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Archive for the ‘Viral diseases’ Category


Immune System Stimulants: Articles of Note @pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

 

  • New Approaches to Immunotherapy

 

New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Three Methods for Design of a Novel Immune Therapy for Cancer: Conceptual Foundation for Development of a Novel Mechanism of Action for a Combination Therapy of Biologics — Password protected

Basic Research in Immune Oncology and Molecular Genomics: Methods to Stimulate Immunity by Alteration of Tumor Antigens – Reporting on R&D @MGH

New insights in cancer, cancer immunogenesis and circulating cancer cells

Perspectives on Anti-metastatic Effects in Cancer Research 2015

 

 

Issues Need to be Resolved With Immuno-Modulatory Therapies: NK cells, mAbs, and adoptive T cells

 

  •  Current Methods of Immuno-Therapy

 

 

Checkpoint inhibitors for gastrointestinal cancers

Immunomodulatory Therapeutic Antibodies for Cancer, August 13-15, 2013 – Boston, MA – Final Agenda

Tang Prize for 2014: Immunity and Cancer

LIVE 10:25 am – 12:00 pm 4/26/2016 Fireside Chat: Robert Bradway, CEO, Amgen & Immunotherapy I: Checkpoint Activation and Cancer Vaccines @2016 World Medical Innovation Forum: CANCER, April 25-27, 2016, Westin Hotel, Boston

Natural Killer Cell Response: Treatment of Cancer

CANCER IMMUNOTHERAPY

Cancer Immunotherapy Conference & Biomarkers for Cancer Immunotherapy Symposium, March 6-11, 2016 | Moscone North Convention Center | San Francisco, CA

Viruses, Vaccines and Immunotherapy

Advances in Cancer Immunotherapy

Perspectives on Anti-metastatic Effects in Cancer Research 2015

 

  • Evolving Approaches including Combination Oncotherapy

 

LIVE – 8:00 am – 12:00 pm 4/25/2016 – First Look: The Next Wave of Cancer Breakthroughs @2016 World Medical Innovation Forum: CANCER, April 25-27, 2016, Westin Hotel, Boston 2016 World Medical Innovation Forum: CANCER, April 25-27, 2016, Partners HealthCare, Boston, at the Westin Hotel, Boston

Brain Cancer Vaccine in Development and other considerations

Rapid regression of HER2 breast cancer

Breakthrough work in cancer

Novel biomarkers for targeting cancer immunotherapy

Humanized Mice May Revolutionize Cancer Drug Discovery

Immunomodulatory Therapeutic Antibodies for Cancer, August 13-15, 2013 – Boston, MA – Final Agenda

Melanoma: Molecule in Immune System Could Help Treat Dangerous Skin Cancer

NIH Study Demonstrates that a New Cancer Immunotherapy Method could be Effective against a wide range of Cancers

Aptamers and Scaffolds

 

  • Microbiological Factors in Cancer Growth

 

Microbe meets cancer

Gut microbiome and anti-tumor response

Malaria Protein Anti-cancer Activity

Retroviruses and Immunity

Oncolytic Viruses in Cancer Therapy @ CHI’s PreClinical Congress, June 14, 2016 Westin Boston Waterfront, Boston

Oncolytic Virus Immuno-Therapy: New Approach for a New Class of Immunotherapy Drugs

 

  • Signaling Pathways in Oncotherapy

 

Protein heals wounds, boosts immunity and protects from cancer – Lactoferrin

Programmed Cell Death and Cancer Therapy

BET Proteins Connect Diabetes and Cancer

Signaling of Immune Response in Colon Cancer

Myc and Cancer Resistance

Renal (Kidney) Cancer: Connections in Metabolism at Krebs cycle and Histone Modulation

Pancreatic Cancer and Crossing Roads of Metabolism

Autophagy-Modulating Proteins and Small Molecules Candidate Targets for Cancer Therapy: Commentary of Bioinformatics Approaches

A Curated Census of Autophagy-Modulating Proteins and Small Molecules Candidate Targets for Cancer Therapy

Biology, Physiology and Pathophysiology of Heat Shock Proteins

Heat Shock Proteins (HSP) and Molecular Chaperones

The Delicate Connection: IDO (Indolamine 2, 3 dehydrogenase) and Cancer Immunology

What is the key method to harness Inflammation to close the doors for many complex diseases?

IDO for Commitment of a Life Time: The Origins and Mechanisms of IDO, indolamine 2, 3-dioxygenase

Confined Indolamine 2, 3 dioxygenase (IDO) Controls the Hemeostasis of Immune Responses for Good and Bad

Insight on Cell Senescence

Neutrophil Serine Proteases in Disease and Therapeutic Considerations

T cell-mediated immune responses & signaling pathways activated by TLRs

 

  • Immunogenetics in Oncotherapy

 

CRISPR/Cas9: Contributions on Endoribonuclease Structure and Function, Role in Immunity and Applications in Genome Engineering

CRISPR-Cas9 and Regenerative Medicine

CRISPR/Cas9 Finds Its Way As an Important Tool For Drug Discovery & Development

GEN Tech Focus: Rethinking Gene Expression Analysis

Gene Expression and Adaptive Immune Resistance Mechanisms in Lymphoma

Serpins: A Review in Human Genomics

Upcoming Meetings on Cancer Immunogenetics

ipilimumab, a Drug that blocks CTLA-4 Freeing T cells to Attack Tumors @DM Anderson Cancer Center

NIH Considers Guidelines for CAR-T therapy: Report from Recombinant DNA Advisory Committee

Cancer Labs at School of Medicine @ Technion: Janet and David Polak Cancer and Vascular Biology Research Center

Host – Tumor Interactions during Cancer Therapy – Dr. Yuval Shaked’s Lab @Technion

Demythologizing sharks, cancer, and shark fins

Naked Mole Rats Cancer-Free

From the Walter and Eliza Hall Institute of Medical Research: Genes Needed for Local Tissue Immune Response

 

  • Immunotherapy Market

 

Next-generation Universal Cell Immunotherapy startup Adicet Bio, Menlo Park, CA is launched with $51M Funding by OrbiMed

Juno Acquires AbVitro for $125M: high-throughput and single-cell sequencing capabilities for Immune-Oncology Drug Discovery

Monoclonal Antibody Therapy and Market

Monoclonal Antibody Therapy: What is in the name or clear description?

Tumor Associated Macrophages: The Double-Edged Sword Resolved?

Targeting Glucose Deprived Network Along with Targeted Cancer Therapy Can be a Possible Method of Treatment

Immunoreactivity of Nanoparticles 

Tofacitinib, an Oral Janus Kinase Inhibitor, in Active Ulcerative Colitis

Acute Lung Injury

Peroxisome proliferator-activated receptor (PPAR-gamma) Receptors Activation: PPARγ transrepression for Angiogenesis in Cardiovascular Disease and PPARγ transactivation for Treatment of Diabetes

Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Cytokines in IBD

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New Class of Immune System Stimulants: Cyclic Di-Nucleotides (CDN): Shrink Tumors and bolster Vaccines, re-arm the Immune System’s Natural Killer Cells, which attack Cancer Cells and Virus-infected Cells

Reporter: Aviva Lev-Ari, PhD, RN

 

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

The initiative was officially launched at an evening reception on March 24, the eve of a one-day symposium at UC Berkeley titled “Harnessing the Immune System to Fight Cancer and Infectious Diseases.” The symposium was jointly sponsored by UC Berkeley’s Henry Wheeler Center for Emerging and Neglected Diseases and Cancer Research Laboratory.

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Aduro Biotech helps launch new immunotherapy, vaccine effort

UC Berkeley cancer immunologists are teaming up with colleagues working on infectious disease to create a new Immunotherapeutics and Vaccine Research Initiative, fueled by $7.5 million in funding from Aduro Biotech Inc., a Berkeley company that develops immunotherapies for cancer and other diseases.

The Immunotherapeutics and Vaccine Research Initiative (IVRI), a UC Berkeley effort funded by Aduro Biotech, Inc.

Aduro’s three years of funding, with the potential for three more, will support work on some of today’s most promising techniques for stimulating the immune system to fight off cancer and infections. These may include investigating a new class of immune system stimulants called cyclic di-nucleotides, which have shown promise in shrinking tumors and bolstering vaccines against tuberculosis, and research that could help re-arm the immune system’s natural killer cells, which normally attack cancer cells and virus-infected cells, to better fight tumors.

“We’re increasingly finding that immune stimulants associated with disease-causing microbes work as cancer therapies, and conversely, that immunotherapies for cancer may have application in fighting infectious disease,” said IVRI director David Raulet, a professor and co-chair of the Department of Molecular and Cell Biology. “Bringing infectious disease and cancer researchers together in a synergistic research effort at UC Berkeley and Aduro Biotech is an exciting and unique idea, and could be where the next generation of therapies will come from.”

Aduro already uses some of UC Berkeley’s technology, including attenuated Listeria monocytogenes mutants and methods to engineer these bacteria to stimulate the immune system as vaccines for immunotherapy. This technology, pioneered by Dan Portnoy, a UC Berkeley professor who has joint appointments in the Department of Molecular and Cell Biology and the infectious diseases and vaccinology division of the School of Public Health, has been further refined by Aduro scientists and is now being employed in Phase IIB clinical trials for vaccines against pancreatic cancer and mesothelioma.

Stephen Isaacs

Stephen Isaacs, CEO of Aduro Biotech, at the launch of the IVRI on March 23. (Peg Skorpinski photos)

“Through this unique collaboration, there is tremendous opportunity to improve our understanding of the immune system’s potential to serve as an important weapon in treating cancer and infectious disease,” said Stephen T. Isaacs, chairman, president and CEO of Aduro Biotech. “By combining UC Berkeley’s leading research and academic resources with innovative technology platforms, such as those developed by Aduro, we are confident that this initiative will lead to an improved understanding of, and potential treatments for, some of the most devastating diseases.”

The initiative was officially launched at an evening reception on March 24, the eve of a one-day symposium at UC Berkeley titled “Harnessing the Immune System to Fight Cancer and Infectious Diseases.” The symposium was jointly sponsored by UC Berkeley’s Henry Wheeler Center for Emerging and Neglected Diseases and Cancer Research Laboratory.

Berkeley research revived cancer immunotherapy

Much of the excitement around combining these two areas — the immunology of cancer and the immunology of infectious disease — comes from the amazing success of immunotherapy against cancer, which started with the work of James Allison when he was a professor of immunology at UC Berkeley and director of the Cancer Research Laboratory from 1985 to 2004. Allison, now at the University of Texas MD Anderson Cancer Center, discovered a way to release a brake on the body’s immune response to cancer that has proved highly successful at unleashing the immune system to attack melanoma and is being tried against other types of cancer.

James Allison

James Allison, whose UC Berkeley work led to the renaissance of cancer immunotherapy.

Allison’s work revived attempts to rev up the immune system to fight cancer, and has led to many new avenues for creating cancer immune-therapies. In addition to Allison’s technique, which uses an antibody that blocks an immune suppressor called CTLA4, antibodies that block another immune suppressor, PD1, have been successful in treating melanoma, renal cancer and a type of lung cancer. Both CTLA4 and PD1 antibodies are now FDA-approved as cancer therapies.

Another promising avenue involves a protein in cells that responds to foreign DNA to launch an innate immune response — the first response of the body’s immune system. The protein, dubbed STING, is triggered by small molecules called cyclic di-nucleotides (CDN), and makes immune cells release interferon and other cytokines that activate disease-fighting T cells and stimulate the production of antibodies that together kill invading pathogens and destroy cancer cells. Listeria bacteria, for example, secrete a CDN directly into infected cells that activates STING.

Russell Vance, a UC Berkeley professor of molecular and cell biology and current head of the Cancer Research Laboratory, discovered several years ago that the chemical structure of these di-nucleotides is critical to their ability to work in humans. Aduro has since developed a CDN designed to work in humans and found that injecting it directly into a tumor in mice caused the tumor to shrink.

“It’s amazing how these discoveries made by infectious disease researchers have given us an exciting new approach to treat cancer,” Raulet said. “These really are areas that have a lot to say to each other.”

Another IVRI-affiliated researcher, Sarah Stanley, an assistant professor of public health, has found evidence that CDNs can help improve the imperfect vaccines we have today against tuberculosis.

Researchers at UC Berkeley will have access to Aduro’s novel technology platforms for research use, including its STING pathway activators, proprietary monoclonal antibodies and the engineered listeria bacteria, referred to as LADD (listeria attenuated double-deleted).

David Raulet

David Raulet, professor of molecular and cell biology and director of IVRI.

Raulet’s own research on natural killer or NK cells of the immune system has contributed to making these cells a new focus of cancer research. Revving up T cells is the goal of most immunotherapies today, but other immune cells, including NK cells, also attack tumors. As tumors advance, NK cells inside the tumors appear to become desensitized, he said. Recent research shows that some cytokines and other immune mediators Raulet discovered are able to “wake them up” and get them to resume their elimination of cancer cells.

“NK cell immunotherapies are very likely to be the next generation to complement T cell immunotherapies,” he predicted.

By focusing on fundamental scientific research to understand the immune system’s biochemical tools and signaling pathways, how the immune system recognizes invaders and how immune cells talk to one another, the IVRI has the potential to discover new ways to selectively target and cure many human diseases.

“The IVRI is a marriage of cancer immunotherapy and infectious disease immunology, where therapies in one area can be effective in the other, and observations in one can be applied to the other,” Raulet said. “It’s exciting to think that drugs tested first in diseases like cancer might have an impact on neglected diseases in the developing world, and that it can work in the other direction too.”

RELATED INFORMATION

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CRISPR/Cas9 and HIV1

Larry H. Bernstein, MD, FCAP, Curator

LPBI

Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus

Hirotaka EbinaNaoko MisawaYuka Kanemura & Yoshio Koyanagi

Scientific Reports 2013; 2510(3)   http://dx.doi.org:/10.1038/srep02510

Even though highly active anti-retroviral therapy is able to keep HIV-1 replication under control, the virus can lie in a dormant state within the host genome, known as a latent reservoir, and poses a threat to re-emerge at any time. However, novel technologies aimed at disrupting HIV-1 provirus may be capable of eradicating viral genomes from infected individuals. In this study, we showed the potential of the CRISPR/Cas9 system to edit the HIV-1 genome and block its expression. When LTR-targeting CRISPR/Cas9 components were transfected into HIV-1 LTR expression-dormant and -inducible T cells, a significant loss of LTR-driven expression was observed after stimulation. Sequence analysis confirmed that this CRISPR/Cas9 system efficiently cleaved and mutated LTR target sites. More importantly, this system was also able to remove internal viral genes from the host cell chromosome. Our results suggest that the CRISPR/Cas9 system may be a useful tool for curing HIV-1 infection.

 

Integration of reverse transcribed viral DNA into the host cell genome is an essential step during the HIV-1 life cycle1. The integrated retroviral DNA is termed a provirus, which serves as the fundamental source of viral protein production. HIV-1 gene expression is regulated by LTR promoter and enhancer activities, where cellular transcription factors such as NF-κB, SP-1 and TBP bind to promote RNA polymerase II processivity. Subsequently, Tat protein is expressed from early double-spliced transcripts and binds to the trans activation response (TAR) region of HIV-1 RNA for its efficient elongation2.

Latent infection occurs when the HIV-1 provirus becomes transcriptionally inactive, resulting in a latent reservoir that has become the main obstacle in preventing viral eradication from HIV-1 infected individuals. However, the mechanisms of viral silencing and reactivation remain incompletely understood3. Previous studies have suggested that the position of the integration site strongly influences viral gene expression and may be one of the determinants of HIV-1 latency4. While highly active anti-retroviral therapy (HARRT) has dramatically decreased mortality from HIV-1 infection, there is currently no effective strategy to target the latent form of HIV-1 proviruses5.

Over the last decade, novel genome-editing methods that utilize artificial nucleases such as zinc finger nucleases (ZFNs)6 and transcription activator like-effector nucleases (TALENs)7 have been developed. These molecularly engineered nucleases recognize and cleave specific nucleotide sequences in target genomes for digestion, resulting in various mutations such as substitutions, deletions and insertions induced by host DNA repair machinery. These technologies have enabled the production of genome-manipulated animals in a wide range of species such as Drosophila8, Zebrafish9 and Rat10. However, ZFNs or TALENs remain somewhat difficult and time-consuming to design, develop, and empirically test in a cellular context11. Recently, a third genome-editing method was developed based on clustered regularly interspaced short palindromic repeat (CRISPR) systems. CRISPR systems were originally identified in bacteria and archaea12 as part of an adaptive immune system, dependent on a complex consisting of CRISPR RNAs (crRNAs) and CRISPR-associated (Cas) proteins to degrade complimentary sequences of invading viral and plasmid DNA. Mali et al. created a novel version of the genome-editing tool applicable to mammalian cells, termed the CRISPR/Cas9 system, which is based on modifications of the Streptococcus pyogenes type II CRISPR system in crRNA fused to trans-encoded tracrRNA13. This CRISPR/Cas9 system is composed of guide RNA (gRNA) and a human codon-optimized Cas9 nuclease that forms an RNA-protein complex to digest unique target sequences matching those of gRNA. The CRISPR/Cas9 system can be utilized by simple transfection of designed gRNA and a humanized Cas9 (hCas9) expression plasmid into target mammalian cells, making it a promising tool for various applications.

In this study, we tested the ability of the CRISPR/Cas9 system to suppress HIV-1 expression by editing HIV-1 integrated proviral DNA. Cas9 and gRNA, designed to target HIV-1 LTR, were transfected and significantly inhibited LTR-driven expression under the control of Tat. This LTR-targeted CRISPR/Cas9 system can also excise provirus from the cellular genome.

 

http://www.nature.com/article-assets/npg/srep/2013/130826/srep02510/images_hires/w926/srep02510-f2.jpg

CRISPR/Cas9 system can target the latent form of HIV-1 provirus in Jurkat cell

Because the putative latently infected cells are CD4+ T cells, we next tested the genome editing potential of the CRISPR/Cas9 system in these cells.

…..

 

In this study, we successfully disrupted the expression of HIV-1 provirus utilizing the CRISPR/Cas9 system (Fig. 1). Importantly, this disruption not only restricted transcriptionally active provirus, it also blocked the expression of latently integrated provirus (Fig. 3). Cas9 proteins are predicted to contain RuvC and HNH motifs15, which possess autonomous ssDNA cleavage activity. Interestingly, mutants lacking one of the motifs become nicking endonucleases16. It is plausible that the independent nicking activity of each domain may enhance efficient access to the heterochromatin state of latently integrated provirus. Another possibility is that Cas9 has a highly efficient target surveillance system similar to what has been previously reported for the Cas3 system17.

T6 gRNA that targeted the NF-κB binding site, also strongly suppressed the LTR promoter activity (Fig. 1). However, the effect was weaker than that of T5 gRNA. In this study we used an LTIG vector modified from the LTR of HIV-1 strain NL4-3 that possesses two adjacent NF-κB binding sites18. The T6 target site is at the end of the 5′ NF-κB binding site, meaning that mutations may not completely render transcription inactive since the 3′ NF-κB binding site may remain functional. On the other hand, T5 gRNA that targeted TAR, is profoundly effective in disrupting HIV-1 gene expression. The putative cleavage site was positioned at the neck of the stem loop region of TAR, which is critical for Cyclin T1-Tat-TAR ternary complex formation19. Therefore, the TAR sequence may be one of the best targets for blocking HIV-1 provirus expression. Target specificity of the CRISPR/Cas system is very high and a single mutation can disrupt targeting20, meaning that some provirus may escape from this genome-editing machinery if mutations arise in target sequences. However, given that the TAR region is relatively conserved and there is little variation among HIV-1 subtypes21, it could still be an appropriate target for the elimination of latently infected provirus.

Perhaps the most important finding in this study is that we could excise provirus from the host genome of HIV-1 infected cells, which may provide a ray of hope to eradicate HIV-1 from infected individuals. However, there are numerous hurdles that must be cleared before utilizing genome editing for HIV-1 eradication therapies such as gene therapy. First, the efficiency of genome-editing and/or proviral excision should be quantified in HIV infected primary cells, including latently infected CD4+ quiescent T cells. Second, an efficient delivery system must be developed. Fortunately, the CRISPR/Cas9 system has the advantage in size compared with TALENs22. Thus, the CRISPR system has the potential to be delivered by lentivirus vectors, whereas TALENs do not because of their large size and repeat sequences23. The final hurdle concerns possible off-target effects, which are pertinent concerns for all genome-editing strategies that may lead to nonspecific gene modification events. If Cas9 has off-target effects, then removal of the off-target activity may be the best approach before utilizing CRISPR/Cas system for anti-HIV treatment.

 

Elimination of HIV-1 Genomes from Human T-lymphoid Cells by CRISPR/Cas9 Gene Editing

Rafal KaminskiYilan ChenTracy FischerEllen TedaldiAlessandro Napoli,Yonggang ZhangJonathan KarnWenhui Hu & Kamel Khalili

Scientific Reports 6, Article number: 22555 (2016)   http://dx.doi.org:/10.1038/srep22555

 

We employed an RNA-guided CRISPR/Cas9 DNA editing system to precisely remove the entire HIV-1 genome spanning between 5′ and 3′ LTRs of integrated HIV-1 proviral DNA copies from latently infected human CD4+ T-cells. Comprehensive assessment of whole-genome sequencing of HIV-1 eradicated cells ruled out any off-target effects by our CRISPR/Cas9 technology that might compromise the integrity of the host genome and further showed no effect on several cell health indices including viability, cell cycle and apoptosis. Persistent co-expression of Cas9 and the specific targeting guide RNAs in HIV-1-eradicated T-cells protected them against new infection by HIV-1. Lentivirus-delivered CRISPR/Cas9 significantly diminished HIV-1 replication in infected primary CD4+ T-cell cultures and drastically reduced viral load in ex vivo culture of CD4+ T-cells obtained from HIV-1 infected patients. Thus, gene editing using CRISPR/Cas9 may provide a new therapeutic path for eliminating HIV-1 DNA from CD4+ T-cells and potentially serve as a novel and effective platform toward curing AIDS.

