases. All patients were negative for anti-DGP IgG and IgA, as well as for anti-tTG and EMA.

Analysis of multiple biopsies is important: patchiness of the lesion has been reported and recent work suggests that different degrees of severity may be present, even in the same bioptic fragment. The biopsies should be taken from the second/third portion of the duodenum and at least one biopsy should be taken from the duodenal bulb. Patients with NCGS do not exhibit significant alterations of the duodenal mucosa; histological negativity is an essential parameter for a diagnosis of NCGS.

The diagnostic criteria proposed by ESPGHAN in 1990 envisaged the performance of gastro-duodenoscopy and histological confirmation of mucosal damage as the conclusive phase of the diagnostic process. These criteria did not indicate which serological tests should be positive, were not applicable to children aged below 2 years, and in any case required other clinical conditions to be ruled out. Therefore, in 2010, the ESPGHAN working group deemed appropriate to set out new criteria based on new knowledge and diagnostic tools developed in the last few years.

The new CD guidelines are revolutionary in two major respects: the crucial role of serological tests in the diagnostic process of symptomatic subjects, and the detection of HLA DQ2/DQ8 in diagnosing asymptomatic subjects belonging to groups at risk of CD.

Concerning the diagnosis of children and adolescents with signs and symptoms suggestive of CD, the ESPGHAN guidelines recommend, as the initial approach to symptomatic patients, testing for anti-tTG IgA antibodies as well as for total serum IgA to exclude IgA deficiency. As an alternative to total serum IgA, direct testing for IgG anti-DGP antibodies can be performed. The decision to perform IgA anti-tTG as the initial test in this population is based on the high sensitivity and specificity of the test, its widespread availability, and low costs compared with the EMA IgA test.

A fundamental aspect of the new guidelines concerns the possibility of not necessarily performing an intestinal biopsy if the anti-tTG antibody levels are very high, as in these cases the specificity of the antibody is absolute. Indeed, pediatric gastroenterologists should discuss with the parents and the patient who is positive for anti-tTG antibody levels N10 times ULN (as appropriate for age) the option of omitting the biopsies and the implications of doing so. If the parents (patient) accept this option, then blood should be drawn for HLA and EMA testing.

Patients with positive anti-tTG antibody levels lower than 10 times the upper limit for the normal population (ULN) given by the manufacturer of this particular test should undergo upper endoscopy with multiple biopsies.

As far as diagnosis methods in asymptomatic pediatric patients belonging to
at-risk groups are concerned, the ESPGHAN guidelines suggest a different procedure. In these patients, HLA-DQ2 and HLA-DQ8 testing as the initial action is probably cost-effective since a significant proportion of the patients can be excluded from further studies because they do not harbor DQ2 or DQ8. In individuals with DQ2 or DQ8 positivity, IgA anti-tTG and total serum IgA determination should be performed. If IgA anti-tTG is negative and IgA deficiency is excluded, then CD is unlikely; however, the disease may still develop later in life. Therefore, serological testing should be repeated at regular intervals. If anti-tTG antibodies are positive, then signs related to CD should be searched for (e.g. anemia, elevated liver enzymes).


Influence of dietary components on Aspergillus niger prolyl endoprotease mediated gluten degradation

Veronica Montserrat, Maaike J. Bruins, Luppo Edens, Frits Koning
Food Chemistry 174 (2015) 440–445

Celiac disease (CD) is caused by intolerance to gluten. Oral supplementation with enzymes like Aspergillus niger propyl-endoprotease (AN-PEP), which can hydrolyse gluten, has been proposed to prevent the harmful effects of ingestion of gluten. The influence of meal composition on AN-PEP activity was investigated using an in vitro model that simulates stomach-like conditions. AN-PEP optimal dosage was 20 proline protease units (PPU)/g gluten. The addition of a carbonated drink strongly enhanced AN-PEP activity because of its acidifying effect. While fat did not affect gluten degradation by AN-PEP, the presence of food proteins slowed down gluten detoxification. Moreover, raw gluten was degraded more efficiently by AN-PEP than baked gluten. We conclude that the meal composition influences the amount of AN-PEP needed for gluten elimination. Therefore, AN-PEP should not be used to replace a gluten free diet, but rather to support digestion of occasional and/or inadvertent gluten consumption.


No Effects of Gluten in Patients with Self-Reported Non-Celiac Gluten Sensitivity after Dietary Reduction of Fermentable, Poorly Absorbed, Short-Chain Carbohydrates

Jessica R. Biesiekierski, Simone L. Peters, Evan D. Newnham, et al.
Gastroenterology 2013;145:320–328

Background & Aims: Patients with non-celiac gluten sensitivity (NCGS) do not have celiac disease but their symptoms improve when they are placed on gluten-free diets. We investigated the specific effects of gluten after dietary reduction of fermentable, poorly absorbed, short-chain carbohydrates (fermentable, oligo-, di-, monosaccharides, and polyols [FODMAPs]) in subjects believed to have NCGS. Methods: We performed a double-blind crossover trial of 37 subjects (aged 2461 y, 6 men) with NCGS and irritable bowel syndrome (based on Rome III criteria), but not celiac disease. Participants were randomly assigned to groups given a 2-week diet of reduced FODMAPs, and were then placed on high-gluten (16 g gluten/d), low-gluten (2 g gluten/d and 14 g whey protein/d), or control (16 g whey protein/d) diets for 1 week, followed by a washout period of at least 2 weeks. We assessed serum and fecal markers of intestinal inflammation/injury and immune activation, and indices of fatigue. Twenty-two participants then crossed over to groups given gluten (16 g/d), whey (16 g/d), or control (no additional protein) diets for 3 days. Symptoms were evaluated by visual analogue scales. Results: In all participants, gastrointestinal symptoms consistently and significantly improved during reduced FODMAP intake, but significantly worsened to a similar degree when their diets included gluten or whey protein. Gluten-specific effects were observed in only 8% of participants. There were no diet-specific changes in any biomarker. During the 3-day rechallenge, participants’ symptoms increased by similar levels among groups. Gluten-specific gastrointestinal effects were not reproduced. An order effect was observed. Conclusions: In a placebo controlled, cross-over rechallenge study, we found no evidence of specific or dose-dependent effects of gluten in patients with NCGS placed diets low in FODMAPs. www.anzctr.org.au.ACTRN12610000524099


Gluten Sensitivity: Not Celiac and Not Certain

See “No effects of gluten in patients with self-reported non-celiac gluten sensitivity after dietary reduction of fermentable, poorlyabsorbed, short-chain carbohydrates,” by Biesiekierski JR, Peters SL, Newnham ED, et al.

