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Archive for the ‘Systemic Inflammatory Response Related Disorders’ Category


Image Source:Koch Institute

 

LIVE – OCTOBER 16 – DAY 1- Koch Institute Immune Engineering Symposium 2017, MIT, Kresge Auditorium

Koch Institute Immune Engineering Symposium 2017

http://kochinstituteevents.cvent.com/events/koch-institute-immune-engineering-symposium-2017/agenda-64e5d3f55b964ff2a0643bd320b8e60d.aspx

 

#IESYMPOSIUM

 

Image Source: Leaders in Pharmaceutical Business Intelligence (LPBI) Group

Aviva Lev-Ari, PhD, RN will be in attendance covering the event in REAL TIME

@pharma_BI

@AVIVA1950

#IESYMPOSIUM

@KOCHINSTITUTE

  • The Immune System, Stress Signaling, Infectious Diseases and Therapeutic Implications: VOLUME 2: Infectious Diseases and Therapeutics and VOLUME 3: The Immune System and Therapeutics (Series D: BioMedicine & Immunology) Kindle Edition – on Amazon.com since September 4, 2017

https://www.amazon.com/dp/B075CXHY1B

SYMPOSIUM SCHEDULE

OCTOBER 16 – DAY 1

7:00 – 8:15 Registration

8:15 – 8:30Introductory Remarks
Darrell Irvine | MIT, Koch Institute; HHMI

  • Stimulating the Immune system not only sustaining it for therapies

K. Dane Wittrup | MIT, Koch Institute

8:30 – 9:45Session I
Moderator: Douglas Lauffenburger | MIT, Biological Engineering and Koch Institute

Garry P. Nolan – Stanford University School of Medicine
Pathology from the Molecular Scale on Up

  • Intracellular molecules,
  • how molecules are organized to create tissue
  • Meaning from data Heterogeneity is an illusion: Order in Data ?? Cancer is heterogeneous, Cells in suspension – number of molecules
  • System-wide changes during Immune Response (IR)
  • Untreated, Ineffective therapy, effective therapy
  • Days 3-8 Tumor, Lymph node…
  • Variation is a Feature – not a bug: Effective therapy vs Ineffective – intercellular modules – virtual neighborhoods
  • ordered by connectivity: very high – CD4 T-cells, CD8 T-cels, moderate, not connected
  • Landmark nodes, Increase in responders
  • CODEX: Multiples epitome detection
  • Adaptable to proteins & mRNA
  • Rendering antibody staining via removal to neighborhood mapping
  • Human tonsil – 42 parameters: CD7, CD45, CD86,
  • Automated Annotations of tissues: F, P, V,
  • Normal BALBs
  • Marker expression defined by the niche: B220 vs CD79
  • Marker expression defines the niche
  • Learn neighborhoods and Trees
  • Improving Tissue Classification and staining – Ce3D – Tissue and Immune Cells in 3D
  • Molecular level cancer imaging
  • Proteomic Profiles: multi slice combine
  • Theory is formed to explain 3D nuclear images of cells – Composite Ion Image, DNA replication
  • Replication loci visualization on DNA backbone – nascent transcriptome – bar code of isotopes – 3D  600 slices
  • use CRISPR Cas9 for Epigenetics

Susan Napier Thomas – Georgia Institute of Technology
Transport Barriers in the Tumor Microenvironment: Drug Carrier Design for Therapeutic Delivery to Sentinel Lymph Nodes

  • Lymph Nodes important therapeutics target tissue
  • Lymphatic flow support passive and active antigen transport to lymph nodes
  • clearance of biomolecules and drug formulations: Interstitial transport barriers influence clearance: Arteriole to Venule –
  • Molecular tracers to analyze in vivo clearance mechanisms and vascular transport function
  • quantifying molecular clearance and biodistribution
  • Lymphatic transport increases tracer concentrations within dLN by orders of magnitude
  • Melanoma growth results in remodeled tumor vasculature
  • passive transport via lymphatic to dLN sustained in advanced tumors despite abrogated cell trafficking
  • Engineered biomaterial drug carriers to enhance sentinel lymph node-drug delivery: facilitated by exploiting lymphatic transport
  • TLR9 ligand therapeutic tumor in situ vaccination – Lymphatic-draining CpG-NP enhanced
  • Sturcutral and Cellular barriers: transport of particles is restriced by
  • Current drug delivery technology: lymph-node are undrugable
  • Multistage delivery platform to overcome barriers to lymphatic uptake and LN targeting
  • nano particles – OND – Oxanorbornade OND Time sensitive Linker synthesized large cargo – NP improve payload
  • OND release rate from nanoparticles changes retention in lymph nodes – Axilliary-Brachial delivery
  • Two-stage OND-NP delivery and release system dramatically – OND acumulate in lymphocyte
  •  delivers payload to previously undraggable lymphe tissue
  • improved drug bioactivity  – OND-NP eliminate LN LYMPHOMAS
  • Engineered Biomaterials

Douglas Lauffenburger – MIT, Biological Engineering and Koch Institute
Integrative Multi-Omic Analysis of Tissue Microenvironment in Inflammatory Pathophysiology

  • How to intervene, in predictive manner, in immunesystem-associated complex diseases
  • Understand cell communication beteen immune cells and other cells, i.e., tumor cells
  • Multi-Variate in Vivo – System Approach: Integrative Experiment & COmputational Analysis
  • Cell COmmunication & Signaling in CHronic inflammation – T-cell transfer model for colitis
  • COmparison of diffrential Regulation (Tcell transfer-elicited vs control) anong data types – relying solely on mRNA can be misleading
  • Diparities in differential responses to T cell transfer across data types yield insights concerning broader multi-organ interactions
  • T cell transfer can be ascertained and validated by successful experimental test
  • Cell COmmunication in Tumor MIcro-Environment — integration of single-cell transcriptomic data and protein interaction
  • Standard Cluster Elucidation – Classification of cell population on Full gene expression Profiles using Training sets: Decision Tree for Cell Classification
  • Wuantification of Pairwise Cell-Cell Receptor/Ligand Interactions: Cell type Pairs vs Receptor/Ligand Interaction
  • Pairwise Cell-Cell Receptor/Ligand Interactions
  • Calculate strength of interaction and its statistical significance
  • How the interaction is related to Phenotypic Behaviors – tumor growth rate, MDSC levels,
  • Correlated the Interactions translated to Phynotypic behavior for Therapeutic interventions (AXL via macrophage and fibroblasts)
  • Mouth model translation to Humans – New machine learning approach
  • Pathways, false negative, tumor negative expression
  • Molecular vs Phynotypical expression
  • Categories of inter-species translation
  • Semi-supervised Learning ALgorithms on Transcriptomic Data can ascertain Key Pathways/Processes in Human IBD from mapping mouse IBD

9:45 – 10:15 Break

10:15 – 11:30Session II
Moderator: Tyler Jacks | MIT, Koch Institute; HHMI

Tyler Jacks – MIT, Koch Institute; HHMI
Using Genetically Engineered Mouse Models to Probe Cancer-Immune Interactions

  • Utility of genetically-engineered mouse models of Cancer:
  1. Immune Response (IR),
  2. Tumor0immune microenvironment
  • Lung adenocarcinoma – KRAS mutation: Genetically-engineered model, applications: CRISPR, genetic interactions
  • Minimal Immune response to KP lung tumors: H&E, T cells (CD3), Bcells (B220) for Lenti-x 8 weeks
  • Exosome sequencing : Modeling loss-and gain-of-function mutations in Lung Cancer by CRISPR-Cas9 – germline – tolerance in mice, In vivo CRISPR-induced knockout of Msh2
  • Signatures of MMR deficient
  • Mutation burden and response to Immunotherapy (IT)
  • Programmed neoantigen expression – robust infiltration of T cells (evidence of IR)
  • Immunosuppression – T cell rendered ineffective
  • Lymphoid infiltration: Acute Treg depletion results in T cell infiltration — this depletion causes autoimmune response
  • Lung Treg from KP tumor-bearing mice have a distinct transcriptional heterogeneity through single cell mRNA sequencing
  • KP, FOXP3+, CD4
  • Treg from no existent to existance, Treg cells increase 20 fold =>>>  Treg activation and effectiveness
  • Single cells cluster by tissue and cell type: Treg, CD4+, CD8+, Tetramer-CD4+
  • ILrl1/II-33r unregulated in Treg at late time point
  • Treg-specific deletion of IL-33r results in fewer effector Tregs in Tumor-bearing lungs
  • CD8+ T cell infiltration
  • Tetramer-positive T cells cluster according to time point: All Lung CD8+ T cells
  • IR is not uniform functional differences – Clones show distinct transcriptional profiles
  • Different phynotypes Exhaustive signature
  • CRISPR-mediated modulation of CD8 T cell regulatory genes
  • Genetic dissection of the tumor-immune microenvironment
  • Single cell analysis, CRISPR – CRISPRa,i, – Drug development

Wendell Lim – University of California, San Francisco

Synthetic Immunology: Hacking Immune Cells

  • Precision Cell therapies – engineered by synthetic biology
  • Anti CD19 – drug approved
  • CAR-T cells still face major problems
  1. success limited to B cells cancers = blood vs solid tumors
  2. adverse effects
  3. OFF-TUMOR effects
  • Cell engineering for Cancer Therapy: User remote control (drug) – user control safety
  • Cell Engineering for TX
  1. new sensors – decision making for
  2. tumor recognition – safety,
  3. Cancer is a recognition issue
  • How do we avoid cross-reaction with bystader tissue (OFF TISSUE effect)
  • Tumor recognition: More receptors & integration
  • User Control
  • synthetic NOTCH receptors (different flavors of synNotch) – New Universal platform for cell-to -cell recognition: Target molecule: Extracellular antigen –>> transciptional instruction to cell
  • nextgen T cell: Engineer T cell recognition circuit that integrates multiple inputs: Two receptors – two antigen priming circuit
  • UNARMED: If antigen A THEN receptor A activates CAR
  • “Bystander” cell single antigen vs “tumor” drug antigen
  • Selective clearance of combinatorial tumor – Boulian formulation, canonical response
  • Cell response: Priming –>> Killing: Spatial & Temporal choreographed cell
  • CAR expression while removed from primed cells deminished
  • Solid Tumor: suppress cell microenvironment: Selected response vs non-natural response
  • Immune stimulator IR IL2, IL12, flagellin in the payload — Ourcome: Immune enhancement “vaccination”
  • Immune suppression –  block
  • Envision ideal situation: Unarmed cells
  • FUTURE: identify disease signatures and vulnerabilities – Precision Medicine using Synthetic Biology

Darrell Irvine – MIT, Koch Institute; HHMI
Engineering Enhanced Cancer Vaccines to Drive Combination Immunotherapies

  • Vaccine to drive IT
  • Intervening in the cancer-immunity cycle – Peptide Vaccines
  • poor physiology  of solute transport to tissue
  • endogenous albumin affinity – Lymphe Node dying
  • Designing Albumin-hitchhiking vaccines
  • Amphiphile-vaccine enhance uptake in lymph nodes in small and large animal models
  • soluble vaccine vs Amphiphile-vaccine
  • DIRECTING Vaccines to the Lymph nodes
  • amph-peptide antigen: Prime, booster, tetramer
  • albimin-mediated LN-targeting of both antigen and adjuvant maximizes IR
  • Immuno-supressed microenvironment will not be overcome by vaccines
  • Replacing adoptive T cell transfer with potent vaccine
  • exploiting albumin biology for mucosal vaccine delivery by amph-vaccines
  • Amph-peptides and -adjuvants show enhanced uptake/retention in lung tissue
  •  Enhancing adoptive T cell therapy: loss of T cell functionality, expand in vivo
  • boost in vivo enhanced adoptive T cell therapy
  • CAR-T cells: Enable T cells to target any cell surface protein
  • “Adaptor”-targeting CAR-T cells to deal with tumor cell heterogeneity
  • Lymph node-targeting Amph as CAR T booster vaccine: prining, production of cytokines
  • Boosting CAR T with amph-caccines: anti FITC CAR-T by DSPE=PEG-FITC coated
  • Targeting FITC to lymph node antigen presenting cells
  • Modulatory Macrophages
  • Amph-FITC expands FITC-CAR T cells in vivo – Adjuvant is needed
  • Hijacking albumin’s natural trafficking pathway

11:30 – 1:00  Lunch Break

1:00 – 2:15Session III
Moderator: Darrell Irvine | MIT, Koch Institute; HHMI

Nicholas P. Restifo – National Cancer Institute
Extracellular Potassium Regulates Epigenetics and Efficacy of Anti-Tumor T Cells

Why T cell do not kill Cancer cells?

  • co-inhibition
  • hostile tumor microenvironment

CAR T – does not treat solid tumors

Somatic mutation

  1. resistence of T cell based IT due to loss of function mutations
  2. Can other genes be lost?