 

AIDS remains a major public health problem, as over 35 million people worldwide are HIV-1-infected and new infections continue at steady rate of greater than two million per year. Antiretroviral therapy (ART) effectively controls viremia in virtually all HIV-1 patients and partially restores the primary host cell (CD4+ T-cells), but fails to eliminate HIV-1 from latently-infected T-cells1,2. In latently-infected CD4+ T cells, integrated proviral DNA copies persist in a dormant state, but can be reactivated to produce replication-competent virus when T-cells are activated, resulting in rapid viral rebound upon interruption of antiretroviral treatment3,4,5,6,7,8. Therefore, most HIV-1-infected individuals, even those who respond very well to ART, must maintain life-long ART due to persistence of HIV-1-infected reservoir cells. During latency HIV infected cells produce little or no viral protein, thereby avoiding viral cytopathic effects and evading clearance by the host immune system. Because the resting CD4+ memory T-cell compartment9is thought to be the most prominent latently-infected cell pool, it is a key focus of research aimed at eradicating latent HIV-1 infection.

Recent efforts to eradicate HIV-1 from this cell population have used primarily a “shock and kill” approach, with the rationale that inducing HIV reactivation in CD4+ memory T-cells may trigger elimination of virus-producing cells by cytolysis or host immune responses. For example, epigenetic modification of chromatin structure is critical for establishing viral reactivation. Consequently, inhibition of histone deacetylase (HDAC) by Trichostatin A (TSA) and vorinostat (SAHA) led to reactivation of latent virus in cell lines10,11,12. Accordingly, other HDACi, including vorinostat, valproic acid, panobinostat and rombidepsin have been tested ex vivo and have led, in the best cases, to transient increases in viremia13,14. Similarly, protein kinase C agonists, can potently reactivate HIV either singly or in combination with HDACi15,16. However, there are multiple limitations of this approach: (i) since a large fraction of HIV genomes in this reservoir are non-functional, not all integrated provirus can produce replication-competent virus17; (ii) total numbers of CD4+ T cells reactivated from resting CD4+ T cell HIV-1 reservoirs, has been found by viral outgrowth assays to be much smaller than the numbers of cells infected, as detected by PCR-based assays, suggesting that not all cells within this reservoir are reactivated18; (iii) the cytotoxic T lymphocyte (CTL) immune response is not sufficiently robust to eliminate the reactivated infected cells19 and (iv) uninfected T-cells are not protected from HIV infection and can therefore sustain viral rebound.

These observations suggest that a cure strategy for HIV-1 infection should include methods that directly eliminate the proviral genome from the majority of HIV-1-positive cells, including CD4+ T-cells, and protect cells from future infection, with little or no harm to the host. The clustered, regularly-interspaced, short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) nuclease has wide utility for genome editing in a broad range of organisms including yeast, Drosophila, zebrafish, C. elegans, and mice, and has been applied in a broad range of in vivo and in vitro studies toward human diseases20,21,22,23,24. Recently we modified the CRISPR/Cas9 system to enable recognition of specific DNA sequences positioned within the HIV-1 promoter spanning the 5′ long terminal sequence (LTR)25,26. Using this modified system, we now demonstrate excision of integrated copies of the proviral DNA fragment from a latently HIV-1-infected human T-lymphoid cell line, completely eliminating HDAC inhibition-elicited viral production. Results of whole-genome sequencing and comprehensive bioinformatic analysis ruled out any genotoxicity to host cell DNA. Further, we found that lentivirally-delivered CRISPR/Cas9 reduces viral replication upon HIV-1 infection of primary cultured CD4+ T-cells. The results point toward this approach as a promising potential therapeutic avenue to eradicating HIV-1 from T reservoir cells of host patients, to prevent AIDS re-emergence.

 

Despite its remarkable therapeutic success and efficacy, ART treatment is unable to eradicate HIV-1 from infected patients who must therefore undergo life-long treatment. A new therapeutic strategy is thus needed in order to achieve permanent remission allowing patients to stop ART and reduce it’s attendant costs and potential long-term side effects. Our findings address key barriers to this goal, as we developed CRISPR/Cas9 techniques that eradicated integrated copies of HIV-1 from human CD4+ T-cells, inhibited HIV-1 infection in primary cultured human CD4+ T-cells, and suppressed viral replication ex vivo in peripheral blood mononuclear cells (PBMCs) and CD4+ T-cells of HIV-1+ patients. They also address a further key issue, providing evidence that such gene editing effectively impedes viral replication without causing genotoxicity to host DNA or eliciting destructive effects via host cell pathways. Prior studies using gene editing based on zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and CRISPR/Cas9 systems prompted much interest in their potential abilities to suppress viral infection, either by altering virus receptors or introducing mutations in the viral genome (for review see26,30). All these studies suggest that gene editing strategies can be engineered for targeting specific regions of the viral genome and once efficiently delivered to infected cells, their robust antiviral activity effectively suppresses viral replication. However, there are several important issues that require close attention including the careful design of the targeting strategy that achieves the highest levels of specificity and safety with optimum efficiency of editing.

In this study, due to the complexity associated with determination of the sites and numbers of randomly integrated proviral HIV-1 DNA in in vitro infected primary cell culture and the difficulty in full scale characterization of the InDel/Excision by Cas9/gRNAs in these cells, as a first step, we chose to use the clonal 2D10 cell line as a human T-cell latency model to establish: (i) the ability of Cas9/gRNA in removing the entire coding sequence of the integrated copies of the HIV-1 DNA using ultradeep whole genome sequencing and (ii) investigate its safely related to off-target effects and cell viability. Once these goals were accomplished, we shifted our attention to primary cell culture as well as patient samples to examine the efficiency of the CRISPR/Cas9 in affecting viral DNA load in a laboratory setting.

We found that CRISPR/Cas9 edited multiple copies of viral DNA scattered among the chromosomes. Combined treatment of latently-infected T cells with Cas9 plus gRNAs A and B that recognize specific DNA motifs within the LTR U3 region efficiently eliminated the entire viral DNA fragment spanning between the two LTRs. The remaining 5′ LTR and 3′ LTR cleavage sites by Cas9 and gRNA B in chromosome 1, and by Cas9 and gRNAs A and B in chromosome 16, were joined by host DNA repair at sites located precisely three nucleotides upstream of the PAM. Genome-wide assessment of CRISPR/Cas9-treated HIV-1-infected 2D10 cells clearly verified complete excision of the integrated copies of viral DNA from the second intron of RSBN1 and exon 2 of MSRB1 genes. To address the critical issue related to its specificity and potential off-target and adverse effects, we analyzed this comprehensively and at an unprecedented level of detail, by whole-genome sequencing and bioinformatic analyses. These revealed many naturally-occurring mutations in the genomes of control cells and gRNAs A- and B-mediated HIV-1 DNA eradication. The mutations discovered included naturally-occurring InDels, base excisions, and base substitutions, all of which are, more or less, expected in rapidly growing cells in culture, including Jurkat 2D10 cells. The critical issue is our discovery that none of these mutations resulted from our gene-editing system, as we identified no sequence identities with either gRNA A or B within 1200 nucleotides of any such mutation site. Further, our method of HIV-1 DNA excision had no adverse effects on proximal or distal cellular genes and showed no impact on cell viability, cell cycle progression or proliferation, and did not induce apoptosis, thus strongly supporting its safety at this translational phase, by all in vitromeasures assessed in cultured cells. We found that the expression levels of Cas9 and the gRNAs diminished after several passages and eventually disappeared, but as long as Cas9 and single or multiplex gRNAs were present, cells remained protected against new HIV-1infection.

Another key translational feasibility question we addressed is whether CRISPR/Cas9-mediated HIV-1 eradication can prevent or suppress HIV-1 infection in the most relevant human and patient target cell populations. We provide a critical new advance, by observing in PBMCs and CD4+ T-cells from HIV-1 infected patients that lentivirally-delivered Cas9/gRNAs A/B significantly decreased viral copy numbers and protein levels. Using primer sets directed within the LTR, we amplified and detected residual viral DNA fragments that were not completely deleted in these cells, yet were affected by Cas9/gRNAs and contained InDel mutants near the PAM sequence. These findings verified that CRISPR/Cas9 exerted efficacious antiviral activity in the PBMCs of HIV-1 patients. We also found that introducing Cas9/gRNAs A/B via lentiviral delivery into primary cultured human CD4+ HIV-1JRFL– or HIV-1NL4-3-infected T-cells significantly reduced viral copy numbers, corroborating earlier findings by us and others that stably-integrated HIV-1-directed Cas9 and gRNAs (distinct from our gRNAs A and B used presently) conferred resistance to HIV-1 infection in cell lines31,32. With the notion that CRISPR/Cas9 can target both integrated, as well as episomal DNA sequences, as evidenced by its editing ability of various human viruses as well as plasmid DNAs in either configuration31,32,33,34,35,36, it is likely that both the integrated as well as pre-integrated, free-floating intracellular HIV-1 DNA are edited by Cas9/gRNA.

As noted, during the course of our studies no ART was included prior to the treatment with CRISPR/Cas9 as our goal in this study was to determine the extent of viral suppression during the productive stage of viral infection. We observed a significant level of suppression suggesting that CRISPR/Cas9 may effectively disable expression of the functionally active integrated copies of HIV-1 DNA in the host chromosome. This notion is supported by our observations using 2D10 CD4+ T-cells where the latent copies of HIV-1 that are integrated in chromosomes 1 and 16 were effectively eliminated by CRISPR/Cas9. Our future studies are aimed to address the impact of CRISPR/Cas9 in in vitro infected CD4+ T-cells where the virus is controlled by ART and a cohort of naïve and ART-treated patient CD4+ T-cells. Results from these studies should determine whether or not, in the context of ART, the virus enters into the latent stage and remains responsive to CRISPR/Cas9. Of note, results from these ex vivo studies using ART treated patient PBMCs and CD4+ T-cells show that CRISPR/Cas9 effectively suppresses viral replication by introducing InDel mutations.

Our findings show comprehensively and conclusively that the entire coding sequence of host-integrated HIV-1 was eradicated in human 2D10 T cells, providing a strong first step of support for potential translatability of such a system to T-cell-directed HIV-1 therapies in patients. The complete absence of genomic and off-target functional effects in all assays also provides critical support for the promise of developing this approach for future therapeutic applications.

When evaluating a therapeutic strategy based on CRISPR/Cas9, it is critical to understand that not only will HIV-1 be eliminated from latently infected cells, but the majority of uninfected cells will become resistant to HIV infection. Thus, there is a high likelihood that rebounding viral infections will be contained by the resistant cells. Still, some formidable challenges remain before this type of strategy can be implemented. First, it will be important to maximize elimination of viral sequences from patients. This will require analysis of the HIV-1 quasi-species harbored by patients’ CD4+ T-cells and design of suitable, i.e. personalized CRISPRs. Second, improved delivery of CRISPR/Cas9 will be required to target the majority of circulating T-cells. In summary, our novel ex vivo findings that our lentiviral delivery-based approach reduced HIV-1 DNA copy numbers and protein levels in PBMCs of HIV-1 infected patients provides strong proof-of-concept evidence that CRISPR/Cas9 can be effectively utilized as part of HIV Cure strategies.

 

The therapeutic application of CRISPR/Cas9 technologies for HIV    PreviewFull text HTML   PDF

Sheena SaaymanabStuart A AlibKevin V MorrisacMarc S Weinberg*abd

Expert Opinion on Biological Therapy 2015;  15(6): 819-830    http://dx.doi.org:/10.1517/14712598.2015.1036736

Introduction: The use of antiretroviral therapy has led to a significant decrease in morbidity and mortality in HIV-infected individuals. Nevertheless, gene-based therapies represent a promising therapeutic paradigm for HIV-1, as they have the potential for sustained viral inhibition and reduced treatment interventions. One new method amendable to a gene-based therapy is the clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein-9 nuclease (Cas9) gene editing system.

Areas covered: CRISPR/Cas9 can be engineered to successfully modulate an array of disease-causing genetic elements. We discuss the diverse roles that CRISPR/Cas9 may play in targeting HIV and eradicating infection. The Cas9 nuclease coupled with one or more small guide RNAs can target the provirus to mediate excision of the integrated viral genome. Moreover, a modified nuclease-deficient Cas9 fused to transcription activation domains may induce targeted activation of proviral gene expression allowing for the purging of the latent reservoirs. These technologies can also be exploited to target host dependency factors such as the co-receptor CCR5, thus preventing cellular entry of the virus.

Expert opinion: The diversity of the CRISPR/Cas9 technologies offers great promise for targeting different stages of the viral life cycle, and have the capacity for mediating an effective and sustained genetic therapy against HIV.

Genetic therapy for HIV/AIDS

Ananthalakshmi PoluriMarc van Maanen & Richard E Sutton

Expert Opinion on Biological Therapy Sept 2003; 3(6):951-963

 

Towards a durable RNAi gene therapy for HIV-AIDS

Ben Berkhout & Olivier ter Brake

Expert Opinion on Biological Therapy  Feb 2009; 9(2): 161-170

 

 

 

 

 

 

 

 

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Inflammatory Disorders: Articles published @ pharmaceuticalintelligence.com

Curators: Larry H. Bernstein, MD, FCAP and Aviva Lev-Ari, PhD, RN

This is a compilation of articles on Inflammatory Disorders that were published 

@ pharmaceuticalintelligence.com, since 4/2012 to date

There are published works that have not been included.  However, there is a substantial amount of material in the following categories:

  1. The systemic inflammatory response
    https://pharmaceuticalintelligence.com/2014/11/08/introduction-to-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/
    https://pharmaceuticalintelligence.com/2014/11/09/summary-and-perspectives-impairments-in-pathological-states-endocrine-disorders-stress-hypermetabolism-cancer/
    https://pharmaceuticalintelligence.com/2015/12/19/neutrophil-serine-proteases-in-disease-and-therapeutic-considerations/
    https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/
    https://pharmaceuticalintelligence.com/2012/08/20/therapeutic-targets-for-diabetes-and-related-metabolic-disorders/
    https://pharmaceuticalintelligence.com/2012/12/03/a-second-look-at-the-transthyretin-nutrition-inflammatory-conundrum/
    https://pharmaceuticalintelligence.com/2012/07/08/zebrafish-provide-insights-into-causes-and-treatment-of-human-diseases/
    https://pharmaceuticalintelligence.com/2016/01/25/ibd-immunomodulatory-effect-of-retinoic-acid-il-23il-17a-axis-correlates-with-the-nitric-oxide-pathway/
    https://pharmaceuticalintelligence.com/2015/11/29/role-of-inflammation-in-disease/
    https://pharmaceuticalintelligence.com/2013/03/06/can-resolvins-suppress-acute-lung-injury/
    https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/
  2. sepsis
    https://pharmaceuticalintelligence.com/2012/10/20/nitric-oxide-and-sepsis-hemodynamic-collapse-and-the-search-for-therapeutic-options/
  3. vasculitis
    https://pharmaceuticalintelligence.com/2015/02/26/acute-lung-injury/
    https://pharmaceuticalintelligence.com/2012/11/26/the-molecular-biology-of-renal-disorders/
    https://pharmaceuticalintelligence.com/2012/11/20/the-potential-for-nitric-oxide-donors-in-renal-function-disorders/
  4. neurodegenerative disease
    https://pharmaceuticalintelligence.com/2013/02/27/ustekinumab-new-drug-therapy-for-cognitive-decline-resulting-from-neuroinflammatory-cytokine-signaling-and-alzheimers-disease/
    https://pharmaceuticalintelligence.com/2016/01/26/amyloid-and-alzheimers-disease/
    https://pharmaceuticalintelligence.com/2016/02/15/alzheimers-disease-tau-art-thou-or-amyloid/
    https://pharmaceuticalintelligence.com/2016/01/26/beyond-tau-and-amyloid/
    https://pharmaceuticalintelligence.com/2015/12/10/remyelination-of-axon-requires-gli1-inhibition/
    https://pharmaceuticalintelligence.com/2015/11/28/neurovascular-pathways-to-neurodegeneration/
    https://pharmaceuticalintelligence.com/2015/11/13/new-alzheimers-protein-aicd-2/
    https://pharmaceuticalintelligence.com/2015/10/31/impairment-of-cognitive-function-and-neurogenesis/
    https://pharmaceuticalintelligence.com/2014/05/06/bwh-researchers-genetic-variations-can-influence-immune-cell-function-risk-factors-for-alzheimers-diseasedm-and-ms-later-in-life/
  5. cancer immunology
    https://pharmaceuticalintelligence.com/2013/04/12/innovations-in-tumor-immunology/
    https://pharmaceuticalintelligence.com/2016/01/09/signaling-of-immune-response-in-colon-cancer/
    https://pharmaceuticalintelligence.com/2015/05/12/vaccines-small-peptides-aptamers-and-immunotherapy-9/
    https://pharmaceuticalintelligence.com/2015/01/30/viruses-vaccines-and-immunotherapy/
    https://pharmaceuticalintelligence.com/2015/10/20/gene-expression-and-adaptive-immune-resistance-mechanisms-in-lymphoma/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
  6. autoimmune diseases: rheumatoid arthritis, colitis, ileitis, …
    https://pharmaceuticalintelligence.com/2016/02/11/intestinal-inflammatory-pharmaceutics/
    https://pharmaceuticalintelligence.com/2016/01/07/two-new-drugs-for-inflammatory-bowel-syndrome-are-giving-patients-hope/
    https://pharmaceuticalintelligence.com/2015/12/16/contribution-to-inflammatory-bowel-disease-ibd-of-bacterial-overgrowth-in-gut-on-a-chip/
    https://pharmaceuticalintelligence.com/2016/02/13/cytokines-in-ibd/
    https://pharmaceuticalintelligence.com/2016/01/23/autoimmune-inflammtory-bowl-diseases-crohns-disease-ulcerative-colitis-potential-roles-for-modulation-of-interleukins-17-and-23-signaling-for-therapeutics/
    https://pharmaceuticalintelligence.com/2014/10/14/autoimmune-disease-single-gene-eliminates-the-immune-protein-isg15-resulting-in-inability-to-resolve-inflammation-and-fight-infections-discovery-rockefeller-university/
    https://pharmaceuticalintelligence.com/2015/03/01/diarrheas-bacterial-and-nonbacterial/
    https://pharmaceuticalintelligence.com/2016/02/11/intestinal-inflammatory-pharmaceutics/
    https://pharmaceuticalintelligence.com/2014/01/28/biologics-for-autoimmune-diseases-cambridge-healthtech-institutes-inaugural-may-5-6-2014-seaport-world-trade-center-boston-ma/
    https://pharmaceuticalintelligence.com/2015/11/19/rheumatoid-arthritis-update/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
    https://pharmaceuticalintelligence.com/2012/09/13/tofacitinib-an-oral-janus-kinase-inhibitor-in-active-ulcerative-colitis/
    https://pharmaceuticalintelligence.com/2013/03/05/approach-to-controlling-pathogenic-inflammation-in-arthritis/
    https://pharmaceuticalintelligence.com/2013/03/05/rheumatoid-arthritis-risk/
    https://pharmaceuticalintelligence.com/2012/07/08/the-mechanism-of-action-of-the-drug-acthar-for-systemic-lupus-erythematosus-sle/
  7. T cells in immunity
    https://pharmaceuticalintelligence.com/2015/09/07/t-cell-mediated-immune-responses-signaling-pathways-activated-by-tlrs/
    https://pharmaceuticalintelligence.com/2015/05/14/allogeneic-stem-cell-transplantation-9-2/
    https://pharmaceuticalintelligence.com/2015/02/19/graft-versus-host-disease/
    https://pharmaceuticalintelligence.com/2014/10/14/autoimmune-disease-single-gene-eliminates-the-immune-protein-isg15-resulting-in-inability-to-resolve-inflammation-and-fight-infections-discovery-rockefeller-university/
    https://pharmaceuticalintelligence.com/2014/05/27/immunity-and-host-defense-a-bibliography-of-research-technion/
    https://pharmaceuticalintelligence.com/2013/08/04/the-delicate-connection-ido-indolamine-2-3-dehydrogenase-and-immunology/
    https://pharmaceuticalintelligence.com/2013/07/31/confined-indolamine-2-3-dehydrogenase-controls-the-hemostasis-of-immune-responses-for-good-and-bad/
    https://pharmaceuticalintelligence.com/2013/04/14/immune-regulation-news/

Proteomics, metabolomics and diabetes

https://pharmaceuticalintelligence.com/2015/11/16/reducing-obesity-related-inflammation/

https://pharmaceuticalintelligence.com/2015/10/25/the-relationship-of-stress-hypermetabolism-to-essential-protein-needs/

https://pharmaceuticalintelligence.com/2015/10/24/the-relationship-of-s-amino-acids-to-marasmic-and-kwashiorkor-pem/

https://pharmaceuticalintelligence.com/2015/10/24/the-significant-burden-of-childhood-malnutrition-and-stunting/

https://pharmaceuticalintelligence.com/2015/04/14/protein-binding-protein-protein-interactions-therapeutic-implications-7-3/

https://pharmaceuticalintelligence.com/2015/03/07/transthyretin-and-the-stressful-condition/

https://pharmaceuticalintelligence.com/2015/02/13/neural-activity-regulating-endocrine-response/

https://pharmaceuticalintelligence.com/2015/01/31/proteomics/

https://pharmaceuticalintelligence.com/2015/01/17/proteins-an-evolutionary-record-of-diversity-and-adaptation/

https://pharmaceuticalintelligence.com/2014/11/01/summary-of-signaling-and-signaling-pathways/

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

https://pharmaceuticalintelligence.com/2014/10/24/diabetes-mellitus/

https://pharmaceuticalintelligence.com/2014/10/16/metabolomics-summary-and-perspective/

https://pharmaceuticalintelligence.com/2014/10/14/metabolic-reactions-need-just-enough/

https://pharmaceuticalintelligence.com/2014/11/03/introduction-to-protein-synthesis-and-degradation/

https://pharmaceuticalintelligence.com/2015/09/25/proceedings-of-the-nyas/

https://pharmaceuticalintelligence.com/2014/10/31/complex-models-of-signaling-therapeutic-implications/

https://pharmaceuticalintelligence.com/2014/03/21/what-is-the-key-method-to-harness-inflammation-to-close-the-doors-for-many-complex-diseases/

https://pharmaceuticalintelligence.com/2013/03/05/irf-1-deficiency-skews-the-differentiation-of-dendritic-cells/

https://pharmaceuticalintelligence.com/2012/11/26/new-insights-on-no-donors/

https://pharmaceuticalintelligence.com/2012/11/20/the-potential-for-nitric-oxide-donors-in-renal-function-disorders/

 

 

 

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Concerns about Viruses

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Zika: The Unexpected Pandemic

by Michael Smith

Sanjay Gupta, MD   Medscape     http://www.medpagetoday.com/InfectiousDisease/GeneralInfectiousDisease/55915

No one really saw Zika virus coming or cared much if it did.

