04 - January 30, 2013.docx

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University of Toronto St. George
Cell and Systems Biology
Gray- Owen

Lecture MIJ485H – January 30, 2012 Immunopathogenesisis of Infectious Diseases: concepts with a focus on HIV-1 How virus infection cause disease – help design to prevent infection – In 1981, presented with dry productive cough Chest X-ray – lungs full of air should be black; greyish material indicating fluid or inflammation in the lungs, indicating pneumonia The autopsy – cause of pneumonia was pneumocystis carinii pneumonia – rare – was seen in a WW2, in Romani orphans – thought that starvation predisposed them to to this infection – in addition, bone marrow transplant patients occasionally came down with it This is found in the soil – if normal immune system, not pathogenic; opportunistic infection; weak infectious agent, unless have deficient immune response Search for common infectious agent – look at lymphocyte count – low – B and T cells – normal B cell counts, CD4 T cells were very low Enlarged lymph glands – immune activation in these people because lymph nodes enlarged Lymph node biopsy – suspect virus etiologic agent – took lymph node cells and occultured with CD4 T cell line – cells started dying and clumping, “sinsitial cells” – cells clumped together Processed supernatant for electron microscopy – saw budding particles, which matched pictures of retroviruses PCR – primers from sequence of HTLV (Gallo) – determined sequence – distinct from HTLV 1 and 2; called type 3 (to be renamed HIV-1) ELISA - check for presence of antibodies to HIV proteins – strong evidence for HIV to be etiologic agent Laboratory works – needlesticks introduced a molecular clone, workers fell sick Global pandemic – most in Africa, suggesting it as the origin of virus Clinical course of HIV infection During chronic phase, may see enlarged lymph nodes – takes 8 years for CD4 T cells to get to less than 200 – Animal models for HIV Loss of CD4 cells how? Can this be reversed? Pathogenesis – what is causing the disease? Etiology of disease process Immunopathogenesis – what is the etiology of disease in the immune system; what is the etiology of disease LCMV causes immune-mediated disease of immune system in mice – RNA virus – Armstrong strain of virus – injected IP – mice gets sick, clear virus Armstrong strain – injected into brain – death Brain tissue – skull is enclosed space – if large amounts of inflammation (edema, swelling), increased pressure in brain, get pressure necrosis – death of brain cells because of increased pressure – brain has hard time tolerating inflammation due to increased pressure b/c skull is limiting the space – easy for brain Brain of BALBc mouse Lots of red blood cells – lymphocytes = black dots – nuclei are single nucleus – not multi-load nuclei Neutrophils have many loads, while lymphocytes Top slides – IF staining using antibody against LCMV protein – virus is where lining is since the lymphocytes are found in the lining, attracting lymphocytes there to try to clear the virus Develop CTL responses to prove this – CTL responses in B graph – A plot – total CTL response – plotted with amount of virus present in body of mice – LCMV is cleared Immune response is cause of death – virus is cleared yet the mouse dies in A To prove this is the cause of death were the following experiments: Newborn mice poor ability to make CTL responses; same result with perforin knock out mice; virus replication in blood – death by kidney disease many months later Death by CTL response in the brain – produces inflammation, cytokines that recruit more inflammatory cells, cause edema – Non-cytopathic virus – directly cause tissue destruction vs cytopathic virus, not causing tissue damage Does not destroy the cells Kidney disease – chronic antigenic stimulation – B cells prouce more antibodies that form complexes with viral proteins and those complexes can precipitate – kidney is filter, immune complexes precipitate, leading to inflammation etc. Immune complex disease due to antibody production Figutre1 – part of filter in kidney has membrane, which is thin, - immune complex deposited in – thicking it, preventing filter capabilitis Immune complexes can activate complement, activate chemotactic factors Look like enormous deposits Can get deposition of immune complexes in the blood vessels If get antibody complexes – include inflammatory cells – get blood vessel destruction – called vasculitis – if all blood vessels get damaged Conclusions Neonatal infections – do not get meningitis Two different pathology caused by immune response due to virus Virus still there but cytokines are causing the inflammatory response Considered autoimmune disease – but indirect effect – own cells attacking own cells Uncontrolled antibody B cells – immune complexes – antigen and antibodies deposit in blood cells – Fc portion of antibody can activate complement pathway – C3a C5a – recruit other immune cells that can cause further damage to the blood vessels – Vasculitis – severe rashes Immune complexes occur Need certain amount of antigen, of antibody present Antigen is probably too low and the antibody levels are too high to make insoluble precipitate – need certain concentration of both to form insoluble antigen-antibody complexes Protection against acute primary infection Cytopathic viruses have shorter life cycle and to protect themselves, need neutralizing antibodies Non-cytopathic viruses need CTL responses Exceptions occur – for cytopathic viruses, need antibodies – cytopathic viruses – only protected against them when get from the mother upon