CRISPR Cas9 – used to identify agents – GeCKOv2 Human library

Two cell-type (2CT) CRISPR assay system for genome-wide mutagenesis

  • work flow for genome-scale SRISPR mutagenesis profiling of genes essential for T cell mediate cytosis
  • sgRNA enrichment at the individual gene level by multiple methods:
  1. subunits of the MHC Class I complex
  2. CRISPR mutagenesis cut germline
  • Measutring the generalizability of resistance mechanism and mice in vivo validation
  • Validation of top gene candidates using libraries: MART-1
  • Checkpoint blockade: cells LOF causes tumor growth and immune escape
  • Weird genesL Large Ribisomal Subunit Proteins are nor all essential for cell survival
  • Bias in enrichment of 60S vs 40S
  • Novel elements of MHC class I antigen processing and presentation
  • Association of top CRISPR hits with response rates to IT – antiCTLA-4
  • CRISPR help identify novel regulators of T cells
  • Analyzed sgRNA – second rarest sgRNA for gene BIRC2 – encoded the Baculoviral Inhibitor
  • Drugs that inhibit BIRC2
  • How T cells can kill tumor cells more efficiently
  • p38kiaseas target for adoptive immunotherapy
  • FACS-based – Mapk14
  • Potent targets p38 – Blockade PD-1 or p38 ??
  • p38 signaling: Inhibition augments expansion and memory-marked human PBMC and TIL cells, N. P. Restifo
  • Tumor killing capacity of human CD19-specific, gene engineered T cells

Jennifer Elisseeff – Johns Hopkins University
The Adaptive Immune Response to Biomaterials and Tissue Repair

  • design scafolds, tissue-specific microenvironment
  • clinical translation of biosynthetic implants for soft tissue reconstruction
  • Local environment affects biomaterials: Epidermis, dermis
  • CD4+ T cells
  • Immune system – first reponders to materials: Natural or Synthetic
  • Biological (ECM) scaffolds to repair muscle injury
  • Which immune cells enter the WOUND?
  • ECM alters Macrophages: CD86, CD206
  • Adaptive system impact on Macrophages: CD86
  • mTOR signaling pathway M2 depend on Th2 Cells in regeneration of cell healing of surgical wounds
  • Systemic Immunological changes
  • Is the response antigen specific? – IL-4 expression in ILN,
  • Tissue reconstruction Clinical Trial: FDA ask to look at what cells infiltrate the scaffold
  • Trauma/biomaterial response – Injury induction of Senescence, anti apoptosis
  • Injury to skin or muscle
  • Is pro-regenerative environment (Th2/M2) pro-tumorigenic?
  • SYNTHETIC Materials for scafolds
  • Biomaterials and Immunology
  1. Immune response to bioscafolds
  2. environment modulate the immune system
  • Regenerative Immunetherapy

Marcela Maus – Massachusetts General Hospital

Engineering Better T Cells

  • Comparing CD19 CARs for Leukemia – anti-CD19- directed CAR T cells with r/r B-cell ALL – age 3-25 – FDA approved Novartis tisagenlecleucel – for pediatric r/r/ ALL
  • Phase II in diffuse large B cell lymphoma. Using T cells – increases prospects for cure
  • Vector retroviral – 30 day expression
  • measuring cytokines release syndrome: Common toxicity with CAR 19
  • neurological toxicity, B-cell aplagia
  • CART issues with heme malignancies
  1. decrease cytokine release
  2. avoid neurological toxicity – homing
  3. new targets address antigene escape variants – Resistance, CD19 is shaded, another target needed
  4. B Cell Maturation Antigen (BCMA) Target
  5. Bluebird Bio: Response duratio up to 54 weeks – Active dose cohort
  6. natural ligand CAR based on April
  7. activated in response to TACI+ target cells – APRIL-based CARs but not BCMA-CAR is able to kill TACI+ target cells
  • Hurdles for Solid Tumors
  1. Specific antigen targets
  2. tumor heterogeneity
  3. inhibitory microenvironment
  • CART in Glioblastoma
  1. rationale for EGFRvIII as therapeutic target
  2. Preclinical Studies & Phase 1: CAR t engraft, not as highly as CD19
  3. Upregulation of immunosuppression and Treg infiltrate in CART EGFRvIII as therapeutic target, Marcela Maus
  • What to do differently?

 

2:15 – 2:45 Break

2:45 – 4:00 Session IV
Moderator: Arup K. Chakraborty | MIT, IMES

Laura Walker – Adimab, LLC
Molecular Dissection of the Human Antibody Response to Respiratory Syncytial Virus

  • prophylactic antibody is available
  • Barriers for development of Vaccine
  • Prefusion and Postfusion RSV structures
  • Six major antigenic sites on RSV F
  • Blood samples Infants less 6 month of age and over 6 month: High abundance RSV F -specific memory B Cells are group  less 6 month

Arup K. Chakraborty – MIT, Institute for Medical Engineering & Science
How to Hit HIV Where it Hurts

  • antibody  – Model IN SILICO
  • Check affinity of each Ab for the Seaman panel of strain
  • Breadth of coverage
  • immmunize with cocktail of variant antigens
  • Mutations on Affinity Maturation: Molecular dynamics
  • bnAb eveolution: Hypothesis – mutations evolution make the antigen binding region more flexible,
  • Tested hypothesisi: carrying out affinity maturation – LOW GERMLINE AFFINITY TO CONSERVE RESIDUES IN 10,000 trials, acquire the mutation (generation 300)

William Schief – The Scripps Research Institute
HIV Vaccine Design Targeting the Human Naive B Cell Repertoire

  • HIV Envelope Trimer Glycan): the Target of neutralizing Antibodies (bnAbs)
  • Proof of principle for germline-targeting: VRC)!-class bnAbs
  • design of a nanoparticle
  • can germline -targeting innumogens prime low frequency precursors?
  • Day 14 day 42 vaccinate
  • Precursor frequency and affinity are limiting for germline center (GC) entry at day 8
  • Germline-targeting immunogens can elicit robust, high quality SHM under physiological conditions of precursor frequency and affinity at day 8, 16, 36
  • Germline-targeting immunogens can lead to production of memory B cells
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Systemic Inflammatory Diseases as Crohn’s disease, Rheumatoid Arthritis and Longer Psoriasis Duration May Mean Higher CVD Risk

Reporter: Aviva Lev-Ari, PhD, RN

Longer Psoriasis Duration May Mean Higher CVD Risk

Effect size ‘similar to that of smoking’

Several studies have shown that methotrexate, which has anti-inflammatory effects, reduces CV risk in patients with rheumatoid arthritis, suggesting that good anti-inflammatory control may be expected to reduce CV risk in patients with psoriasis.

Menter has worked closely with the senior author of the current study, Nehal Mehta, MD, of the University of Pennsylvania in Philadelphia, to identify cardiovascular issues in the psoriasis population. In one recent study, investigators found that the prevalence of moderate-to-severe coronary calcification was similar between patients with psoriasis and those with type 2 diabetes, and approximately five times greater than healthy controls.

Investigators found that moderate-to-severe psoriasis was a significantly stronger predictor of coronary calcification than type 2 diabetes, and the effect was independent of known CV and cardiometabolic risk factors.

 

SOURCE

https://www.medpagetoday.com/Dermatology/Psoriasis/68429?xid=nl_mpt_cardiodaily_2017-10-09&eun=g99985d0r

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Nutrition: Articles of Note @PharmaceuticalIntelligence.com

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

 

 

Nutrition and Wellbeing

 

Introduction 

Larry H. Bernstein, MD, FCAP

 

The chapters that follow are divided into three parts, but they are also a summary of 25 years of work with nutritional support research and involvement with nutritional support teams in Connecticut and New York, attendance and presentations at the American Association for Clinical Chemistry and the American Society for Parenteral and Enteral Nutrition, and long term collaborations with the surgeons Walter Pleban and Prof. Stanley Dudrick, and Prof. Yves Ingenbleek at the Laboratory of Nutrition, Department of Pharmacy, University Louis Pasteur, Strasbourg, Fr.   They are presented in the order: malnutrition in childhood; cancer, inflammation, and nutrition; and vegetarian diet and nutrition role in alternative medicines. These are not unrelated as they embrace the role of nutrition throughout the lifespan, the environmental impact of geo-ecological conditions on nutritional wellbeing and human development, and the impact of metabolism and metabolomics on the outcomes of human disease in relationship to severe inflammatory disorders, chronic disease, and cancer. Finally, the discussion emphasizes the negative impact of a vegan diet on long term health, and it reviews the importance of protein sources during phases of the life cycle.

Malnutrition in Childhood

 

Protein Energy Malnutrition and Early Child Development

Curator: Larry H. Bernstein, MD, FCAP

 

The Significant Burden of Childhood Malnutrition and Stunting

Curator: Larry H. Bernstein, MD, FCAP

 

Is Malnutrition the Cost of Civilization?

Curation: Larry H. Bernstein, MD, FCAP

 

Malnutrition in India, High Newborn Death Rate and Stunting of Children Age Under Five Years

Curator: Larry H Bernstein, MD, FCAP

 

Under Nutrition Early in Life may lead to Obesity

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Protein Malnutrition

Reporter and Curator: Dr. Sudipta Saha, Ph.D.

 

Cancer, Inflammation and Nutrition

 

A Second Look at the Transthyretin Nutrition Inflammatory Conundrum

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

 

Cancer and Nutrition

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

 

The history and creators of total parenteral nutrition

Curator: Larry H. Bernstein, MD, FCAP

 

Nutrition Plan

Curator: Larry H. Bernstein, MD, FCAP

 

Nutrition and Aging

Curator: Larry H Bernstein, MD, FCAP

 

Vegetarian Diet and Nutrition Role in Alternative Medicines

 

Plant-based Nutrition, Neutraceuticals and Alternative Medicine: Article Compilation the Journal PharmaceuticalIntelligence.com

Curator: Larry H. Bernstein, MD, FCAP

 

Metabolomics, Metabonomics and Functional Nutrition: the next step in nutritional metabolism and biotherapeutics

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

 

2014 Epidemiology and Prevention, Nutrition, Physical Activity and Metabolism Conference: San Francisco, Ca. Conference Dates: San Francisco, CA 3/18-21, 2014

Reporter: Aviva Lev-Ari, PhD, RN

 

Metabolomics: its Applications in Food and Nutrition Research

Reporter and Curator: Sudipta Saha, Ph.D.

 

Summary

Larry H. Bernstein, MD, FCAP 

The interest in human malnutrition became a major healthcare issue in the 1980’s with the publication of several seminal papers on hospital malnutrition. However, the basis for protein-energy malnutrition that focused on the distinction between kwashiorkor and marasmus was first identified in seminal papers by Ingenbleek and others:

Ingenbleek Y. La malnutrition protein-calorique chez l’enfant en bas age. Repercussions sur la function thyroidienne et les protein vectrices du serum. PhD Thesis. Acco Press. 1997. Univ Louvain.

Ingenbleek Y, Carpentier YA. A prognostic inflammatory and nutrition index scoring critically ill patients. Internat J Vit Nutr Res 1985; 55:91-101.

Ingenbleek Y, Young VR. Transthyretin (prealbumin) in health and disease. Nutritional implications. Ann Rev Nutr 1994; 14:495-533.

Ingenbleek Y, Hardillier E, Jung L. Subclinical protein malnutrition is a determinant of hyperhomocysteinemia. Nutrition 2002; 18:40-46.

It was these early papers that transfixed my attention, and drove me to establish early the transthyretin test by immunodiffusion and later by automated immunoassay at Bridgeport Hospital.