In general, it has been regarded “clinically inconsequential,” Anthony Fauci, MD, director of the National Institute of Allergy and Infectious Diseases, told MedPage Today — so much so that it wasn’t even on a recent World Health Organization list of pathogens that need urgent research to prevent epidemics.

And — absent its apparent association with a spike in cases of microcephaly in Brazil — it probably still would be thought of as a minor nuisance, experts told MedPage Today.

But Zika virus illustrates a worrisome fact — the pace of emerging infectious diseases is both increasing and unpredictable.

Zika is a flavivirus, discovered in 1947, that is carried mainly by the mosquito Aedes aegypti. It causes a mild, self-limiting febrile illness in 20% to 25% of the people it infects; most people would never know they had it.

Until recently, it was pretty much confined to its ancestral home in Africa. Then in 2007 it was found in Micronesia and in 2013 ongoing transmission was documented in French Polynesia.

Early last year, it made its appearance in Brazil and it now appears to be established in 20 countries or territories in the Americas, including Puerto Rico.

Given that much of the region also has endemic dengue fever and chikungunya — with similar but more serious symptoms and also carried by A. aegypti — the appearance of Zika virus was originally just recorded with the notation that it would be nice to know more about these concurrent infections.

Then in September 2015, reports emerged of a spike in cases of microcephaly in the region of Brazil where the outbreak of Zika had been noted. It was an alarm bell, even though there’s still no definitive proof that Zika infection causes microcephaly.

“Microcephaly is obviously where the significant global public health concern is,” according to Michael Diamond, MD, PhD, of the Washington University School of Medicine in St. Louis.

But, he told MedPage Today, there have also been reports of a spike in Guillain-Barré syndrome during the Polynesian outbreak. Again, it’s an association with nothing to prove that Zika was responsible.

Still, there are now two clinical syndromes that have appeared at the same time as a Zika outbreak. It might be coincidence but health officials are urging precautions anyway.

And it’s yet another instance of a pathogen emerging from the shadows.

‘Emerging’ Pathogens?

The term “emerging diseases” is widely used but it’s often bit of a misnomer. Many such pathogens are bugs that have moved into new places, while a few are actually novel. In the latter group, put HIV, SARS and MERS. But Zika — like dengue, chikungunya, and West Nile virus — is a traveller.

Previously known or not, the list of such pathogens has grown in recent years. Consider a partial list: West Nile in 1999, SARS in 2002, the H1N1 pandemic influenza in 2009, MERS in 2012, Ebola in 2013, chikungunya in 2013.

And now Zika.

Is the apparent increase real? If so, what’s causing it? And what can we do?

The answers, experts told MedPage Today, are:

  • Yes, it’s probably a real phenomenon
  • It has multiple causes
  • And while there are steps we can take to minimize the effects — if we have the will and the cash — emerging diseases are going to be a continuing problem

“We’re definitely seeing more, there’s no question about it,” according to James LeDuc, PhD, director of the Galveston National Laboratory.

And it really shouldn’t be a surprise, he told MedPage Today: The National Academy of Sciences warned in 1992 that infectious disease had not been conquered and that — as a consequence of human activities — we were likely to see more and more pathogens spreading beyond their ancestral ranges.

The causes, that 1992 report said, include:

  • Increasing human populations, often pushing into new places and coming in contact with new pathogens
  • More and faster travel
  • Growing urbanization
  • Erosion of some traditional public health infrastructure, such as mosquito control programs

To those, we might have to add climate change, LeDuc said. For some of the mosquito-borne diseases especially, climate change might expand or move their ranges, as temperate regions become semi-tropical.

What propels Zika into the headlines is the link with birth defects. And however nuanced health officials try to be — it’s only an association, we still need more research, there might be other causes — just making the link creates fear.

“We’re still trying to figure out what’s going on with Zika and microcephaly,” commentedHeidi Brown, PhD, of the University of Arizona in Tucson. That’s going to take a lot of study and some time.

Put simply, an outbreak of disease needs three pro-conditions, Brown told MedPage Today: “You need the vector, you need the virus, and you need a human population that is susceptible.”

In the case of Zika, as well as dengue fever and chikungunya, the vector is A. aegypti, a mosquito that historically was implicated in the spread of yellow fever. In the early part of the last century, huge public health programs aimed to eradicate A. aegypti, with some success at reducing yellow fever.

But it’s the fate of successful public health programs to wither once they’ve achieved success and A. aegypti has made a comeback.

And, Brown said, A. aegypti is an “urban mosquito” — it likes to feed on people and to breed in the pools of standing water we all-too-often leave around our dwellings. The increasingly large cities of South and Central America, usually with slums where people can’t afford window screens or other protection against mosquito bites, offer a huge pool of targets.

Then an outbreak is just one plane ride away. “It’s very easy now for an infected person or an infected mosquito to move from one area to another,” Diamond said.

Of course, local conditions play an important role. Fauci told MedPage Today that it’s unlikely Zika will make huge inroads into the U.S. for two reasons. In the first place, most of the country has a severe enough winter to cause the mosquitoes to die off. And second, he said, “we can do vector control if we want to do vector control” — a capability that some other countries in the Americas don’t have.

Other experts noted that our cities are less densely populated than those in South and Central America and conditions are better — there’s air conditioning and household screens.

The same applies to other pathogens carried by A. aegypti, like dengue. But not every pathogen needs a mosquito. It’s still not clear what is the animal reservoir for Ebola, for instance, but in the recent epidemic, the vector was good old Homo sapiens. And other pathogens have intermediate hosts that don’t necessarily die off in the winter.

If the pathogens are likely to keep coming, what can we do? Slowing the pace and speed of travel is a nonstarter, we’re not going to stop living in cities, and our numbers, while growth is slowing, continue to rise.

In other words, the third of Brown’s triad — the pool of susceptible people — is going to remain.

That leaves the vector or the pathogen.

Mosquito Control

“Mosquitoes, in the end, don’t contribute much to society,” Diamond said, so A. aegypti is an obvious target if we want to prevent Zika, dengue, chikungunya, or yellow fever. And it’s something we know how to do, noted LeDuc, citing the mass eradication campaigns of the 20th century.

But that effort used “armies of people,” LeDuc noted. “That kind of commitment is just not economically feasible today,” he said.

On the other hand, the modern age has brought new tools. For instance, Australian researchers, focusing on dengue, think they can use Wolbachia, bacteria found in many insects, as a way to reduce the ability of A. aegypti to transmit viruses.

And the Brazilian city of Piracicaba is working with a British company, Oxitec, to release male mosquitoes genetically modified so their offspring don’t survive. The males don’t bite, so they can’t transmit disease, but if they outcompete normal males for mates, the net result would be a reduction in adult mosquitoes.

A similar program to prevent screwworm among livestock has been working in the U.S. since the 1950s, Brown said, so it’s not a pipe dream. But neither approach is a “silver bullet,” she said, and will need to be used in combination with other approaches.

Some approaches are decidedly low-tech. Eliminate sources of standing water. Wear insect repellent if you’re somewhere with mosquitoes. Ditto long sleeves and long pants. Put up bednets.

Those have the advantage that they work against all mosquito species, Diamond said, and therefore lots of pathogens.

No Help

A recurring theme in the story of emerging diseases is that there are no specific treatments and no vaccines. And if you think about it, that makes perfect sense — if we don’t know something is coming (because it’s emerging, after all), how can we have a vaccine or a therapy?

So consider the current Zika outbreak. Most people working in the field would not have predicted it for the next viral epidemic in the Americas and if they had would not have been especially worried.

“It took a lot of people by surprise and they were perhaps a little bit dismissive,” Brown said.

Other pathogens — Lassa fever, Rift Valley fever, Marburg, and MERS among them — might well have been higher on the priority list. Indeed, they are higher on the WHO’s blueprint for future research into epidemic prevention.

And who pays for the research? It’s not as if there is a huge commercial market for a vaccine or treatment for Zika, which in most cases causes mild or no illness. There might be a better market for other pathogens but how do you know where to focus?

The problem with vaccinology in this field, LeDuc said, is that vaccines generally have to be pathogen-specific and they are costly to develop. They’re also technically challenging; work on a dengue vaccine has been going on for years, he noted.

That said, Fauci commented, researchers on West Nile virus have developed a “platform” for a flavivirus vaccine that might be quickly adaptable to Zika. The issue then would be getting it through the regulatory hurdles and into the field — a long expensive process.

Even if a vaccine were available, how would it be used?

Writing with a colleague recently in the New England Journal of Medicine, Fauci noted that outbreaks are unpredictable, so vaccinating a population against a given pathogen would not be cost-effective, while stockpiling a vaccine for later deployment might be too slow to stop an epidemic.

And, of course, both approaches depend on knowing the pathogen is there or on its way.

The Ebola epidemic, which left thousands dead in West Africa, was missed for months because health officials in the region weren’t expecting it and didn’t recognize it when it arrived. In the case of Zika, the silent circulation of the virus in asymptomatic people makes it hard for surveillance systems to pick it up.

Then there’s treatment.

Broad-Spectrum Antivirals?

There is a specific therapy for just a handful of viruses, Diamond noted: hepatitis C, HIV, herpes simplex, and influenza. Such drugs are not easy to develop, especially in the throes of reacting to a crisis.

But LeDuc, for one, is “quite optimistic” that broad-spectrum antivirals can be developed. “The more we understand how pathogens cause disease,” he said, “the more we see common pathways” that might be avenues for intervention.

Once again, though, we run into the issue of getting drugs to people when they need it. Even if a Zika treatment were available, the vast majority of infected people would not take it because they would never know they were infected.

“One of the biggest challenges is diagnosis — and early diagnosis — so that we have a chance to intervene,” LeDuc said.

Mug’s Game

So what’s next?

Predicting the next outbreak is a mug’s game, as the case of Zika illustrates.

“There are many viruses that could emerge,” Diamond said, but whether they do or not depends on a host of variables, such things as the presence or absence of a vector and the titer needed to cause infection.

But the world could do better at being prepared, he said. “You can be reactionary or you can be proactive,” Diamond said.

The reactionary approach is to wait until something happens and then wheel out the fire trucks to put out the blaze. But we’d be better off, Diamond said, investing in “ways to make your house fireproof.”

Those investments would certainly include better surveillance, drugs, and vaccines, he said.

But first on the list, Diamond said, should be basic research on the nature of viruses so that we are “prepared to deal not just with the pandemic du jour but to really respond to any virus that comes up.”

 

HIV Growing Resistant to Common Treatment

Ryan Bushey, Associate Editor    http://www.dddmag.com/news/2016/01/hiv-growing-resistant-common-treatment

http://www.dddmag.com/sites/dddmag.com/files/ddd1601_HIV.jpg

Scientists from the University College London made a new discovery regarding the HIV virus.

The researchers learned the common HIV therapy tenofovir was less effective against certain strains of the pathogen after studying an estimated 2,000 patients, writes theBBC. Tenofovir is typically used in combination with other medication to suppress the growth of this infection.

A comparison was done between HIV patients in Africa versus those in Europe. Individuals in Africa were 60 percent more resistant to tenofovir whereas European patients experienced 20 percent more resistance.

Irregular dosing of the drug was partly to blame as well as sub-standard administration of the medication.

Lead author Dr. Ravi Gupta told the BBC, “If the right levels of the drug are not taken, as in they are too low or not regularly maintained, the virus can overcome the drug and become resistant.”

Gupta added that there should be a simultaneous global initiative and cash investment to improve facilities and disease monitoring in African countries.

 

Zika Update

The virus continues to spread as countries issue pregnancy advisories and drug firms pick up on vaccine development.

By Kerry Grens | January 28, 2016

As the mosquito-borne Zika virus has now spread to at least 23 countries in the Americas in recent months, the World Health Organization (WHO) is convening an emergency meeting on International Health Regulations Monday (February 1), Director-General Margaret Chan announced today (January 28).

Meanwhile, four countries—Ecuador, El Salvador, Jamaica, and Colombia—have asked women to delay getting pregnant for fear the virus can cause severe brain damage in fetuses. And some airlines have offered refund to pregnant travelers who booked trips to countries where Zika is circulating.

President Obama chimed in this week, calling for an acceleration of “research efforts to make available better diagnostic tests, to develop vaccines and therapeutics, and to ensure that all Americans have information about the Zika virus and steps they can take to better protect themselves from infection,” according to a White House statement.

There is currently no immunization or cure for Zika, but several pharmaceutical companies and academic labs have expressed interest in developing a vaccine. The University of Texas Medical Branch has already begun work on a Zika vaccine, which could be ready for testing in a year or two.

“What would take the longest time would be the process of passing it through the [US Food and Drug Administration] and other regulatory agencies to allow it for public use and that may take up to 10 to 12 years,” Nikos Vasilakis, who is working on the vaccine, told BBC News. His team is also advancing diagnostics that could help answer questions about the risks of fetal Zika exposure. (See “New Tests for Zika in the Works,” The Scientist, January 25, 2016.)

Sanofi, which has had a dengue shot recently approved in several countries, and GlaxoSmithKline have expressed interest in starting a vaccine program, while another firm, Inovio Pharmaceuticals, announcedMonday (January 25) it was beginning work on a Zika vaccine.

On Tuesday (January 26), the US Centers for Disease Control and Prevention (CDC) issued guidelines for screening babies whose mothers may have contracted Zika while pregnant. Infants should be tested for Zika if their mother tested positive or if they have microcephaly and their mothers were in a country with circulating virus while they were pregnant.

Microcephaly is not the only concern for exposed fetuses; these babies should also be screened for hearing and vision impairments, the CDC urged. “One rationale is we don’t know the spectrum of problems that perhaps are related to Zika virus, so we want to do a lot of screenings of infants out of an abundance of caution,” Cynthia Moore, the director of the CDC’s division of birth defects and developmental disabilities, told The New York Times. “We worry because other intrauterine infections may have some effects that last or show up after birth.”

Meanwhile, US health officials this week said a massive outbreak stateside is unlikely, given the geographic range of the mosquito species that transmit the virus and Americans’ housing conditions, with screens and air conditioning. “If you look at historically what we’ve seen, I think we can say that it’s a remote possibility and unlikely to happen,” Anthony Fauci, head of the National Institutes of Allergy and Infectious Disease, told NPR’s Shots.

 

Interim Guidelines for the Evaluation and Testing of Infants with Possible Congenital Zika Virus Infection — United States, 2016

The Lancet Infectious Diseases   JANUARY 28, 2016

Global epidemiology of drug resistance after failure of WHO recommended first-line regimens for adult HIV-1 infection: a multicentre retrospective cohort study

Summary  
Background

Antiretroviral therapy (ART) is crucial for controlling HIV-1 infection through wide-scale treatment as prevention and pre-exposure prophylaxis (PrEP). Potent tenofovir disoproxil fumarate-containing regimens are increasingly used to treat and prevent HIV, although few data exist for frequency and risk factors of acquired drug resistance in regions hardest hit by the HIV pandemic. We aimed to do a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART.

Methods

The TenoRes collaboration comprises adult HIV treatment cohorts and clinical trials of HIV drug resistance testing in Europe, Latin and North America, sub-Saharan Africa, and Asia. We extracted and harmonised data for patients undergoing genotypic resistance testing after virological failure with a first-line regimen containing tenofovir plus a cytosine analogue (lamivudine or emtricitabine) plus a non-nucleotide reverse-transcriptase inhibitor (NNRTI; efavirenz or nevirapine). We used an individual participant-level meta-analysis and multiple logistic regression to identify covariates associated with drug resistance. Our primary outcome was tenofovir resistance, defined as presence of K65R/N or K70E/G/Q mutations in the reverse transcriptase (RT) gene.   Findings

We included 1926 patients from 36 countries with treatment failure between 1998 and 2015. Prevalence of tenofovir resistance was highest in sub-Saharan Africa (370/654 [57%]). Pre-ART CD4 cell count was the covariate most strongly associated with the development of tenofovir resistance (odds ratio [OR] 1·50, 95% CI 1·27–1·77 for CD4 cell count <100 cells per μL). Use of lamivudine versus emtricitabine increased the risk of tenofovir resistance across regions (OR 1·48, 95% CI 1·20–1·82). Of 700 individuals with tenofovir resistance, 578 (83%) had cytosine analogue resistance (M184V/I mutation), 543 (78%) had major NNRTI resistance, and 457 (65%) had both. The mean plasma viral load at virological failure was similar in individuals with and without tenofovir resistance (145 700 copies per mL [SE 12 480] versus 133 900 copies per mL [SE 16 650; p=0·626]).

Interpretation

We recorded drug resistance in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Effective surveillance for transmission of drug resistance is crucial.

Introduction

More than 35 million people worldwide are living with HIV-1.1 There is no effective vaccine and therefore control of the HIV pandemic relies heavily on combination antiretroviral therapy (cART). WHO treatment guidelines for adult HIV-1 infection recommend the nucleotide reverse-transcriptase inhibitor (NRTI) tenofovir for first-line ART, in combination with lamivudine or emtricitabine and the non-nucleoside reverse-transcriptase inhibitor (NNRTI) efavirenz.2 Older NRTIs such as the thymidine analogue drugs are being replaced by tenofovir and the NNRTI nevirapine, although mentioned in WHO guidelines, is being phased out from first-line regimens.2

The global scale-up of cART has now reached 15 million treated individuals.1 The administration of cART at the time individuals with HIV-1 are initially diagnosed prevents immunological deterioration as early as possible and interrupts the spread of HIV-1 from newly diagnosed individuals.3 This strategy, referred to as treatment as prevention, is being studied especially in high-incidence regions and nearly always includes the use of first-line tenofovir-containing ART regimens. Likewise, the strategy of pre-exposure prophylaxis (PrEP) depends entirely on the administration of tenofovir or tenofovir and emtricitabine to uninfected individuals at high risk of HIV-1 infection.4

In individuals receiving tenofovir, HIV-1 develops phenotypically and clinically significant resistance usually as a result of one mutation at position 65 (lysine to arginine; K65R) in the reverse transcriptase (RT) gene.5 Data from clinical trials and cohorts in high-income settings using tenofovir combined with NNRTI have reported low prevalence of tenofovir resistance at viral failure,6, 7, 8 in stark contrast with reports from low-income and middle-income countries where prevalence seems to be much higher.9, 10 Similarly, high-level resistance to NNRTI and the cytosine analogue component (emtricitabine and lamivudine) arise through changes to one aminoacid, which suggests a low genetic barrier to resistance for these drugs as well. In view of the pivotal role of tenofovir-containing ART as both treatment and prophylaxis, and the striking potential for drug resistance, we did a global assessment of drug resistance after virological failure with first-line tenofovir-containing ART.

Research in context

Evidence before this study

We searched PubMed for studies of the prevalence of tenofovir resistance after failure of first-line antiretroviral therapy with efavirenz or nevirapine (non-nucleoside reverse-transcriptase inhibitors [NNRTIs]) in patients with HIV-1, published between January, 1999, and June, 2015, using the search terms “HIV” AND “tenofovir” AND “resistance”. We identified studies done in untreated adults (age >15 years) in which either efavirenz or nevirapine was combined with tenofovir and either emtricitabine or lamivudine as first line antiretroviral therapy. Several studies reported resistance data for tenofovir when the drug was started after initial use of stavudine or zidovudine; these studies were not reviewed further. We also excluded studies that reported tenofovir use without NNRTI because standard first-line antiretroviral therapy under a public health approach is based on NNRTI in adults.

We identified randomised controlled trials and a meta-analysis comparing NNRTI with protease inhibitors, in combination with tenofovir, which reported resistance data. Patients in high-income settings reported tenofovir resistance in 0–25% of virological failures and those in sub-Saharan Africa in 28–50%. The only other prospective study in sub-Saharan Africa was PASER-M, and was limited by few resistance data for patients given tenofovir plus NNRTI-based combination antiretroviral therapy (cART). The remaining studies were largely from South Africa and reported a wide range of prevalence (between 23% and 70%) of tenofovir resistance after virological failure. In west Africa, one study reported that 57% of virological failures were tenofovir resistant in a very small sample of 23 patients. Although aforementioned studies also reported NNRTI and cytosine analogue resistance, they were unable to quantify to what extent tenofovir resistance was a marker for high-level compromise of the regimen. We found no studies that specifically reported resistance data for patients given first-line tenofovir in east Africa. No study reported resistance data from more than one continent, and none seemed adequately powered to establish the effect of co-administered reverse-transcriptase inhibitors on the emergence of tenofovir resistance.

Added value of this study

This study reports the most comprehensive assessment of HIV-1 drug resistance after scale-up of first-line WHO recommended tenofovir-based antiretroviral regimens, showing that tenofovir resistance is surprisingly common in patients with treatment failure across many studies in all low-income regions. Importantly, these individuals also have notable resistance to other drugs in their regimen, leading to almost complete compromise of combination treatment. Challenging current perceptions in the specialty, our findings show that tenofovir resistant viruses have substantial transmission potential. Furthermore, our results show that viral strain affects tenofovir resistance in Europe but is not the main driver for resistance in viruses circulating in sub-Saharan Africa. Newly identified risk factors for resistance to tenofovir and NNRTI drugs include pre-treatment CD4 cell count (but not viral load) and co-administered antiretrovirals.

Implications of all the available evidence

Improvements in the quality of HIV care and viral load monitoring could mitigate the emergence and spread of tenofovir resistance, thereby prolonging the lifetime of tenofovir-containing regimens for both treatment and prophylaxis. Surveillance of tenofovir and NNRTI resistance should be a priority both in untreated and treated populations.

Methods …..

Results

The TenoRes collaboration included 1926 individuals from 36 countries (figure 1 and appendix).Table 1 summarises the median size and year of ART initiation for the cohorts comprising the collaboration. Viral load monitoring was done in about 50% of the cohorts including nearly all of cohorts from upper-income regions and from a small proportion of the cohorts in low-income and middle-income countries (appendix shows income status for each cohort; table 1).

 

http://www.thelancet.com/cms/attachment/2045482109/2056813674/gr1.sml

Figure 1

(A) Countries contributing data to resistance analysis and HIV-1 subtype distribution, (B) prevalence of drug resistance by mutation and by region

NNRTI=non-nucleotide reverse-transcriptase inhibitor. TDF=tenofovir disoproxil fumarate. *24% (n=462) of participants had tenofovir resistance when genotypes from viral load >1000 copies HIV-1 RNA per mL were considered.