birth; non-cytopathic viruses can survive in host without killing it, chronic disease caused, protection CT8 response sufficient LCMV – model of immunopathogenesis – give Armstrong IP, virus cleared Cloen 13, docile – no clearance – WE Traub – animals – amount of virus is not as high as clone 13 – symptoms similar; some deaths Srains differ only by a few sequence nucleotides Not a good model for HIV infection in AIDS LCMV-docile – using these models – characterized CTL response – Smelt et al Correlate viral load with amount of virus given Armstrong viruses – able to clear virus regardless of viral inoculum Other strains – virus never cleared Why? Spleen – large lymph node involved with immune clearance of blood – filters it Architecture – red pulp where blood vessels go through; white pulp is the lymphoid tissue In lymphoid tissue – B cell area – maybe different LCMV affect spleen differently – Looked at spleen – found different patterns of infection depending on virus subtype used IF against common epitope found on all LCMV strains Clone thirteen goes into the white pulp, where immune cells are; Armstrong into the red pulp, infecting different cell type Type of cell found in lymphoid tissue – FRC – supporting cell important in maintaining structure of lymph nodes – these cells supply growth factors that allow other lymphocytes to survive in structure of lymph node – In A, TR7 strain – against FRC cells Colocalization of virus and FRC for clone 13 – not for Armstrong Clone 13 suggested to be infecting the FRC cells D – flow cytometry – positive for both antibodies for Clone 13 ***Two possibilities – direct cytopathic effect AND__________________ Different strains of envelope from different viruses seemed to bind to alpha DG proteins differentially – affinity analysis where they used different amounts of protein, able to bind to infected cell – clone 13 seems to have higher binding affinity to this receptor, alpha DG – bad virus seems to bind to that receptor with stronger avidity Where is alpha DG being expressed? In the white pulp Alpha DG expressed on cells that are CD11c positive – more easily infected by clone 13 than Armstrong strain Virus is targeting the DC, which are somehow destroyed See CTL Response – are activated and then killed – all DCs and FRCs get infected, present antigen to CD8 cells, which expand and kill the DC – cannot make more immune response – resulting in severe immune suppression – mainly with Docile/Clone 13 strain LCMV and immune suppression this is mechanism of disease For viruses that are not cytopathic – many do cause disease – certain factors that appear to be responsible for getting rid of virus – then do not get disease – but if virus persists then can get immunity-mediated disease because virus is not supposed to be cytopathic LCMV model – prove that LCMV is not cytopathic for DC and FRC if want to prove that it is immune response that is causing the disease – immune-mediated disease – can depend on many factors – ex. Amount of virus used to cause infection Expose to high er amount of virus, maybe harder to clear the virus – virus may affect the immune response differently – if the virus replicates quickly and is not cytopathic, can allow virus to constantly escape from the immune response, that could cause persistence of virus antigen, triggering ongoing immune response, immune damage If virus has high mutation rate, can allow escape immune response –c ause immune mediated damage Certain HLA are better at getting rid of viruses are better than other HLAs Successful anti-viral immune response – many of viral disease in humans caused by viruses jnown to be cytopathic If no immune response to virus, NO DISEASE If strong immune response against virus, and virus is unable to mutate away from that immune response, clear the virus – get disease? If less effective immune response, controsl virus but allows replication, SEES DISEASE A lot of virus-induced diseases from non cytopathic viruses are due to oderate immune response against virus but virus still able to persist, so get disease Replication cycle of HIV – virus needs to bind to two virus receptors – CD4 molecule and co-receptor which is derived from chemokine receptor family – once in, replicates RNA, DNA - integrate into nucleus – get proviral DNA – transcribed to produce new viral RNA, which makes proteins and assembles to make new virus particle – need integration of genome into chromosomes of nucleus – Integration into cell chromosome – difficult to eliminate this – but if kill the cell, can get rid of chromosome Reverse transcriptase has high mutation rate – so every time virus replicates genome – makes at least one error Tag and Rev are two factors that allows integrated DNA to become genomic RNA Promoter activation of host gene (NF kappa B) – promoters can bind to NF kappa B, a generic transcription factor for immune cells – HIV can activate from proviral stage by NF kappa B TNF-alpha good at producing activated NF kappa B transcripton factor Lymph nodes – infected is enlarged; more B cell follicles in infected lymph nodes; every region of lymphoid tissue is expanded See extensive immune activation in lymph nodes of HIV infected people Immune activation in terms of HIV – stimulate HIV – infected lymph node would have more NF kappa B Early on in infection - enlarged LN then see gradual destruction of LN architecture Like LCMV Set point viremia predicts progression to aIDS More virus, more disease – How does HIV cause CD4 destruction? Direct or indirect? 1995 – effect of giving a single drug on HIV replication in pla
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