Among the important studies often referred to with respect to hospital malnutrition are:

  1. Hill GL, Blackett RL, Pickford I, Burkinshaw L, Young GA, Warren JV. Malnutrition in surgical patients: An unrecognised problem. Lancet.1977; 310:689–692. [PubMed]
  2. Bistrian BR, Blackburn GL, Vitale J, Cochrane D, Naylor J. Prevalence of malnutrition in general medical patients. JAMA. 1976; 235:1567–1570. [PubMed]
  3. Butterworth CE. The skeleton in the hospital closet. Nutrition Today.1974; 9:4–8.
  4. Buzby GP, Mullen JL, Matthews DC, Hobbs CL, Rosato EF. Prognostic nutritional index in gastrointestinal surgery. Am. J. Surg. 1980; 139:160–167.[PubMed]
  5. Dempsey DT, Mullen JL, Buzby GP. The link between nutritional status and clinical outcomes: can nutritional intervention modify it? Am. J. Clin. Nutr. 1988; 47:352–356. [PubMed]
  6. Detsky AS, Mclaughlin JR, Baker JP, Johnston N, Whittaker S, Mendleson RA, Jeejeebhoy KN. What is subjective global assessment of nutritional status? JPEN J Parenter Enteral Nutr. 1987; 11:8–13. [PubMed]
  7. Scrimshaw NS, DanGiovanni JP. Synergism of nutrition, infection and immunity, an overview. J. Nutr. 1997; 133:S316–S321.
  8. Chandra RK. Nutrition and the immune system: an introduction. Am. J. Clin. Nutr. 1997; 66:460S–463S. [PubMed]
  9. Hill GL. Body composition reserach: Implications for the practice of clinical nutrition. JPEN J. Parenter. Enteral Nutr. 1992; 16:197. [PubMed]
  10. Smith PE, Smith AE. High-quality nutritional interventions reduce costs.Healthc. Financ. Manage. 1997; 5:66–69. [PubMed]
  11. Gallagher-Allred CR, Voss AC, Finn SC, McCamish MA. Malnutrition and clinical outcomes. J. Am. Diet. Assoc. 1996; 96:361–366. [PubMed]
  12. Ferguson M. Uncovering the skeleton in the hoapital closet. What next? Aust. J. Nutr. Diet. 2001; 58:83–84.
  13. Waitzberg DL, Caiaffa WT, Correia MITD. Hospital malnutrition: The Brazilian national survey (IBRANUTRI): a study of 4000 patients. Nutrition.2001; 17:573–580. [PubMed]

The work on hospital (and nursing home) treatment of malnutrition described in this series led to established standards. It first requires identifying a patient at malnutrition risk to be identified via either screening or assessment. This needs to be done on admission, and it has been made mandatory by health care accrediting bodies. In order to achieve this, dietitians need to have the confidence and knowledge to detect malnutrition, which is ideally done using a validated assessment for patient outcomes and financial benefits to be realized.

There is a worldwide relationship between ecological conditions, religious practices, soil conditions, availability of animal food sources, and altitude and river flows has not received the attention that evidence requires. We have seen that the emphasis on the Hindu tradition of not eating beef or having dairy is possibly problematic in the Ganges River basin. There may be other meat sources, but it is questionable that sufficient animal protein is available for the large population. The additional problem of water pollution is an aggravating situation. However, it is this region that is one of the most affected by stunting of children. We have a situation here and in other poor societies where veganism is present, and there is also voluntary veganism in western societies. This is not a practice that leads to any beneficial effect, and it has been shown to lead to a hyperhomocystenemia with the associated risk of arterial vascular disease. For those who voluntarily choose veganism, this is an unexpected result.

Met is implicated in a large spectrum of metabolic and enzyme activities and participates in the conformation of a large number of molecules of survival importance. Due to the fact that plant products are relatively Met-deficient, vegan subjects are more exposed than omnivorous to develop hyperhomocysteinemia – related disorders. Dietary protein restriction may promote supranormal Hcy concentrations which appears as the dark side of adaptive attempts developed by the malnourished and/or stressed body to preserve Met homeostasis.  Summing up, we assume that the low TTR concentrations reported in the blood and CSF of AD or MID patients result in impairment of their normal scavenging capacity and in the excessive accumulation of Hcy in body fluids, hence causing direct harmful damage to the brain and cardiac vasculature.

The content of these discussions has also included nutrition and cancer. This is perhaps least well understood. Reasons for such an association may well include chronic exposure to radiation damage, or persistent focal chronic inflammatory conditions. These would result in a cirumferential and repeated cycle of injury and repair combined with an underlying hypoxia. I have already established a fundamental relationship between inflammation, the cytokine storm, the decreased hepatic synthesis of essential plasma proteins, such as, albumin, transferrin, retinol-binding protein, and transthyretin, and the surge of steroid hormones. This results in an imbalance in the protein and free protein equilibrium of essential vitamins, the retinoids, and other circulating ligands transported. This is discussed in the ‘nutrition-inflammatory conundrum”. As stated, whatever the nutritional status and the disease condition, the actual transthyretin (TTR) plasma level is determined by opposing influences between anabolic and catabolic alterations. Rising TTR values indicate that synthetic processes prevail over tissue breakdown with a nitrogen balance (NB) turning positive as a result of efficient nutritional support and / or anti-inflammatory therapy. Declining TTR values are associated with an effect of maladjusted dietetic management and / or further worsening of the morbid condition.

Inflammatory disorders of any cause are initiated by activated leukocytes releasing a shower of cytokines working as autocrine, paracrine and endocrine molecules. Cytokines regulate the overproduction of acute-phase proteins (APPs), notably that of CRP, 1-acid glycoprotein (AGP), fibrinogen, haptoglobin, 1-antitrypsin and antichymotrypsin. APPs contribute in several ways to defense and repair mechanisms, being characterized by proper kinetic and functional properties. Interleukin-6 (IL-6) is regarded as a key mediator governing both the acute and chronic inflammatory processes, as documented by data recorded on burn, sepsis and AIDS patients. IL-6-NF possesses a high degree of homology with C/EBP-NF1 and competes for the same DNA response element of the IL-6 gene. IL-6-NF is not expressed under normal circumstances, explaining why APP concentrations are kept at baseline levels. In stressful conditions, IL-6-NF causes a dramatic surge in APP values with a concomitant suppressed synthesis of TTR.

Inadequate nutritional management, multiple injuries, occurrence of severe sepsis and metabolic complications result in persistent proteolysis and subnormal TTR concentrations. The evolutionary patterns of urinary N output and of TTR thus appear as mirror images of each other, which supports the view that TTR might well reflect the depletion of TBN in both acute and chronic disease processes. Even in the most complex stressful conditions, the synthesis of visceral proteins is submitted to opposing anabolic or catabolic influences yielding ultimately TTR as an end-product reflecting the prevailing tendency. Whatever the nutritional and/or inflammatory causal factors, the actual TTR plasma level and its course in process of time indicates the exhaustion or restoration of the body N resources, hence its likely (in)ability to assume defense and repair mechanisms.

In westernized societies, elderly persons constitute a growing population group. A substantial proportion of them may develop a syndrome of frailty characterized by weight loss, clumsy gait, impaired memory and sensorial aptitudes, poor physical, mental and social activities, depressive trends. Hallmarks of frailty combine progressive depletion of both structural and metabolic N compartments. Sarcopenia and limitation of muscle strength are naturally involutive events of normal ageing which may nevertheless be accelerated by cytokine-induced underlying inflammatory disorders. Depletion of visceral resources is substantiated by the shrinking of FFM and its partial replacement by FM, mainly in abdominal organs, and by the down-regulation of indices of growth and protein status. Due to reduced tissue reserves and diminished efficiency of immune and repair mechanisms, any stressful condition affecting old age may trigger more severe clinical impact whereas healing processes require longer duration with erratical setbacks. As a result, protein malnutrition is a common finding in most elderly patients with significantly increased morbidity and mortality rates.

TTR has proved to be a useful marker of nutritional alterations with prognostic implications in large bowel cancer, bronchopulmonary carcinoid tumor, ovarian carcinoma and squamous carcinoma of bladder. Many oncologists have observed a rapid TTR fall 2 or 3 months prior to the patient’s death. In cancer patients submitted to surgical intervention, most postoperative complications occurred in subjects with preoperative TTR  180 mg/L. Two independent studies came to the same conclusion that a TTR threshold of 100 mg/L is indicative of extremely weak survival likelihood and that these terminally ill patients better deserve palliative care rather than aggressive therapeutic strategies.

Thyroid hormones and retinoids indeed function in concert through the mediation of common heterodimeric motifs bound to DNA response elements. The data also imply that the provision of thyroid molecules within the CSF works as a relatively stable secretory process, poorly sensitive to extracerebral influences as opposed to the delivery of retinoid molecules whose plasma concentrations are highly dependent on nutritional and/or inflammatory alterations. This last statement is documented by mice experiments and clinical investigations showing that the level of TTR production by the liver operates as a limiting factor for retinol transport. Defective TTR synthesis determines the occurrence of secondary hyporetinolemia which nevertheless results from entirely different kinetic mechanisms in the two quoted studies.

Points to consider:

Protein energy malnutrition has an unlikely causal relationship to carcinogenesis. Perhaps the opposite is true. However, cancer has a relationship to protein energy malnutrition without any doubt. PEM is the consequence of cachexia, whether caused by dietary insufficiency, inflammatory or cancer.

Protein energy malnutrition leads to hyperhomocysteinemia, and by that means, the relationship of dietary insufficiency of methionine has a relationship to heart disease. This is the significant link between veganism and cardiovascular disease, whether voluntary or by unavailability of adequate source.

The last portion of these chapters deals with metabolomics and functional nutrition. This is an emerging and important area of academic interest. There is a significant relationship between these emerging studies and pathways to understanding natural products medicinal chemistry.

 

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Selye’s Riddle solved

Larry H. Bernstein, mD, FCAP, Curator

LPBI

 

Mathematicians Solve 78-year-old Mystery

Mathematicians developed a solution to Selye's riddle which has puzzled scientists for almost 80 years.
Mathematicians developed a solution to Selye’s riddle which has puzzled scientists for almost 80 years.

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted. In 1938, Hans Selye introduced “adaptation energy” and found strong experimental arguments in favor of this hypothesis. However, this term has caused much debate because, as it cannot be measured as a physical quantity, adaptation energy is not strictly energy.

 

Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death

Alexander N. Gorbana, , Tatiana A. Tyukinaa, Elena V. Smirnovab, Lyudmila I. Pokidyshevab,

Highlights

•   We formalize Selye׳s ideas about adaptation energy and dynamics of adaptation.
•   A hierarchy of dynamic models of adaptation is developed.
•   Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation.
•   The optimal distribution of resources for neutralization of harmful factors is studied.
•   The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted.       

In previous research, it was suggested that adaptation of an animal to different factors looks like spending of one resource, and that the animal dies when this resource is exhausted.

In 1938, Selye proposed the notion of adaptation energy and published ‘Experimental evidence supporting the conception of adaptation energy.’ Adaptation of an animal to different factors appears as the spending of one resource. Adaptation energy is a hypothetical extensive quantity spent for adaptation. This term causes much debate when one takes it literally, as a physical quantity, i.e. a sort of energy. The controversial points of view impede the systematic use of the notion of adaptation energy despite experimental evidence. Nevertheless, the response to many harmful factors often has general non-specific form and we suggest that the mechanisms of physiological adaptation admit a very general and nonspecific description.

We aim to demonstrate that Selye׳s adaptation energy is the cornerstone of the top-down approach to modelling of non-specific adaptation processes. We analyze Selye׳s axioms of adaptation energy together with Goldstone׳s modifications and propose a series of models for interpretation of these axioms. Adaptation energy is considered as an internal coordinate on the ‘dominant path’ in the model of adaptation. The phenomena of ‘oscillating death’ and ‘oscillating remission’ are predicted on the base of the dynamical models of adaptation. Natural selection plays a key role in the evolution of mechanisms of physiological adaptation. We use the fitness optimization approach to study of the distribution of resources for neutralization of harmful factors, during adaptation to a multifactor environment, and analyze the optimal strategies for different systems of factors.

In this work, an international team of researchers, led by Professor Alexander N. Gorban from the University of Leicester, have developed a solution to Selye’s riddle, which has puzzled scientists for almost 80 years.

Alexander N. Gorban, Professor of Applied Mathematics in the Department of Mathematics at the University of Leicester, said: “Nobody can measure adaptation energy directly, indeed, but it can be understood by its place already in simple models. In this work, we develop a hierarchy of top-down models following Selye’s findings and further developments. We trust Selye’s intuition and experiments and use the notion of adaptation energy as a cornerstone in a system of models. We provide a ‘thermodynamic-like’ theory of organism resilience that, just like classical thermodynamics, allows for economics metaphors, such as cost and bankruptcy and, more importantly, is largely independent of a detailed mechanistic explanation of what is ‘going on underneath’.”

Adaptation energy is considered as an internal coordinate on the “dominant path” in the model of adaptation. The phenomena of “oscillating death” and “oscillating remission,” which have been observed in clinic for a long time, are predicted on the basis of the dynamical models of adaptation. The models, based on Selye’s idea of adaptation energy, demonstrate that the oscillating remission and oscillating death do not need exogenous reasons. The developed theory of adaptation to various factors gives the instrument for the early anticipation of crises.

Professor Alessandro Giuliani from Istituto Superiore di Sanità in Rome commented on the work, saying: “Gorban and his colleagues dare to make science adopting the thermodynamics style: they look for powerful principles endowed with predictive ability in the real world before knowing the microscopic details. This is, in my opinion, the only possible way out from the actual repeatability crisis of mainstream biology, where a fantastic knowledge of the details totally fails to predict anything outside the test tube.1

Citation: Alexander N. Gorban, Tatiana A. Tyukina, Elena V. Smirnova, Lyudmila I. Pokidysheva. Evolution of adaptation mechanisms: Adaptation energy, stress, and oscillating death. Journal of Theoretical Biology, 2016; DOI:10.1016/j.jtbi.2015.12.017. Voosen P. (2015) Amid a Sea of False Findings NIH tries Reform, The Chronicle of Higher Education.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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High blood pressure can damage the retina’s blood vessels and limit the retina’s function. It can also put pressure on the optic nerve.