Rohini Vanga, Daniel A. Leffler

The past few years have seen a flurry of clinical and basic research studies targeting NCGS, the results of which seem determined to thwart any attempt to come to broad consensus regarding what NCGS is or is not, what causes it, and who it might affect.

Some studies suggest that NCGS generally belongs on the spectrum of functional bowel disorders. Other studies are more suggestive that NCGS may actually fit better within the spectrum of celiac disease. For example, in contrast with the studies by Biesiekierski et al. and Saponi et al., a number of studies have reported that nonceliac individuals with gluten-responsive symptoms are more likely to carry human leukocyte antigen (HLA)-DQ2/8. Taking a somewhat different tack, Carroccio et al. reported that NCGS patients with negative wheat IgE allergy testing developed greater symptoms with wheat exposure compared with placebo (P < .0001). The presence of anemia, weight loss, self-reported wheat intolerance, history of food allergy in infancy, and coexistent atopic diseases were more frequent in wheat-sensitive patients than in non–gluten-responsive IBS controls. There was also a higher frequency of positive serum assays for IgG/IgA anti-gliadin and greater association with DQ2 or DQ8 haplotype than controls.

In this issue of Gastroenterology, Biesiekierski et al. return with another double-blind, randomized, controlled trial on NCGS. Although in many ways this work seems to have been designed as a more thorough follow-on study to their prior work, the most significant variation from the prior study was the recommendation that participants restrict to low-fermentable, poorly absorbed, short-chain carbohydrates (FODMAPs) throughout the study. With the changing patterns of food intake and dietary behaviors over the last 20 years so-called westernization, FODMAPs have constituted significant proportion in food consumption. FODMAPs have been identified as important triggers for functional gut symptoms in people with visceral hypersensitivity or abnormal motility responses, largely by inducing luminal distension via a combination of osmotic effects and gas production related to their rapid fermentation by bacteria in the small and proximal large intestines. This seems to have been the rationale behind the addition of a low FODMAP diet in the current study, limiting alternate dietary triggers that could confound results. In the current study,  subjects with NCGS defined as “IBS fulfilling Rome III criteria that self-reportedly improved with a GFD” after exclusion of celiac disease were enrolled into the trial.

This study calls into question the very existence of NCGS as a discrete entity and suggests that FODMAPs, rather than gluten or other wheat proteins, might be the mediator by which low-gluten diets improve gastrointestinal symptoms. As noted, there are many potential ways in which FODMAPs may elicit gastrointestinal symptoms in predisposed individuals; however, limited as our understanding of NCGS is, investigation into FODMAPs in gastrointestinal disease has been nearly nonexistent outside of a few small studies published by this same group. The other clear possibility is that NCGS is a real entity but confounded by a low FODMAP diet by an unclear mechanism. In either case, it is tempting to say that everything seems to be at a standstill and therefore NCGS remains a controversial topic. Overall, these studies have highlighted the great potential of specific dietary interventions in gastrointestinal disorders outside of celiac disease. Although few facets of NCGS are clear, it is apparent that only the combination of larger, high-quality clinical trials on the role of specific diets in patients with chronic gastrointestinal symptoms, and translational studies evaluating mechanisms and potential biomarkers of NCGS and other food sensitivities, will allow us to make advances on this elusive entity.


Predictors of dietary gluten avoidance in adults without a prior diagnosis of celiac disease

Pornthep Tanpowpong , S Broder-Fingert, AJ. Katz, CA. Camargo Jr.
Nutrition 31 (2015) 236–238

Objective: Prior studies have shown that dietary gluten avoidance (DGA) is relatively common in children without previously diagnosed celiac disease (CD), and several clinical predictors of DGA have been found. However, available data on predictors of DGA in adults without diagnosed CD are limited. The aim of this study was to determine the independent predictors of DGA in this population. Methods: We performed a structured medical record review of 376 patients, ages 20 y, who had never been formally diagnosed with CD, presenting for an initial CD evaluation (ICD-9-CM 579.0) between January 2000 and December 2010 at two large Boston teaching hospitals. We collected data including demographic characteristics, medical history, history of CD serology before referral, and self-reported DGA. Predictors of DGA were determined using multivariable logistic regression. Results: Mean age was 47 (SD ¼ 17) years. We found that 41 patients (10.9%; 95% confidence interval [CI], 7.9–14.5) had avoided gluten at some time in their lives. Most patients had subjective abdominal complaints or bowel movement changes. History of CD seropositivity before referral was noted in 14%. Independent predictors of DGA (P < 0.05) were lactose intolerance (odds ratio [OR], 2.8; 95% CI, 1.1–7.5), food allergy (OR, 3.8; 95% CI, 1.04–13.7), and history of positive serology of less-specific CD markers before the referral (OR, 3.2; 95% CI, 1.3–7.9). Conclusions: Gluten avoidance is common in a clinic population of adults without prior CD diagnosis. The recognized predictors suggest that DGA may associate with conditions presenting with nonspecific gastrointestinal complaints and perhaps with the perceived benefits of DGA among patients with prior history of positive CD serology.

