Treatment with a JAK1/2 inhibitor ameliorates murine autoimmune cholangitis induced by IFN overexpression.

The interferon (IFN) signaling pathways are major immunological checkpoints with clinical significance in the pathogenesis of autoimmunity. We have generated a unique murine model named ARE-Del, with chronic overexpression of IFNγ, by altering IFNγ metabolism. Importantly, these mice develop an immunologic and clinical profile similar to patients with primary biliary cholangitis, including high titers of autoantibodies and portal inflammation. We hypothesized that the downregulation of IFN signaling pathways with a JAK1/2 inhibitor would inhibit the development and progression of cholangitis. To study this hypothesis, ARE-Del+/- mice were treated with the JAK1/2 inhibitor ruxolitinib and serially studied. JAK inhibition resulted in a significant reduction in portal inflammation and bile duct damage, associated with a significant reduction in splenic and hepatic CD4+ T cells and CD8+ T cells. Functionally, ruxolitinib inhibited the secretion of the proinflammatory cytokines IFNγ and TNF from splenic CD4+ T cells. Additionally, ruxolitinib treatment also decreased the frequencies of germinal center B (GC B) cells and T follicular helper (Tfh) cells and led to lower serological AMA levels. Of note, liver and peritoneal macrophages were sharply decreased and polarized from M1 to M2 with a higher level of IRF4 expression after ruxolitinib treatment. Mechanistically, ruxolitinib inhibited the secretion of IL-6, TNF and MCP1 and the expression of STAT1 but promoted the expression of STAT6 in macrophages in vitro, indicating that M1 macrophage polarization to M2 occurred through activation of the STAT6-IRF4 pathway. Our data highlight the significance, both immunologically and clinically, of the JAK/STAT signaling pathway in autoimmune cholangitis.

Autoinflammatory and autoimmune conditions at the crossroad of COVID-19.

Coronavirus disease 2019 (COVID-19) has been categorized as evolving in overlapping phases. First, there is a viral phase that may well be asymptomatic or mild in the majority, perhaps 80% of patients. The pathophysiological mechanisms resulting in minimal disease in this initial phase are not well known. In the remaining 20% of cases, the disease may become severe and/or critical. In most patients of this latter group, there is a phase characterized by the hyperresponsiveness of the immune system. A third phase corresponds to a state of hypercoagulability. Finally, in the fourth stage organ injury and failure occur. Appearance of autoinflammatory/autoimmune phenomena in patients with COVID-19 calls attention for the development of new strategies for the management of life-threatening conditions in critically ill patients. Antiphospholipid syndrome, autoimmune cytopenia, Guillain-Barré syndrome and Kawasaki disease have each been reported in patients with COVID-19. Here we present a scoping review of the relevant immunological findings in COVID-19 as well as the current reports about autoinflammatory/autoimmune conditions associated with the disease. These observations have crucial therapeutic implications since immunomodulatory drugs are at present the most likely best candidates for COVID-19 therapy. Clinicians should be aware of these conditions in patients with COVID-19, and these observations should be considered in the current development of vaccines.

Recommendations for coronavirus infection in rheumatic diseases treated with biologic therapy.

The Coronavirus-associated disease, that was first identified in 2019 in China (CoViD-19), is a pandemic caused by a bat-derived beta-coronavirus, named SARS-CoV2. It shares homology with SARS and MERS-CoV, responsible for past outbreaks in China and in Middle East. SARS-CoV2 spread from China where the first infections were described in December 2019 and is responsible for the respiratory symptoms that can lead to acute respiratory distress syndrome. A cytokine storm has been shown in patients who develop fatal complications, as observed in past coronavirus infections. The management includes ventilatory support and broad-spectrum antiviral drugs, empirically utilized, as a targeted therapy and vaccines have not been developed. Based upon our limited knowledge on the pathogenesis of CoViD-19, a potential role of some anti-rheumatic drugs may be hypothesized, acting as direct antivirals or targeting host immune response. Antimalarial drugs, commonly used in rheumatology, may alter the lysosomal proteases that mediates the viral entry into the cell and have demonstrated efficacy in improving the infection. Anti-IL-1 and anti-IL-6 may interfere with the cytokine storm in severe cases and use of tocilizumab has shown good outcomes in a small cohort. Baricitinib has both antiviral and anti-inflammatory properties. Checkpoints inhibitors such as anti-CD200 and anti-PD1 could have a role in the treatment of CoViD-19. Rheumatic disease patients taking immunosuppressive drugs should be recommended to maintain the chronic therapy, prevent infection by avoiding social contacts and pausing immunosuppressants in case of infection. National and international registries are being created to collect data on rheumatic patients with CoViD-19.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.