 

Table 1

Characteristics of resistance studies included in analysis

Data are n, range, or n (%). cART=combination antiretroviral therapy.

*Multinational studies were treated as separate studies within each country.

 

The region-level pre-ART median CD4 cell count ranged from 44 to 104  cells per μL in sub-Saharan Africa, Asia, and Latin America (table 2). As expected, in north America pre-ART median CD4 cell count was 144 cells per μL and 190  cells per μL in Europe. The proportion of individuals using emtricitabine (vs lamivudine) and efavirenz (vs nevirapine) varied significantly by region. Emtricitabine was used significantly more than lamivudine in Europe, North America, and west and central Africa, and efavirenz was used significantly more than nevirapine in all regions apart from east and west and central Africa. The median duration of ART ranged from 11 to 26 months. Pre-treatment viral load ranged between 4·80 and 5·58 log copies per mL and was significantly higher in eastern and western and central Africa and Latin America than the other regions (table 2).

 

 

Table 2

Participant characteristics and details of antiretroviral therapy

Data are n (%) or median (IQR). TDF=tenofovir disoproxil fumarate.

 

Crude prevalence of tenofovir resistance in patients with treatment failure was highest in low-income and middle-income regions (figure 1). Prevalence of cytosine analogue resistance (M184V/I) was highest in sub-Saharan Africa and Latin America and lowest in western Europe. By contrast, resistance to NNRTI did not show this pattern (figure 1). Furthermore, the M184V/I mutation was less common than NNRTI resistance across all regions except in eastern Africa. Of the 700 patients with tenofovir resistance in the dataset, 457 (65%) had resistance to both remaining drugs. Participants with tenofovir resistant viruses were likely to be resistant to one or both accompanying drugs and therefore have profound compromise of their regimen, as compared with those without tenofovir resistance (figure 1).

Low baseline CD4 cell count was consistently associated with a higher prevalence of tenofovir resistance across regions. The pooled OR for tenofovir in individuals with a CD4 cell count of less than 100 cells per μL versus 100  cells per μL was 1·50 (95% CI 1·27–1·77; figure 2). By contrast, a high baseline viral load was only associated with a small, not significant increase in tenofovir resistance (OR for viral load ≥100 000 copies per mL vs <100 000 copies per mL was 1·17, 95% CI 0·94–1·44;appendix). We compared tenofovir resistance by use of co-administered antiretrovirals with tenofovir as first-line therapy. Use of lamivudine rather than emtricitabine (NRTIs) was associated with a higher prevalence of tenofovir resistance (OR 1·48, 95% CI 1·20–1·82), as was use of the NNRTI nevirapine rather than efavirenz (OR 1·46, 1·28–1·67; appendix). Subgroup analysis showed that as well as associations being consistent across regions, they were also generally similar across a range of study settings (appendix), although there was some evidence of a greater effect size of baseline CD4 when efavirenz was co-administered with tenofovir, as compared with nevirapine.

Figure 2

Pooled odds ratios for tenofovir resistance after viral failure for baseline CD4 cell count <100 vs≥100 × 106 cells per μL

TDF+ denotes presence of tenofovir resistance. TDF=tenofovir disoproxil fumarate.

 

When considering the effect of baseline CD4, baseline viral load (figure 3), and co-administered antiretrovirals (appendix) on cytosine analogue and NNRTI resistance, we noted that the magnitude of associations were smaller than those recorded for tenofovir resistance.

 

http://www.thelancet.com/cms/attachment/2045482109/2056813671/gr3.sml

Figure 3

Odds ratios for NNRTI resistance for (A) baseline CD4 cell count <100 vs ≥100 cells per μL, (B) viral load ≥100 000 vs <100 000 copies HIV-1 RNA per mL

NNRTI=non-nucleotide reverse-transcriptase inhibitor.

We also assessed the relation between viral subtype C on acquisition of tenofovir resistance. We restricted this analysis to western European studies in view of the consistent standard of care available in this region and relatively lower level of subtype diversity in other regions (figure 1A). We also limited the comparison to subtypes found in immigrant populations to minimise bias due to socioeconomic factors (thereby excluding subtype B infections mainly recorded in participants born in western Europe). Tenofovir resistance was higher in subtype C compared with non-C, non-B infections with a pooled OR of 2·44 (1·66–3·59).

As a sensitivity analysis we studied risk factors for tenofovir resistance using univariate (adjusted only for region) and multivariate logistic regression analyses (appendix). We noted a dose-response relationship for baseline CD4, which was not markedly altered by adjustment for baseline viral load, viral subtype, or type of co-administered drug used (appendix). Baseline viral load of 100 000 or more copies of HIV-1 RNA per mL was not significantly associated with tenofovir resistance (OR 1·31, 95% CI 0·91–1·91) and we noted no clear trend across increasing viral loads (appendix). Adjustment for several risk factors also had little effect on associations with tenofovir resistance of emtricitabine versus lamivudine and nevirapine versus efavirenz.

Finally, we compared the viral load at treatment failure in the presence and absence of tenofovir-associated mutations. The mean plasma viral load at treatment failure was not different in the presence or absence of tenofovir associated mutations (145 700 copies HIV RNA per mL [SE 12 480]vs 133 900 copies [SE 16 650]; p=0·626; figure 4 shows the within-study viral load by region). These results did not change when analysis was restricted to individuals who had evidence of the K65R mutation, either with or without M184V/I (appendix). Mutations at aminoacids K65 and M184 in the RT gene have been associated with suboptimum replication.13

http://www.thelancet.com/cms/attachment/2045482109/2056813672/gr4.sml

Figure 4

Boxplot of log viral load by presence (TDF-positive) or absence (TDF-negative) of tenofovir resistance at viral failure in studies with at least ten patients with TDF resistance and a viral load measurement at treatment failure

We restricted to studies with at least ten TDF-resistant mutations to help with graphical clarity, although the pattern of similar distributions of failure viral load in the presence or absence of TDF resistance was true for all studies. TDF=tenofovir disoproxil fumarate. Blue dots represent outliers.

 

Discussion

Our study has three main findings relating to the prevalence, risk factors for, and transmissibility of tenofovir resistance. First, we noted that levels of tenofovir resistance in individuals with viral failure ranged from 20% in Europe to more than 50% in sub-Saharan Africa. Second, a CD4 cell count of less than 100 cells per μL, treatment with nevirapine rather than efavirenz, and treatment with lamivudine rather than emtricitabine, were consistently associated with a 50% higher odds of tenofovir resistance in those with viral failure. Third, we noted that in patients with viral failure, viral loads were similar in the presence or absence of tenofovir resistance.

Our findings are important in view of the fact that following WHO recommendations,2 tenofovir is replacing thymidine analogues (zidovudine and stavudine) as part of the NRTI backbone in first-line regimens in resource-limited settings. Every drug in these regimens can be compromised by one aminoacid mutation, and the combination therapy is therefore potentially fragile. In view of the crucial role of tenofovir-containing ART in both treatment and prevention of new infections, restriction of drug resistance in high-burden settings is of paramount importance. Understanding how common tenofovir resistance is, and how and why it varies, is key to its prevention. Although our risk factors are only associated with a modest 50% increase in odds, this translates to a roughly 10% increase in resistance in those who fail when the overall tenofovir resistance prevalence is about 50% (as recorded in sub-Saharan Africa).

We hypothesise that the regional differences in tenofovir resistance are due to the frequency of viral load monitoring with close patient follow-up and feedback of results. For example, although viral load monitoring is not routinely done in most low-income and middle-income countries, in high-income countries viral load is tested three to four times per year with close patient follow-up and adherence support. Such an approach is likely to lead to earlier detection of viral failure, before selection of drug resistance mutations against tenofovir has occurred.14 This view is supported by the uncommon detection of drug resistance mutations in specimens with low viral load (400–1000 copies per mL) from patients given tenofovir in both high-income settings (figure 1; see higher prevalence of tenofovir resistance where viral load >1000 copies per mL is used as threshold in western Europe)15 and sub-Saharan Africa (Chunfu Yang, Centres for Disease Control, Atlanta, GA, USA, personal communication). Tenofovir resistance could be limited by viral load monitoring,16with rapid feedback to clinicians followed by adherence counselling to preserve first line, or switch to second line when this approach fails. Furthermore, pre-ART (baseline) resistance testing for key NNRTI mutations could potentially protect against tenofovir resistance by avoiding use of partly active treatment regimens. In our report, transmitted NNRTI resistance was low in the regions studied (<10%),17 and therefore not likely to be a major driver of wide variation in drug resistance across income settings.

Other factors that vary geographically could also affect success of ART and should be noted. Treatment failure is associated not only with drug resistance, but also side-effects. Efavirenz is associated with CNS side-effects such as sleep disturbance and is associated with treatment discontinuation.18 Furthermore, drug stock-outs and other indicators of quality of HIV services that have shown geographic variation would also predispose to treatment failure.19 The issue of regional variation in adherence levels has received considerable attention, with data from several studies suggesting that adherence is not worse in sub-Saharan Africa compared with North America.20, 21

With regards to increased tenofovir resistance in individuals with low baseline CD4 counts, this finding is consistent with results from the ACTG 5202 trial22 suggesting higher frequency of RT mutations in patients given ART with low CD4 cell counts, and offer a benefit of CD4 cell count measurement after diagnosis of HIV infection beyond establishing prophylaxis against opportunistic infections.23 Lamivudine warrants further study in first-line regimens in view of data presented in our study and the conflicting reports regarding virological efficacy of lamivudine versus emtricitabine.24,25, 26 Of note, the differences between lamivudine and emtricitabine might become less important in high-income regions where implementation of the second generation integrase inhibitor dolutegravir occurs, in view of the fact that this agent has not been associated with any cytosine analogue resistance at virological failure.27

Viral load has been associated with transmission risk.28 Despite evidence for diminished replication of tenofovir resistant viruses (containing the K65R mutation in the RT gene) in vitro, we noted similar viral loads in participants with and without tenofovir resistance. Therefore, there might be substantial potential for onward transmission to uninfected individuals,29 despite little evidence of K65R transmission up to now.30 This finding reinforces the need for drug resistance surveillance activities in both untreated and treated HIV-positive individuals.

There are several important limitations of our study. First, because we only included patients with virological failure related to existing study cohorts,1 our estimates of the prevalence of tenofovir resistance might not be representative in certain high-burden regions. Although this situation might have biased our findings on absolute prevalences of tenofovir resistance, it is unlikely to have affected associations with baseline CD4 or co-administered drugs. Second, we only included patients at failure so were unable to assess overall rates of tenofovir resistance in all patients starting first-line treatment. We used this method because many of the contributing studies had no clear denominator, especially those done in resource-limited settings. However, extensive WHO-led analysis reported that 15–35% (on treatment vs intention to treat) of patients in sub-Saharan Africa have virological failure by 12 months.31 Therefore, using a conservative 50% prevalence of tenofovir resistance at failure from our analysis, we suggest that it is likely that 7·5–17·5% of individuals given tenofovir plus cytosine analogue plus efavirenz will develop tenofovir resistance within 1 year of treatment initiation under present practices in sub-Saharan Africa.

Third, our findings on risk factors for tenofovir resistance were derived from an unadjusted meta-analysis involving very different study populations. Although this enhances the generalisability of results, it has the potential to lead to biased comparisons. However, we took measures to minimise biases. We exclusively used within-study and within-country comparisons for our primary analyses, thereby ensuring that comparisons were for participants undergoing similar treatment monitoring practices. We tested associations between risk factors and found that they were generally weak. For example, baseline CD4 cell count and viral load were only weakly associated with one another and neither was strongly associated with type of co-administered drug. Additionally, we undertook sensitivity analyses, which suggested that adjustment for other covariates had minimum effect on estimated associations. Lastly, our data tended to be consistent with previous studies—eg, our findings of higher resistance in subtype C patients are consistent with in-vitro data suggesting subtype C viruses are more susceptible to developing the K65R mutation.32

Fourth, despite our analysis being the largest drug resistance study ever undertaken after failure of first-line tenofovir-containing cART, patient numbers were somewhat limited by the slow uptake of tenofovir-based regimens in west and central Africa, eastern Europe, and Asia (in particular China and Russia), and information about baseline viral load in these settings was uncommon. As a result, European countries, Thailand, and South Africa contributed substantially to the analysis.

In summary, extensive drug resistance emerges in a high proportion of patients after virological failure on a tenofovir-containing first-line regimen across low-income and middle-income regions. Optimisation of treatment programmes and effective surveillance for transmission of drug resistance is therefore crucial.

Correspondence to: Dr Ravindra K Gupta, UCL, Department of Infection, London WC1E 6BT, UK ravindra.gupta@ucl.ac.uk

Cross-Reactive Ebola Antibodies

Human monoclonal antibodies induced during Ebola infection are able to neutralize related viral species, scientists show.

By Anna Azvolinsky | January 21, 2016

http://www.the-scientist.com//?articles.view/articleNo/45146/title/Cross-Reactive-Ebola-Antibodies/

 

http://www.the-scientist.com/images/News/January2016/620_Ebola%20Ab.jpg

Structure of Bundibugyo survivor antibodies (colors) bound to viral glycoproteinSCRIPPS RESEARCH INSTITUTE; CHARLES MURIN, ANDREW WARD

From blood samples of survivors of a 2007 Ebola outbreak in Uganda, researchers have isolated antibodies that are protective and can neutralize two other species of Ebolavirus, including ­­Zaire ebolavirus—the one responsible for the massive 2014 outbreak in West Africa. The binding specificities of these human monoclonal antibodies and their activity in animal models of Ebolavirus appeared today (January 21) in Cell.

“What is exciting is that the authors demonstrated that cross-reactive antibodies exist in survivors, and these antibodies are protective in animal models,”Larry Zeitlin, president of Mapp Biopharmaceutical, who was not involved in the current study but is collaborating with its authors as part of a National Institutes of Health (NIH)-funded project to develop and test vaccines and therapeutics against Ebolaviruses, told The Scientist in an email. “This gives real hope that a single product could be developed for treating all the Ebolavirus species.”

“This is a very good paper,” said Lisa Hensley, who has worked on the pathogenesis of viruses including Ebola and is associate director for science at the NIH’s Integrated Research Facility in Maryland. “Looking in people who survived Bundibugyo ebolavirus,” the species behind the 2007 outbreak, “brings our understanding of these viruses a step further. The study moved what we only knew anecdotally into convincing and strong data,” Hensley said.

Within the genus, there are currently three identified viral species that have caused deadly human outbreaks: Z. ebolavirus, B. ebolavirus, and Sudan ebolavirus.

Isolating peripheral blood B cells from seven survivors of B. ebolavirus, James Crowe, Jr., a viral immunologist and director of the Vanderbilt University Vaccine Center in Nashville, Tennessee, and his colleagues identified 90 antibodies that bound to the virus’s outer glycoprotein. Characterizing these human antibodies, the researchers found that 63 percent of them bound the glycoprotein of at least two of the three Ebolavirus species in vitro. Thirty-one (34 percent) of these antibodies were able to neutralize B. ebolavirus in an assay that is commonly used as a surrogate of an in vivo infection, and seven of these neutralized all three viral species, binding to one of three highly conserved regions of the glycoprotein.

The researchers found two different neutralizing antibodies, each used separately to treat Ebola-infected mice, effective. A single treatment with one of these antibodies successfully rescued Ebola-infected guinea pigs, while a combination of two antibodies resulted in complete protection of guinea pigs infected with Z. ebolavirus, the scientists showed.

“Protection against Ebola virus achieved in the guinea pig model is quite predictive of what can happen in humans, and in this model we can achieve protection,” said study coauthor Alexander Bukreyev, a virologist at the University of Texas Medical Branch in Galveston. “The key [result] is that the human immune system does produce protective antibodies. We just need to choose the right ones and give them at a high concentration.”

Crowe agreed. “Some of [the human antibodies] are extremely potent, some of the most potent antivirus antibodies ever isolated. Some of these antibodies possess the two major qualities you want to see in a therapeutic treatment—potent neutralization and breadth of activity against multiple Ebolavirus species.”

A recent study showed that serum isolated from Ebola survivors did not improve patient prognosis. But Mapp Biopharmaceutical’s cocktail of three mouse-derived, humanized monoclonal antibodies against Z. ebolavirus, ZMapp, was last year shown to be an effective treatment in Ebola-infected macaques. “Our goal for the second generation ZMapp product is a pan-Ebola virus antibody cocktail,” Zeitlin told The Scientist.The results of the present study, he added, “provides proof-of-concept for our effort.”

“The previously developed mouse monoclonal antibodies that were part of ZMapp were a solid start,” said Crowe. “But now, this study with antibodies from human survivors shows that we may have been underestimating the ability of antibodies to kill Ebola.”

The Ebola epitopes to which the cross-reactive antibodies bind will not only be helpful to develop new antibody cocktails as treatments but also point to a vaccine that could be used for multiple Ebola species. “This is the deeper implication of this work,” said Crowe.

Crowe, Bukreyev, and their colleagues are now isolating and analyzing antibodies against Z. ebolavirusstrains from the most recent outbreak. The researchers are also looking to combine the two types of therapies that have so far been shown to be most potent against Ebola, for a one-two punch: antibodies, which can neutralize already formed viral particles, and small interfering RNAs (siRNAs) that block viral replication.

The ultimate test will be whether the antibodies might be used in a protective vaccine or to treat people infected in the next outbreak. “That is everyone’s goal,” said Hensley. “What we do in animal models is nice, but how does it reflect what will happen in humans?”

A.I. Flyak et al., “Cross-reactive and potent neutralizing antibody responses in human survivors of natural Ebolavirus infection,” Cell, doi:10.1016/j.cell.2015.12.022, 2016.

 

 

 

 

Lung Cancer

http://www.cancernetwork.com/lung-cancer

The term “HER2-positive lung cancer” may actually refer to two distinct entities, according to a new study. HER2 mutations and HER2 amplifications were found in similar numbers of lung adenocarcinoma cases, but they did not occur in the same samples, suggesting HER2-targeted agents should differentiate between mutation and amplification.

Read Full Post »


Neutrophil Serine Proteases in Disease and Therapeutic Considerations

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

SERPINB1 Regulates the activity of the neutrophil proteases elastase, cathepsin G, proteinase-3, chymase,
chymotrypsin, and kallikrein-3. Belongs to the serpin family. Ov-serpin subfamily. Note: This description may
include information from UniProtKB.
Chromosomal Location of Human Ortholog: 6p25
Cellular Component: extracellular space; membrane; cytoplasm
Molecular Function: serine-type endopeptidase inhibitor activity
Reference #:  P30740 (UniProtKB)
Alt. Names/Synonyms: anti-elastase; EI; ELANH2; ILEU; LEI; Leukocyte elastase inhibitor; M/NEI; MNEI; Monocyte/neutrophil elastase inhibitor; Peptidase inhibitor 2; PI-2; PI2; protease inhibitor 2 (anti-elastase), monocyte/neutrophil derived; serine (or cysteine) proteinase inhibitor, clade B (ovalbumin), member 1; Serpin B1; serpin peptidase inhibitor, clade B (ovalbumin), member 1; SERPINB1
Gene Symbols: SERPINB1
Molecular weight: 42,742 Da
 

SERPIN PEPTIDASE INHIBITOR, CLADE B (OVALBUMIN), MEMBER 1; SERPINB1

Alternative titles; symbols
PROTEASE INHIBITOR 2, MONOCYTE/NEUTROPHIL DERIVED; ELANH2
ELASTASE INHIBITOR, MONOCYTE/NEUTROPHIL; EI
HGNC Approved Gene Symbol: SERPINB1
Cloning and Expression
Monocyte/neutrophil elastase inhibitor (EI) is a protein of approximately 42,000 Mr with serpin-like functional properties.
Remold-O’Donnell et al. (1992) cloned EI cDNA and identified 3 EI mRNA species of 1.5, 1.9, and 2.6 kb in monocyte-like cells
and no hybridizing mRNA in lymphoblastoid cells lacking detectable EI enzymatic activity. The cDNA open reading frame encoded
a 379-amino acid protein. Its sequence established EI as a member of the serpin superfamily. Sequence alignment indicated that
the reactive center P1 residue is cys-344, consistent with abrogation of elastase inhibitory activity by iodoacetamide and making
EI a naturally occurring cys-serpin.
 

 

Mapping

In the course of studying 4 closely linked genes encoding members of the ovalbumin family of serine proteinase inhibitors
(Ov-serpins) located on 18q21.3, Schneider et al. (1995) investigated the mapping of elastase inhibitor. They prepared PCR
primer sets of the gene, and by using the NIGMS monochromosomal somatic cell hybrid panel, showed that the EI gene maps
to chromosome 6.

By amplifying DNA of a somatic cell hybrid panel, Evans et al. (1995) unambiguously localized ELANH2 to chromosome 6.
With the use of a panel of radiation and somatic cell hybrids specific for chromosome 6, they refined the localization to
the short arm telomeric of D6S89, F13A (134570), and D6S202 at 6pter-p24.

http://www.phosphosite.org/getImageAction.do?id=27292293

 

 

REFERENCES
Evans, E., Cooley, J., Remold-O’Donnell, E. Characterization and chromosomal localization of ELANH2, the gene encoding human
monocyte/neutrophil elastase inhibitor. Genomics 28: 235-240, 1995. [PubMed: 8530031related citations] [Full Text]
Remold-O’Donnell, E., Chin, J., Alberts, M. Sequence and molecular characterization of human monocyte/neutrophil elastase inhibitor.
Proc. Nat. Acad. Sci. 89: 5635-5639, 1992. [PubMed: 1376927related citations][Full Text]
Schneider, S. S., Schick, C., Fish, K. E., Miller, E., Pena, J. C., Treter, S. D., Hui, S. M., Silverman, G. A. A serine proteinase inhibitor locus at
18q21.3 contains a tandem duplication of the human squamous cell carcinoma antigen gene. Proc. Nat. Acad. Sci. 92: 3147-3151, 1995.
[PubMed: 7724531,related citations] [Full Text]

 

Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Short name: Serpin_B1

Family relationships

  • Serpin family (IPR000215)
    • Leukocyte elastase inhibitor (serpin B1) (IPR015557)

Description

Leukocyte elastase inhibitor is also known as serpin B1. Serpins (SERine Proteinase INhibitors) belong to MEROPS inhibitor family I4 (clan ID)
[PMID: 14705960].