Solubilization of gliadins for use as a source of nitrogen in the selection of bacteria with gliadinase activity

Patricia Alvarez-Sieiro, Begoña Redruello, Victor Ladero, Elena Cañedo, et al.
Food Chemistry 168 (2015) 439–444

For patients with celiac disease, gliadin detoxification via the use of gliadinases may provide an alternative to a gluten-free diet. A culture medium, in which gliadins were the sole source of nitrogen, was developed for screening for microorganisms with gliadinase activity. The problem of gliadin insolubility was solved by mild acid treatment, which renders an acid-hydrolysed gliadin/peptide mixture (AHG). This medium provided a sensitive and reliable means of detecting proteases, compared to the classical spectrophotometric method involving azocasein. When a sample of fermented wheat (a source of bacteria) was plated on an AHG-based culture medium, strains with gliadinase activity were isolated. These strains’ gliadinase profiles were determined using an AHG-based substrate in zymographic analyses.


Sustained in vivo signaling by long-lived IL-2 induces prolonged increases of regulatory T cells

Charles J.M. Bell, Yongliang Sun, Urszula M. Nowak, Jan Clark, et al.
Journal of Autoimmunity 56 (2015) 66e80

Regulatory T cells (Tregs) expressing FOXP3 are essential for the maintenance of self-tolerance and are deficient in many common autoimmune diseases. Immune tolerance is maintained in part by IL-2 and deficiencies in the IL-2 pathway cause reduced Treg function and an increased risk of autoimmunity. Recent studies expanding Tregs in vivo with low-dose IL-2 achieved major clinical successes highlighting the potential to optimize this pleiotropic cytokine for inflammatory and autoimmune disease indications. Here we compare the clinically approved IL-2 molecule, Proleukin, with two engineered IL-2 molecules with long half-lives owing to their fusion in monovalent and bivalent stoichiometry to a non-FcRg binding human IgG1. Using nonhuman primates, we demonstrate that single ultra-low doses of IL-2 fusion proteins induce a prolonged state of in vivo activation that increases Tregs for an extended period of time similar to multiple-dose Proleukin. One of the common pleiotropic effects of high dose IL-2 treatment, eosinophilia, is eliminated at doses of the IL-2 fusion proteins that greatly expand Tregs. The long half-lives of the IL-2 fusion proteins facilitated a detailed characterization of an IL-2 dose response driving Treg expansion that correlates with increasingly sustained, supra-threshold pSTAT5α induction and subsequent sustained increases in the expression of CD25, FOXP3 and Ki-67 with retention of Treg-specific epigenetic signatures at FOXP3 and CTLA4.

Over the last 20 years we have progressed from discovering that IL-2 and IL-2RA are genetically associated with autoimmune diabetes and the functional state of Tregs to seeing dramatic clinical success with IL-2 in chronic GVHD. The central role of IL-2 in the maintenance of self-tolerance and Treg function is now immunological canon and many attempts are being made to harness Tregs to combat a variety of autoimmune and inflammatory diseases. The recent clinical successes with Proleukin are noteworthy since pharmacologically it is a drug with limitations: its short half-life requires daily or every other day injection and the doses used to date stimulate CD4+ T effector cells, NK cells and eosinophils in addition to Tregs. Our goal was to develop and characterize IL-2 molecules with improved pharmacologic profiles that could be delivered less frequently and at lower doses than Proleukin and selectively expand Tregs that maintained their epigenetic profiles at FOXP3 and CTLA4.

Increasing the in vivo half-life of IL-2 by fusion to IgG1, i.e. IgG-IL-2, results in a molecule that can induce a 4-fold increase in Tregs after a single dose in cynomolgus, a response that multiple-dose, but no single dose, of Proleukin can achieve. Increasing the stoichiometry and hence the avidity, i.e. IgG-(IL-2)2, increases the potency and stimulates a similar increase in Tregs albeit at a 5-fold lower dose than IgG-IL-2. A detailed characterization of the in vivo dose responses for Proleukin and IgG-(IL-2)2 highlights that the magnitude and duration of Treg expansion, defined by its AUC, correlates with the magnitude and duration of pSTAT5α upregulation, also defined by its AUC. Single doses of Proleukin that increase pSTAT5α for one day have a minimal AUC and as a consequence little impact on Treg numbers; whereas single dose IgG-IL-2 and IgG-(IL-2)2 or multiple-dose Proleukin stimulate pSTAT5α that is  sustained for 4 days resulting in 3-4-fold larger pSTAT5α AUCs and corresponding increases in Tregs and the AUCs of Treg/mm3. Intermediate levels and duration of pSTAT5α induction result in moderate increases in Tregs. Following in vivo activation with Proleukin and IgG-(IL-2)2, Treg cell surface CD25 as well as intracellular FOXP3 and Ki-67, increased in a dose-dependent manner and persisted longer than the corresponding pSTAT5α responses; the effects of IgG-(IL-2)2 were >10-fold more potent and persisted longer than those induced by Proleukin. Of particular significance, the cynomolgus Tregs present after IgG-IL-2 and IgG-(IL-2)2-induced in vivo expansion retain their fully demethylated FOXP3 and CTLA4 epigenetic signatures indicating a functional suppressive phenotype.

The ability of cynomolgus to respond and differentiate amongst different forms and doses of IL-2 with varying degrees of activation and increases in Tregs speaks to their utility as a translational preclinical species. In fact, single doses of IgG-IL-2 and IgG-(IL-2)2 replicated the increased number of Tregs seen in GVHD patients given daily Proleukin. Furthermore, Proleukin given to cynomolgus following the same multiple-dose protocol at the human equivalent dose achieved the same increases in Tregs and eosinophils as patients with type 1 diabetes.

The long half-lives of IgG-IL-2 and IgG-(IL-2)2 enable the detection of receptor-mediated clearance of IL-2 in vivo; the half-lives of the fusion proteins are five times longer in mice in the absence of the high affinity IL-2 receptor. The competition for injected IL-2 by different cell populations and the upregulation of IL-2 receptors in response to injections of the cytokine are important considerations when interpreting IL-2 doses required for preferential Treg expansion.  The failure of low-dose IL-2 to expand cynomolgus NK cells in vivo means that this aspect of IL-2 immunotherapy using novel, long-lived molecules will need to be addressed in future human studies. Despite these differences, the pharmacokinetic and pharmacodynamic analyses in this cynomolgus study strongly support the hypothesis that increasing the half-life of IL-2 allows for lower doses of IL-2 to be delivered far less frequently thereby favoring prolonged Treg-specific cell expansion.