MAdCAM costimulation through Integrin-α4ß7 promotes HIV replication.

Human gut-associated lymphoid tissues (GALT) play a key role in the acute phase of HIV infection. The propensity of HIV to replicate in these tissues, however, is not fully understood. Access and migration of naive and memory CD4+ T cells to these sites is mediated by interactions between integrin α4ß7, expressed on CD4+ T cells, and MAdCAM, expressed on high endothelial venules. We report here that MAdCAM delivers a potent costimulatory signal to naive and memory CD4+ T cells following ligation with α4ß7. Such costimulation promotes high levels of HIV replication. An anti-α4ß7 mAb that prevents mucosal transmission of SIV blocks MAdCAM signaling through α4ß7 and MAdCAM-dependent viral replication. MAdCAM costimulation of memory CD4+ T cells is sufficient to drive cellular proliferation and the upregulation of CCR5, while naive CD4+ T cells require both MAdCAM and retinoic acid to achieve the same response. The pairing of MAdCAM and retinoic acid is unique to the GALT, leading us to propose that HIV replication in these sites is facilitated by MAdCAM-α4ß7 interactions. Moreover, complete inhibition of MAdCAM signaling by an anti-α4ß7 mAb, an analog of the clinically approved therapeutic vedolizumab, highlights the potential of such agents to control acute HIV infection.

Preliminary studies on the use of pertussis toxin for the modulation of intravaginal SIV transmission in rhesus macaques.

BACKGROUND: Pertussis toxin (PTX) blocks GPCR signaling resulting in the inhibition of chemotaxis/cell adhesion. It was reasoned that inhibition of cell trafficking may be an approach to prevent HIV/SIV transmission. METHODS: In this study, PTX in HEC gel was applied to the vaginal wall of monkeys that were then challenged intravaginally with SIVmac251. RESULTS: Results of these studies showed that 2 of 4 animals were resistant to infection. Furthermore, infection was correlated with a marked increase in the plasma and cervicovaginal lavage levels of select chemokines and cytokines. CONCLUSIONS: Results from this preliminary feasibility study dictate that further studies that include a larger number of animals are required to optimize this protocol and establish the efficacy of this approach. In addition, such future studies will provide important information on the role of specific chemokines that play a role in lymphocyte trafficking within the genital tract and serve as additional therapeutic targets.

Early initiation of antiretroviral treatment postSIV infection does not resolve lymphoid tissue activation.

OBJECTIVE: Germinal center resident follicular helper T (TFH) cells in lymphoid follicles are a potential sanctuary for HIV/simian immunodeficiency virus (SIV) replication. But the dynamics of germinal centers upon early initiation of antiretroviral therapy (ART) and their potential role in the formation of viral sanctuaries post-SIV infection are not fully understood. DESIGN: Sequential lymph node biopsies (n = 10) were collected from SIVmac239-infected rhesus macaques before infection, at 5 weeks postinfection/pre-ART, 6 and 12 weeks following ART initiation. These tissues and cells were analyzed for frequencies of TFH cells and assignment of germinal center scores. RESULTS: Modest but significant increases in TFH cells and hyperplastic follicles with large germinal centers were noted during the acute phase of SIV infection (week 5/pre-ART). However, 6 weeks after ART initiation, substantial increases in germinal center TFH cells, germinal center B cells, hyperplastic follicles with large germinal centers, and abundant local IL-21 production were observed, whereas levels of SIV RNA and DNA of lymph nodes had decreased to barely detectable values along with barely detectable levels of SIV antibody-producing cells. An additional 6 weeks of ART did not appreciably decrease germinal center TFH or germinal center scores. CONCLUSION: Thus, although early ART rapidly controls SIV replication, it does not regulate early lymphoid activation, which may contribute to the seeding and magnitude of viral reservoirs.

Simian Immunodeficiency Virus Targeting of CXCR3+ CD4+ T Cells in Secondary Lymphoid Organs Is Associated with Robust CXCL10 Expression in Monocyte/Macrophage Subsets.