Sourced through Scoop.it from: www.healthline.com

See on Scoop.itCardiovascular Disease: PHARMACO-THERAPY

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Cytokines in IBD

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

 

Revised 2/14/2016

 

The following presentation explores the application of antisense oligonucleotide agents that modulate the activity of Il17 and Il23 signaling activity in the cell.

IL 17 & 23

United States Patent 9,238,042
Schnell ,   et al. January 19, 2016

Antisense modulation of interleukins 17 and 23 signaling
Provided are antisense oligonucleotides and other agents that target and modulate IL-17 and/or IL-23 signaling activity in a cell, compositions that comprise the same, and methods of use thereof. Also provided are animal models for identifying agents that modulate 17 and/or IL-23 signaling activity.

Abes et al., “Arginine-rich cell penetrating peptides: Design, structure-activity, and applications to alter pre-mRNA splicing by steric-block oligonucleotides,” J Pept Sci 14: 455-460, 2008. cited by applicant .
Abes et al., “Delivery of steric block morpholino oligomers by (R-X-R).sub.4 peptides: structure-activity studies,” Nucleic Acids Research 36(20): 6343-6354, Sep. 16, 2008. cited by applicant .
Abes et al., “Vectorization of morpholino oligomers by the (R-Ahx-R).sub.4 peptide allows efficient splicing correction in the absence of endosomolytic agents,” Journal of Controlled Release 116: 304-313, 2006. cited by applicant .
Lebleu et al., “Cell penetrating peptide conjugates of steric block oligonucleotides,” Advanced Drug Delivery Reviews 60: 517-529, 2008. cited by applicant .
Marshall et al., “Arginine-rich cell-penetrating peptides facilitate delivery of antisense oligomers into murine leukocytes and alter pre-mRNA splicing,” Journal of Immunological Methods 325: 114-126, 2007. cited by applicant .
Moulton et al., “Cellular Uptake of Antisense Morpholino Oligomers Conjugated to Arginine-Rich Peptides,” Bioconjugate Chem 15: 290-299, 2004. cited by applicant .
Summerton et al., “Morpholino Antisense Oligomers: Design, Preparation, and Properties,” Antisense & Nucleic Acid Drug Development 7: 187-195, 1997. cited by applicant .
Wright et al., “The Human IL-17F/IL-17A Heterodimeric Cytokine Signals through the IL-17RA/IL-17RC Receptor Complex,” The Journal of Immunology 181: 2799-2805, 2008. cited by applicant .

 

Immunity. 2015 Oct 20;43(4):739-50. doi: 10.1016/j.immuni.2015.08.019. Epub 2015 Sep 29.

Differential Roles for Interleukin-23 and Interleukin-17 in Intestinal Immunoregulation.

Maxwell JR1Zhang Y1Brown WA1Smith CL1Byrne FR2Fiorino M2Stevens E3Bigler J4Davis JA5Rottman JB6Budelsky AL1Symons A1Towne JE7.

 

Interleukin-23 (IL-23) and IL-17 are cytokines currently being targeted in clinical trials. Although inhibition of both of these cytokines is effective for treating psoriasis, IL-12 and IL-23 p40 inhibition attenuates Crohn’s disease, whereas IL-17A or IL-17 receptor A (IL-17RA) inhibition exacerbates Crohn’s disease. This dichotomy between IL-23 and IL-17 was effectively modeled in the multidrug resistance-1a-ablated (Abcb1a(-/-)) mouse model of colitis. IL-23 inhibition attenuated disease by decreasing colonic inflammation while enhancing regulatory T (Treg) cell accumulation. Exacerbation of colitis by IL-17A or IL-17RA inhibition was associated with severe weakening of the intestinal epithelial barrier, culminating in increased colonic inflammation and accelerated mortality. These data show that IL-17A acts on intestinal epithelium to promote barrier function and provide insight into mechanisms underlying exacerbation of Crohn’s disease when IL-17A or IL-17RA is inhibited.

 

Immunity. 2015 Oct 20;43(4):727-38. doi: 10.1016/j.immuni.2015.09.003. Epub 2015 Sep 29.

Interleukin-23-Independent IL-17 Production Regulates Intestinal Epithelial Permeability.

Lee JS1Tato CM1Joyce-Shaikh B1Gulan F2Cayatte C1Chen Y1Blumenschein WM1Judo M1Ayanoglu G1McClanahan TK1Li X2Cua DJ3.

Whether interleukin-17A (IL-17A) has pathogenic and/or protective roles in the gut mucosa is controversial and few studies have analyzed specific cell populations for protective functions within the inflamed colonic tissue. Here we have provided evidence for IL-17A-dependent regulation of the tight junction protein occludin during epithelial injury that limits excessive permeability and maintains barrier integrity. Analysis of epithelial cells showed that in the absence of signaling via the IL-17 receptor adaptor protein Act-1, the protective effect of IL-17A was abrogated and inflammation was enhanced. We have demonstrated that after acute intestinal injury, IL-23R(+) γδ T cells in the colonic lamina propria were the primary producers of early, gut-protective IL-17A, and this production of IL-17A was IL-23 independent, leaving protective IL-17 intact in the absence of IL-23. These results suggest that IL-17-producing γδ T cells are important for the maintenance and protection of epithelial barriers in the intestinal mucosa.

 

Gastroenterology. 2008 Apr;134(4):1038-48. doi: 10.1053/j.gastro.2008.01.041. Epub 2008 Jan 17.

Regulation of gut inflammation and th17 cell response by interleukin-21.

Fina D1Sarra MFantini MCRizzo ACaruso RCaprioli FStolfi CCardolini IDottori MBoirivant MPallone FMacdonald TT,Monteleone G.

Interleukin (IL)-21, a T-cell-derived cytokine, is overproduced in inflammatory bowel diseases (IBD), but its role in the pathogenesis of gut inflammation remains unknown. We here examined whether IL-21 is necessary for the initiation and progress of experimental colitis and whether it regulates specific pathways of inflammation.

Both dextran sulfate sodium colitis and trinitrobenzene sulfonic acid-relapsing colitis were induced in wild-type and IL-21-deficient mice. CD4(+)CD25(-) T cells from wild-type and IL-21-deficient mice were differentiated in T helper cell (Th)17-polarizing conditions, with or without IL-21 or an antagonistic IL-21R/Fc. We also examined whether blockade of IL-21 by anti-IL-21 antibody reduced IL-17 in cultures of IBD lamina propria CD3(+) T lymphocytes. Cytokines were evaluated by real-time polymerase chain reaction and/or enzyme-linked immunosorbent assay.

High IL-21 was seen in wild-type mice with dextran sulfate sodium- and trinitrobenzene sulfonic acid-relapsing colitis. IL-21-deficient mice were largely protected against both colitides and were unable to up-regulate Th17-associated molecules during gut inflammation, thus suggesting a role for IL-21 in controlling Th17 cell responses. Indeed, naïve T cells from IL-21-deficient mice failed to differentiate into Th17 cells. Treatment of developing Th17 cells from wild-type mice with IL-21R/Fc reduced IL-17 production. Moreover, in the presence of transforming growth factor-beta1, exogenous IL-21 substituted for IL-6 in driving IL-17 induction. Neutralization of IL-21 reduced IL-17 secretion by IBD lamina propria lymphocytes.

These results indicate that IL-21 is a critical regulator of inflammation and Th17 cell responses in the gut.

 

Neurochem Res. 2010 Jun;35(6):940-6. doi: 10.1007/s11064-009-0091-9. Epub 2009 Nov 14.

Synergy of IL-23 and Th17 cytokines: new light on inflammatory bowel disease.

Shen W1Durum SK.

Inflammatory bowel diseases (IBDs), including Crohn’s disease and ulcerative colitis, involve an interplay between host genetics and environmental factors including intestinal microbiota. Animal models of IBD have indicated that chronic inflammation can result from over-production of inflammatory responses or deficiencies in key negative regulatory pathways. Recent research advances in both T-helper 1 (Th1) and T-helper 17 (Th17) effect responses have offered new insights on the induction and regulation of mucosal immunity which is linked to the development of IBD. Th17 cytokines, such as IL-17 and IL-22, in combination with IL-23, play crucial roles in intestinal protection and homeostasis. IL-23 is expressed in gut mucosa and tends to orchestrate T-cell-independent pathways of intestinal inflammation as well as T cell dependent pathways mediated by cytokines produced by Th1 and Th17 cells. Th17 cells, generally found to be proinflammatory, have specific functions in host defense against infection by recruiting neutrophils and macrophages to infected tissues. Here we will review emerging data on those cytokines and their related regulatory networks that appear to govern the complex development of chronic intestinal inflammation; we will focus on how IL-23 and Th17 cytokines act coordinately to influence the balance between tolerance and immunity in the intestine.

 

Eur J Immunol. 2007 Oct;37(10):2680-2.

IL-23 and IL-17 have a multi-faceted largely negative role in fungal infection.

Cooper AM1.

The role of IL-23 and IL-17 in the response to fungal infection has been the focus of recent reports. In this issue of the European Journal of Immunology there is an article that reports an important role for IL-23 and IL-17 in limiting fungal control, promoting neutrophillic inflammation and regulating the killing activity of neutrophils. In the fungal model it appears that IL-23 and IL-17 are counter-productive for protection.

 

IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases

MWL TengEP BowmanJJ McElwee,…, AM Cooper & DJ Cua
Nature Med July 2016; 21(7):719–729
http://www.nature.com/nm/journal/v21/n7/full/nm.3895.html

The cytokine interleukin-12 (IL-12) was thought to have a central role in T cell–mediated responses in inflammation for more than a decade after it was first identified. Discovery of the cytokine IL-23, which shares a common p40 subunit with IL-12, prompted efforts to clarify the relative contribution of these two cytokines in immune regulation. Ustekinumab, a therapeutic agent targeting both cytokines, was recently approved to treat psoriasis and psoriatic arthritis, and related agents are in clinical testing for a variety of inflammatory disorders. Here we discuss the therapeutic rationale for targeting these cytokines, the unintended consequences for host defense and tumor surveillance and potential ways in which these therapies can be applied to treat additional immune disorders.

IL-12 and IL-23 are produced by inflammatory myeloid cells and influence the development of TH1 cell and IL-17–producing T helper (TH17) cell responses, respectively. The rationale for developing IL-12 antagonists was prompted by observations that mice deficient in IL-12p40 are resistant to experimentally induced autoimmune conditions, including paralysis induction after immunization with brain-derived antigens, arthritis inflammation after immunization with a joint antigen, ocular disease after immunization with a retinal antigen and multiple gut disease models. This suggested that IL-12 could be an effective therapeutic target1, 2, 3, 4, 5. Studies of neutralizing antibodies to IL-12p40 in multiple mouse strains seemed to confirm the importance of therapeutically targeting IL-12 to decrease immune pathology6, 7. However, mice deficient in the other IL-12 subunit, IL-12p35, showed no protection or showed exacerbated disease in some models1, 2. Following the recognition, in 2000, that IL-12 and IL-23 share the IL-12p40 subunit but only IL-23 uses the p19 subunit8, it was determined that mice deficient in IL-23 but not IL-12 are resistant to experimental immune-mediated disease1, 2, 3, 4, 5. By 2000, the first anti–IL-12p40 therapy targeting IL-12—subsequently recognized to target IL-23 as well—was under evaluation in patients with Crohn’s disease9. Currently, at least 10 therapeutic agents targeting IL-12, IL-23 or IL-17A are being tested in the clinic for more than 17 immune-mediated diseases (Table 1). Here we discuss the preclinical and clinical data validating these therapeutic strategies and the potential consequences of targeting these immune pathways.

Figure 1: Schematic representation of IL-12 and IL-23, and their receptors and downstream signaling pathways

Schematic representation of IL-12 and IL-23, and their receptors and downstream signaling pathways.

IL-12 is made up of the IL-12/23p40 and IL-12p35 subunits, and IL-23 comprises IL-23p19 and IL-12/23p40. IL-12 signals through the IL-12Rβ1 and IL-12Rβ2 subunits, and IL-23 signals through IL-12Rβ1 and IL-23R. IL-12 stimulation of JAK2…

Figure 4: Schematic representation of the mechanisms by which IL-23 indirectly or directly promotes tumorigenesis, growth and metastasis.

 

Schematic representation of the mechanisms by which IL-23 indirectly or directly promotes tumorigenesis, growth and metastasis.