T cell subsets and their signature cytokines in autoimmune and inflammatory diseases

Itay Raphael, Saisha Nalawade, Todd N. Eagar, Thomas G. Forsthuber
Cytokine xxx (2014) xxx–xxx

CD4+ T helper (Th) cells are critical for proper immune cell homeostasis and host defense, but are also major contributors to pathology of autoimmune and inflammatory diseases. Since the discovery of the Th1/Th2 dichotomy, many additional Th subsets were discovered, each with a unique cytokine profile, functional properties, and presumed role in autoimmune tissue pathology. This includes Th1, Th2, Th17, Th22, Th9, and Treg cells which are characterized by specific cytokine profiles. Cytokines produced by these Th subsets play a critical role in immune cell differentiation, effector subset commitment, and in directing the effector response. Cytokines are often categorized into proinflammatory and anti-inflammatory cytokines and linked to Th subsets expressing them. This article reviews the different Th subsets in terms of cytokine profiles, how these cytokines influence and shape the immune response, and their relative roles in promoting pathology in autoimmune and inflammatory diseases. Furthermore, we will discuss whether Th cell pathogenicity can be defined solely based on their cytokine profiles and whether rigid definition of a Th cell subset by its cytokine profile is helpful.

T helper cell subsets differentiate and express their protective and pathogenic roles of their lineage-signature cytokines. The signature cytokines for each subset are as follows: IL-12 induces the expression of T-bet and differentiation into the Th1 subset which produces IFN-c and TNF; Th2 differentiation and GATA3 expression is induced by IL-4, leading to the production of IL-4, IL-5 and IL-13, whereas TGF- T helper-cell subset differentiation and the protective and pathogenic roles of their lineage-signature cytokines. The signature cytokines for each subset is as follows:  IL-12 induces the expression of T-β and differentiation into the Th1 subset which produces IFN-c and TNF; Th2 differentiation and GATA3 expression is induced by IL-4, leading to the production of IL-4, IL-5 and IL-13, whereas TGF-β and IL-4 induce PU.1 expression. This causes differentiation into the Th9 subset and leads to the production of IL-9. TGF-β induces the expression of Foxp3, which leads to differentiation into the Treg lineage; Th17 differentiation is a result of RORct expression induced by TGF-β, IL-6 and IL-23, leading to the production of IL-17, IL-22, IL-21, IL-25 and IL-26 (human); IL-6 and TNF induce AHR and differentiation into the Th22 subset and production of IL-22. STAT: Signal transducer and activator of transcription; RORc: RAR related orphan receptor gamma, AHR: Aryl hydrocarbon receptor, Foxp3: forkhead box P3 and IL-4 induce PU.1 expression which causes differentiation into the Th9 subset leading to the production of IL-9. TGF-β induces the expression of Foxp3, which leads to differentiation into the Treg lineage; Th17 differentiation is a result of RORct expression induced by TGF-β, IL-6 and IL-23, leading to the production of IL-17, IL-22, IL-21, IL-25 and IL-26 (human); IL-6 and TNF induce AHR and differentiation into the Th22 subset and production of IL-22. STAT: Signal transducer and activator of transcription; RORc: RAR related orphan receptor gamma, AHR: Aryl hydrocarbon receptor, Foxp3: forkhead box P3.

In autoimmune diseases, Th2 cells were initially described as anti-inflammatory based on their ability to suppress cell-mediated or Th1 models of disease. Th2 cells have been described in lesions of MS patients, and IL-4 and IL-4R expression has been reported in several cell types in close proximity to active demyelinating lesions. Over the years, however, a number of reports established a role for Th2 cells in tissue inflammation and implicated their cytokines in immunopathology.  Genain et al. reported that in marmoset monkeys with EAE the cytokine production was shifted from a Th1 to a Th2 pattern, and titers of autoantibodies to myelin oligodendrocyte glycoprotein (MOG) were enhanced. They concluded that induction of Th2 responses may exacerbate autoimmunity by enhancing production of pathogenic autoantibodies.

The involvement of Th2 cells and pathogenic antibodies contrast the prevailing models of murine EAE which are considered to be Th1 and Th17-effector T cell-mediated diseases. However, pathogenic roles for Th2 cells have also been reported in murine EAE. Lafaille et al. showed that adoptive transfer of Th2-polarized MBP-specific T effector cells elicited EAE in immunocompromised recipient mice (RAG-1 or TCRα deficient), but not immune-sufficient hosts. When compared with other T effector subsets, mice receiving Th2 cells developed EAE with delayed onset and milder symptoms. Jager et al. have also reported that 2D2 MOG-specific Th2 cells can induce EAE with delayed onset and low severity. Taken together, these reports support that Th2 cells can promote pathogenicity, but ensuing disease may be less severe. Alternatively, but not mutually exclusive, development of EAE may not have been mediated by ‘‘Th2’’ cytokines, but might have been due to the switch of Th2 cells to a Th1-like phenotype and secretion of proinflammatory cytokines such as IFN-c. Th2 cytokines are associated with the pathogenesis of antibody-mediated autoimmune diseases.

The expression of one signature cytokine, such as IL-17, may not tell the full story about Th subset commitment, since the stability of its expression may be influenced by different factors as mentioned above. Along these lines, IL-17 is enhanced by IL-23, which promotes the pathogenic potential of Th17 cells and enhances the expression of IL-17 by these cells. Thus, adoptive transfer of IL-23-induced Th17 cells results in severe EAE, and in the absence of IL-23 signaling the mice are resistant to EAE. However, the disease resistance seen in the absence of IL-23 signals was not due to the lack of expression of IL-17 or IL-22 by Th17 cells, but rather by the failure of these cells to produce GM-CSF, a cytokine that was initially believed to be produced by encephalitogenic, IFN-c producing Th1 cells. Indeed both Th1 cells and Th17 cells can produce GM-CSF. Interestingly, induction of GM-CSF expression by human Th cells is constrained by the IL-23/ROR-ct/Th17 cell axis but promoted by the IL-12/T-bet/Th1 cell axis. Thus the enigma remains as to why IL-23-induced Th17 cells are indispensable for the induction of EAE. As it turns out, IL-23-induced Th17 cells not only produce GM-CSF, but are also producing IFN-c. The observation of IFN-c producing Th17 cells lead to the realization that IL-17 and IFN-c double-producing cells, belonging to the Th17 subset, developed under the influence of IL-23 and converted into IL-17 producing Th1-like cells, and later to ‘‘exTh17’’ cells, while discontinuing the production of IL-17.