Glycosylation of Env defines pathogenic properties of simian immunodeficiency virus (SIV). We previously demonstrated that pathogenic SIVmac239 and a live-attenuated, quintuple deglycosylated Env mutant (Δ5G) virus target CD4+ T cells residing in different tissues during acute infection. SIVmac239 and Δ5G preferentially infected distinct CD4+ T cells in secondary lymphoid organs (SLOs) and within the lamina propria of the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Here, we studied the host responses relevant to SIV targeting of CXCR3+ CCR5+ CD4+ T cells in SLOs. Genome-wide transcriptome analyses revealed that Th1-polarized inflammatory responses, defined by expression of CXCR3 chemokines, were distinctly induced in the SIVmac239-infected animals. Consistent with robust expression of CXCL10, CXCR3+ T cells were depleted from blood in the SIVmac239-infected animals. We also discovered that elevation of CXCL10 expression in blood and SLOs was secondary to the induction of CD14+ CD16+ monocytes and MAC387+ macrophages, respectively. Since the significantly higher levels of SIV infection in SLOs occurred with a massive accumulation of infiltrated MAC387+ macrophages, T cells, dendritic cells (DCs), and residential macrophages near high endothelial venules, the results highlight critical roles of innate/inflammatory responses in SIVmac239 infection. Restricted infection in SLOs by Δ5G also suggests that glycosylation of Env modulates innate/inflammatory responses elicited by cells of monocyte/macrophage/DC lineages.IMPORTANCE We previously demonstrated that a pathogenic SIVmac239 virus and a live-attenuated, deglycosylated mutant Δ5G virus infected distinct CD4+ T cell subsets in SLOs and the small intestine, respectively (C. Sugimoto et al., J Virol 86:9323-9336, 2012, https://doi.org/10.1128/JVI.00948-12). Accordingly, infections with SIVmac239, but not with Δ5G, deplete CXCR3+ CCR5+ CD4+ T (Th1) cells during the primary infection, thereby compromising the cellular immune response. Thus, we hypothesized that distinct host responses are elicited by the infections with two different viruses. We found that SIVmac239 induced distinctly higher levels of inflammatory Th1 responses than Δ5G. In particular, SIVmac239 infection elicited robust expression of CXCL10, a chemokine for CXCR3+ cells, in CD14+ CD16+ monocytes and MAC387+ macrophages recently infiltrated in SLOs. In contrast, Δ5G infection elicited only modest inflammatory responses. These results suggest that the glycosylation of Env modulates the inflammatory/Th1 responses through the monocyte/macrophage subsets and elicits marked differences in SIV infection and clinical outcomes.

Checkpoint-based immunotherapy for autoimmune diseases - Opportunities and challenges.

Immune checkpoint pathways modulate the T cell response to ensure their dual function in maintaining health, eradicating or controlling malignancy and infection, as well as tolerating host autoantigens. Dysregulation of the checkpoint pathways may lead to development of cancer or persistent infection, or alternately, autoimmune diseases. Currently available treatments for autoimmune diseases, such as steroid and nonsteroid immunosuppressive medicines, have limited efficacy. Checkpoint-based immunotherapy, designed to directly block the activation pathway or agitate the inhibitory pathway of autoreactive T cells, is a promising therapeutic strategy for restoring T cell tolerance in autoimmune diseases. A major challenge in developing the checkpoint-based therapy for autoimmune diseases is identifying pathways that will minimize the induction of adverse global immune suppression that impairs the host protective immunity against cancer and microbial pathogens. Therefore research efforts should focus on increasing the organ or antigen specificity of such therapies.

Sustained virologic control in SIV+ macaques after antiretroviral and α4ß7 antibody therapy.

Antiretroviral drug therapy (ART) effectively suppresses replication of both the immunodeficiency viruses, human (HIV) and simian (SIV); however, virus rebounds soon after ART is withdrawn. SIV-infected monkeys were treated with a 90-day course of ART initiated at 5 weeks post infection followed at 9 weeks post infection by infusions of a primatized monoclonal antibody against the α4ß7 integrin administered every 3 weeks until week 32. These animals subsequently maintained low to undetectable viral loads and normal CD4+ T cell counts in plasma and gastrointestinal tissues for more than 9 months, even after all treatment was withdrawn. This combination therapy allows macaques to effectively control viremia and reconstitute their immune systems without a need for further therapy.

Heat Shock Enhances the Expression of the Human T Cell Leukemia Virus Type-I (HTLV-I) Trans-Activator (Tax) Antigen in Human HTLV-I Infected Primary and Cultured T Cells.