Serpin B1 regulates the activity of neutrophil serine proteases such as elastase, cathepsin G and proteinase-3 and may play a regulatory role to
limit inflammatory damage due to proteases of cellular origin [PMID: 11747453]. It also functions as a potent intracellular inhibitor of granzyme
H [PMID: 23269243]. In mouse, four different homologues of human serpin B1 have been described [PMID: 12189154].

 

The neutrophil serine protease inhibitor SerpinB1 protects against inflammatory lung injury and morbidity in influenza virus infection

Dapeng Gong1,2, Charaf Benarafa1,2, Kevan L Hartshorn3 and Eileen Remold-O’Donnell1,2
J Immunol April 2009; 182(Meeting Abstract Supplement) 43.10
http://www.jimmunol.org/cgi/content/meeting_abstract/182/1_MeetingAbstracts/43.10

SerpinB1 is an efficient inhibitor of neutrophil serine proteases. SerpinB1-/- mice fail to clear bacterial lung infection with increased inflammation and neutrophil death. Here, we investigated the role of serpinB1 in influenza virus infection, where infiltrating neutrophils and monocytes facilitate virus clearance but can also cause tissue injury. Influenza virus (H3N2 A/Phil/82) infection caused greater and more protracted body weight loss in serpinB1-/- vs. WT mice (20% vs. 15%; nadir on day 4 vs. day 3). Increased morbidity was not associated with defective virus clearance. Cytokines (IFN, TNF, IL-17, IFN, G-CSF) and chemokines (MIP-1, KC, MIP-2) were increased in serpinB1-/- mice vs. WT on days 2-7 post-infection but not on day 1. In WT mice, histology indicated large infiltration of neutrophils peaking on day 1 and maximal airway injury on day 2 that resolved on day 3 coincident with the influx of monocytes/macrophages. In serpinB1-/- mice, neutrophils also peaked on day 1; epithelial injury was severe and sustained with accumulation of dead cells on day 2 and 3. Immunophenotyping of lung digests on day 2 and 3 showed delayed recruitment of monocytes, macrophages and DC in serpinB1-/- mice, but increase of activated CD4 (day 2-3) and CD8 (day 3) T cells. Our findings demonstrate that serpinB1 protects against morbidity and inflammatory lung injury associated with influenza infection.

 

The neutrophil serine protease inhibitor serpinb1 preserves lung defense functions in Pseudomonas aeruginosainfection

Charaf Benarafa 1 , 2 Gregory P. Priebe 3 , 4 , and Eileen Remold-O’Donnell 1 , 2
JEM July 30, 2007; 204(8): 1901-1909   http://dx.doi.org:/10.1084/jem.20070494

Neutrophil serine proteases (NSPs; elastase, cathepsin G, and proteinase-3) directly kill invading microbes. However, excess NSPs in the lungs play a central role in the pathology of inflammatory pulmonary disease. We show that serpinb1, an efficient inhibitor of the three NSPs, preserves cell and molecular components responsible for host defense against Pseudomonas aeruginosa. On infection, wild-type (WT) and serpinb1-deficient mice mount similar early responses, including robust production of cytokines and chemokines, recruitment of neutrophils, and initial containment of bacteria. However, serpinb1−/− mice have considerably increased mortality relative to WT mice in association with late-onset failed bacterial clearance. We found that serpinb1-deficient neutrophils recruited to the lungs have an intrinsic defect in survival accompanied by release of neutrophil protease activity, sustained inflammatory cytokine production, and proteolysis of the collectin surfactant protein–D (SP-D). Coadministration of recombinant SERPINB1 with the P. aeruginosa inoculum normalized bacterial clearance inserpinb1−/− mice. Thus, regulation of pulmonary innate immunity by serpinb1 is nonredundant and is required to protect two key components, the neutrophil and SP-D, from NSP damage during the host response to infection.

 

Neutrophils are the first and most abundant phagocytes mobilized to clear pathogenic bacteria during acute lung infection. Prominent among their antimicrobial weapons, neutrophils carry high concentrations of a unique set of serine proteases in their granules, including neu trophil elastase (NE), cathepsin G (CG), and proteinase-3. These neutrophil serine proteases (NSPs) are required to kill phagocytosed bacteria and fungi (12). Indeed, neutrophils lacking NE fail to kill phagocytosed pathogens, and mice deficient for NE and/or CG have increased mortality after infection with pulmonary pathogens (34). However, NSPs in the lung airspace can have a detrimental effect in severe inflammatory lung disease through degradation of host defense and matrix proteins (57). Thus, understanding of the mechanisms that regulate NSP actions during lung infections associated with neutrophilia will help identify strategies to balance host defense and prevent infection-induced tissue injury.

 

SERPINB1, also known as monocyte NE inhibitor (8), is an ancestral serpin super-family protein and one of the most efficient inhibitors of NE, CG, and proteinase-3 (910). SERPINB1 is broadly expressed and is at particularly high levels in the cytoplasm of neutrophils (1112). SERPINB1 has been found complexed to neutro phil proteases in lung fluids of cystic fibrosis patients and in a baboon model of bronchopulmonary dysplasia (1314). Although these studies suggest a role for SERPINB1 in regulating NSP activity, it is unclear whether these complexes reflect an important physiological role for SERPINB1 in the lung air space.

RESULTS

To define the physiological importance of SERPINB1 in shaping the outcome of bacterial lung infection, we generated mice deficient for serpinb1 (serpinb1−/−) by targeted mutagenesis in embryonic stem (ES) cells (Fig. 1, A–C). Crossings of heterozygous mice produced WT (+/+), heterozygous (+/−), and KO (−/−) mice for serpinb1 at expected Mendelian ratios (25% +/+, 51% +/−, and 24% −/−; n = 225; Fig. 1 D), indicating no embryonic lethality. Bone marrow neutrophils of serpinb1−/− mice lacked expression of the protein, whereas heterozygous serpinb1+/− mice had reduced levels compared with WT mice (Fig. 1 E). Importantly, levels of the cognate neutrophil proteases NE and CG, measured as antigenic units, were not altered by deletion of serpinb1 (Fig. 1 F). When maintained in a specific pathogen-free environment, serpinb1−/− mice did not differ from WT littermates in growth, litter size, or life span (followed up to 12 mo), and no gross or histopathological defects were observed at necropsy in 8-wk-old mice.

6–8-wk-old animals were intranasally inoculated with the nonmucoid Pseudomonas aeruginosa strain PAO1. Using two infection doses (3 × 106 and 7 × 106 CFU/mouse),serpinb1−/− mice had a significantly lower survival probability and a shorter median survival time compared with WT mice (Fig. 2 A). Further groups of infected mice were used to evaluate bacterial clearance. At 6 h after infection, the bacteria were similarly restricted in mice of the two genotypes, suggesting that the serpinb1−/− mice have a normal initial response to infection. At 24 h, the median bacterial count in the lungs of serpinb1−/− mice was five logs higher than that of the WT mice (P < 0.001), and the infection had spread systemically in serpinb1−/− mice but not in WT mice, as shown by high median CFU counts in the spleen (Fig. 2 B). Histological examination at 24 h after infection revealed abundant neutrophil infiltration in the lungs of both WT and serpinb1−/− mice, and consistent with the bacteriological findings, numerous foci of bacterial colonies and large areas of alveolar exudates were found in serpinb1−/− mice only (Fig. 2 C). When challenged with the mucoid P. aeruginosa clinical strain PA M57-15 isolated from a cystic fibrosis patient, WT mice cleared >99.9% of the inoculum within 24 h, whereas serpinb1-deficient mice failed to clear the infection (Fig. 2 D). Thus, the NSP inhibitor serpinb1 is essential for maximal protection against pneumonia induced by mucoid and nonmucoid strains of P. aeruginosa.

Figure 2.

Serpinb1−/− mice fail to clear P. aeruginosalung infection. (A) Kaplan-Meier survival curves of WT (+/+) and serpinb1-deficient (−/−) mice intranasally inoculated with nonmucoid P. aeruginosa strain PAO1. Increased mortality of serpinb1−/− mice was statistically significant (P = 0.03 at 3 × 106CFU/mouse; P < 0.0001 at 7 × 106CFU/mouse). (B) CFUs per milligram of lung (left) and splenic (right) tissue determined 6 and 24 h after inoculation with 3 × 106 CFUP. aeruginosa PAO1 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Each symbol represents a value for an individual mouse. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test. Data below the limit of detection (dotted line) are plotted as 0.5 CFU × dilution factor. (C) Lung sections stained with hematoxylin and eosin show bacterial colonies (arrowheads) and alveolar exudate in lungs of serpinb1−/− mice 24 h after infection with P. aeruginosa PAO1. Bars, 50 μm. (D) Total CFUs in the lung and spleen 24 h after inoculation with 2 × 108 CFU of the mucoid P. aeruginosa strain PA M57-15 in WT (+/+, filled circles) and serpinb1−/− (−/−, open circles) mice. Differences between median values (horizontal lines) were analyzed by the Mann-Whitney U test.

To verify specificity of the gene deletion, we tested whether delivering rSERPINB1 would correct the defective phenotype. Indeed, intranasal instillation of rSERPINB1 to serpinb1−/− mice at the time of inoculation significantly improved clearance of P. aeruginosa PAO1 from the lungs assessed at 24 h and reduced bacteremia compared with infectedserpinb1−/− mice that received PBS instead of the recombinant protein (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20070494/DC1). We have previously demonstrated that rSERPINB1 has no effect on the growth of P. aeruginosa in vitro (15) and does not induce bacterial aggrega tion (16). Also, rSERPINB1 mixed with PAO1 had no effect on adherence of the bacteria to human bronchial epithelial and corneal epithelial cell lines (unpublished data). Therefore, the improved bacterial clearance in treated serpinb1−/− mice is not related to a direct antibacterial role for rSERPINB1 but rather to reducing injury induced by excess neutrophil proteases. In addition, previous in vivo studies in WT rats showed that rSERPINB1 can protect against elastase-induced lung injury (17) and accelerate bacterial clearance two- to threefold in the Pseudomonas agar bead model (15).

Evidence of excess NSP action was examined in the lungs of infected serpinb1−/− mice by measuring surfactant protein–D (SP-D). SP-D, a multimeric collagenous C-type lectin produced by alveolar epithelial cells, is highly relevant as a host defense molecule, because it functions as an opsonin in microbial clearance (18) and acts on alveolar macrophages to regulate pro- and antiinflammatory cytokine production (19). SP-D is also relevant as an NSP target because it is degraded in vitro by trace levels of each of the NSPs (1620). SP-D levels in lung homogenates of WT and serpinb1−/− mice were similar 6 h after P. aeruginosa infection. At 24 h, SP-D levels were reduced in the lungs ofserpinb1−/− mice compared with WT mice, as indicated by immunoblots. A lower molecular mass band indicative of proteolytic degradation is also apparent (Fig. 3 A). Densitometry analysis of the 43-kD SP-D band relative to β-actin indicated that the reduction of SP-D level was statistically significant (+/+, 45 ± 6 [n = 8]; −/−, 10 ± 2 [n = 8]; P < 0.0001 according to the Student’s t test). Furthermore, rSERPINB1 treatment ofP. aeruginosa–infected serpinb1−/− mice partly prevented the degradation of SP-D in lung homogenates compared with nontreated mice (Fig. S1 B). As a further test of the impact of serpinb1 deletion on NSP activity, isolated neutrophils of serpinb1−/− mice were treated with LPS and FMLP and tested for their ability to cleave recombinant rat SP-D (rrSP-D) in vitro. The extent of rrSP-D cleavage by serpinb1−/− neutrophils was fourfold greater than by WT neutrophils, as determined by densitometry. The cleavage was specific for NSPs because it was abrogated by rSERPINB1 and diisopropyl fluorophosphate (Fig. 3 B). Collectively, these findings indicate a direct role for serpinb1 in regulating NSP activity released by neutrophils and in preserving SP-D, an important-host defense molecule.

Efficient clearance of P. aeruginosa infection requires an early cytokine and chemokine response coordinated by both resident alveolar macrophages and lung parenchymal cells (2122). The IL-8 homologue keratinocyte-derived chemokine (KC) and the cytokines TNF-α, IL-1β, and G-CSF were measured in cell-free bronchoalveolar (BAL) samples. Although the tested cytokines were undetectable in sham-infected mice of both genotypes (unpublished data), comparable induc tion of these cytokines was observed in BAL of WT and serpinb1−/− mice at 6 h after infection, demonstrating that there is no early defect in cytokine production in serpinb1−/− mice. At 24 h, levels of TNF-α, KC, and IL-1β were sustained or increased in serpinb1−/− mice and significantly higher than cytokine levels in WT mice. G-CSF levels at 24 h were elevated to a similar extent in BAL of WT and KO mice (Fig. 3 C). However, G-CSF levels were significantly higher in the serum of serpinb1−/− mice (WT, 336 ± 80 ng/ml; KO, 601 ± 13 ng/ml; n = 6 of each genotype; P < 0.01). In addition, serpinb1−/− mice that were treated at the time of infection with rSERPINB1 had cytokine levels in 24-h lung homogenates that were indistinguishable from those of infected WT mice (Fig. S1 C). The increased cytokine production in the lungs of infected serpinb1−/− mice may be caused by failed bacterial clearance but also by excess NSPs, which directly induce cytokine and neutrophil chemokine production in pulmonary parenchymal cells and alveolar macrophages (2324).

Neutrophil recruitment to the lungs was next examined as a pivotal event of the response to P. aeruginosa infection (25). Lung homogenates were assayed for the neutrophil-specific enzyme myeloperoxidase (MPO) to quantify marginating, interstitial, and alveolar neutrophils. Neutrophils in BAL fluid were directly counted as a measure of neutrophil accumulation in the alveolar and airway lumen. MPO in lung homo genates was undetectable in uninfected mice and was comparably increased in mice of both genotypes at 6 h, suggesting normal early serpinb1−/− neutrophil margination and migration into the interstitium. However, by 24 h after infection, MPO levels in lung homogenates remained high in WT mice but were significantly decreased in serpinb1−/− mice (Fig. 4 A). Importantly, the content of MPO per cell was the same for isolated neutrophils of WT andserpinb1−/− mice (+/+, 369 ± 33 mU/106 cells; −/−, 396 ± 27 mU/106 cells). The numbers of neutrophils in BAL were negligible in uninfected mice and were similarly increased in WT and serpinb1−/− mice at 6 h after infection. Neutrophil counts in BAL further increased at 24 h, but the mean BAL neutrophil numbers were significantly lower in serpinb1−/− mice compared with WT mice (Fig. 4 B). The evidence from the 6-h quantitation of MPO in homogenates and neutrophils in BAL strongly suggests that neutrophil recruitment is not defective in infected serpinb1−/− mice. Moreover, the high levels of cytokines and neutrophil chemoattractant KC in serpinb1−/− mice at 24 h (Fig. 3 C) also suggest that, potentially, more neutrophils should be recruited. Therefore, to examine neutrophil recruitment in serpinb1−/− mice, we used a noninfectious model in which neutrophils are mobilized to migrate to the lung after intranasal delivery of P. aeruginosa LPS. MPO levels in lung homogenate and neutrophil numbers in BAL were not statistically different in WT and serpinb1−/− mice 24 h after LPS instillation (Fig. 4, C and D). Furthermore, the number of circulating blood neutrophils and recruited peritoneal neutrophils after injection of sterile irritants glycogen and thioglycollate did not differ in WT and serpinb1−/− mice (unpublished data). Alveolar macrophage numbers were similar in uninfected mice of both genotypes (∼5 × 105 cells/mouse) and did not substantially change upon infection. Collectively, these findings show that neutrophil recruitment to the lungs in response to P. aeruginosa infection is not defective in serpinb1−/− mice, and therefore, the recovery of lower numbers of serpinb1−/− neutrophils at 24 h after infection suggests their decreased survival.

To examine the putative increased death of serpinb1−/− neutrophils in the lungs after P. aeruginosa infection, lung sections were analyzed by immunohistochemistry. Caspase-3–positive leukocytes were more relevant in the alveolar space of serpinb1−/− mice compared with WT mice at 24 h after infection, suggesting increased neutrophil apoptosis (Fig. 5 A). The positive cells were counted in 50 high power fields (hpf’s), and mean numbers of caspase-3–stained cells were increased in the lungs of serpinb1/− mice (1.8 ± 0.2 cells/hpf) compared with WT mice (0.4 ± 0.1 cells/hpf; P < 0.0001). To characterize neutrophils in the alveoli and airways, neutrophils in BAL were identified in flow cytometry by forward scatter (FSC) and side scatter and were stained with annexin V (AnV) and propidium iodide (PI). At 24 h after infection, the proportion of late apoptotic/necrotic neutrophils (AnV+PI+) was increased at the expense of viable neutrophils (AnVPI) in the BAL of serpinb1−/− mice compared with WT mice (Fig. 5 B). Neutrophil fragments in BAL were also identified in flow cytometry by low FSC (FSClow) within the neutrophil population defined by the neutrophil marker Gr-1. The number of neutrophil fragments (FSClow, Gr-1+) relative to intact neutrophils was increased two- to threefold at 24 h after infection for serpinb1−/− compared with WT mice (Fig. 5 C). Moreover, free MPO in BAL supernatants was increased in serpinb1−/− mice compared with WT mice at 24 h after infection, indicating increased PMN lysis or degranulation (Fig. 5 D).

Finally, we questioned whether the enhanced death of serpinb1−/− pulmonary neutrophils was a primary effect of gene deletion or a secondary effect caused by, for example, bacteria or components of inflammation. To address this, neutrophils were collected using the noninfectious LPS recruitment model and were cultured in vitro to allow for spontaneous cell death. After 24 h, the percentages of apoptotic and necrotic neutrophils evaluated by microscopy were increased in serpinb1−/− neutrophils compared with WT neutrophils (Fig. 6, A–C). A similar increase in apoptotic cells was observed using AnV/PI staining and measurements of hypodiploid DNA (unpublished data). Moreover, live cell numbers from serpinb1−/− mice remaining in culture after 24 h were significantly decreased compared with WT mice (Fig. 6 D). The in vitro findings indicate that enhanced death of pulmonary neutrophils of infected serpinb1−/− mice is at least in part a cell-autonomous defect likely mediated by unchecked NSP actions.

 

In this paper, we have demonstrated that serpinb1, an intracellular serpin family member, regulates the innate immune response and protects the host during lung bacterial infection. Serpinb1 is among the most potent inhibitors of NSPs and is carried at high levels within neutrophils. Serpinb1-deficient mice fail to clear P. aeruginosa PAO1 lung infection and succumb from systemic bacterial spreading. The defective immune function in serpinb1−/− mice stems at least in part from an increased rate of neutrophil necrosis, reducing the number of phagocytes and leading to increased NSP activity in the lungs with proteolysis of SP-D. In addition, serpinb1-deficient mice also have impaired clearance of the mucoid clinical strain PA M57-15. Interestingly, mucoid strains of P. aeruginosa are cleared with a very high efficiency from the lungs of WT and cystic fibrosis transmembrane conductance regulator–deficient mice (26). The phenotype of serpinb1−/− mice reproduces major pathologic features of human pulmonary diseases characterized by excessive inflammation, massive neutrophil recruitment to the air space, and destruction of cellular and molecular protective mechanisms. Importantly, serpinb1 deficiency may be helpful as an alternative or additional model of the inflammatory lung pathology of cystic fibrosis.

The present study documents a key protective role for serpinb1 in regulating NSP actions in the lung. This role has previously been attributed to the NSP inhibitors α1-antitrypsin and secretory leukocyte protease inhibitor, which are found in the airway and alveolar lining fluid (2728). However, patients with α1-antitrypsin deficiency do not present with pulmonary infection secondary to innate immune defects despite increased NSP activity that leads to reduced lung elasticity and emphysema. Moreover, there is so far no evidence that deficiency in secretory leukocyte protease inhibitor results in failure to clear pulmonary infection. Because synthesis and storage of NSPs in granules is an event that exclusively takes place in bone marrow promyelocytes (29), the regulation of NSPs in the lung relies entirely on NSP inhibitors. Thus, the extent of the innate immune defect inserpinb1−/− mice and the normalization of bacterial clearance with topical rSERPINB1 treatment indicate that serpinb1 is required to regulate NSP activity in the airway fluids and that, during acute lung infection associated with high neutrophilic recruitment, there is insufficient compensation by other NSP inhibitors. The devastating effects of NSPs when released in the lungs by degranulating and necrotic neutrophils are well documented in human pulmonary diseases (5630). Therefore, our findings clearly establish a physiological and nonredundant role for serpinb1 in regulating NSPs during pulmonary infection.

NSPs also cleave molecules involved in apoptotic cell clearance, including the surfactant protein SP-D and the phosphatidylserine receptor on macrophages (3132), thereby tipping the balance further toward a detrimental outcome. The increased numbers of leukocytes with active caspase-3 in the alveolar space of P. aeruginosa–infectedserpinb1−/− mice suggest that the removal of apoptotic cells may be inadequate during infection. SP-D has been shown to stimulate phagocytosis of P. aeruginosa by alveolar macrophages in vitro (33), and SP-D–deficient mice were found to have defective early (6-h) clearance of P. aeruginosa from the lung (34). Although the destruction of SP-D alone may not entirely account for the defective phenotype of serpinb1−/− mice, loss of SP-D likely diminishes bacterial clearance and removal of apop totic neutrophils.

Given that NSPs also mediate bacterial killing, why would NSP excess lead to a failed bacterial clearance? In the NE KO mice, the decreased killing activity of neutrophils is a direct consequence of the loss of the bactericidal activity of NE. The absence of an early bacterial clearance defect at 6 h after infection in serpinb1−/− mice suggests that there is initially normal bacterial killing. The current understanding is that the compartmentalization of the NSPs is crucial to the outcome of their actions: on the one hand, NSPs are protective when killing microbes within phagosomes, and on the other hand, extracellular NSPs destroy innate immune defense molecules such as lung collectins, immunoglobulins, and complement receptors. We have shown that the regulation of NSP activity is essential and that cytoplasmic serpinb1 provides this crucial shield. Neutrophils undergoing cell death gradually transition from apoptosis, characterized by a nonpermeable plasma membrane, to necrosis and lysis, where cellular and granule contents, including NSPs, are released. The increased pace of serpinb1−/− neutrophil cell death strongly suggests that unopposed NSPs may precipitate neutrophil demise and, therefore, reduce the neutrophil numbers leading to a late-onset innate immune defect. High levels of G-CSF, a prosurvival cytokine for neutrophils, also indicate that increased cell death is likely independent or downstream of G-CSF.