The concept of a specialized subset of T lymphocytes with suppressive function has been around since the early 1970s. In the mid-1990s a novel subset of Th cells with ‘‘regulatory’’ function was identified and designated Tregs. Tregs were later found to express the signature Foxp3 transcription factor, which is critical for their development, lineage commitment, and regulatory functions. Foxp3 expressing Treg subsets include thymically derived or natural Tregs (nTregs) and Tregs that are induced via post-thymic maturation (iTregs). Later, iTregs were further discriminated into Foxp3+ cells (Th3) and Foxp3 cells (Tr1). Numerous studies have identified Tregs as important immunoregulators in many inflammatory and autoimmune disease conditions including asthma, MS, and type-I diabetes.

Several mechanisms of Treg-mediated immune suppression have been identified, including: the secretion of anti-inflammatory  cytokines, expression of inhibitory receptors, and cytokine deprivation. For the purpose of this review we will focus on regulatory cytokine production. The two cytokines mostly associated with Tregs are IL-10 and TGF-β. Importantly, Tregs can themselves secrete these cytokines and use them to carry out their suppressive function. TGF-β is produced by both nTreg and Th3 cells, however other cells including B cells, macrophages, DCs, and many other non-immune cells, can also produce this cytokine. TGF-β is required for the generation of iTregs by inducing the expression of Foxp3 in a paracrine feedback loop that will convert naïve T cells (Th0) to differentiate into iTregs. The positive feedback loop between TGF-β and Foxp3 plays a critical role in maintaining peripheral tolerance and is key to the generation and maintenance of Tregs. In vivo, TGF-β producing Tregs have been shown to suppress EAE by inhibiting autoimmune T cell responses in the CNS of EAE mice.

Not shown.  A proposed model reveals an immune switch point from pathogenic Th17 cells to suppressive ex-Th17 cells in EAE. TGF-β, IL-6 and IL-23 induce the differentiation of Th17 cells in the immune periphery. In the CNS, signaling by IL-23 induces the expression of GM-CSF and IFN-c in Th17 cells, thereby rendering these cells pathogenic. In an autocrine signaling loop, IFN-c suppresses the expression of RORct and the production of GM-CSF (as well as IL-17) by pathogenic Th17 cells, thereby inducing a switch to ‘‘suppressive’’ exTh17 cells.

ExTh17 cells are expressing the transcription factor T-bet and as a result IFN-c, in an IL-23 dependent manner, which is important for the pathogenic potential of exTh17 cells. Furthermore, IFN-c acts as a potent negative regulator of ROR-ct, the master regulator of the Th17 subset that drives the production of GM-CSF. Similar observations were made in other inflammatory and autoimmune conditions, illustrating the transition of Th17 cells into Th1-like cells. These observations further support the view of a switch point at which anti-inflammatory pathways are activated by the same Th subsets that initially promoted pathogenicity. In this scenario, IFN-c inhibits GM-CSF production by Th17 cells in the target tissues. We propose a possible model for a switch point for GM-CSF production by ‘‘pathogenic’’ Th-17 cells which is mediated by IL-23 and IFN-c in EAE.

Taken together, the one cytokine, one pathogenic Th cell, does not fit the bill anymore. The discovery of Th1-like Th17 cells, exTh17 cells, etc. complicates the question as to whether targeting a single cytokine or pathogenic T cell subset will ever result in the cure for autoimmune diseases.

The immune system seems to favor a balance between pathogenic and protective Th cells via dual roles for ‘‘subset-specific’’, or ‘‘signature cytokines’’, as well as allowing plasticity for subset differentiation and expression of ‘‘signature’’ cytokine(s) by other Th subsets. The observation that many Th subsets can convert into IFN-c secreting Th1-like cells illustrates this fact since IFN-c can be both pathogenic and protective. Targeting cytokines as therapy for autoimmune and/or inflammatory disorders remains a conceptual challenge more than ever. Clearly, cytokine therapy proved successful in some cases, such as anti-TNF therapy of RA, with the caveat that surprising adverse effects were observed in some patients indicative of the additional roles of this cytokine.


Regulatory T-cells in autoimmune diseases: Challenges, controversies and—yet—unanswered question

Charlotte R. Grant, R Liberal, G Mieli-Vergani, D Vergani, MS Longhi
Autoimmunity Reviews 14 (2015) 105–116

Regulatory T cells (Tregs) are central to the maintenance of self-tolerance and tissue homeostasis. Markers commonly used to define human Tregs in the research setting include high expression of CD25, FOXP3 positivity and low expression/negativity for CD127. Many other markers have been proposed, but none unequivocally identifies bona fide Tregs. Tregs are equipped with an array of mechanisms of suppression, including the modulation of antigen presenting cell maturation and function, the killing of target cells, the disruption of metabolic pathways and the production of anti-inflammatory cytokines. Treg impairment has been reported in a number of human autoimmune conditions and includes Treg numerical and functional defects and conversion into effector cells in response to inflammation. In addition to intrinsic Treg impairment, resistance of effector T cells to Treg control has been described. Discrepancies in the literature are common, reflecting differences in the choice of study participants and the technical challenges associated with investigating this cell population. Studies differ in terms of the methodology used to define and isolate putative regulatory cells and to assess their suppressive function. In this review we outline studies describing Treg frequency and suppressive function in systemic and organ specific autoimmune diseases, with a specific focus on the challenges faced when investigating Tregs in these conditions.

