Survey
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Hygiene hypothesis wikipedia , lookup
DNA vaccination wikipedia , lookup
Molecular mimicry wikipedia , lookup
Monoclonal antibody wikipedia , lookup
Polyclonal B cell response wikipedia , lookup
Psychoneuroimmunology wikipedia , lookup
Innate immune system wikipedia , lookup
Sjögren syndrome wikipedia , lookup
Adoptive cell transfer wikipedia , lookup
Immunosuppressive drug wikipedia , lookup
XMRV, A New Human Pathogenic Retrovirus: Detection In Chronic Diseases • • • • Picture of cell art Picture of cell under a microscope Daniel L. Peterson, MD Whittemore Peterson Institute Daniel L. Peterson, MD Whittemore Peterson Institute October 29th, 2009 Chronic Fatigue Syndrome (CFS) • • • • • • • • • • A debilitating disorder of unknown etiology that is estimated A debilitating disorder of unknown etiology that is estimated to affect 17 million people worldwide to affect 17 million people worldwide CDC Criteria (Fakuda, 1994) Persistent or relapsing fatigue of 6 mo or longer in duration,generally with a distinct onset or relapsing fatigue of 6 mo longerdiagnosis in duration, Other Persistent known medical conditions excluded byor clinical generally with a distinct onset Patients have at least 4 of the following symptoms: Impaired memory or concentration Other known medical conditions excluded by clinical diagnosis CDC Criteria (Fakuda, 1994) Sore throat at least of the following symptoms: TenderPatients cervical orhave axillary lymph4 nodes Muscle pain pain Impaired memory or Multi-joint pain Multi-joint concentration New headaches New headaches Sore throat Un-refreshing sleep Un-refreshing sleep Tender cervical or axillary lymph Post exertional malaise Post exertional malaise nodes lasting > 24 hrs lasting > 24 hrs Muscle pain CFS Clinical Research Findings CFS is a heterogeneous, multi-system disorder manifested by • CFS is a heterogeneous, multi-system disorder inflammatory sequelae including: manifested by inflammatory sequelae including: antiviral enzyme (RNase L) dysfunction low natural killer (NK) cell numbers and function • • • antiviral enzyme (RNase L) dysfunction increased numbers activated T cells and function low natural killer (NK)ofcell numbers increased production of inflammatory innate immune activation cytokines/chemokines innate immune activation – increased numbers of activated T cells – increased production of inflammatory Could these patients’ immune cells be infected with XMRV? Could these patients’ immune cells be infected with XMRV? Gamma (Type-C) retrovirus XMRV Envelope proteins p15E gp70 • Picture of Envelope proteins Core proteins and core proteins p30 p15 p12 p10 Core proteins Envelope proteins • XMRV is a simple retrovirus—it encodes only for structural proteins • Retroviruses are NOT ubiquitous and NOT benign • They are all associated with disease such as cancer and neurological disease • There are three known human exogenous retroviruses: Budding XMRV from a – HIV, HTLV-1 (bothcell complex •XMRV is a simple retrovirus—it encodes only for structural proteinsand XMRV retroviruses) •Retroviruses are NOT ubiquitous and NOT benign Budding XMRV from a cell •They are all associated with disease such as cancer and neurological disease •There are three known human exogenous retroviruses: 4 •HIV, HTLV-1 (both complex retroviruses) and XMRV Presence of XMRV Sequences in Human DNA • These results are representative of the 101 patients tested. • They reflect the presence of virus (DNA PCR). 68/101 (67%) • The importance of this slide- in the normals 3.75% is 10 million Americans! • This gel is representative gel- 11 of the 101 are shown • Joy Das Gupta in the Silverman lab obtained full length sequence from 3 patient samples • To explore the relationship between XMRV in prostate cancer 12 /320 (3.75%) and the on in CFS These results are representative of the 101 patients tested. They reflect the presence of virus (DNA PCR). Retroviral Life Cycle: Latent vs. Active infection • In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins. • Distinguished by qPCR (DNA) and qRT-PCR (RNA) In latent infection- retroviral genome is present but is not transcribing viral genome or mRNA for structural proteins. Distinguished by qPCR (DNA) and qRT-PCR (RNA) Phylogenetic analysis revealed that XMRV isolates from prostate cancer and CFS form a distinct branch within non ecotronic MLVs • Not a mouse contaminant • XMRV is a new human retrovirus • Not prostate cancer XMRV •Not a mouse contaminant •XMRV is a new human retrovirus •Not prostate cancer XMRV Detection of Infectious Xenotropic MuLV-Related Virus (XMRV)in Blood Cells From Patients With Chronic Fatigue Syndrome • • • • • • University Of Nevada, Reno Vincent Lombardi Judy Mikovits Daniel Peterson Kathryn Hagen Max Pfost Cleveland Clinic – Bob Silverman and Javdip Das Gupta University of Nevada, Reno • National Cancer Institute • Sandra Ruscetti • Frank