IL-23 is produced by myeloid cells in response to exogenous or endogenous signals such as damage-associated molecular patterns (DAMPs), pathogen-associated molecular patterns (PAMPs) or tumor-secreted factors such as prostaglandin E2 (PGE2). IL-23 can act directly on tumor cells to promote their transformation, proliferation and/or metastasis. In mice, IL-23R is expressed on several innate and adaptive immune cell types, which are found in various proportions in tumors. Stimulation of IL-23R on these immune cells leads to production of cytokines such as IL-17 and/or IL-22, which can have direct proliferative effects on stromal or tumor cells. IL-17 and/or IL-22 also elicit a range of factors from various hematopoietic and nonhematopoietic cells, which can have direct effects on tumor proliferation and metastasis or induce the production of additional inflammatory cytokines, chemokines and mediators such as IL-6, IL-8, matrix metallopeptidases (MMPs) and vascular endothelial growth factor (VEGF), all of which can contribute to the generation of a tumor microenvironment in which CD8 and NK cell effector functions are suppressed. DC, dendritic cell; Mφ, macrophage.

IL-12 and IL-23 cytokines: from discovery to targeted therapies for immune-mediated inflammatory diseases

Michele W L TengEdward P Bowman,…., & Daniel J Cua

Nature Medicine 21, 719–729 (2015) doi:10.1038/nm.3895

Familial genetic studies, large-scale genome-wide association studies (GWAS) and next-generation sequencing approaches have highlighted therapeutic indications where IL-23 may contribute to inflammatory disease risk. For example, a psoriasis GWAS reported a protective association for the single-nucleotide polymorphism (SNP) rs11209026 (c.1142G>A; p.Arg381Gln) residing in the IL-23R protein-coding sequence with a modest odds ratio (OR) of 0.67 (P = 7 × 10−7)25. A GWAS in ileal Crohn’s disease also showed an association with rs11209026 (ref. 26), with the minor glutamine variant protective for Crohn’s disease risk with an OR of 0.26–0.45. The protective association of this variant (and other SNPs in linkage disequilibrium with it) in Crohn’s disease was also shown in ulcerative colitis27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 41. The largest meta-analysis of all inflammatory bowel disease GWAS to date (~40,000 cases and ~40,000 controls) indicates that carriage of the glutamine variant gives a modest reduction for disease risk (OR = 0.43, P = 8 × 10−161) (ref. 36). The rs11209026 allele is also associated with protection from ankylosing spondylitis42, 43, psoriatic arthritis44, 45, 46, 47 and graft-versus-host disease48, 49, 50, 51. Notably, this IL-23R variant has not been reliably associated with other common inflammatory diseases such as rheumatoid arthritis, type 1 diabetes or multiple sclerosis in GWAS powered to detect protective effects similar to those seen in Crohn’s disease and psoriasis52, 53, 54. Although these GWAS findings are compelling, it is important to keep in mind the limitations of such studies; these common loci tend to additively explain only a small proportion of the narrow-sense heritability of disease risk55.

Treatment of inflammatory disease with any immunosuppressive agent carries the theoretical risk of impaired host defense responses to pathogens and/or decreased tumor surveillance. Emerging data from human loss-of-function variants and mouse preclinical studies have informed the relative risks of targeting IL-12 and/or IL-23.

The theoretical risk of compromised immunity are of particular concern owing to immune defects discovered in patients with autosomal recessive deficiencies in IL-12/23p40 and IL-12Rβ1 (refs.105,106,107) (Fig. 3). Both deficiencies are genetic etiologies of Mendelian susceptibility to mycobacterial disease (MSMD) (genes involved in MSMD are listed at http://www.biobase-international.com), a rare condition in otherwise healthy patients who have a selective infection predisposition to weakly virulent mycobacteria such as Bacillus Calmette-Guerin (BCG) vaccines, nontuberculous environmental mycobacteria and virulent Mycobacterium tuberculosis (OMIM209950)108, 109, 110, 111, 112, 113. Half of patients with MSMD also have nontyphoidal and, to a lesser extent, typhoidal Salmonella infection.

Owing to the roles of IL-12 and/or IL-23 in host defense and tumor surveillance, particular attention has been focused on infectious disease–related adverse events after anti–IL-12/23p40 treatment in humans. Meta-analysis of briakinumab’s phase 2, phase 3 and open-label extension (OLE) psoriasis databases in 2010 identified 14 cases of candidiasis (including mucocutaneous esophageal and oral candidiasis); no reports of mycobacteria or Salmonella were noted. With regard to the roles of IL-12 and/or IL-23 in tumorigenesis, malignancies were observed at a rate of 1.7 events per 100 patient years (PY), and were cancers commonly seen in the general population.

Concluding remarks

Clinical testing of IL-23 and IL-17A inhibitors have confirmed the initial hypotheses that IL-23–TH17 pathways are indispensable in promoting immune-mediated diseases, and agents targeting these pathways work particularly well in specific disease settings. However, it is not clear why IL-17A and IL-17RA antagonists work well for psoriasis but exacerbate Crohn’s disease95, 96. It appears that different classes of inhibitor targeting IL-23 and IL-17 pathways may have unique nonoverlapping attributes in different clinical settings. Investigators are still learning where the overlap occurs and what the differences are between targeting IL-23 and targeting other related pathway cytokines. For example, mouse innate lymphoid cells constitutively produce gut protective IL-17A and IL-22 in an IL-23–independent manner. The constitutive IL-17A and IL-22 expression levels generated in response to commensal gut organisms seem to be crucial for maintenance of epithelial barrier function185 and tight junction formation (D.J.C., unpublished observation). However, high levels of IL-17A and IL-22 induced by IL-23 can be pathogenic during tissue injury responses in the presence of additional inflammatory cytokines such as IL-1, IL-6, GM-CSF and TNF. Therefore, targeting IL-23 via anti–IL-23p19 will partially suppress IL-17A and reduce inflammation, whereas anti–IL-17A therapy will neutralize all protective IL-17A.

The immune system’s function is to maintain balance in the face of insult from external pathogens and accumulation of genetic errors leading to cancer. Disruption of this balance toward immune-exuberance can lead to autoimmunity and immunopathology after infection, whereas inadequate immunity can allow pathogen evasion and breakdown in tumor surveillance. The common thread that connects autoimmunity, infection and cancer is inflammation, and the drivers of inflammation are intercellular messengers that enable cross-talk between immune cells and surrounding stromal tissues. We have underscored the importance of innate cell-produced IL-12 and IL-23 as intermediaries that act on T cells and NK cells to promote inflammation and highlighted that IL-12 and IL-23 have overlapping cellular immune functions. Whereas IL-12 is important in driving STAT1- and STAT4-mediated immune surveillance against specific intracellular pathogens and immunity against neoplasm, IL-23 promotes STAT3-dependent antifungal immunity and drives ‘sterile’ wound-healing responses in psoriatic lesions, which have a gene signature similar to that of many autoinflammatory conditions186, 187. Strikingly, this signature of uncontrolled wound-healing response is also observed in many cancers188. Although there is insufficient clinical data to determine the long-term safety of IL-23 inhibitors, preclinical models suggest that IL-23 paradoxically promotes tumorigenesis by enhancing skin and mucosal tissue inflammation associated with immune evasion mechanisms.

As the roles of IL-12 and IL-23 were elucidated in preclinical models, there was concern that inhibiting these factors could lead to profound immune suppression. Is it better to target factors capable of regulating a broad range of immune function and may leave patients unprotected against pathogens and cancers or to aim for a restricted pathway that may have limited efficacy for treatment of immune disorders? Although the efficacy and safety profiles of IL-12/23p40, IL-23p19 and IL-17A and IL-17RA therapies become clearer with each clinical trial, the decisions to progress these targets were made many years in advance, on the basis of limited data. Animal studies are important for elucidating the cellular and molecular mechanisms, but clinical testing is required to determine whether a specific disease mechanism also operates in humans. Immunological research is at an inflection point, where the basic concepts of molecular and cellular immunology are being translated into effective therapies for diseases that were considered intractable only a few years ago. Despite the challenges, efforts to translate basic disease mechanisms to the clinic are finally paying off. Although much work remains to be done, the fundamental question of which immune target will benefit which patient population is now being clarified. We optimistically await the answers that will change the lives of patients with serious immune-mediate conditions.

 

Cytokines in Crohn’s colitis.

Sher ME1D’Angelo AJStein TABailey BBurns GWise L.
Am J Surg. 1995 Jan; 169(1):133-6.

Increasing evidence points to a pathologic role for cytokines in Crohn’s colitis. Levels of cytokines are increased in diseased segments of colon in Crohn’s colitis, but no one has studied the concentration of cytokines in clinically and histologically nondiseased segments.

Mucosal biopsies were obtained from 7 patients with active segmental Crohn’s colitis and from 7 controls without inflammatory bowel disease. The concentration of Interleukin (IL)-1 beta, IL-2, IL-6, and IL-8 in patients and controls were determined using enzyme linked immunosorbent assay and compared. Histologic sections were also performed to confirm diseased and nondiseased segments of colon.

The concentrations of IL-1 beta, IL-6, and IL-8 were significantly higher in the involved segments of colon (10.3 +/- 4.1, 3.7 +/- 1.0, 34.4 +/- 6.9 picograms [pg] per mg) when compared to controls (1.8 +/- 0.5, 1.1 +/- 0.5, 5.3 +/- 1.0 pg/mg). The concentrations of IL-1 beta, IL-2, and IL-8 (8.5 +/- 2.9, 5.3 +/- 1.2, 26.3 +/- 8.8 pg/mg) in normal appearing segments of colon of patients with Crohn’s colitis were also significantly higher than in controls, whose IL-2 level was 2.0 +/- 0.5 pg/mg. IL-1 beta and IL-8 were significantly more concentrated in both the involved and uninvolved colonic segments of patients with Crohn’s colitis compared to controls. IL-2 and IL-6 were also more concentrated in Crohn’s patients than in controls, but not significantly. The differences in interleukin concentrations between involved and uninvolved segments of colon in patients with segmental Crohn’s colitis were not significant.

Although Crohn’s colitis is often a segmental disease, concentrations of IL-1 beta and IL-8 are increased throughout the entire colon. These observations reinforce the hypothesis that Crohn’s colitis involves the whole colon even when this is not apparent clinically or histologically.

 

Clin Exp Immunol. 2000 May;120(2):241-6.

Increased production of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 by inflamed mucosa in inflammatory bowel disease.

Louis E1Ribbens CGodon AFranchimont DDe Groote DHardy NBoniver JBelaiche JMalaise M.

Inflammatory bowel diseases (IBD) are characterized by a sustained inflammatory cascade that gives rise to the release of mediators capable of degrading and modifying bowel wall structure. Our aims were (i) to measure the production of matrix metalloproteinase-3 (MMP-3), and its tissue inhibitor, tissue inhibitor of metalloproteinase-1 (TIMP-1), by inflamed and uninflamed colonic mucosa in IBD, and (ii) to correlate their production with that of proinflammatory cytokines and the anti-inflammatory cytokine, IL-10. Thirty-eight patients with IBD, including 25 with Crohn’s disease and 13 with ulcerative colitis, were included. Ten controls were also studied. Biopsies were taken from inflamed and uninflamed regions and inflammation was graded both macroscopically and histologically. Organ cultures were performed for 18 h. Tumour necrosis factor-alpha (TNF-alpha), IL-6, IL-1beta, IL-10, MMP-3 and TIMP-1 concentrations were measured using specific immunoassays. The production of both MMP-3 and the TIMP-1 were either undetectable or below the sensitivity of our immunoassay in the vast majority of uninflamed samples either from controls or from those with Crohn’s disease or ulcerative colitis. In inflamed mucosa, the production of these mediators increased significantly both in Crohn’s disease (P < 0.01 and 0.001, respectively) and ulcerative colitis (P < 0.001 and 0.001, respectively). Mediator production in both cases was significantly correlated with the production of proinflammatory cytokines and IL-10, as well as with the degree of macroscopic and microscopic inflammation. Inflamed mucosa of both Crohn’s disease and ulcerative colitis show increased production of both MMP-3 and its tissue inhibitor, which correlates very well with production of IL-1beta, IL-6, TNF-alpha and IL-10.

 

Gut. 1997 Apr;40(4):475-80.

In vitro effects of oxpentifylline on inflammatory cytokine release in patients with inflammatory bowel disease.

Reimund JM1Dumont SMuller CDKenney JSKedinger MBaumann RPoindron PDuclos B.

Inflammatory cytokines, including tumour necrosis factor-alpha (TNF-alpha) and interleukin (IL)-1 beta, have been implicated as primary mediators of intestinal inflammation in inflammatory bowel disease.

To investigate the in vitro effects of oxpentifylline (pentoxifylline; PTX; a phosphodiesterase inhibitor) on inflammatory cytokine production (1) by peripheral mononuclear cells (PBMCs) and (2) by inflamed intestinal mucosa cultures from patients with Crohn’s disease and patients with ulcerative colitis.

PBMCs and mucosal biopsy specimens were cultured for 24 hours in the absence or presence of PTX (up to 100 micrograms/ml), and the secretion of TNF-alpha, IL-1 beta, IL-6, and IL-8 determined by enzyme linked immunosorbent assays (ELISAs).