In conclusion, serpinb1 deficiency unleashes unbridled proteolytic activity during inflammation and thereby disables two critical components of the host response to bacterial infection, the neutrophil and the collectin SP-D. The phenotype of the infectedserpinb1-deficient mouse, characterized by a normal early antibacterial response that degenerates over time, highlights the delicate balance of protease–antiprotease systems that protect the host against its own defenses as well as invading microbes during infection-induced inflammation.

 

 

Proteinase 3 and neutrophil elastase enhance inflammation in mice by inactivating antiinflammatory progranulin

K Kessenbrock,1 LFröhlich,2 M Sixt,3 …., A Belaaouaj,5 J Ring,6,7 M Ollert,6 R Fässler,3 and DE. Jenne1
J Clin Invest. 2008 Jul 1; 118(7): 2438–2447.   http://dx.doi.org:/10.1172/JCI34694

Neutrophil granulocytes form the body’s first line of antibacterial defense, but they also contribute to tissue injury and noninfectious, chronic inflammation. Proteinase 3 (PR3) and neutrophil elastase (NE) are 2 abundant neutrophil serine proteases implicated in antimicrobial defense with overlapping and potentially redundant substrate specificity. Here, we unraveled a cooperative role for PR3 and NE in neutrophil activation and noninfectious inflammation in vivo, which we believe to be novel. Mice lacking both PR3 and NE demonstrated strongly diminished immune complex–mediated (IC-mediated) neutrophil infiltration in vivo as well as reduced activation of isolated neutrophils by ICs in vitro. In contrast, in mice lacking just NE, neutrophil recruitment to ICs was only marginally impaired. The defects in mice lacking both PR3 and NE were directly linked to the accumulation of antiinflammatory progranulin (PGRN). Both PR3 and NE cleaved PGRN in vitro and during neutrophil activation and inflammation in vivo. Local administration of recombinant PGRN potently inhibited neutrophilic inflammation in vivo, demonstrating that PGRN represents a crucial inflammation-suppressing mediator. We conclude that PR3 and NE enhance neutrophil-dependent inflammation by eliminating the local antiinflammatory activity of PGRN. Our results support the use of serine protease inhibitors as antiinflammatory agents.

 

Neutrophils belong to the body’s first line of cellular defense and respond quickly to tissue injury and invading microorganisms (1). In a variety of human diseases, like autoimmune disorders, infections, or hypersensitivity reactions, the underlying pathogenic mechanism is the formation of antigen-antibody complexes, so-called immune complexes (ICs), which trigger an inflammatory response by inducing the infiltration of neutrophils (2). The subsequent stimulation of neutrophils by C3b-opsonized ICs results in the generation of ROS and the release of intracellularly stored proteases leading to tissue damage and inflammation (3). It is therefore important to identify the mechanisms that control the activation of infiltrating neutrophils.

Neutrophils abundantly express a unique set of neutrophil serine proteases (NSPs), namely cathepsin G (CG), proteinase 3 (PR3; encoded by Prtn3), and neutrophil elastase (NE; encoded by Ela2), which are stored in the cytoplasmic, azurophilic granules. PR3 and NE are closely related enzymes, with overlapping and potentially redundant substrate specificities different from those of CG. All 3 NSPs are implicated in antimicrobial defense by degrading engulfed microorganisms inside the phagolysosomes of neutrophils (48). Among many other functions ascribed to these enzymes, PR3 and NE were also suggested to play a fundamental role in granulocyte development in the bone marrow (911).

While the vast majority of the enzymes is stored intracellularly, minor quantities of PR3 and NE are externalized early during neutrophil activation and remain bound to the cell surface, where they are protected against protease inhibitors (1213). These membrane presented proteases were suggested to act as path clearers for neutrophil migration by degrading components of the extracellular matrix (14). This notion has been addressed in a number of studies, which yielded conflicting results (1517). Thus, the role of PR3 and NE in leukocyte extravasation and interstitial migration still remains controversial.

Emerging data suggest that externalized NSPs can contribute to inflammatory processes in a more complex way than by simple proteolytic tissue degradation (18). For instance, recent observations using mice double-deficient for CG and NE indicate that pericellular CG enhances IC-mediated neutrophil activation and inflammation by modulating integrin clustering on the neutrophil cell surface (1920). Because to our knowledge no Prtn3–/– mice have previously been generated, the role of this NSP in inflammatory processes has not been deciphered. Moreover, NE-dependent functions that can be compensated by PR3 in Ela2–/–animals are still elusive.

One mechanism by which NSPs could upregulate the inflammatory response has recently been proposed. The ubiquitously expressed progranulin (PGRN) is a growth factor implicated in tissue regeneration, tumorigenesis, and inflammation (2123). PGRN was previously shown to directly inhibit adhesion-dependent neutrophil activation by suppressing the production of ROS and the release of neutrophil proteases in vitro (23). This antiinflammatory activity was degraded by NE-mediated proteolysis of PGRN to granulin (GRN) peptides (23). In contrast, GRN peptides may enhance inflammation (23) and have been detected in neutrophil-rich peritoneal exudates (24). In short, recent studies proposed PGRN as a regulator of the innate immune response, but the factors that control PGRN function are still poorly defined and its relevance to inflammation needs to be elucidated in vivo.

In the present study, we generated double-deficient Prtn3–/–Ela2–/– mice to investigate the role of these highly similar serine proteases in noninfectious neutrophilic inflammation. We established that PR3 and NE are required for acute inflammation in response to subcutaneous IC formation. The proteases were found to be directly involved in early neutrophil activation events, because isolated Prtn3–/–Ela2–/– neutrophils were poorly activated by ICs in vitro. These defects in Prtn3–/–Ela2–/– mice were accompanied by accumulation of PGRN. We demonstrated that PGRN represents a potent inflammation-suppressing factor that is cleaved by both PR3 and NE. Our data delineate what we believe to be a previously unknown proinflammatory role for PR3 and NE, which is accomplished via the local inactivation of antiinflammatory PGRN.

 

Generation of Prtn3–/–Ela2–/– mice.

To analyze the role of PR3 and NE in neutrophilic inflammation, we generated a Prtn3–/–Ela2–/– mouse line by targeted gene disruption in embryonic stem cells (see Supplemental Figure 1; supplemental material available online with this article; doi: 10.1172/JCI34694DS1). Positive recombination of the Prtn3/Ela2locus was proven by Southern blotting of embryonic stem cell clones (Figure ​(Figure1A).1A). Prtn3–/–Ela2–/– mice showed no expression of mRNA for PR3 and NE in bone marrow cells, as assessed by RT-PCR (Figure ​(Figure1B).1B). The successful elimination of PR3 and NE was confirmed at the level of proteolytic activity in neutrophil lysates using a PR3/NE-specific chromogenic substrate (Supplemental Figure 3) as well as by casein zymography (Figure ​(Figure1C).1C). The substantially reduced casein degradation by heterozygous neutrophils indicates gene-dosage dependence of PR3/NE activities. Furthermore, PR3 and NE deficiency was proven by Western blotting using cell lysates from bone marrow–derived neutrophils, while other enzymes stored in azurophilic granula, such as CG and myeloperoxidase (MPO), were normally detected (Figure ​(Figure1D).1D). Crossing of heterozygous Prtn3+/–Ela2+/– mice resulted in regular offspring of WT, heterozygous, and homozygous genotype according to the Mendelian ratio. Despite the absence of 2 abundant serine proteases, and in contrast to expectations based on previous reports (911), we found unchanged neutrophil morphology (Figure ​(Figure1E)1E) and regular neutrophil populations in the peripheral blood of the mutant mice, the latter as assessed via flow cytometry to determine the differentiation markers CD11b and Gr-1 (Figure ​(Figure1F)1F) (2526). Moreover, Prtn3–/–Ela2–/– mice demonstrated normal percentages of the leukocyte subpopulations in the peripheral blood, as determined by the Diff-Quick staining protocol and by hemocytometric counting (Supplemental Figure 2, A and B). Hence, the proteases are not crucially involved in granulopoiesis, and ablating PR3 and NE in the germ line represents a valid approach to assess their biological significance in vivo.

 

Figure 1

Generation and characterization of Prtn3–/–Ela2–/– mice.

PR3 and NE are dispensable for neutrophil extravasation and interstitial migration.

To examine neutrophil infiltration into the perivascular tissue, we applied phorbol esters (croton oil) to the mouse ears. At 4 h after stimulation, we assessed the neutrophil distribution in relation to the extravascular basement membrane (EBM) by immunofluorescence microscopy of fixed whole-mount specimens (Figure ​(Figure2A).2A). We found that Prtn3–/–Ela2–/– neutrophils transmigrated into the interstitium without retention at the EBM (Figure ​(Figure2B),2B), resulting in quantitatively normal and widespread neutrophil influx compared with WT mice (Figure ​(Figure2C).2C). Moreover, we analyzed chemotactic migration of isolated neutrophils through a 3-dimensional collagen meshwork in vitro (Supplemental Video 1) and found unhampered chemotaxis toward a C5a gradient, based on the directionality (Figure ​(Figure2D)2D) and velocity (Figure ​(Figure2E)2E) of Prtn3–/–Ela2–/–neutrophils. These findings led us to conclude that PR3 and NE are not principally required for neutrophil extravasation or interstitial migration.

 

Figure 2

PR3 and NE are not principally required for neutrophil extravasation and interstitial migration.

Reduced inflammatory response to ICs in Prtn3–/–Ela2–/– mice.

The formation of ICs represents an important trigger of neutrophil-dependent inflammation in many human diseases (2). To determine the role of PR3 and NE in this context, we induced a classic model of subcutaneous IC-mediated inflammation, namely the reverse passive Arthus reaction (RPA) (27). At 4 h after RPA induction, we assessed the cellular inflammatory infiltrates by histology using H&E-stained skin sections (Figure ​(Figure3A).3A). Neutrophils, which were additionally identified by Gr-1 immunohistochemistry, made up the vast majority of all cellular infiltrates (Figure ​(Figure3A).3A). We found that neutrophil infiltration to the sites of IC formation was severely diminished in Prtn3–/–Ela2–/– mice. Indeed, histological quantification revealed significantly reduced neutrophil influx in Prtn3–/–Ela2–/– mice compared with WT mice, while Ela2–/– mice showed marginally reduced neutrophil counts (Figure ​(Figure3B).3B). These results indicate that PR3 and NE fulfill an important proinflammatory function during IC-mediated inflammation.

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Figure 3

Impaired inflammatory response to locally formed ICs inPrtn3–/–Ela2–/– mice.

(A) Representative photomicrographs of inflamed skin sections 4 h after IC formation. Neutrophils were identified morphologically (polymorphic nucleus) in H&E stainings and by Gr-1 staining (red). The cellular infiltrates were located to the adipose tissue next to the panniculus carnosus muscle (asterisks) and were primarily composed of neutrophil granulocytes. Scale bars: 200 μm. (B) Neutrophil infiltrates in lesions from Prtn3–/–Ela2–/– mice were significantly diminished compared with Ela2–/– mice and WT mice. Neutrophil influx in Ela2–/–mice was slightly, but not significantly, diminished compared with WT mice. Results are mean ± SEM infiltrated neutrophils per HPF. *P < 0.05.

PR3 and NE enhance neutrophil activation by ICs in vitro.

PR3 and NE enhance neutrophil activation by ICs in vitro.

Because PR3 and NE were required for the inflammatory response to IC (Figure ​(Figure3),3), but not to phorbol esters (Figure ​(Figure2),2), we considered the enzymes as enhancers of the neutrophil response to IC. We therefore assessed the oxidative burst using dihydrorhodamine as a readout for cellular activation of isolated, TNF-α–primed neutrophils in the presence of ICs in vitro. While both WT and Prtn3–/–Ela2–/– neutrophils showed a similar, approximately 20-min lag phase before the oxidative burst commenced, the ROS production over time was markedly reduced, by 30%–40%, in the absence of PR3 and NE (Figure ​(Figure4A).4A). In contrast, oxidative burst triggered by 25 nM PMA was not hindered in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure4B),4B), which indicated no general defect in producing ROS. We also performed a titration series ranging from 0.1 to 50 nM PMA and found no reduction in oxidative burst activity in Prtn3–/–Ela2–/– neutrophils at any PMA concentration used (Supplemental Figure 4). These data are consistent with our in vivo experiments showing that neutrophil influx to ICs was impaired (Figure ​(Figure3),3), whereas the inflammatory response to phorbol esters was normal (Figure ​(Figure2,2, A–C), in Prtn3–/–Ela2–/– mice. To compare neutrophil priming in WT and Prtn3–/–Ela2–/–neutrophils, we analyzed cell surface expression of CD11b after 30 min of incubation at various concentrations of TNF-α and found no difference (Supplemental Figure 5). Moreover, we observed normal neutrophil adhesion to IC-coated surfaces (Supplemental Figure 6A) and unaltered phagocytosis of opsonized, fluorescently labeled E. coli bacteria (Supplemental Figure 6, B and C) in the absence of both proteases. We therefore hypothesized that PR3 and NE enhance early events of adhesion-dependent neutrophil activation after TNF-α priming and binding of ICs. It is important to note that Ela2–/– neutrophils were previously shown to react normally in the same setup (20). Regarding the highly similar cleavage specificities of both proteases, we suggested that PR3 and NE complemented each other during the process of neutrophil activation and inflammation.

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Figure 4

Impaired oxidative burst and PGRN degradation by IC-activatedPrtn3–/–Ela2–/– neutrophils.

Oxidative burst as the readout for neutrophil activation by ICs was measured over time. (A) While no difference was observed during the initial 20-min lag phase of the oxidative burst, Prtn3–/–Ela2–/– neutrophils exhibited diminished ROS production over time compared with WT neutrophils. (B) Bypassing receptor-mediated activation using 25 nM PMA restored the diminished oxidative burst of Prtn3–/–Ela2–/–neutrophils. Results are presented as normalized fluorescence in AU (relative to maximum fluorescence produced by WT cells). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate. (C) Isolated mouse neutrophils were activated by ICs in vitro and tested for PGRN degradation by IB. In the cellular fraction, the PGRN (~80 kDa) signal was markedly increased in Prtn3–/–Ela2–/–cells compared with WT and Ela2–/– neutrophils. Intact PGRN was present in the supernatant (SN) of IC-activated Prtn3–/–Ela2–/–neutrophils only, not of WT or Ela2–/– cells. (D and E) Exogenous administration of 100 nM PGRN significantly reduced ROS production of neutrophils activated by ICs (D), but not when activated by PMA (E). Data (mean ± SD) are representative of 3 independent experiments each conducted in triplicate.

Antiinflammatory PGRN is degraded by PR3 and NE during IC-mediated neutrophil activation.

PGRN inhibits neutrophil activation by ICs in vitro.

Both PR3 and NE process PGRN in vitro.

Figure 5

PR3 and NE are major PGRN processing enzymes of neutrophils.

PGRN inhibits IC-mediated inflammation in vivo.

Figure 6

PGRN is a potent inhibitor of IC-stimulated inflammation in vivo.

PR3 and NE cleave PGRN during inflammation in vivo.

Finally, we aimed to demonstrate defective PGRN degradation in Prtn3–/–Ela2–/– mice during neutrophilic inflammation in vivo. For practical reasons, we harvested infiltrated neutrophils from the inflamed peritoneum 4 h after casein injection and subjected the lysates of these cells to anti-PGRN Western blot. Intact, inhibitory PGRN was detected in Prtn3–/–Ela2–/– neutrophils, but not in WT cells (Figure ​(Figure6D).6D). These data prove that neutrophilic inflammation is accompanied by proteolytic removal of antiinflammatory PGRN and that the process of PGRN degradation is essentially impaired in vivo in the absence of PR3 and NE.

 

Chronic inflammatory and autoimmune diseases are often perpetuated by continuous neutrophil infiltration and activation. According to the current view, the role of NSPs in these diseases is mainly associated with proteolytic tissue degradation after their release from activated or dying neutrophils. However, recent observations suggest that NSPs such as CG may contribute to noninfectious diseases in a more complex manner, namely as specific regulators of inflammation (18). Here, we demonstrate that PR3 and NE cooperatively fulfilled an important proinflammatory role during neutrophilic inflammation. PR3 and NE directly enhanced neutrophil activation by degrading oxidative burst–suppressing PGRN. These findings support the use of specific serine protease inhibitors as antiinflammatory agents.

Much attention has been paid to the degradation of extracellular matrix components by NSPs. We therefore expected that ablation of both PR3 and NE would cause impaired neutrophil extravasation and interstitial migration. Surprisingly, we found that the proteases were principally dispensable for these processes:Prtn3–/–Ela2–/– neutrophils migrated normally through a dense, 3-dimensional collagen matrix in vitro and demonstrated regular extravasation in vivo when phorbol esters were applied (Figure ​(Figure2).2). This finding is in agreement with recent reports that neutrophils preferentially and readily cross the EBM through regions of low matrix density in the absence of NE (28).

Conversely, we observed that PR3 and NE were required for the inflammatory response to locally formed ICs (Figure ​(Figure3).3). Even isolated Prtn3–/–Ela2–/– neutrophils were challenged in performing oxidative burst after IC stimulation in vitro (Figure ​(Figure4A),4A), showing that the proteases directly enhanced the activation of neutrophils also in the absence of extracellular matrix. However, when receptor-mediated signal transduction was bypassed by means of PMA, neutrophils from Prtn3–/–Ela2–/– mice performed normal oxidative burst (Figure ​(Figure4B),4B), indicating that the function of the phagocyte oxidase (phox) complex was not altered in the absence of PR3 and NE. These findings substantiate what we believe to be a novel paradigm: that all 3 serine proteases of azurophilic granules (CG, PR3, and NE), after their release in response to IC encounter, potentiate a positive autocrine feedback on neutrophil activation.

In contrast to CG, the highly related proteases PR3 and NE cooperate in the effacement of antiinflammatory PGRN, leading to enhanced neutrophil activation. Previous studies already demonstrated that PGRN is a potent inhibitor of the adhesion-dependent oxidative burst of neutrophils in vitro, which can be degraded by NE (23). Here, we showed that PR3 and NE play an equally important role in the regulation of PGRN function. Ela2–/– neutrophils were sufficiently able to degrade PGRN. Only in the absence of both PR3 and NE was PGRN degradation substantially impaired, resulting in the accumulation of antiinflammatory PGRN during neutrophil activation in vitro (Figure ​(Figure4C)4C) and neutrophilic inflammation in vivo (Figure ​(Figure6D).6D). Moreover, we provided in vivo evidence for the crucial role of PGRN as an inflammation-suppressing mediator, because administration of recombinant PGRN potently inhibited the neutrophil influx to sites of IC formation (Figure ​(Figure6,6, A–C). Hence, the cooperative degradation of PGRN by PR3 and NE is a decisive step for the establishment of neutrophilic inflammation.

The molecular mechanism of PGRN function is not yet completely understood, but it seems to interfere with integrin (CD11b/CD18) outside-in signaling by blocking the function of pyk2 and thus dampens adhesion-related oxidative burst even when added after the initial lag phase of oxidase activation (23). PGRN is produced by neutrophils and stored in highly mobile secretory granules (29). It was recently shown that PGRN can bind to heparan-sulfated proteoglycans (30), which are abundant components of the EBM and various cell surfaces, including those of neutrophils. Also, PR3 and NE are known to interact with heparan sulfates on the outer membrane of neutrophils, where the enzymes appear to be protected against protease inhibitors (121331). These circumstantial observations support the notion that PGRN cleavage by PR3 and NE takes place at the pericellular microenvironment of the neutrophil cell surface.

Impaired outside-in signaling most likely reduced the oxidative burst in Prtn3–/–Ela2–/– neutrophils adhering to ICs. In support of this hypothesis, we excluded an altered response to TNF-α priming (Supplemental Figure 5) as well as reduced adhesion to immobilized ICs and defective endocytosis of serum-opsonized E. coli in Prtn3–/–Ela2–/– neutrophils (Supplemental Figure 6). MPO content and processing was also unchanged in Prtn3–/–Ela2–/– neutrophils (Figure ​(Figure1D);1D); hence, the previously discussed inhibitory effect of MPO on phox activity (3233) does not appear to be stronger in neutrophils lacking PR3 and NE. Because there was no difference in the lag phase of the oxidative burst, initial IC-triggered receptor activation was probably not affected by either PRGN or PR3/NE. Our concept is consistent with all these observations and takes into account that PGRN unfolds its suppressing effects in the second phase, when additional membrane receptors, endogenous PGRN, and some PR3/NE from highly mobile intracellular pools are translocated to the cell surface. The decline and cessation of ROS production suggested to us that outside-in signaling was not sustained and that active oxidase complexes were no longer replenished in the absence of PR3 and NE. Our present findings, however, do not allow us to exclude other potential mechanisms, such as accelerated disassembly of the active oxidase complex.

 

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Proposed function of PR3 and NE in IC-mediated inflammation.

TNF-α–primed neutrophils extravasate from blood vessels, translocate PR3/NE to the cellular surface, and discharge PGRN to the pericellular environment (i). During transmigration of interstitial tissues (ii), neutrophil activation is initially suppressed by relatively high pericellular levels of antiinflammatory PGRN (green shading), which is also produced locally by keratinocytes and epithelial cells of the skin. Until IC depots are reached, neutrophil activation is inhibited by PGRN. Surface receptors (e.g., Mac-1) recognize ICs, which results in signal transduction (black dotted arrow) and activation of the phox. The molecular pathway of PGRN-mediated inhibition is not completely understood, but it may interfere with integrin signaling after IC encounter (green dotted line inside the cell). Adherence of neutrophils to ICs (iii) further increases pericellular PR3 and NE activity. PR3 and NE cooperatively degrade PGRN in the early stage of neutrophilic activation to facilitate optimal neutrophil activation (red shading), resulting in sustained integrin signaling (red arrow) and robust production of ROS by the phox system. Subsequently, neutrophils release ROS together with other proinflammatory mediators and chemotactic agents, thereby enhancing the recruitment of further neutrophils and establishing inflammation (iv). In the absence of PR3/NE, the switch from inflammation-suppressing (ii) to inflammation-enhancing (iii) conditions is substantially delayed, resulting in diminished inflammation in response to ICs (iv).