Ruscetti • Rachel Bagni Sandra • Ruscetti Cari Petrow-Sadowski Frank • Ruscetti Bert Gold Rachel Bagni • Michael Dean Cari Petrow-Sadowski Bert Gold Michael Dean Vincent Lombardi Judy Mikovits Daniel Peterson Kathryn Hagen Max Pfost Bob Silverman Jaydip Das Gupta CFS Study Cohort Reported in Science: • Study cohort from the WPI national repository. • Repository samples include samples from NV, CA, OR, FL, NC and NY as well as international CFS patients. • Repository inclusion criteria: – CFS diagnosis, regardless of severity – 19-75 years of age • Study characteristic: – 67% women, reflecting gender incidence of CFS – Mean age: 55 – 320 control samples from same geographic locations Questions • What cell types are infected? • How is XMRV transmitted? • Do infected individuals make an immune response? • What are the interactions between XMRV and the innate immune system? • Does XMRV infection alter the risk of cancer development in CFS? • Can we develop immune based therapies for CFS based on XMRV? XMRV Protein Expression in Purified Activated T and B Lymphocytes from CFS Patients • See using a flow cytometry assay expression of 3 goat poly clonal antibodies to purified viral proteins • in both T and B cells but not in T cells from a normal donor .. • We have shown expression of XMRV proteins but next wanted to ask if that reflected the presence of infectious and transmissable virus • 3 goat polyclonal antibodies Rat α-MuLV p30 Gag mAbto purified viral proteins Transmission of XMRV from Activated PBMCs of CFS Patients to the Human Prostate Cancer Cell Line LNCaP • Prostate cancer cell line Cell Free Transmission of XMRV from CFS Patients Plasma to LNCap • Using a technique known as spinoculation we transmitted virus from plasma and confirmed infectious particles by TEM next slide 21 positive of 25 (84%) Transmission Electron Micrograph of C-type Retrovirus Particles Transmitted from CFS patient T-Cells to LNCaP • Early and late buds type C morphology • Mature extracellular viral particles with condensed centrally located nucleoid surrounded by an outer membrane separated by an electron lucent area • Having detected the presence of XMRV viral proteins and particles in the PBMC of CFS patients, we next asked if we could detect an immune response to XMRV in CFS patients Infectious whole virus budding from the cell membrane Infectious whole virus budding from the cell membrane Antibodies in CFS Patients Plasma to XMRV Env WB: αSFFV Env mAb SFFV Env BaF3-ER BaF3-ER-SFFV Env BaF3-ER BaF3-ER-SFFV Env Evidence for the presence of XMRV in 33 PCR Negative US CFS Patients • 19/33 Antibodies in the plasma • 30/33 Transmissable virus in the plasma • 10/33 Protein expression in Decitibine (5Aza2DC) treated PBMC • Thus, since the submission to Science we determined 99 of 101 US patients show evidence of XMRV infection XMRV Expression in NC/FL Cohort Since the submission to Science, we to haveScience, replicated atwe the NCI/CC findings in other Since the submission have our +-replicated cohorts not in the WPI repository. at the NCI/CC our findings in other cohorts not in the WPI repository. (60%) positive for XMRV gag DNA from fresh • 9/15 9/15 PBMC (60%) positive for XMRV gag DNA from fresh PBMC (86.7%) positive by western forby XMRV Env • 13/15 13/15 (86.7%) positive western for XMRV Env and Gag upon co-culture of plasma or PBMC with LNCaP • and Gag upon co-culture of plasma or PBMC with LNCaP 8/15 (53%) plasma samples contain antibody to XMRV Env 8/15 (53%) plasma samples contain antibody to XMRV Env Familial Transmission of XMRV From Plasma from patients with Childhood Alzheimer’s • Acute flulike infection in the family • Mother/Father XMRV Env/Antibody positive (no active virus detected) XMRV envelope protein in LNCaP from children’s plasma XMRV Associated Neuroimmune Diseases (XAND): Potential Candidates: • Atypical MS: 3/3 positive for XMRV ENV protein and gag DNA • Fibromyalgia: 12/20 (60%) positive for XMRV gag DNA • Autism: 6/15 (40%) positive for XMRV gag DNA – 4/7 ( 57%) positive for serum Antibody to XMRV Env • Gulf War Illness : Not Tested • Samples were taken from family members of XMRV positive CFS subjects with these neuroimmune diseases. TCR g Clonality in Nevada CFS Cohort • ~ 77 of the Nevada cohort have a clonal TCRg rearrangement • More than 100 of these patients have clonal populations of gamma delta T cells • Gamma delta T cells play an active role in the regulation and resolution of pathogen induced immune responses. They accumulate at sites of inflammation associated with viral, bacterial and parasitic infections and in auto immune diseases Interestingly, Gamma Delta T cells up-regulate MIP1lalpha and Beta , TNF alpha, WPI Repository CFS Subjects with Cancer ID# XMRV status Clonal TCRg Lymphoma/cancer • XMRV status determined by the WPI since the submission to Science. XMRV status 1103 positive positive MCL 1109 positive negative Thymoma 1118 positive