PTX inhibited the release of TNF-alpha by PBMCs from patients with inflammatory bowel disease and the secretion of TNF-alpha and IL-1 beta by organ cultures of inflamed mucosa from the same patients. Secretion of TNF-alpha by PBMCs was inhibited by about 50% at a PTX concentration of 25 micrograms/ml (IC50). PTX was equally potent in cultures from controls, patients with Crohn’s disease, and those with ulcerative colitis. The concentrations of IL-6 and IL-8 were not significantly modified in PBMCs, but IL-6 increased slightly in organ culture supernatants.

PTX or more potent related compounds may represent a new family of cytokine inhibitors, potentially interesting for treatment of inflammatory bowel disease.

 

Inflamm Bowel Dis. 2015 May;21(5):973-84. doi: 10.1097/MIB.0000000000000353.

Neutralizing IL-23 is superior to blocking IL-17 in suppressing intestinal inflammation in a spontaneous murine colitis model.

Wang R1Hasnain SZTong HDas IChe-Hao Chen AOancea IProctor MFlorin THEri RDMcGuckin MA.

IL-23/T(H)17 inflammatory responses are regarded as central to the pathogenesis of inflammatory bowel disease, but clinically IL-17A antibodies have shown low efficacy and increased infections in Crohn’s disease. Hence, we decided to closely examine the role of the IL-23/T(H)17 axis in 3 models of colitis.

IL-17A(-/-) and IL-17Ra(-/-) T cells were transferred into Rag1 and RaW mice to assess the role of IL-17A-IL-17Ra signaling in T cells during colitis. In Winnie mice with spontaneous colitis due to an epithelial defect, we studied the progression of colitis in the absence of IL-17A and the efficacy of neutralizing antibodies against the IL-17A or IL-23p19 cytokines.

In transfer colitis models, IL-17A-deficient T cells failed to ameliorate disease, and IL-17Ra-deficient T cells were more colitogenic than wild-type T cells. In Winnie mice with an epithelial defect and spontaneous T(H)17-dominated inflammation, genetic deficiency of IL-17A did not suppress initiation of colitis but limited colitis progression. Furthermore, inhibition of IL-17A by monoclonal antibodies did not reduce colitis severity. In contrast, neutralizing IL-23 using an anti-p19 antibody significantly alleviated both emerging and established colitis, downregulating T(H)17 proinflammatory cytokine expression and diminishing neutrophil infiltration.

Our results support clinical studies showing that IL-17 neutralization is not therapeutic but that targeting IL-23 suppresses intestinal inflammation. Effects of IL-23 distinct from its effects on maturation of IL-17A-producing lymphocytes may underlie the protection from inflammatory bowel disease conveyed by hypomorphic IL-23 receptor polymorphisms and contribute to the efficacy of IL-23 neutralizing antibodies in inflammatory bowel disease.

  1. Luger, D. et al. Either a Th17 or a Th1 effector response can drive autoimmunity: conditions of disease induction affect dominant effector category. J. Exp. Med. 205, 799810 (2008).
  2. Yen, D. et al. IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 13101316 (2006).
  3. Uhlig, H.H. et al. Differential activity of IL-12 and IL-23 in mucosal and systemic innate immune pathology. Immunity 25, 309318 (2006).

 

IL-17A signaling in colonic epithelial cells inhibits pro-inflammatory cytokine production by enhancing the activity of ERK and PI3K.

Guo X1Jiang X2Xiao Y3Zhou T2Guo Y4Wang R2Zhao Z2Xiao H2Hou C2Ma L3Lin Y2Lang X2Feng J2Chen G2Shen B2Han G2Li Y2.
PLoS One. 2014 Feb 25;9(2):e89714. doi: 10.1371/journal.pone.0089714. eCollection 2014.

Our previous data suggested that IL-17A contributes to the inhibition of Th1 cell function in the gut. However, the underlying mechanisms remain unclear. Here we demonstrate that IL-17A signaling in colonic epithelial cells (CECs) increases TNF-α-induced PI3K-AKT and ERK phosphorylation and inhibits TNF-α induced expression of IL-12P35 and of a Th1 cell chemokine, CXCL11 at mRNA level. In a co-culture system using HT-29 cells and PBMCs, IL-17A inhibited TNF-α-induced IL-12P35 expression by HT-29 cells and led to decreased expression of IFN-γ and T-bet by PBMCs. Finally, adoptive transfer of CECs from mice with Crohn’s Disease (CD) led to an enhanced Th1 cell response and exacerbated colitis in CD mouse recipients. The pathogenic effect of CECs derived from CD mice was reversed by co-administration of recombinant IL-17A. Our data demonstrate a new IL-17A-mediated regulatory mechanism in CD. A better understanding of this pathway might shed new light on the pathogenesis of CD.

 

J Immunol. 2008 Aug 15;181(4):2799-805.

The human IL-17F/IL-17A heterodimeric cytokine signals through the IL-17RA/IL-17RC receptor complex.

Wright JF1Bennett FLi BBrooks JLuxenberg DPWhitters MJTomkinson KNFitz LJWolfman NMCollins MDunussi-Joannopoulos KChatterjee-Kishore MCarreno BM.

IL-17A and IL-17F, produced by the Th17 CD4(+) T cell lineage, have been linked to a variety of inflammatory and autoimmune conditions. We recently reported that activated human CD4(+) T cells produce not only IL-17A and IL-17F homodimers but also an IL-17F/IL-17A heterodimeric cytokine. All three cytokines can induce chemokine secretion from bronchial epithelial cells, albeit with different potencies. In this study, we used small interfering RNA and Abs to IL-17RA and IL-17RC to demonstrate that heterodimeric IL-17F/IL-17A cytokine activity is dependent on the IL-17RA/IL-17RC receptor complex. Interestingly, surface plasmon resonance studies indicate that the three cytokines bind to IL-17RC with comparable affinities, whereas they bind to IL-17RA with different affinities. Thus, we evaluated the effect of the soluble receptors on cytokine activity and we find that soluble receptors exhibit preferential cytokine blockade. IL-17A activity is inhibited by IL-17RA, IL-17F is inhibited by IL-17RC, and a combination of soluble IL-17RA/IL-17RC receptors is required for inhibition of the IL-17F/IL-17A activity. Altogether, these results indicate that human IL-17F/IL-17A cytokine can bind and signal through the same receptor complex as human IL-17F and IL-17A. However, the distinct affinities of the receptor components for IL-17A, IL-17F, and IL-17F/IL-17A heterodimer can be exploited to differentially affect the activity of these cytokines.

 

Am J Surg. 1995 Jan;169(1):133-6.

Cytokines in Crohn’s colitis.

Sher ME1D’Angelo AJStein TABailey BBurns GWise L.

Increasing evidence points to a pathologic role for cytokines in Crohn’s colitis. Levels of cytokines are increased in diseased segments of colon in Crohn’s colitis, but no one has studied the concentration of cytokines in clinically and histologically nondiseased segments.

Mucosal biopsies were obtained from 7 patients with active segmental Crohn’s colitis and from 7 controls without inflammatory bowel disease. The concentration of Interleukin (IL)-1 beta, IL-2, IL-6, and IL-8 in patients and controls were determined using enzyme linked immunosorbent assay and compared. Histologic sections were also performed to confirm diseased and nondiseased segments of colon.

The concentrations of IL-1 beta, IL-6, and IL-8 were significantly higher in the involved segments of colon (10.3 +/- 4.1, 3.7 +/- 1.0, 34.4 +/- 6.9 picograms [pg] per mg) when compared to controls (1.8 +/- 0.5, 1.1 +/- 0.5, 5.3 +/- 1.0 pg/mg). The concentrations of IL-1 beta, IL-2, and IL-8 (8.5 +/- 2.9, 5.3 +/- 1.2, 26.3 +/- 8.8 pg/mg) in normal appearing segments of colon of patients with Crohn’s colitis were also significantly higher than in controls, whose IL-2 level was 2.0 +/- 0.5 pg/mg. IL-1 beta and IL-8 were significantly more concentrated in both the involved and uninvolved colonic segments of patients with Crohn’s colitis compared to controls. IL-2 and IL-6 were also more concentrated in Crohn’s patients than in controls, but not significantly. The differences in interleukin concentrations between involved and uninvolved segments of colon in patients with segmental Crohn’s colitis were not significant.

Although Crohn’s colitis is often a segmental disease, concentrations of IL-1 beta and IL-8 are increased throughout the entire colon. These observations reinforce the hypothesis that Crohn’s colitis involves the whole colon even when this is not apparent clinically or histologically.

 

Protein Pept Lett. 2015;22(7):570-8.

An Overview of Interleukin-17A and Interleukin-17 Receptor A Structure, Interaction and Signaling.

Krstic JObradovic HKukolj TMojsilovic SOkic-Dordevic IBugarski DSantibanez JF1.

Interleukin-17A (IL-17A) and its receptor (IL-17RA) are prototype members of IL-17 ligand/receptor family firstly identified in CD4+ T cells, which comprises six ligands (IL-17A to IL- 17F) and five receptors (IL-17RA to IL-17RE). IL-17A is predominantly secreted by T helper 17 (Th17) cells, and plays important roles in the development of autoimmune and inflammatory diseases. IL-17RA is widely expressed, and forms a complex with IL-17RC. Binding of IL-17A to this receptor complex triggers the activation of several intracellular signaling pathways. In this review, we aimed to summarize literature data about molecular features of IL-17A and IL-17RA from gene to mature protein. We are also providing insight into regulatory mechanisms, protein structural conformation, including ligand-receptor interaction, and an overview of signaling pathways. Our aim was to compile the data on molecular characteristics of IL-17A and IL-17RA which may help in the understanding of their functions in health and disease.

 

Gut. 2014 Dec;63(12):1902-12. doi: 10.1136/gutjnl-2013-305632. Epub 2014 Feb 17.

Involvement of interleukin-17A-induced expression of heat shock protein 47 in intestinal fibrosis in Crohn’s disease.

Honzawa Y1Nakase H1Shiokawa M1Yoshino T1Imaeda H2Matsuura M1Kodama Y1Ikeuchi H3Andoh A2Sakai Y4Nagata K5Chiba T1.

Intestinal fibrosis is a clinically important issue in Crohn’s disease (CD). Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone involved in fibrotic diseases. The molecular mechanisms of HSP47 induction in intestinal fibrosis related to CD, however, remain unclear. Here we investigated the role of interleukin (IL)-17A-induced HSP47 expression in intestinal fibrosis in CD.

Expressions of HSP47 and IL-17A in the intestinal tissues of patients with IBD were determined. HSP47 and collagen I expressions were assessed in intestinal subepithelial myofibroblasts (ISEMFs) isolated from patients with IBD and CCD-18Co cells treated with IL-17A. We examined the role of HSP47 in IL-17A-induced collagen I expression by administration of short hairpin RNA (shRNA) to HSP47 and investigated signalling pathways of IL-17A-induced HSP47 expression using specific inhibitors in CCD-18Co cells.

Gene expressions of HSP47 and IL-17A were significantly elevated in the intestinal tissues of patients with active CD. Immunohistochemistry revealed HSP47 was expressed in α-smooth muscle actin (α-SMA)-positive cells and the number of HSP47-positive cells was significantly increased in the intestinal tissues of patients with active CD. IL-17A enhanced HSP47 and collagen I expressions in ISEMFs and CCD-18Co cells. Knockdown of HSP47 in these cells resulted in the inhibition of IL-17A-induced collagen I expression, and analysis of IL-17A signalling pathways revealed the involvement of c-Jun N-terminal kinase in IL-17A-induced HSP47 expression.

IL-17A-induced HSP47 expression is involved in collagen I expression in ISEMFs, which might contribute to intestinal fibrosis in CD.

 

 

Biochem Biophys Res Commun. 2011 Jan 14;404(2):599-604. doi: 10.1016/j.bbrc.2010.12.006. Epub 2010 Dec 6.

Role of heat shock protein 47 in intestinal fibrosis of experimental colitis.

Kitamura H1Yamamoto SNakase HMatsuura MHonzawa YMatsumura KTakeda YUza NNagata KChiba T.

Intestinal fibrosis is a clinically important issue of inflammatory bowel disease (IBD). It is unclear whether or not heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, plays a critical role in intestinal fibrosis. The aim of this study is to investigate the role of HSP47 in intestinal fibrosis of murine colitis.

HSP47 expression and localization were evaluated in interleukin-10 knockout (IL-10KO) and wild-type (WT, C57BL/6) mice by immunohistochemistry. Expression of HSP47 and transforming growth factor-β1 (TGF-β1) in colonic tissue was measured. In vitro studies were conducted in NIH/3T3 cells and primary culture of myofibroblasts separated from colonic tissue of IL-10KO (PMF KO) and WT mice (PMF WT) with stimulation of several cytokines. We evaluated the inhibitory effect of administration of small interfering RNA (siRNA) targeting HSP47 on intestinal fibrosis in IL-10KO mice in vivo.