 

NSPs are strongly implicated as effector molecules in a large number of destructive diseases, such as emphysema or the autoimmune blistering skin disease bullous pemphigoid (143537). Normally, PR3/NE activity is tightly controlled by high plasma levels of α1-antitrypsin. This balance between proteases and protease inhibitors is disrupted in patients with genetic α1-antitrypsin deficiency, which represents a high risk factor for the development of emphysema and certain autoimmune disorders (38). The pathogenic effects of NSPs in these diseases have so far been associated with tissue destruction by the proteases after their release from dying neutrophils. Our findings showed that PR3 and NE were already involved in much earlier events of the inflammatory process, because the enzymes directly regulated cellular activation of infiltrating neutrophils by degrading inflammation-suppressing PGRN. This concept is further supported by previous studies showing increased inflammation in mice lacking serine protease inhibitors such as SERPINB1 or SLPI (3940). Blocking PR3/NE activity using specific inhibitors therefore represents a promising therapeutic strategy to treat chronic, noninfectious inflammation. Serine protease inhibitors as antiinflammatory agents can interfere with the disease process at 2 different stages, because they attenuate both early events of neutrophil activation and proteolytic tissue injury caused by released NSPs.

 

 

 

 

Editorial: Serine proteases, serpins, and neutropenia

David C. Dale

J Leuko Biol July 2011;  90(1): 3-4   http://dx.doi.org:/10.1189/jlb.1010592

Cyclic neutropenia and severe congenital neutropenia are autosomal-dominant diseases usually attributable to mutations in the gene for neutrophil elastase orELANE. Patients with these diseases are predisposed to recurrent and life-threatening infections [1]. Neutrophil elastase, the product of the ELANE gene, is a serine protease that is synthesized and packaged in the primary granules of neutrophils. These granules are formed at the promyelocytes stage of neutrophil development. Synthesis of mutant neutrophil elastase in promyelocytes triggers the unfolded protein response and a cascade of intracellular events, which culminates in death of neutrophil precursors through apoptosis [2]. This loss of cells causes the marrow abnormality often referred to as “maturation arrest” [34].

Neutrophil elastase is one of the serine proteases normally inhibited by serpinB1. In this issue of JLB, Benarafa and coauthors [5] present their intriguing studies of serpinB1 expression in human myeloid cells and their extensive investigations ofSERPINB1−/− mice. They observed that serpinB1 expression parallels protease expression. The peak of serpinB1 expression occurs in promyelocytes. Benarafa et al. [5] found that SERPINB1−/− mice have a deficiency of postmitotic neutrophils in the bone marrow. This change was accompanied by an increase in the plasma levels of G-CSF. The decreased supply of marrow neutrophils reduced the number of neutrophils that could be mobilized to an inflammatory site. Using colony-forming cell assays, they determined that the early myeloid progenitor pool was intact. Separate assays showed that maturing myeloid cells were being lost through accelerated apoptosis of maturing neutrophils in the marrow. The authors concluded that serpinB1 is required for maintenance of a healthy reserve of marrow neutrophils and a normal acute immune response [5].

This paper provides new and fascinating insights for understanding the mechanism for neutropenia. It also suggests opportunities to investigate potential therapies for patients with neutropenia and prompts several questions. As inhibition of the activity of intracellular serine proteases is the only known function of serpinB1, the findings reported by Benarafa et al. [5] suggest that uninhibited serine proteases perturbed neutrophil production severely. The SERPINB1−/− mice used in their work have accelerated apoptosis of myeloid cells, a finding suggesting that uninhibited serine proteases or mutant neutrophil elastase perturb myelopoiesis by similar mechanisms. It is now important to determine whether the defect in the SERPINB1−/− mice is, indeed, attributable to uninhibited activity of normal neutrophil elastase, other neutrophil proteases, or another mechanism. ″Double-knockout″ studies in mice deficient in neutrophil elastase and serpinB1 might provide an answer.

This report provides evidence regarding the intracellular mechanisms for the apoptosis of myeloid cells and indicates that other studies are ongoing. The key antiapoptotic proteins, Mcl-1, Bcl-XL, and A1/Bfl-I, are apparently not involved. A more precise understanding of the mechanisms of cell death is important for development of targeted therapies for neutropenia. It is also important to discover whether only cells of the neutrophil lineage are involved or whether monocytes are also affected. In cyclic and congenital neutropenia, patients failed to produce neutrophils, but they can produce monocytes; in fact, they overproduce monocytes and have significantly elevated blood monocyte counts. Neutropenia with monocytosis is probably attributable to differences in the expression of ELANE in the two lineages. Benarafa et al. [5] reported that human bone marrow monocytes contain substantially less serpinB1 than marrow neutrophils, suggesting that the expression of serpinB1 and the serine proteases are closely coordinated.

This report shows the importance of the marrow neutrophil reserves in the normal response to infections. Compared with humans, healthy mice are always neutropenic, but they have a bigger marrow neutrophil reserve, and their mature neutrophils in the marrow and blood look like human band neutrophils. These differences are well known, but they are critical for considering the clinical inferences that can be made from this report. For example, although theSERPINB1−/− mice were not neutropenic, human SERPINB1−/− might cause neutropenia because of physiological differences between the species. If some but not all mutations in SERPINB1 cause neutropenia, we might gain a better understanding about how serpinB1 normally inhibits the neutrophil’s serine proteases.

We do not know if some or all of the mutant neutrophil elastases can be inhibited by serpinB1. We do not know whether cyclic or congenital neutropenia are attributable to defects in this interaction. However, we do know that there are chemical inhibitors of neutrophil elastase that can abrogate apoptosis of myeloid cells in a cellular model for congenital neutropenia [6]. It would be interesting to see if these chemical inhibitors can replace the natural inhibitor and normalize neutrophil production in the SERPINB1−/− mice. This would provide evidence to support use of chemical protease inhibitors as a treatment for cyclic and congenital neutropenia.

Concerns with the use of G-CSF for the treatment of cyclic and congenital neutropenia are how and why some of these patients are at risk of developing leukemia. Are the SERPINB1−/− mice with a hyperproliferative marrow and high G-CSF levels also at risk of developing myeloid leukemia?

This is a very provocative paper, and much will be learned from further studies of the SERPINB1−/− mice.

 

SerpinB1 is critical for neutrophil survival through cell-autonomous inhibition of cathepsin G

Mathias Baumann1,2, Christine T. N. Pham3, and Charaf Benarafa1

Blood May 9, 2013; 121(19)   http://www.bloodjournal.org/content/121/19/3900

Key Points

  • Serine protease inhibitor serpinB1 protects neutrophils by inhibition of their own azurophil granule protease cathepsin G.
  • Granule permeabilization in neutrophils leads to cathepsin G–mediated death upstream and independent of apoptotic caspases.

Abstract

Bone marrow (BM) holds a large reserve of polymorphonuclear neutrophils (PMNs) that are rapidly mobilized to the circulation and tissues in response to danger signals. SerpinB1 is a potent inhibitor of neutrophil serine proteases neutrophil elastase (NE) and cathepsin G (CG). SerpinB1 deficiency (sB1−/−) results in a severe reduction of the BM PMN reserve and failure to clear bacterial infection. Using BM chimera, we found that serpinB1 deficiency in BM cells was necessary and sufficient to reproduce the BM neutropenia ofsB1−/− mice. Moreover, we showed that genetic deletion of CG, but not NE, fully rescued the BM neutropenia in sB1−/− mice. In mixed BM chimera and in vitro survival studies, we showed that CG modulates sB1−/− PMN survival through a cell-intrinsic pathway. In addition, membrane permeabilization by lysosomotropic agent L-leucyl-L-leucine methyl ester that allows cytosolic release of granule contents was sufficient to induce rapid PMN death through a CG-dependent pathway. CG-mediated PMN cytotoxicity was only partly blocked by caspase inhibition, suggesting that CG cleaves a distinct set of targets during apoptosis. In conclusion, we have unveiled a new cytotoxic function for the serine protease CG and showed that serpinB1 is critical for maintaining PMN survival by antagonizing intracellular CG activity.

Introduction

Polymorphonuclear neutrophil (PMN) granulocytes are essential components of the innate immune response to infection. PMNs are relatively short-lived leukocytes that originate from hematopoietic stem cells in the bone marrow (BM) in a process called granulopoiesis. Granulopoiesis proceeds through a proliferative phase followed by a maturation phase. After maturation, the BM retains a large reserve of mature PMNs, which includes over 90% of the mature PMNs in the body while only a small proportion (1%-5%) is in the blood.1,2 Even in noninflammatory conditions, granulopoiesis is remarkable as >1011 PMNs are produced daily in an adult human, only to be disposed of, largely unused, a few hours later.3 There is evidence that the majority of PMNs produced never reach circulation and die within the BM.4 Congenital or acquired forms of neutropenia are associated with the highest risks of bacterial and fungal infection,5 indicating a strong evolutionary pressure to maintain granulopoiesis at high levels and sustain a large mobilizable pool of PMNs in the BM.

In steady state, PMNs die by apoptosis, a form of programmed cell death that allows for the safe disposal of aging PMNs and their potentially toxic cargo. Like in other cells, caspases participate in the initiation, amplification, and execution steps of apoptosis in PMNs.6,7 Interestingly, noncaspase cysteine proteases calpain and cathepsin D were reported to induce PMN apoptosis through activation of caspases.811 In addition, PMNs carry a unique set of serine proteases (neutrophil serine proteases [NSPs]) including elastase (NE), cathepsin G (CG), and proteinase-3 (PR3) stored active in primary granules. There is strong evidence for a role of NSPs in killing pathogens and inducing tissue injury when released extracellularly.1214 In contrast, the function of NSPs in PMN homeostasis and cell death remains elusive. In particular, no defects in granulopoiesis or PMN homeostasis have been reported in mice deficient in cathepsin G (CG−/−),15 neutrophil elastase (NE−/−),16,17 or dipeptidylpeptidase I (DPPI−/−), which lack active NSPs.18 We have recently shown that mice lacking the serine protease inhibitor serpinB1 (sB1−/−) have reduced PMN survival in the lungs following Pseudomonas infection and that these mice have a profound reduction in mature PMN numbers in the BM.19,20SerpinB1, also known as monocyte NE inhibitor, is expressed at high levels in the cytoplasm of PMNs and is one of the most potent inhibitors of NE, CG, and PR3.21,22 In this study, we tested the hypothesis that serpinB1 promotes PMN survival by inhibiting 1 or several NSPs, and we discovered a novel regulatory pathway in PMN homeostasis in vivo.

 

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Figure 1

Defective PMN reserve in BM chimera depends on serpinB1 deficiency in the hematopoietic compartment. Flow cytometry analysis of major BM leukocyte subsets of lethally irradiated mice was performed 8 to 10 weeks after BM transfer. (A) Irradiated WT (CD45.1) mice were transferred with WT (●) or sB1−/− (○) BM cells. (B) Irradiated WT (●) andsB1−/− (○) mice both CD45.2 were transferred with WT (CD45.1) BM cells. Each circle represents leukocyte numbers for 1 mouse and horizontal line indicates the median. Median subsets numbers were compared by the Mann-Whitney test (*P < .05; ***P < .001).

CG regulates neutrophil numbers in the BM

Because serpinB1 is an efficient inhibitor of NE, CG, and PR3, we then examined PMN numbers in mice deficient in 1 or several NSPs in combination with serpinB1 deletion. As expected, sB1−/− mice had significantly reduced numbers and percentage of mature PMNs in the BM compared with WT and heterozygous sB1+/− mice. In addition, PMN numbers were normal in mice deficient in either DPPI, NE, or CG (Figure 2A). DPPI is not inhibited by serpinB1 but is required for the activation of all NSPs, and no NSP activity is detectable in DPPI−/− mice.18,23 PMN counts in DPPI−/−.sB1−/− BM were significantly higher than in sB1−/− BM, suggesting that 1 or several NSPs contribute to the PMN survival defect. To examine the role of NSPs in this process, we crossed several NSP-deficient strains with sB1−/− mice. We found that NE.CG.sB1−/− mice had normal PMN numbers indicating that these NSPs play a key role in the defective phenotype of sB1−/− PMNs (Figure 2A). Furthermore, CG.sB1−/− mice showed normal PMN numbers whereasNE.sB1−/− mice retained the BM neutropenia phenotype indicating that CG, but not NE, plays a significant role in the death of sB1−/− PMNs (Figure 2A). In addition, the double-deficient NE.sB1−/− mice had significantly lower BM myelocyte numbers than sB1−/− mice while the myelocyte numbers in singly deficient NE−/− and sB1−/− BM were normal (Figure 2B). These results suggest that NE may promote myeloid cell proliferation, an activity that is revealed only when serpinB1 is absent. This complex interaction between sB1 and NE requires further investigation. On the other hand, B-cell and monocyte numbers and relative percentage in the BM were largely similar in all genotypes (supplemental Figure 2). Total numbers of blood leukocytes, erythrocytes, and platelets were normal in mice deficient in NSPs and/or serpinB1 (supplemental Figure 3). PMN numbers in blood were normal insB1−/− mice in steady state and combined deficiency of NSPs did not significantly alter these numbers (Figure 2C). Taken together, our results indicate that serpinB1 likely sustains the survival of postmitotic PMNs through its interaction with CG.

Figure 2

PMN and myelocyte numbers in BM and blood of mice deficient in NSPs and serpinB1.

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CG-mediated PMN death proceeds independent of caspase activity

Figure 4

sB1−/− PMN death mediated by CG does not require caspase activity

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Granule membrane permeabilization induces CG-mediated death in PMNs

To test whether granule disruption contributes to the serpinB1-regulated CG-dependent cell death, BM cells were treated with the lysosomotropic agent LLME. LLME accumulates in lysosomes where the acyl transferase activity of DPPI generates hydrophobic (Leu-Leu)n-OMe polymers that induce lysosomal membrane permeabilization (LMP) and cytotoxicity in granule-bearing cells such as cytotoxic T lymphocytes, NK cells, and myeloid cells.29,30

Figure 5

LMP induces CG-mediated death in PMNs

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G-CSF therapy increases sB1−/− PMN numbers via enhanced granulopoiesis

G-CSF therapy is an effective long-term treatment in many cases of severe congenital neutropenia and it is also used to prevent chemotherapy-induced febrile neutropenia by enhancing PMN production. In addition, G-CSF delays neutrophil apoptosis by differentially regulating proapoptotic and antiapoptotic factors.10 To test whether G-CSF could rescue sB1−/− PMN survival defect, WT and sB1−/− mice were treated with therapeutic doses of G-CSF or saline for 5 days and BM and blood PMNs were analyzed 24 hours after the last injection. Total counts of myelocytes and PMNs were significantly increased in the BM of treated mice compared with their respective untreated genotype controls (Figure 6A-B). The increase in myelocyte numbers was identical in G-CSF–treated WT and sB1−/− mice, indicating that G-CSF–induced granulopoiesis proceeds normally in sB1−/−myeloid progenitors (Figure 6B).

Figure 6

In vivo G-CSF therapy increases PMN numbers in BM of sB1−/− mice.

 

SerpinB1 is a member of the clade B serpins, a subfamily composed of leaderless proteins with nucleocytoplasmic localization. Clade B serpins are often expressed in cells that also carry target proteases, which led to the hypothesis that intracellular serpins protect against misdirected granule proteases and/or protect bystander cells from released proteases.31 We previously reported that deficiency in serpinB1 is associated with reduced PMN survival in the BM and at inflammatory sites.19,20 The evidence presented here demonstrates that the cytoprotective function of serpinB1 in PMNs is based on the inhibition of granule protease CG. Deficiency in CG was sufficient to rescue the defect of sB1−/− mice as illustrated by normal PMN counts in the BM of double knockout CG.sB1−/− mice. We also showed that the protease-serpin interaction occurred within PMNs. Indeed, WT PMNs had a greater survival over sB1−/− PMNs in mixed BM chimera, whereas the survival of CG.sB1−/− PMNs was similar to WT PMNs after BM transfer. SerpinB1 is an ancestral clade B serpin with a conserved specificity determining reactive center loop in all vertebrates.32 Furthermore, human and mouse serpinB1 have the same specificity for chymotrypsin-like and elastase-like serine proteases.21,22 Likewise, human and mouse CG have identical substrate specificities and the phenotype of CG−/− murine PMN can be rescued by human CG.33 Therefore, it is highly likely that the antagonistic functions of CG and serpinB1 in cellular homeostasis observed in mice can be extended to other species.

Extracellular CG was previously reported to promote detachment-induced apoptosis (anoikis) in human and mouse cardiomyocytes.34 This activity is mediated through the shedding and transactivation of epidermal growth factor receptor and downregulation of focal adhesion signaling.35,36 In our study, exogenous human CG also induced PMN death in vitro but these effects were not enhanced in sB1−/− PMNs and the neutropenia associated with serpinB1 deficiency was principally cell intrinsic. How intracellular CG induces PMN death remains to be fully investigated. However, our studies provide some indications on the potential pathways. Like other NSPs, the expression of CG is transcriptionally restricted to the promyelocyte stage during PMN development and NSPs are then stored in active form in primary azurophil granules.37 Because serpinB1 is equally efficient at inhibiting NE, CG, and PR3, it was surprising that deletion of CG alone was sufficient to achieve a complete reversal of the PMN survival defect in CG.sB1−/− mice. A possible explanation would be that CG gains access to targets more readily than other granule proteases. There is evidence that binding to serglycin proteoglycans differs between NE and CG resulting in altered sorting of NE but not CG into granules of serglycin-deficient PMNs.38 Different interactions with granule matrix may thus contribute to differential release of CG from the granules compared with other NSPs. However, because sB1−/− PMNs have similar levels of CG and NE as WT PMNs20 and because LLME-induced granule permeabilization likely releases all granule contents equally, we favor an alternative interpretation where CG specifically targets essential cellular components that are not cleaved by the other serpinB1-inhibitable granule proteases. Upon granule permeabilization, we found that CG can induce cell death upstream of caspases as well as independent of caspases. CG was previously shown to activate caspase-7 in vitro and it functions at neutral pH, which is consistent with a physiological role in the nucleocytoplasmic environment.39 Cell death induced by lysosomal/granule membrane permeabilization has previously been linked to cysteine cathepsins in other cell types. However, these proteases appear to depend on caspase activation to trigger apoptosis and they function poorly at neutral pH, questioning their potential role as regulators of cell death.40 In contrast, CG-mediated cell death is not completely blocked by caspase inhibition, which is a property reminiscent of granzymes in cytotoxic T cells.41 In fact, CG is phylogenetically most closely related to serine proteases granzyme B and H.42 Granzymes have numerous nuclear, mitochondrial, and cytoplasmic target proteins leading to cell death41 and we anticipate that this may also be the case for CG.

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G-CSF therapy is successfully used to treat most congenital and acquired neutropenia through increased granulopoiesis, mobilization from the BM, and increased survival of PMNs. Prosurvival effects of G-CSF include the upregulation of antiapoptotic Bcl-2 family members, which act upstream of the mitochondria and the activation of effector caspases. In sB1−/− mice, G-CSF levels in serum are fourfold higher than in WT mice in steady state and this is accompanied by an upregulation of the antiapoptotic Bcl-2 family member Mcl-1 in sB1−/− PMNs.19 Here, G-CSF therapy significantly increased granulopoiesis in both WT and sB1−/− mice. However, the PMN numbers in treated sB1−/− BM and blood were significantly lower than those of treated WT mice, indicating only a partial rescue of the survival defect. This is consistent with our findings that CG-mediated death can proceed independent of caspases and can thus bypass antiapoptotic effects mediated by G-CSF.

CG has largely been studied in association with antimicrobial and inflammatory functions due to its presence in PMNs.1214,49 In this context, we have previously shown that serpinB1 contributes to prevent increased mortality and morbidity associated with production of inflammatory cytokines upon infection with Pseudomonas aeruginosa and influenza A virus.20,50 In this study, we demonstrate that serpinB1 inhibition of the primary granule protease CG in PMNs is essential for PMN survival and this ultimately regulates PMN numbers in vivo. Our findings also extend the roles of CG from antimicrobial and immunoregulatory functions to a novel role in inducing cell death.

 

Neutrophil Elastase, Proteinase 3, and Cathepsin G as Therapeutic Targets in Human Diseases

Brice KorkmazMarshall S. HorwitzDieter E. Jenne and Francis Gauthier
Pharma Rev Dec 2010; 62(4):726-759  http://dx.doi.org:/10.1124/pr.110.002733

Polymorphonuclear neutrophils are the first cells recruited to inflammatory sites and form the earliest line of defense against invading microorganisms. Neutrophil elastase, proteinase 3, and cathepsin G are three hematopoietic serine proteases stored in large quantities in neutrophil cytoplasmic azurophilic granules. They act in combination with reactive oxygen species to help degrade engulfed microorganisms inside phagolysosomes. These proteases are also externalized in an active form during neutrophil activation at inflammatory sites, thus contributing to the regulation of inflammatory and immune responses. As multifunctional proteases, they also play a regulatory role in noninfectious inflammatory diseases. Mutations in the ELA2/ELANE gene, encoding neutrophil elastase, are the cause of human congenital neutropenia. Neutrophil membrane-bound proteinase 3 serves as an autoantigen in Wegener granulomatosis, a systemic autoimmune vasculitis. All three proteases are affected by mutations of the gene (CTSC) encoding dipeptidyl peptidase I, a protease required for activation of their proform before storage in cytoplasmic granules. Mutations of CTSC cause Papillon-Lefèvre syndrome. Because of their roles in host defense and disease, elastase, proteinase 3, and cathepsin G are of interest as potential therapeutic targets. In this review, we describe the physicochemical functions of these proteases, toward a goal of better delineating their role in human diseases and identifying new therapeutic strategies based on the modulation of their bioavailability and activity. We also describe how nonhuman primate experimental models could assist with testing the efficacy of proposed therapeutic strategies.

 

Human polymorphonuclear neutrophils represent 35 to 75% of the population of circulating leukocytes and are the most abundant type of white blood cell in mammals (Borregaard et al., 2005). They are classified as granulocytes because of their intracytoplasmic granule content and are characterized by a multilobular nucleus. Neutrophils develop from pluripotent stem cells in the bone marrow and are released into the bloodstream where they reach a concentration of 1.5 to 5 × 109 cells/liter. Their half-life in the circulation is only on the order of a few hours. They play an essential role in innate immune defense against invading pathogens and are among the primary mediators of inflammatory response. During the acute phase of inflammation, neutrophils are the first inflammatory cells to leave the vasculature, where they migrate toward sites of inflammation, following a gradient of inflammatory stimuli. They are responsible for short-term phagocytosis during the initial stages of infection (Borregaard and Cowland, 1997Hampton et al., 1998Segal, 2005). Neutrophils use complementary oxidative and nonoxidative pathways to defend the host against invading pathogens (Kobayashi et al., 2005).