negative myelodysplasia 1125 positive Positive + IGH MCL 1186 positive positive Lymphoma 1199 positive positive Previous Lymphoma 1150 positive positive Lymphoma 1320 Not tested Not tested Thymoma 1321 Not tested Not tested MCL 1174 positive positive Thymoma 1205 positive Not tested lymphoma 1172 positive positive MCL 1135 positive positive suspicious 1204 positive Positive + IGH suspicious 1113 positive positive CLL 1322 Not tested Not tested MCL 1181 positive Not tested CLL 1188 positive positive CLL 1189 positive positive MCL 1190 positive positive suspicious determined by the WPI since the submission to Science. WPI-1125 CFS Diagnosis One Decade Prior to MCL • 1988---Seen 1988- seen atforNIH at NIH CFS for CFS to decrease aggressiveness. • 1998---Splenectomy 1998- Splenectomy to decrease 2000---Seen at NIH for mantle cell lymphoma. Given Rituxan aggressiveness. and Velcade with at adult stemfor cellsmantle cell lymphoma. • 2004---BMT 2000- seen NIH 2008---Blast crisis MCL … death Given Rituxan and Velcade • 2004- BMT with adult stem cells • 2008- Blast crisis MCL…. death MCL cell line developed 2008 Neither XMRV infection nor a diagnosis of CFS correlate to RNase L genetic variation R462Q • Relationship between RNaseL variant genotype and XMRV expression in CFS patients Dysfunction of Natural Killer Activity in a Family with Chronic Fatigue Syndrome Hypothesis of XMRV disease progression: NK dysregulation Relative time-scale of the virological and immunological events during XMRV infection CFS develops Env antigen Relative time-scale of the virological and immunological events during XMRV infection NK cells kill targets that do not express HLA class I Methods for addressing the NK cell dysregulation • PBMCs from XMRV infected patients with low NK cell function were activated with the mitogen PHA and treated with Ampligen • The effects on NK cell (CD56+) phenotype were determined by flow cytomentry • Signaling changes due to the treatment were detected via cytokine analysis • The change in XMRV copy number was detected with qRT-PCR Preliminary results from 8 study subjects • Ampligen significantly amplifies the degranulation of Ampligen significantly amplifies the degranulation of NK cells NK cells Ampligen may increase the ability of CD56+ cells to degranulate when – Ampligen may increase the encountering a target ability of CD56+ cells to degranulate when encountering a target • Ampligen significantly increases the production of Ampligen significantly the production of Perforin and GranzymeB Perforin andincreases GranzymeB Ampligen may promote synthesis of cytotoxic proteins- perforin and – Ampligen may promote synthesis of cytotoxic proteinsGranzymeB perforin and GranzymeB • Ampligen may regulate NK and inflammatory signaling molecules Ampligen may regulate NK and inflammatory signaling molecules Anti-viral as wellas as immune-stimulating cytokines are upregulated – Anti-viral well as immune-stimulating cytokines are upregulated Preliminary Results Cont. XMRV copy number is modulated by Ampligen • When treated with Ampligen, qRT-PCR indicates a decrease in some patients and an increase in others. • This may suggest why Ampligen worsens some CFS patients and not others. • Stratification for Ampligen treatment must consider XMRV status. There is a highly significant • Infectious XMRV was detected in lymphocytes association between the and plasma from >75% of CFS patients XMRV retrovirus and Chronic Fatigue • CFS-XMRV can be transmitted cell-free from patient Syndrome plasma to human prostate and T cell lines and to primary T cells Infectious XMRV was detected in lymphocytes and plasma from >75% of CFS patients response to the virus was detected in a • An immune CFS-XMRV can beCFS transmitted cell-free from patient plasma to human majority of subjects prostate and T cell lines and to primary T cells • XMRV inresponse CFS and cancer closely related An immune to theprostate virus was detected in a are majority of CFSform subjectsa distinct phylogenetic branch and XMRV in CFS and prostate cancer are closely related and form a distinct phylogenetic branch Acknowledgements: • • • • • • • • • • • • • • • • Cancer and Inflammation Program: Frank Ruscetti Cancer and Inflammation Program: Mike Dean Frank Ruscetti Bert Gold Mike Dean Dan Bertolette Bert Ying Gold Huang Dan Bertolette Ying Huang Laboratory of Cancer Prevention: Sandra Ruscetti Charlotte Hanson Laboratory of Cancer Prevention: Jami Troxler Sandra Ruscetti Charlotte Hanson Jami Troxler Cari Petrow-Sadowski Rachel Bagni Kunio Nagashima Cari Petrow-Sadowski Rachel Bagni Kunio Nagashima • • • • • From Judy AWPI Mikovits Judy A Mikovits Vincent Lombardi Vincent Lombardi Max Pfost Max Pfost Kathryn Hagen Kathryn Hagen • • • • • From Cleveland Clinic Robert Silverman Robert Silverman Jaydip Das Gupta Jaydip Das Gupta Robert Silverman Robert Silverman Jaydip Das Gupta Jaydip Das Gupta The CFS patients in the US