Immunohistochemistry revealed HSP47 positive cells were observed in the mesenchymal and submucosal area of both WT and IL-10 KO mice. Gene expressions of HSP47 and TGF-β1 were significantly higher in IL-10KO mice than in WT mice and correlated with the severity of inflammation. In vitro experiments with NIH3T3 cells, TGF-β1 only induced HSP47 gene expression. There was a significant difference of HSP47 gene expression between PMF KO and PMF WT. Administration of siRNA targeting HSP47 remarkably reduced collagen deposition in colonic tissue of IL-10KO mice.

Our results indicate that HSP47 plays an essential role in intestinal fibrosis of IL-10KO mice, and may be a potential target for intestinal fibrosis associated with IBD.

 

Kidney Int. 2003 Sep;64(3):887-96.

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats.

Nishino T1Miyazaki MAbe KFurusu AMishima YHarada TOzono YKoji TKohno S.

Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG).

HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry.

In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels.

Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.

 

Trends Mol Med. 2007 Feb;13(2):45-53. Epub 2006 Dec 13.

The collagen-specific molecular chaperone HSP47: is there a role in fibrosis?

Taguchi T1Razzaque MS.

Heat shock protein 47 (HSP47) is a collagen-specific molecular chaperone that is required for molecular maturation of various types of collagens. Recent studies have shown a close association between increased expression of HSP47 and excessive accumulation of collagens in scar tissues of various human and experimental fibrotic diseases. It is presumed that the increased levels of HSP47 in fibrotic diseases assist in excessive assembly and intracellular processing of procollagen molecules and, thereby, contribute to the formation of fibrotic lesions. Studies have also shown that suppression of HSP47 expression can reduce accumulation of collagens to delay the progression of fibrotic diseases in experimental animal models. Because HSP47 is a specific chaperone for collagen synthesis, it provides a selective target to manipulate collagen production, a phenomenon that might have enormous clinical impact in controlling a wide range of fibrotic diseases. Here, we outline the fibrogenic role of HSP47 and discuss the potential usefulness of HSP47 as an anti-fibrotic therapeutic target.

 

Arthritis Rheum. 2013 May;65(5):1347-56. doi: 10.1002/art.37860.

Interleukin-17A+ cell counts are increased in systemic sclerosis skin and their number is inversely correlated with the extent of skin involvement.

Truchetet ME1Brembilla NCMontanari ELonati PRaschi EZeni SFontao LMeroni PLChizzolini C.

Levels of interleukin-17A (IL-17A) have been found to be increased in synovial fluid from individuals with systemic sclerosis (SSc). This study was undertaken to investigate whether IL-17A-producing cells are present in affected SSc skin, and whether IL-17A exerts a role in the transdifferentiation of myofibroblasts.

Skin biopsy samples were obtained from the involved skin of 8 SSc patients and from 8 healthy control donors undergoing plastic surgery. Immunohistochemistry and multicolor immunofluorescence techniques were used to identify and quantify the cell subsets in vivo, including IL-17A+, IL-4+, CD3+, tryptase-positive, α-smooth muscle actin (α-SMA)-positive, myeloperoxidase-positive, and CD1a+ cells. Dermal fibroblast cell lines were generated from all skin biopsy samples, and quantitative polymerase chain reaction, Western blotting, and solid-phase assays were used to quantify α-SMA, type I collagen, and matrix metalloproteinase 1 (MMP-1) production by the cultured fibroblasts.

IL-17A+ cells were significantly more numerous in SSc skin than in healthy control skin (P = 0.0019) and were observed to be present in both the superficial and deep dermis. Involvement of both T cells and tryptase-positive mast cells in the production of IL-17A was observed. Fibroblasts positive for α-SMA were found adjacent to IL-17A+ cells, but not IL-4+ cells. However, IL-17A did not induce α-SMA expression in cultured fibroblasts. In the presence of IL-17A, the α-SMA expression induced in response to transforming growth factor β was decreased, while MMP-1 production was directly enhanced. Furthermore, the frequency of IL-17A+ cells was higher in the skin of SSc patients with greater severity of skin fibrosis (lower global skin thickness score).

IL-17A+ cells belonging to the innate and adaptive immune system are numerous in SSc skin. IL-17A participates in inflammation while exerting an inhibitory activity on myofibroblast transdifferentiation. These findings are consistent with the notion that IL-17A has a direct negative-regulatory role in the development of dermal fibrosis in humans.

 

Gut. 2014 Dec;63(12):1902-12. doi: 10.1136/gutjnl-2013-305632. Epub 2014 Feb 17.

Involvement of interleukin-17A-induced expression of heat shock protein 47 in intestinal fibrosis in Crohn’s disease.

Honzawa Y1Nakase H1Shiokawa M1Yoshino T1Imaeda H2Matsuura M1Kodama Y1Ikeuchi H3Andoh A2Sakai Y4Nagata K5Chiba T1.

Intestinal fibrosis is a clinically important issue in Crohn’s disease (CD). Heat shock protein (HSP) 47 is a collagen-specific molecular chaperone involved in fibrotic diseases. The molecular mechanisms of HSP47 induction in intestinal fibrosis related to CD, however, remain unclear. Here we investigated the role of interleukin (IL)-17A-induced HSP47 expression in intestinal fibrosis in CD.

Expressions of HSP47 and IL-17A in the intestinal tissues of patients with IBD were determined. HSP47 and collagen I expressions were assessed in intestinal subepithelial myofibroblasts (ISEMFs) isolated from patients with IBD and CCD-18Co cells treated with IL-17A. We examined the role of HSP47 in IL-17A-induced collagen I expression by administration of short hairpin RNA (shRNA) to HSP47 and investigated signalling pathways of IL-17A-induced HSP47 expression using specific inhibitors in CCD-18Co cells.

Gene expressions of HSP47 and IL-17A were significantly elevated in the intestinal tissues of patients with active CD. Immunohistochemistry revealed HSP47 was expressed in α-smooth muscle actin (α-SMA)-positive cells and the number of HSP47-positive cells was significantly increased in the intestinal tissues of patients with active CD. IL-17A enhanced HSP47 and collagen I expressions in ISEMFs and CCD-18Co cells. Knockdown of HSP47 in these cells resulted in the inhibition of IL-17A-induced collagen I expression, and analysis of IL-17A signalling pathways revealed the involvement of c-Jun N-terminal kinase in IL-17A-induced HSP47 expression.

IL-17A-induced HSP47 expression is involved in collagen I expression in ISEMFs, which might contribute to intestinal fibrosis in CD.

 

Kidney Int. 2003 Sep;64(3):887-96.

Antisense oligonucleotides against collagen-binding stress protein HSP47 suppress peritoneal fibrosis in rats.

Nishino T1Miyazaki MAbe KFurusu AMishima YHarada TOzono YKoji TKohno S.

Peritoneal fibrosis is a serious complication in patients on continuous ambulatory peritoneal dialysis (CAPD), but the molecular mechanism of this process remains unclear. Heat shock protein 47 (HSP47), a collagen-specific molecular chaperone, is essential for biosynthesis and secretion of collagen molecules, and is expressed in the tissue of human peritoneal fibrosis. In the present study, we examined the effect of HSP47 antisense oligonucleotides (ODNs) on the development of experimental peritoneal fibrosis induced by daily intraperitoneal injections of chlorhexidine gluconate (CG).

HSP47 antisense or sense ODNs were injected simultaneously with CG from day 14, after injections of CG alone. Peritoneal tissue was dissected out 28 days after CG injection. The expression patterns of HSP47, type I and type III collagen, alpha-smooth muscle actin (alpha-SMA), as a marker of myofibroblasts, ED-1 (as a marker of macrophages), and factor VIII were examined by immunohistochemistry.

In rats treated with CG alone, the submesothelial collagenous compact zone was thickened, where the expression levels of HSP47, type I and type III collagen and alpha-SMA were increased. Marked macrophage infiltration was also noted and the number of vessels positively stained for factor VIII increased in the CG-treated group. Treatment with antisense ODNs, but not sense ODNs, abrogated CG-induced changes in the expression of HSP47, type I and III collagen, alpha-SMA, and the number of infiltrating macrophages and vessels.

Our results indicate the involvement of HSP47 in the progression of peritoneal fibrosis and that inhibition of HSP47 expression might merit further clinical investigation for the treatment of peritoneal fibrosis in CAPD patients.

 

Zhong Nan Da Xue Xue Bao Yi Xue Ban. 2007 Aug;32(4):650-5.

[Effect of heat shock protein 47 on the expression of collagen I induced by TGF-beta(1) in hepatic stellate cell-T6 cells].

[Article in Chinese]

Li Y1Wu WJiang YFWang KK.

To determine the effect of heat shock protein 47 (HSP47) on the expression of collagen I induced by transforming growth factor beta(1) (TGF-beta(1)) in hepatic stellate cell-T6 (HSC-T6) cells.

We used 1 ng/mL and 10 ng/mL recombinant human TGF-beta(1) to stimulate the cultured HSC-T6 cells. Heat shock response (HSR) and antisense oligonucleotides of HSP47 were used to induce and block the expression of HSP47, respectively. The expressions of HSP47 and collagen I were detected by Western blot and the cell viability was observed by MTT assay.

Both HSP47 and collagen I were expressed in normal HSC-T6 cells. Collagen I and HSP47 expression could be induced by both 1 ng/mL and 10 ng/mL TGF-beta(1) and collagen I was expressed the most after the treatment with 10 ng/mL TGF-beta(1). Although HSR could not affect the synthesis of collagen I as it induced the HSP47 expression, HSR could promote the expression of collagen I induced by TGF-beta(1). With no effect on the cell viability, antisense oligonucleotides could significantly inhibit HSR-mediated HSP47 expression and TGF-beta(1)-induced collagen I synthesis.

Over-expression of HSP47 enhances TGF-beta(1)-induced expression of collagen I in HSC-T6 cells, and HSP47 may play important roles in the process of hepatic fibrosis

 

Fibrogenesis Tissue Repair. 2013 Jul 8;6(1):13. doi: 10.1186/1755-1536-6-13.

The role of interleukin 17 in Crohn’s disease-associated intestinal fibrosis.

Biancheri P1Pender SLAmmoscato FGiuffrida PSampietro GArdizzone SGhanbari ACurciarello RPasini AMonteleone G,Corazza GRMacdonald TTDi Sabatino A.

Interleukin (IL)-17A and IL-17E (also known as IL-25) have been implicated in fibrosis in various tissues. However, the role of these cytokines in the development of intestinal strictures in Crohn’s disease (CD) has not been explored. We investigated the levels of IL-17A and IL-17E and their receptors in CD strictured and non-strictured gut, and the effects of IL-17A and IL-17E on CD myofibroblasts.

IL-17A was significantly overexpressed in strictured compared with non-strictured CD tissues, whereas no significant difference was found in the expression of IL-17E or IL-17A and IL-17E receptors (IL-17RC and IL-17RB, respectively) in strictured and non-strictured CD areas. Strictured CD explants released significantly higher amounts of IL-17A than non-strictured explants, whereas no difference was found as for IL-17E, IL-6, or tumor necrosis factor-α production. IL-17A, but not IL-17E, significantly inhibited myofibroblast migration, and also significantly upregulated matrix metalloproteinase (MMP)-3, MMP-12, tissue inhibitor of metalloproteinase-1 and collagen production by myofibroblasts from strictured CD tissues.

Our results suggest that IL-17A, but not IL-17E, is pro-fibrotic in CD. Further studies are needed to clarify whether the therapeutic blockade of IL-17A through the anti-IL-17A monoclonal antibody secukinumab is able to counteract the fibrogenic process in CD.

 

Int J Colorectal Dis. 2013 Jul;28(7):915-24. doi: 10.1007/s00384-012-1632-2. Epub 2012 Dec 28.

Role of N-acetylcysteine and GSH redox system on total and active MMP-2 in intestinal myofibroblasts of Crohn’s disease patients.

Romagnoli C1Marcucci TPicariello LTonelli FVincenzini MTIantomasi T.

Intestinal subepithelial myofibroblasts (ISEMFs)(1) are the predominant source of matrix metalloproteinase-2 (MMP-2) in gut, and a decrease in glutathione/oxidized glutathione (GSH/GSSG) ratio, intracellular redox state index, occurs in the ISEMFs of patients with Crohn’s disease (CD). The aim of this study is to demonstrate a relationship between MMP-2 secretion and activation and changes of GSH/GSSG ratio in ISEMFs stimulated or not with tumor necrosis factor alpha (TNFα).

ISEMFs were isolated from ill and healthy colon mucosa of patients with active CD. Buthionine sulfoximine, GSH synthesis inhibitor, and N-acetylcysteine (NAC), precursor of GSH synthesis, were used to modulate GSH/GSSG ratio. GSH and GSSG were measured by HPLC and MMP-2 by ELISA Kit.