There are four basic mechanisms that Tregs use to suppress immune responses:

  1. the modulation of antigen presenting cell (APC) maturation and function,
  2. the killing of target cells,
  3. the disruption of metabolic pathways and
  4. the production of anti-inflammatory cytokines

Fig not shown. A) Regulatory T cellmechanisms of suppression. Regulatory T cell (Treg) can suppress by four basicmechanisms. The interaction between cytotoxic T lymphocyte antigen-4 (CTLA4) and CD80/CD86, expressed by antigen presenting cells (APCs), leads to CD80/CD86 down-regulation. Removal of these co-stimulatory molecules modulates APC function, limiting the initiation of an adaptive immune response. Tregs induce effector T cell (Teff) apoptosis by the interaction between Galectin-9 (Gal-9) and the T cell immunoglobulin and mucin domain-3 (TIM-3), and by the release of granzymes which enter Teffs via perforin pores. Tregs release the anti-inflammatory cytokines TGFβ, IL10 and IL35. Treg expression of the ecto-enzymes CD39 and CD73 enables the hydrolysis of pro-inflammatory adenosine triphosphate (ATP) into anti-inflammatory adenosine (ADO). B) Regulatory T cell defects in autoimmunity. In health, Tregs maintain tolerance by exerting suppression of effector T cells. In organ specific autoimmune disease, Tregs fail to suppress autoreactive effector T cells, therefore leading to target cell death. Reported reasons for this include inadequate numbers of Tregs, impaired suppressive ability, Treg conversion into effector cells and resistance of effector T cells to Treg-mediated suppression.

In the following sections, studies investigating the frequency and suppressive function of Tregs in the archetypal non-organ specific autoimmune disease SLE, and the organ specific autoimmune diseases MS, T1D, RA, autoimmune thyroid disease, psoriasis and IBD will be discussed.

Treg defects are frequently reported in autoimmune disease. There are, however, often discrepancies in the literature, which can be accounted for by the choice of study participants and the techniques used to study this challenging population of cells. The search for new markers that could unequivocally identify bona fide human Tregs—for the purposes of both phenotypic and functional analysis—will greatly facilitate our understanding of the role of Tregs in autoimmune disease. Studies suggest that the nature of the Treg impairment differs according to the autoimmune disease under investigation. There are reports of numerical and functional Treg impairments, of resistance of effector T cells to Treg suppression and of conversion of Tregs to effector cells. It is, therefore, important to consider numerical, phenotypic and functional defects affecting a range of Treg subsets. Moreover, current evidence strongly implies that systemic or regional factors can confine Treg impairments to the target organ. Treg studies would, therefore, benefit from more thorough investigation of the inflammatory site.

Take-home message

  • Tregs are central to tolerance maintenance and tissue homeostasis.
  • Treg impairment has been reported in several autoimmune diseases.
  • Systemic or regional factors can confine Treg impairment to the target organ.
  • Challenges remain when defining and investigating Tregs in autoimmune diseases.






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In focus: Melanoma therapeutics


Author and Curator: Ritu Saxena, Ph.D.

In the last post of Melanoma titled “In focus: Melanoma Genetics”, I discussed the clinical characteristics and the genetics involved in Melanoma.  This post would discuss melanoma therapeutics, both current and novel.

According to the American Cancer Society, more than 76,000 new cases and more than 9100 deaths from melanoma were reported in the United States in 2012[1] Melanoma develops from the malignant transformation of melanocytes, the pigment-producing cells that reside in the basal epidermal layer in human skin. Although most melanomas arise in the skin, they may also arise from mucosal surfaces or at other sites to which neural crest cells migrate.

Melanoma therapeutics

Surgical treatment of cutaneous melanoma employs specific surgical margins depending on the depth of invasion of the tumor and there are specific surgical treatment guidelines of primary, nodal, and metastatic melanoma that surgeons adhere to while treatment. Melanoma researchers have been focusing on developing adjuvant therapies for that would increase the survival post-surgery.


Among traditional chemotherapeutic agents, only dacarbazine is FDA approved for the treatment of advanced melanoma (Eggermont AM and Kirkwood JM, Eur J Cancer, Aug 2004;40(12):1825-36). Dacarbazine is a triazene derivative and alkylates and cross-links DNA during all phases of the cell cycle, resulting in disruption of DNA function, cell cycle arrest, and apoptosis. Currently, 17 clinical trials are underway to test the efficacy and effectiveness of dacarbazine against melanoma as either a single agent or in combination chemotherapy regimens with other anti-cancer chemotherapeutic agents such as cisplatin, paclitaxel. Temozolomide is a triazene analog of dacarbazine and is approved for the treatment of malignant gliomas. At physiologic pH, it is converted to a short-lived active cytotoxic compound, monomethyl triazeno imidazole carboxamide (MTIC). MTIC methylates DNA at the O6 and N7 positions of guanine, resulting in inhibition of DNA replication. Unlike dacarbazine, which is metabolized to MITC only in the liver, temozolomide is metabolized to MITC at all sites. Temozolomide is administered orally and penetrates well into the central nervous system. Temozolomide is being tested in many combination regimens for patients with melanoma metastatic to the brain (Douglas JG and Margolin K, Semin Oncol, Oct 2002;29(5):518-24).


Melanoma and the immune system are closely related. Hence, immunotherapy has been explored in the treatment of the disease. The two most widely investigated immunotherapy drugs for melanoma are Interferon (IFN)-alpha and Interleukin-2 (IL-2).

The role of IFNalpha-2b in the adjuvant therapy of patients with localized melanoma at high risk for relapse was established by the results of three large randomized trials conducted by the US Intergroup; all three trials demonstrated an improvement in relapse-free survival and two in overall survival. One of these trials, a large randomized multicenter trial performed by the Eastern Cooperative Oncology Group (ECOG), in high-risk melanoma patients showed significant improvements in relapse-free and overall survival with adjuvant IFN-α-2b therapy, compared with standard observation (ECOG 1684). The results of the study led to FDA approval of IFN-α-2b for treatment of melanoma. This study was performed on patients with deep primary tumors without lymph node involvement and node-positive melanomas. In other studies, little antitumor activity has been demonstrated in IFN-α-2b–treated metastatic stage IV melanoma.