The three serine proteases neutrophil elastase (NE1), proteinase 3 (PR3), and cathepsin G (CG) are major components of neutrophil azurophilic granules and participate in the nonoxidative pathway of intracellular and extracellular pathogen destruction. These neutrophil serine proteases (NSPs) act intracellularly within phagolysosomes to digest phagocytized microorganisms in combination with microbicidal peptides and the membrane-associated NADPH oxidase system, which produces reactive oxygen metabolites (Segal, 2005). An additional extracellular antimicrobial mechanism, neutrophil extracellular traps (NET), has been described that is made of a web-like structure of DNA secreted by activated neutrophils (Papayannopoulos and Zychlinsky, 2009) (Fig. 1). NETs are composed of chromatin bound to positively charged molecules, such as histones and NSPs, and serve as physical barriers that kill pathogens extracellularly, thus preventing further spreading. NET-associated NSPs participate in pathogen killing by degrading bacterial virulence factors extracellularly (Brinkmann et al., 2004;Papayannopoulos and Zychlinsky, 2009).

http://pharmrev.aspetjournals.org/content/62/4/726/F1.small.gif

Fig. 1.

Polymorphonuclear neutrophil. Quiescent (A) and chemically activated (B) neutrophils purified from peripheral blood. C, PMA-activated neutrophils embedded within NET and neutrophil spreading on insoluble elastin.

In addition to their involvement in pathogen destruction and the regulation of proinflammatory processes, NSPs are also involved in a variety of inflammatory human conditions, including chronic lung diseases (chronic obstructive pulmonary disease, cystic fibrosis, acute lung injury, and acute respiratory distress syndrome) (Lee and Downey, 2001Shapiro, 2002Moraes et al., 2003Owen, 2008b). In these disorders, accumulation and activation of neutrophils in the airways result in excessive secretion of active NSPs, thus causing lung matrix destruction and inflammation. NSPs are also involved in other human disorders as a consequence of gene mutations, altered cellular trafficking, or, for PR3, autoimmune disease. Mutations in the ELA2/ELANE gene encoding HNE are the cause of human cyclic neutropenia and severe congenital neutropenia (Horwitz et al., 19992007). Neutrophil membrane-bound proteinase 3 (mPR3) is the major target antigen of anti-neutrophil cytoplasmic autoantibodies (ANCA), which are associated with Wegener granulomatosis (Jenne et al., 1990). All three proteases are affected by mutation of the gene (CTSC) encoding dipeptidyl peptidase I (DPPI), which activates several granular hematopoietic serine proteases (Pham and Ley, 1999Adkison et al., 2002). Mutations of CTSC cause Papillon-Lefèvre syndrome and palmoplantar keratosis (Hart et al., 1999Toomes et al., 1999).

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Fully processed mature HNE, PR3, and CG isolated from azurophilic granules contain, respectively, 218 (Bode et al., 1986Sinha et al., 1987), 222 (Campanelli et al., 1990b), and 235 (Salvesen et al., 1987Hof et al., 1996) residues. They are present in several isoforms depending on their carbohydrate content, with apparent mass of 29 to 33 kDa upon SDS-polyacrylamide gel electrophoresis (Twumasi and Liener, 1977Watorek et al., 1993). HNE and PR3 display two sites of N-glycosylation, whereas CG possesses only one. NSPs are stored mainly in neutrophil azurophilic granules, but HNE is also localized in the nuclear envelope, as revealed by immunostaining and electron microscopy (Clark et al., 1980;Benson et al., 2003), whereas PR3 is also found in secretory vesicles (Witko-Sarsat et al., 1999a). Upon neutrophil activation, granular HNE, PR3, and CG are secreted extracellularly, although some molecules nevertheless remain at the cell surface (Owen and Campbell, 1999Owen, 2008a). The mechanism through which NSPs are sorted from the trans-Golgi network to the granules has not been completely defined, even though an intracellular proteoglycan, serglycin, has been identified as playing a role in elastase sorting and packaging into azurophilic granules (Niemann et al., 2007). Unlike HNE and CG, PR3 is constitutively expressed on the membranes of freshly isolated neutrophils (Csernok et al., 1990Halbwachs-Mecarelli et al., 1995). Stimulation of neutrophils at inflammatory sites triggers intracytoplasmic granules to translocate to the phagosomes and plasma membrane, thereby liberating their contents. The first step of the translocation to the target membrane depends on cytoskeleton remodeling and microtubule assembly (Burgoyne and Morgan, 2003). This is followed by a second step of granule tethering and docking, which are dependent on the sequential intervention of SNARE proteins (Jog et al., 2007).

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Exposure of neutrophils to cytokines (TNF-α), chemoattractants (platelet-activating factor, formyl-Met-Leu-Phe, or IL-8), or bacterial lipopolysaccharide leads to rapid granule translocation to the cell surface with secretion of HNE, PR3, and CG into the extracellular medium (Owen and Campbell, 1999). A fraction of secreted HNE, PR3, and CG is detected at the surface of activated neutrophils (Owen et al., 1995a1997Campbell et al., 2000). Resting purified neutrophils from peripheral blood express variable amounts of PR3 on their surface. A bimodal, apparently genetically determined, distribution has been observed with two populations of quiescent neutrophils that express or do not express the protease at their surface (Halbwachs-Mecarelli et al., 1995Schreiber et al., 2003). The percentage of mPR3-positive neutrophils ranges from 0 to 100% of the total neutrophil population within individuals. Furthermore, the percentage of mPR3-positive neutrophils remains stable over time and is not affected by neutrophil activation (Halbwachs-Mecarelli et al., 1995).

The mechanism through which HNE and CG are associated with the outer surface of the plasma membrane of neutrophils mainly involves electrostatic interactions with the sulfate groups of chondroitin sulfate- and heparan sulfate-containing proteoglycans (Campbell and Owen, 2007). These two proteases are released from neutrophil cell surfaces by high concentrations of salt (Owen et al., 1995b1997;Korkmaz et al., 2005a) and after treatment with chondroitinase ABC and heparinase (Campbell and Owen, 2007). Membrane PR3 is not solubilized by high salt concentrations, which means that its membrane association is not charge dependant (Witko-Sarsat et al., 1999aKorkmaz et al., 2009). Unlike HNE and CG, PR3 bears at its surface a hydrophobic patch formed by residues Phe166, Ile217, Trp218, Leu223, and Phe224 that is involved in membrane binding (Goldmann et al., 1999Hajjar et al., 2008) (Fig. 3B). Several membrane partners of PR3 have been identified, including CD16/FcγRIIIb (David et al., 2005Fridlich et al., 2006), phospholipid scramblase-1, a myristoylated membrane protein with translocase activity present in lipid rafts (Kantari et al., 2007), CD11b/CD18 (David et al., 2003), and human neutrophil antigen NB1/CD177 (von Vietinghoff et al., 2007Hu et al., 2009), a 58- to 64-kDa glycosyl-phosphatidylinositol anchored surface receptor belonging to the urokinase plasminogen activator receptor superfamily (Stroncek, 2007). NB1 shows a bimodal distribution that superimposes with that of PR3 on purified blood neutrophils (Bauer et al., 2007). Active, mature forms of PR3 but not pro-PR3 can bind to the surface of NB1-transfected human embryonic kidney 293 cells (von Vietinghoff et al., 2008) and Chinese hamster ovary cells (Korkmaz et al., 2008b). Interaction involves the hydrophobic patch of PR3 because specific amino acid substitutions disrupting this patch in the closely related gibbon PR3 prevent binding to NB1-transfected cells (Korkmaz et al., 2008b). Decreased interaction of pro-PR3 with NB1-transfected cells is explained by the topological changes affecting the activation domain containing the hydrophobic patch residues. Together, these results support the hydrophobic nature of PR3-membrane interaction.

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Roles in Inflammatory Process Regulation

NSPs are abundantly secreted into the extracellular environment upon neutrophil activation at inflammatory sites. A fraction of the released proteases remain bound in an active form on the external surface of the plasma membrane so that both soluble and membrane-bound NSPs are able to proteolytically regulate the activities of a variety of chemokines, cytokines, growth factors, and cell surface receptors. Secreted proteases also activate lymphocytes and cleave apoptotic and adhesion molecules (Bank and Ansorge, 2001Pham, 2006Meyer-Hoffert, 2009). Thus, they retain pro- and anti-inflammatory activities, resulting in a modulation of the immune response at sites of inflammation.

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Processing of Cytokines, Chemokines, and Growth Factors.

Processing and Activation of Cellular Receptors.

Induction of Apoptosis by Proteinase 3.

Physiological Inhibitors of Elastase, Proteinase 3, and Cathepsin G

During phagocytosis and neutrophil turnover, HNE, PR3, and CG are released into the extracellular space as active proteases. The proteolytic activity of HNE, PR3, and CG seems to be tightly regulated in the extracellular and pericellular space to avoid degradation of connective tissue proteins including elastin, collagen, and proteoglycans (Janoff, 1985). Protein inhibitors that belong to three main families, the serpins, the chelonianins, and the macroglobulins, ultimately control proteolytic activity of HNE, PR3, and CG activities. The individual contributions of these families depend on their tissue localization and that of their target proteases. The main characteristics of HNE, PR3, and CG physiological inhibitors are presented in Table 2.

 

Serine Protease Inhibitors

Serpins are the largest and most diverse family of protease inhibitors; more than 1000 members have been identified in human, plant, fungi, bacteria, archaea, and certain viruses (Silverman et al., 2001Mangan et al., 2008). They share a similar highly conserved tertiary structure and similar molecular weight of approximately 50 kDa. Human serpins belong to the first nine clades (A–I) of the 16 that have been described based on phylogenic relationships (Irving et al., 2000Silverman et al., 2001Mangan et al., 2008). For historical reasons, α1-protease inhibitor (α1-PI) was assigned to the first clade. Clade B, also known as the ov-serpin clan because of the similarity of its members to ovalbumin (a protein that belongs to the serpin family but lacks inhibitory activity), is the second largest clan in humans, with 15 members identified so far. Ov-serpin clan members are generally located in the cytoplasm and, to a lesser extent, on the cell surface and nucleus (Remold-O’Donnell, 1993).

Serpins play important regulatory functions in intracellular and extracellular proteolytic events, including blood coagulation, complement activation, fibrinolysis, cell migration, angiogenesis, and apoptosis (Potempa et al., 1994). Serpin dysfunction is known to contribute to diseases such as emphysema, thrombosis, angioedema, and cancer (Carrell and Lomas, 1997Lomas and Carrell, 2002). Most inhibitory serpins target trypsin-/chymotrypsin-like serine proteases, but some, termed “cross-class inhibitors,” have been shown to target cysteine proteases (Annand et al., 1999). The crystal structure of the prototype plasma inhibitor α1-PI revealed the archetype native serpin fold (Loebermann et al., 1984). All serpins typically have three β-sheets (termed A, B, and C) and eight or nine α-helices (hA–hI) arranged in a stressed configuration. The so-called reactive center loop (RCL) of inhibitory molecules determines specificity and forms the initial encounter complex with the target protease (Potempa et al., 1994Silverman et al., 2001). Serpins inhibit proteases by a suicide substrate inhibition mechanism. The protease initially recognizes the serpin as a potential substrate using residues of the reactive center loop and cleaves it between P1 and P1′ This cleavage allows insertion of the cleaved RCL into the β-sheet A of the serpin, dragging the protease with it and moving it over 71 Å to the distal end of the serpin to form a 1:1 stoichiometric covalent inhibitory complex (Huntington et al., 2000). Such cleavage generates a ∼4-kDa C-terminal fragment that remains noncovalently bound to the cleaved serpin. Displacement of the covalently attached active site serine residue from its catalytic partner histidine explains the loss of catalytic function in the covalent complex. The distortion of the catalytic site structure prevents the release of the protease from the complex, and the structural disorder induces its proteolytic inactivation (Huntington et al., 2000). Covalent complex formation between serpin and serine proteases triggers a number of conformational changes, particularly in the activation domain loops of the bound protease (Dementiev et al., 2006).

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Pathophysiology of Elastase, Proteinase 3 and Cathepsin G in Human Diseases

In many instances, the initiation and propagation of lung damage is a consequence of an exaggerated inappropriate inflammatory response, which includes the release of proteases and leukocyte-derived cytotoxic products (Owen, 2008b;Roghanian and Sallenave, 2008). Inflammation is a physiological protective response to injury or infection consisting of endothelial activation, leukocyte recruitment and activation, vasodilation, and increased vascular permeability. Although designed to curtail tissue injury and facilitate repair, the inflammatory response sometimes results in further injury and organ dysfunction. Inflammatory chronic lung diseases, chronic obstructive pulmonary disease, acute lung injury, acute respiratory distress syndrome, and cystic fibrosis are syndromes of severe pulmonary dysfunction resulting from a massive inflammatory response and affecting millions of people worldwide. The histological hallmark of these chronic inflammatory lung diseases is the accumulation of neutrophils in the microvasculature of the lung. Neutrophils are crucial to the innate immune response, and their activation leads to the release of multiple cytotoxic products, including reactive oxygen species and proteases (serine, cysteine, and metalloproteases). The physiological balance between proteases and antiproteases is required for the maintenance of the lung’s connective tissue, and an imbalance in favor of proteases results in lung injury (Umeki et al., 1988Tetley, 1993). A number of studies in animal and cell culture models have demonstrated a contribution of HNE and related NSPs to the development of chronic inflammatory lung diseases. Available preclinical and clinical data suggest that inhibition of NSP in lung diseases suppresses or attenuates the contribution of NSP to pathogenesis (Chughtai and O’Riordan, 2004Voynow et al., 2008Quinn et al., 2010). HNE could also participate in fibrotic lung remodeling by playing a focused role in the conversion of latent transforming growth factor-β into its biologically active form (Chua and Laurent, 2006Lungarella et al., 2008).

Anti-Neutrophil Cytoplasmic Autoantibody-Associated Vasculitides

ANCA-associated vasculitides encompasses a variety of diseases characterized by inflammation of blood vessels and by the presence of autoantibodies directed against neutrophil constituents. These autoantibodies are known as ANCAs (Kallenberg et al., 2006). In Wegener granulomatosis (WG), antibodies are mostly directed against PR3. WG is a relatively uncommon chronic inflammatory disorder first described in 1931 by Heinz Karl Ernst Klinger as a variant of polyarteritis nodosa (Klinger, 1931). In 1936, the German pathologist Friedrich Wegener described the disease as a distinct pathological entity (Wegener, 19361939). WG is characterized by necrotizing granulomatous inflammation and vasculitis of small vessels and can affect any organ (Fauci and Wolff, 1973Sarraf and Sneller, 2005). The most common sites of involvement are the upper and lower respiratory tract and the kidneys. WG affects approximately 1 in 20,000 people; it can occur in persons of any age but most often affects those aged 40 to 60 years (Walton, 1958Cotch et al., 1996). Approximately 90% of patients have cold or sinusitis symptoms that fail to respond to the usual therapeutic measures and that last considerably longer than the usual upper respiratory tract infection. Lung involvement occurs in approximately 85% of the patients. Other symptoms include nasal membrane ulcerations and crusting, saddle-nose deformity, inflammation of the ear with hearing problems, inflammation of the eye with sight problems, and cough (with or without hemoptysis).

Hereditary Neutropenias

Neutropenia is a hematological disorder characterized by an abnormally low number of neutrophils (Horwitz et al., 2007). The normal neutrophil count fluctuates across human populations and within individual patients in response to infection but typically lies in the range of 1.5 to 5 × 109 cells/liter. Neutropenia is categorized as severe when the cell count falls below 0.5 × 109 cells/liter. Hence, patients with neutropenia are more susceptible to bacterial infections and, without prompt medical attention, the condition may become life-threatening. Common causes of neutropenia include cancer chemotherapy, drug reactions, autoimmune diseases, and hereditary disorders (Berliner et al., 2004Schwartzberg, 2006).

Papillon-Lefèvre Syndrome

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New Strategies for Fighting Neutrophil Serine Protease-Related Human Diseases

Administration of therapeutic inhibitors to control unwanted proteolysis at inflammation sites has been tested as a therapy for a variety of inflammatory and infectious lung diseases (Chughtai and O’Riordan, 2004). Depending on the size and chemical nature of the inhibitors, they may be administered orally, intravenously, or by an aerosol route. Whatever the mode of administration, the access of therapeutic inhibitors to active proteases is often hampered by physicochemical constraints in the extravascular space and/or by the partitioning of proteases between soluble and solid phases.

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Concluding Remarks

NSPs were first recognized as protein-degrading enzymes but have now proven to be multifunctional components participating in a variety of pathophysiological processes. Thus, they appear as potential therapeutic targets for drugs that inhibit their active site or impair activation from their precursor. Overall, the available preclinical and clinical data suggest that inhibition of NSPs using therapeutic inhibitors would suppress or attenuate deleterious effects of inflammatory diseases, including lung diseases. Depending on the size and chemical nature of inhibitors, those may be administered orally, intravenously, or by aerosolization. But the results obtained until now have not been fully convincing because of the poor knowledge of the biological function of each protease, their spatiotemporal regulation during the course of the disease, the physicochemical constraints associated with inhibitor administration, or the use of animal models in which NSP regulation and specificity differ from those in human. Two different and complementary approaches may help bypass these putative problems. One is to target active proteases by inhibitors at the inflammatory site in animal models in which lung anatomy and physiology are close to those in human to allow in vitro and in vivo assays of human-directed drugs/inhibitors. The other is to prevent neutrophil accumulation at inflammatory sites by impairing production of proteolytically active NSPs using an inhibitor of their maturation protease, DPPI. Preventing neutrophil accumulation at the inflammatory sites by therapeutic inhibition of DPPI represents an original and novel approach, the exploration of which has just started (Méthot et al., 2008). Thus pharmacological inactivation of DPPI in human neutrophils could well reduce membrane binding of PR3 and, as a consequence, neutrophil priming by pathogenic auto-antibodies in WG. In addition, it has been recognized that the intracellular level of NSPs depends on their correct intracellular trafficking. In the future, pharmacological targeting of molecules specifically involved in the correct intracellular trafficking of each NSP could possibly regulate their production and activity, a feature that could be exploited as a therapeutic strategy for inflammatory diseases.

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Flu Virus Transmission

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Mystery behind flu virus transmission solved

lu virus, influenza virus, soft palate

The epidemiological success of flu viruses relies on successful airborne transmission from person to person. But the viral properties governing the airborne transmission of flu viruses are complex. A new study reveals that the soft palate at the back of the roof of the mouth plays a key role in the flu viruses’ ability to transmit through air. Previous research had shown that airborne transmissibility is dependent on the viral surface hemagluttinin (HA) glycoprotein’s ability to bind to receptors on human respiratory cells. Some viral strains bind better to alfa 2-6 glycan receptors found primarily in humans and other mammals while others are better suited to bind alfa 2-3 glycan receptors found in birds.

In the current study, researchers made 4 mutations in the HA protein of the flu virus which made it better suited to bind the bird receptors than the human receptors. They then used this strain to infect ferrets that are often used as models of human influenza infection. In theory the mutated virus should not have spread but it traveled through the air just as well as the wild type virus strain. Upon sequencing the virus genome, the scientists found that it had undergone a genetic reversion that allowed its HA protein to bind to the bird as well as human receptors. This experiment validated that gain of binding to the human receptor is critical for aerosol transmission. On examining the different parts of the respiratory tract, scientists discovered that viruses that genetically reverted were most abundantly found in the soft palate. The researchers are next trying to figure out how this genetic reversion takes place and why particularly in the soft palate. They hypothesize that the viruses outcompete each other in the soft palate from which they can spread by packaging themselves into mucus droplets produced by cells in the soft palate.

From a pandemic point of view, this study enables the systematic evaluation of highly transmissible viruses. The findings published in Nature will enable scientists better understand how the flu virus develops airborne transmissibility while helping monitor strains that acquire the potential to cause Influenza outbreaks.

 

 

The soft palate is an important site of adaptation for transmissible influenza viruses (Sep 2015)

Lakdawala SS1, Jayaraman A2, Halpin RA3, Lamirande EW1, Shih AR1, Stockwell TB3, Lin X3, Simenauer A3, Hanson CT, et al.
Nature. 2015 Oct 1; 526(7571):122-5.    http://dx.doi.org:/10.1038/nature15379. Epub 2015 Sep 23.      http://www.ncbi.nlm.nih.gov/pubmed/26416728

Influenza A viruses pose a major public health threat by causing seasonal epidemics and sporadic pandemics. Their epidemiological success relies on airborne transmission from person to person; however, the viral properties governing airborne transmission of influenza A viruses are complex. Influenza A virus infection is mediated via binding of the viral haemagglutinin (HA) to terminally attached α2,3 or α2,6 sialic acids on cell surface glycoproteins. Human influenza A viruses preferentially bind α2,6-linked sialic acids whereas avian influenza A viruses bind α2,3-linked sialic acids on complex glycans on airway epithelial cells. Historically, influenza A viruses with preferential association with α2,3-linked sialic acids have not been transmitted efficiently by the airborne route in ferrets. Here we observe efficient airborne transmission of a 2009 pandemic H1N1 (H1N1pdm) virus (A/California/07/2009) engineered to preferentially bind α2,3-linked sialic acids. Airborne transmission was associated with rapid selection of virus with a change at a single HA site that conferred binding to long-chain α2,6-linked sialic acids, without loss of α2,3-linked sialic acid binding. The transmissible virus emerged in experimentally infected ferrets within 24 hours after infection and was remarkably enriched in the soft palate, where long-chain α2,6-linked sialic acids predominate on the nasopharyngeal surface. Notably, presence of long-chain α2,6-linked sialic acids is conserved in ferret, pig and human soft palate. Using a loss-of-function approach with this one virus, we demonstrate that the ferret soft palate, a tissue not normally sampled in animal models of influenza, rapidly selects for transmissible influenza A viruses with human receptor (α2,6-linked sialic acids) preference.

 

 

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