In cells, stimulated or not with TNFα, a significant increase in MMP-2 secretion and activation, related to increased oxidative stress, due to low GSH/GSSG ratio, was detected. NAC treatment, increasing this ratio, reduced MMP-2 secretion and exhibited a direct effect on the secreted MMP-2 activity. In NAC-treated and TNFα-stimulated ISEMFs of CD patients’ MMP-2 activity were restored to physiological value. The involvement of c-Jun N-terminal kinase pathway on redox regulation of MMP-2 secretion has been demonstrated.

For the first time, in CD patient ISEMFs, a redox regulation of MMP-2 secretion and activation related to GSH/GSSG ratio and inflammatory state have been demonstrated. This study suggests that compounds able to maintain GSH/GSSG ratio to physiological values can be useful to restore normal MMP-2 levels reducing in CD patient intestine the dysfunction of epithelial barrier.

 

BMC Pulm Med. 2012 Jun 13;12:24. doi: 10.1186/1471-2466-12-24.

Pirfenidone inhibits TGF-β1-induced over-expression of collagen type I and heat shock protein 47 in A549 cells.

Hisatomi K1Mukae HSakamoto NIshimatsu YKakugawa THara SFujita HNakamichi SOku HUrata YKubota HNagata K,Kohno S.

Pirfenidone is a novel anti-fibrotic and anti-inflammatory agent that inhibits the progression of fibrosis in animal models and in patients with idiopathic pulmonary fibrosis (IPF). We previously showed that pirfenidone inhibits the over-expression of collagen type I and of heat shock protein (HSP) 47, a collagen-specific molecular chaperone, in human lung fibroblasts stimulated with transforming growth factor (TGF)-β1 in vitro. The increased numbers of HSP47-positive type II pneumocytes as well as fibroblasts were also diminished by pirfenidone in an animal model of pulmonary fibrosis induced by bleomycin. The present study evaluates the effects of pirfenidone on collagen type I and HSP47 expression in the human alveolar epithelial cell line, A549 cells in vitro.

The expression of collagen type I, HSP47 and E-cadherin mRNAs in A549 cells stimulated with TGF-β1 was evaluated by Northern blotting or real-time PCR. The expression of collagen type I, HSP47 and fibronectin proteins was assessed by immunocytochemical staining.

TGF-β1 stimulated collagen type I and HSP47 mRNA and protein expression in A549 cells, and pirfenidone significantly inhibited this process. Pirfenidone also inhibited over-expression of the fibroblast phenotypic marker fibronectin in A549 cells induced by TGF-β1.

We concluded that the anti-fibrotic effects of pirfenidone might be mediated not only through the direct inhibition of collagen type I expression but also through the inhibition of HSP47 expression in alveolar epithelial cells, which results in reduced collagen synthesis in lung fibrosis. Furthermore, pirfenidone might partially inhibit the epithelial-mesenchymal transition.

 

 

 

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H2S-mediated protein sulfhydration in stress reveals metabolic reprogramming

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

 

Quantitative H2S-mediated protein sulfhydration reveals metabolic reprogramming during the Integrated Stress Response

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>Bo-JhihGuan, 

Ilya Bederman
Department of Pediatrics, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>IlyaBederman, 

Mithu Majumder
Department of Pharmacology, Case Western Reserve University, Cleveland, United States
No competing interests declared

” data-author-inst=”CaseWesternReserveUniversityUnitedStates”>MithuMajumder, et al.
eLife 2015;10.7554/eLife.10067    

http://elifesciences.org/content/early/2015/11/23/eLife.10067http://dx.doi.org/10.7554/eLife.10067

The sulfhydration of cysteine residues in proteins is an important mechanism involved in diverse biological processes. We have developed a proteomics approach to quantitatively profile the changes of sulfhydrated cysteines in biological systems. Bioinformatics analysis revealed that sulfhydrated cysteines are part of a wide range of biological functions. In pancreatic β cells exposed to endoplasmic reticulum (ER) stress, elevated H2S promotes the sulfhydration of enzymes in energy metabolism and stimulates glycolytic flux. We propose that transcriptional and translational reprogramming by the Integrated Stress Response (ISR) in pancreatic β cells is coupled to metabolic alternations triggered by sulfhydration of key enzymes in intermediary metabolism.
Posttranslational modification is a fundamental mechanism in the regulation of structure and function of proteins. The covalent modification of specific amino acid residues influences diverse biological processes and cell physiology across species. Reactive cysteine residues in proteins have high nucleophilicity and low pKa values and serve as a major target for oxidative modifications, which can vary depending on the subcellular environment, including the type and intensity of intracellular or environmental cues. Oxidative environments cause different post-translational cysteine modifications, including disulfide bond formation (-S-S-), sulfenylation (-S-OH), nitrosylation (-S-NO), glutathionylation (-S-SG), and sulfhydration (-S-SH) (also called persulfidation) (Finkel, 2012; Mishanina et al., 2015). In the latter, an oxidized cysteine residue included glutathionylated, 60 sulfenylated and nitrosylated on a protein reacts with the sulfide anion to form a cysteine persulfide. The reversible nature of this modification provides a mechanism to fine tune biological processes in different cellular redox states. Sulfhydration coordinates with other post-translational protein modifications such as phosphorylation and nitrosylation to regulate cellular functions (Altaany et al., 2014; Sen et al., 2012). Despite great progress in bioinformatics and advanced mass spectroscopic techniques (MS), identification of different cysteine-based protein modifications has been slow compared to other post-translational modifications. In the case of sulfhydration, a small number of proteins have been identified, among them the glycolytic enzyme glyceraldehyde phosphate dehydrogenase, GAPDH (Mustafa et al., 2009). Sulfhydrated GAPDH at Cys150 exhibits an increase in its catalytic activity, in contrast to the inhibitory effects of nitrosylation or glutathionylation of the same cysteine residue (Mustafa et al., 2009; Paul and Snyder, 2012). The biological significance of the Cys150 modification by H2S is not well-studied, but H2S could serve as a biological switch for protein function acting via oxidative modification of specific cysteine residues in response to redox homeostasis (Paul and Snyder, 2012). Understanding the physiological significance of protein sulfhydration requires the development of genome-wide innovative experimental approaches. Current methodologies based on the modified biotin switch technique do not allow detection of a broad spectrum of sulfhydrated proteins (Finkel, 2012). Guided by a previously reported strategy (Sen et al., 2012), we developed an experimental approach that allowed us to quantitatively evaluate the sulfhydrated proteome and the physiological consequences of H2S synthesis during chronic ER stress. The new methodology allows a quantitative, close-up view of the integrated cellular response to environmental and intracellular cues, and is pertinent to our understanding of human disease development.
The ER is an organelle involved in synthesis of proteins followed by various modifications. Disruption of this process results in the accumulation of misfolded proteins, causing ER stress (Tabas and Ron, 2011; Walter and Ron, 2011), which is associated with development of many diseases ranging from metabolic dysfunction to neurodegeneration (Hetz, 2012). ER stress induces transcriptional, translational, and metabolic reprogramming, all of which are interconnected through the transcription factor Atf4. Atf4 increases expression of genes promoting adaptation to stress via their protein products. One such gene is the H2S-producing enzyme, γ-cystathionase (CTH), previously shown to be involved in the signaling pathway that negatively regulates the activity of the protein tyrosine phosphatase 1B (PTP1B) via sulfhydration (Krishnan et al., 2011). We therefore hypothesized that low or even modest levels of reactive oxygen species (ROS) during ER stress may reprogram cellular metabolism via H2S-mediated protein sulfhydration (Figure 1A).
In summary, sulfhydration of specific cysteines in proteins is a key function of H2S (Kabil and Banerjee, 2010; Paul and Snyder, 2012; Szabo et al., 2013). Thus, the development of tools that can quantitatively measure genome-wide protein sulfhydration in physiological or pathological conditions is of central importance. However, a significant challenge in studies of the biological significance of protein sulfhydration is the lack of an approach to selectively detect sulfhydrated cysteines from other modifications (disulfide bonds, glutathionylated thiols and sulfienic acids) in complex biological samples. In this study, we introduced the BTA approach that allowed the quantitative assessment of changes in the sulfhydration of specific cysteines in the proteome and in individual proteins. BTA is superior to other reported methodologies that aimed to profile cysteine modifications, such as the most commonly used, a modified biotin switch technique (BST). BST was originally designed to study protein nitrosylation and postulated to differentiate free thiols and persulfides (Mustafa et al., 2009). A key advantage of BTA over the existing methodologies, is that the experimental approach has steps to avoid false-positive and negative results, as target proteins for sulfhydration. BST is commonly generating such false targets for cysteine modifications (Forrester et al., 2009; Sen et al., 2012). Using mutiple validations, our data support the specificity and reliability of the BTA assay for analysis of protein sulfhydration both in vitro and in vivo. With this approach, we found that ATF4 is the master regulator of protein sulfhydration in pancreatic β cells during ER stress, by means of its function as a transcription factor. A large number of protein targets have been discovered to undergo sulfhydration in β cells by the BTA approach. Almost 1,000 sulfhydrated cysteine- containing peptides were present in the cells under the chronic ER stress condition of treatment with Tg for 18 h. Combined with the isotopic-labeling strategy, almost 820 peptides on more than 500 proteins were quantified in the 405 cells overexpressing ATF4. These data show the potential of the BTA method for further systematic studies of biological events. To our knowledge, the current dataset encompasses most known sulfhydrated cysteine residues in proteins in any organism. Our bioinformatics analyses revealed sulfhydrated cysteine residues located on a variety of structure-function domains, suggesting the possibility of regulatory mechanism(s) mediated by protein sulfhydration. Structure and sequence analysis revealed consensus motifs that favor sulfhydration; an arginine residue and alpha-helix dipoles are both contributing to stabilize sulfhydrated cysteine thiolates in the local environment.
Pathway analyses showed that H2S-mediated sulfhydration of cysteine residues is that part of the ISR with the highest enrichment in proteins involved in energy metabolism. The metabolic flux revealed that H2S promotes aerobic glycolysis associated with decreased oxidative phosphorylation in mitochondria during ER stress in β cells. The TCA cycle revolves by the action of the respiratory chain that requires oxygen to operate. In response to ER stress, mitochondrial function and cellular respiration are down-regulated to limit oxygen demand and to sustain mitochondria. When ATP production from the TCA cycle becomes limited and glycolytic flux increases, there is a risk of accumulation of lactate from pyruvate. One way to escape accumulation of lactate is the mitochondrial conversion of pyruvate to oxalacetic acid (OAA) by pyruvate carboxylase. This latter enzyme was found to be sulfhydrated, consistent with the notion that sulfhydration is linked to metabolic reprogramming towards glycolysis.
The switch of energy production from mitochondria to glycolysis is known as a signature of hypoxic conditions. This metabolic switch has also been observed in many cancer cells characterized as the Warburg effect, which contributes to tumor growth. The Warburg effect provides advantages to cancer cell survival via the rapid ATP production through glycolysis, as well as the increased conversion of glucose into anabolic biomolecules (amino acid, nucleic acid and lipid biosynthesis) and reducing power (NADPH) for regeneration of antioxidants. This metabolic response of tumor cells contributes to tumor growth and metastasis (Vander Heiden et al., 2009). By analogy, the aerobic glycolysis trigged by increased H2S production could give β cells the capability to acquire ATP and nutrients to adapt their cellular metabolism towards maintaining ATP levels in the ER (Vishnu et al., 2014), increasing synthesis of glycerolphospholipids, glycoproteins and protein (Krokowski et al., 2013b), all important components of the ISR. Similar to hypoxic conditions, a phenotype associated with most tumors, the decreased mitochondria function in β cells during ER stress, can also be viewed as an adaptive response by limiting mitochondria ROS and mitochondria-mediated apoptosis. We therefore view that the H2S-mediated increase in glycolysis is an adaptive mechanism for survival of β cells to chronic ER stress, along with the improved ER function and insulin production and folding, both critical factors controlling hyperglycemia in diabetes. Future work should determine which are the key proteins targeted by H2S and thus contributing to metabolic reprogramming of β cells, and if and how insulin synthesis and secretion is affected by sulfhydration of these proteins during ER stress.
Abnormal H2S metabolism has been reported to occur in various diseases, mostly through the deregulation of gene expression encoding for H2S-generating enzymes (Wallace and Wang, 2015). An increase of their levels by stimulants is expected to have similar effects on sulfhydration of proteins like the ATF4- induced CTH under conditions of ER stress. It is the levels of H2S under oxidative conditions that influence cellular functions. In the present study, ER stress in β cells induced elevated Cth levels, whereas CBS was unaffected. The deregulated oxidative modification at cysteine residues by H2S may be a major contributing factor to disease development. In this case, it would provide a rationale for the design of therapeutic agents that would modulate the activity of the involved enzymes.

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