Recombinant IL-2 showed an overall response rate of 15-20% in metastatic melanoma and was capable of producing complete and durable remissions in about 6% of patients treated. Based upon these data, the US FDA has approved the use of high-dose IL-2 for the therapy of patients with metastatic melanoma. Aldesleukin (Brand name: Proleukin) is a recombinant analog of the endogenous cytokine interleukin-2 (IL-2). It binds to and activates the IL-2 receptor (IL-2R), followed by heterodimerization of the IL-2R beta and gamma(c) cytoplasmic chains; activation of Jak3; and phosphorylation of tyrosine residues on the IL-2R beta chain, resulting in an activated receptor complex (NCI). The activated complex recruits several signaling molecules that act as substrates for regulatory enzymes associated with the complex. It is administered intravenously and stimulates lymphokine-activating killer (LAK) cells, natural killer (NK) cells and the production of cytokines such as gamma interferon (nm|OK). Several clinical trials are currently underway using Aldesleukin to determine the efficacy of combination treatment in melanoma patients.

Another anti-cancer immunotherapeuty-based mechanism involved inhibition of inhibitory signal of cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a molecule on T-cells that plays a critical role in regulating natural immune responses. Ipilimumab (Brand name: Yervoy) was by FDA for melanoma treatment.  It is a human monoclonal antibody (MAb) T-cell potentiator that specifically blocks CTLA-4. It is approved for inoperable advanced (Stage III) or metastatic (Stage IV) melanoma in newly diagnosed or previously treated patients (nm|OK). The approval, March 25, 2011, was based on a randomized (3:1:1) double-blind double-dummy clinical trial (MDX010-20) in patients with unresectable or metastatic melanoma who had received at least one prior systemic treatment for melanoma. Patients were randomly assigned to receive either ipilimumab, 3 mg/kg intravenously, in combination with the tumor vaccine (n=403); ipilimumab plus vaccine placebo (n=137); or tumor vaccine with placebo (n=136). Patients treated with ipilimumab alone had a median overall survival (OS) of 10 months while those treated with tumor vaccine had a median OS of 6 months. The trial also demonstrated a statistically significant improvement in OS for patients treated with the combination of ipilimumab plus tumor vaccine compared with patients treated with tumor vaccine alone. For more information on the trial, check the clinical trials website, www.clinicaltrials.gov

Signaling pathway inhibitors

Approximately 90% of BRAF gene mutations involve valine (V) to glutamic acid (E) mutation at number 600 residue (V600E). The resulting oncogene product, BRAF (V600E) kinase is highly active and exhibits elevated MAPK pathway. The BRAF(V600E) gene mutation occurs in approximately 60% of melanomas indicating that it could be therapeutically relevant. Vemurafenib (Brand name: Zelboraf) is a novel small-molecule inhibitor of BRAF (V600E) kinase. It selectively binds to the ATP-binding site and inhibits the activity of BRAF (V600E) kinase. Vemurafebib inhibits over active MAPK pathway by inhibiting the mutated BRAF kinase, thereby reducing tumor cell proliferation (NCI). Encouraging results of phase III randomized, open-label, multicenter trial were reported recently at the 2011 ASCO meeting (Chapman PB, et al, ASCO 2011, Abstract LBA4).  The trial compared the novel BRAF inhibitor vemurafenib with dacarbazine in patients with BRAF-mutated melanoma. Previously untreated, unresectable stage IIIC or stage IV melanoma that tested positive for BRAF mutation were randomized (1:1) to vemurafenib or dacarbazine. The response rate (RR) was significantly high (48.4%) in vemurafenib treated patients as compared to 5.5% in dacarbazine among the 65% of patients evaluable for RR to date. In addition, vemurafenib was associated with significantly improved OS and PFS compared to dacarbazine in patients with previously untreated BRAF (V600E) mutation bearing patients with metastatic melanoma.


Biochemothreapy combine traditional chemotherapy with immunotherapies, such as IL-2 and IFN-α-2b. These combination therapies seemed promising in phase II trials, however, seven large studies failed to show statistically significant increased overall survival rates for various biochemotherapy regimens in patients with stage IV metastasis (Margolin KA, et al, Cancer, 1 Aug 2004;101(3):435-8). Owing to inconsistent results of the available studies with regard to benefit including RR, OS and progression time, and consistently high toxicity rates, clinical practice guideline do not recommend biochemotherapy for the treatment of metastatic melanoma (Verma S, et al, Curr Oncol, April 2008; 15(2): 85–89).


The use of therapeutic vaccines is an ongoing area of research, and clinical trials of several types of vaccines (whole cell, carbohydrate, peptide) are being conducted in patients with intermediate and late-stage melanoma. Vaccines are also being tested in patients with metastatic melanoma to determine their immune effects and to define their activity in combination with other immunotherapeutic agents such as IL-2 or IFNalpha (Agarwala S, Am J Clin Dermatol, 2003;4(5):333-46). In fact, recently investigators at the Indiana University Health Goshen Center for Cancer Care (Goshen, IN) conducted a randomized, multicenter phase III trial involving 185 patients with stage IV or locally advanced stage III cutaneous melanoma. The patients were assigned into treatment groups with IL-2 alone or with vaccine (gp100) followed by IL-2. The vaccine-IL-2 group had a significantly improved OR as compared to the IL-2-only group (16% Vs. 6%) and longer progression free survival (2.2 months Vs. 1.6 months). The median overall survival was also longer in the vaccine-interleukin-2 group than in the interleukin-2-only group (17.8 months Vs. 11.1 months). Thus, a combination of vaccine and immunotherapy showed a better response rate and longer progression-free survival than with interleukin-2 alone in patients with advanced melanoma (Schwartzentruber DJ, et al, N Engl J Med, 2 Jun 2011;364(22):2119-27).

Which Treatment When?

Earlier, there were essentially two main options for patients suffering from advanced melanoma, dacarbazine and IL-2. Dacarbazine, a chemotherapeutic agent produces modest improvements in survival or symptomatic benefits in most patients. Interleukin-2 -based drugs, on the other hand, induce long-term remissions in a small group of patients but are highly toxic. Recently, FDA approved ipilimumab and vemurafenib for patients with metastatic melanoma. Apart from these, therapies are also aiming at starving the tumor by inhibiting angiogenesis or depleting nutrients essential for cancer growth. Of the antiangiogenic compounds, VEGFR inhibitors SU5416 and AG-013736 demonstrated broad-spectrum antitumor activity in mice bearing xenografts of human cancer cell lines originating from various tissues, including melanoma. In addition, several trials are currently underway to test the efficacy of the drugs in combination. In the future, personalized medicine-based recommendations of novel and existing drugs for melanoma patients might be the way to go.


  1. Eggermont AM and Kirkwood JM, Eur J Cancer, Aug 2004;40(12):1825-36
  2. Douglas JG and Margolin K, Semin Oncol, Oct 2002;29(5):518-24
  3. Chapman PB, et al, ASCO 2011, Abstract LBA4
  4. Margolin KA, et al, Cancer, 1 Aug 2004;101(3):435-8
  5. Verma S, et al, Curr Oncol, April 2008; 15(2): 85–89
  6. Agarwala S, Am J Clin Dermatol, 2003;4(5):333-46
  7. Schwartzentruber DJ, et al, N Engl J Med, 2 Jun 2011;364(22):2119-27
  8. Chudnovsky Y, et al, J Clin Invest, Apr 2005;115(4):813-24.
  9. National Cancer Institute (National Institute of Health)
  10. Clinical Trials reported on the U.S. Institute of Health
  11. New Medicine Oncology KnowledgeBase (nm|OK)

Related articles on Melanoma on this Open Access Online Scientific Journal: 

  1. In focus: Melanoma Genetics Curator- Ritu Saxena, Ph.D.
  2. Thymosin alpha1 and melanoma Author/Editor- Tilda Barliya, Ph.D.
  3. A New Therapy for Melanoma  Reporter- Larry H Bernstein, M.D.
  4. Melanoma: Molecule in Immune System Could Help Treat Dangerous Skin Cancer Reporter: Prabodh Kandala, Ph.D.
  5. Why Braf inhibitors fail to treat melanoma. Reporter: Prabodh Kandala, Ph.D.


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A revolutionary microchip-based human disease model for testing drugs

Reporter: Ritu Saxena, Ph.D.

Researchers at the Wyss Institute for Biologically Inspired Engineering at Harvard University, Boston, have developed lung-on-a-microfluid chip and shown that it mimic human lung function in response to Interluekin-2 (IL-2) and mechanical strain. Authors describe it as “a “lung-on-a-chip” that reconstituted the alveolar-capillary interface of the human lung and exposed it to physiological mechanical deformation and flow; in other words, it breathed rhythmically much like a living lung”.

The model was developed by Hu et al and reported earlier in the journal Science in 2010. The group has now been successful in demonstrating that lung-on-a-chip can act as a drug-testing model for pulmonary edema. Infact, Hu et al were able to predict the activity of a new drug, GSK2193874, for edema. Authors stated “These studies also led to identification of potential new therapeutics, including angiopoietin-1 (Ang-1) and a new transient receptor potential vanilloid 4 (TRPV4) ion channel inhibitor (GSK2193874), which might prevent this life-threatening toxicity of IL-2 in the future.” The findings have been published recently in the November 7 issue of Science Translational Medicine.


To recreate lung on the microchip, the authors cultured two toes of human lung cells in parallel microchannels separated by a thin membrane. It was observed that the upper channel (alveolar) was filled with air, while the lower channel (microvascular) was filled with liquid. The observation was similar to what occurs in human lung. Breathing motion of the lung was mimicked on the chip by applying vacuum cyclically to the sides of the channels.

Mimicking pulmonary edema

Pulmonary edema is a condition characterized by the abnormal buildup of fluid in the air sacs of the lungs, which leads to shortness of breath. It is often caused when the heart is not able to pump blood to the body efficiently, it can back up into the veins that take blood through the lungs to the left side of the heart. As the pressure in these blood vessels increases, fluid is pushed into the air spaces (alveoli) in the lungs. This fluid reduces normal oxygen movement through the lungs. This and the increased pressure can lead to shortness of breath.

Hu and colleagues observed that when IL-2 was added to the microvascular channel, the fluid started to leak into the alveolar compartment of the chip. This process is a reproduction of what happens in edema. Further, adding cyclic mechanical strain along with IL-2 compromised the pulmonary barrier even further and leading to a threefold increase in leakage.

Drug-testing model

Once the authors established the pulmonary disease model on the microchip, they tested against a novel pharmacological agent, GSK2193874, which blocks certain ion channels activated by mechanical strain. This drug was able to inhibit leakage suggesting that it might be a viable treatment option for patients with pulmonary edema who are being mechanically ventilated. A major advantage of using this model is avoiding the use of animal models for research.

Future perspective

The lung-on-a-chip model developed by Hu et al could be used to test novel agents for pulmonary edema.

Editorial note on the article in Science translational medicine article states “The next step is to hook this lung up to other chip-based organs− heart, liver, pancreas, etc.−with the goal of one day being able to rapidly screen many drugs and conditions that could affect patient health.”


Journal articles

Hul D, et al. A Human Disease Model of Drug Toxicity−Induced Pulmonary Edema in aLung-on-a-Chip. Microdevice Sci Transl Med. 2012 Nov 7;4(159):159ra147.http://www.ncbi.nlm.nih.gov/pubmed/23136042

Hul D et al Reconstituting organ-level lung functions on a chipScience. 2010 Jun 25;328(5986):1662-8. http://www.ncbi.nlm.nih.gov/pubmed/20576885

News brief

Video link to lung-on-a-chip http://wyss.harvard.edu/viewpage/240/

Sciencedaily report, November 7, 2012 http://www.sciencedaily.com/releases/2012/11/121107141044.htm

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