The environmental factors that lead to the reactivation of human T cell leukemia virus type-1 (HTLV-I) in latently infected T cells in vivo remain unknown. It has been previously shown that heat shock (HS) is a potent inducer of HTLV-I viral protein expression in long-term cultured cell lines. However, the precise HTLV-I protein(s) and mechanisms by which HS induces its effect remain ill-defined. We initiated these studies by first monitoring the levels of the trans-activator (Tax) protein induced by exposure of the HTLV-I infected cell line to HS. HS treatment at 43 °C for 30 min for 24 h led to marked increases in the level of Tax antigen expression in all HTLV-I-infected T cell lines tested including a number of HTLV-I-naturally infected T cell lines. HS also increased the expression of functional HTLV-I envelope gp46 antigen, as shown by increased syncytium formation activity. Interestingly, the enhancing effect of HS was partially inhibited by the addition of the heat shock protein 70 (HSP70)-inhibitor pifithlin-µ (PFT). In contrast, the HSP 70-inducer zerumbone (ZER) enhanced Tax expression in the absence of HS. These data suggest that HSP 70 is at least partially involved in HS-mediated stimulation of Tax expression. As expected, HS resulted in enhanced expression of the Tax-inducible host antigens, such as CD83 and OX40. Finally, we confirmed that HS enhanced the levels of Tax and gp46 antigen expression in primary human CD4⁺ T cells isolated from HTLV-I-infected humanized NOD/SCID/γc null (NOG) mice and HTLV-I carriers. In summary, the data presented herein indicate that HS is one of the environmental factors involved in the reactivation of HTLV-I in vivo via enhanced Tax expression, which may favor HTLV-I expansion in vivo.

MHC and KIR Polymorphisms in Rhesus Macaque SIV Infection.

Natural killer lymphocytes are essentially involved as the first line of defense against agents such as viruses and malignant cells. The activity of these cells is regulated via interaction of specific and diverse killer cell immunoglobulin-like receptors (KIR) with the highly polymorphic cognate MHC class I proteins on target cells. Genetic variability of both KIR and MHC-I ligands has been shown to be associated with resistance to many diseases, including infection with the immunodeficiency virus. Disease course and progression to AIDS after infection with human immunodeficiency virus-1 (HIV-1) is essentially influenced by the presence of the stimulatory KIR3DS1 receptor in combination with HLA-Bw4. Knowledge of such genetic interactions that contribute to not only disease resistance but also susceptibility are just as important. Such combined genetic factors were recently reported in the rhesus macaque AIDS model. Here, we review the rhesus macaque MHC class I and KIR gene systems and the role of their polymorphisms in the SIV infection model.

Phenotypic and Functional Characterization of Monoclonal Antibodies with Specificity for Rhesus Macaque CD200, CD200R and Mincle.

Lectin-like molecules and their receptors are cell surface molecules that have been shown to play a role in either facilitating infection or serving as transporters of HIV/SIV in vivo. The role of these lectin-like molecules in the pathogenesis of HIV/SIV infection continues to be defined. In efforts to gain further insight on the potential role of these lectin-like molecules, our laboratory generated monoclonal antibodies (mAb) against the human analogs of rhesus macaque CD200, CD200R and Mincle, since the rhesus macaques are accepted as the most reliable animal model to study human HIV infection. The characterization of the cell lineages from the blood and various tissues of rhesus macaques that express these lectin-like molecules are described herein. Among the mononuclear cells, the cells of the myeloid lineage of rhesus macaques are the predominant cell lineages that express readily detectable levels of CD200, CD200R and Mincle that is similar to the expression of Siglec-1 and Siglec-3 reported by our laboratory earlier. Subset analysis revealed that a higher frequency of the CD14+/CD16- subset from normal rhesus macaques express CD200, CD200R and Mincle. Differences in the frequencies and density of expression of these molecules by the gated population of CD14+ cells from various tissues are noted with PBMC and bone marrow expressing the highest and the mononuclear cells isolated from the colon and ileum expressing the lowest levels. While a significant frequency of pDCs and mDCs express Siglec-1/Siglec-3, a much lower frequency expresses CD200, CD200R and Mincle in PBMCs from rhesus macaques. The mAb against CD200 and CD200R but not Mincle appear to inhibit the infection of macrophage tropic SIV/SHIV in vitro. We conclude that these mAbs may have potential to be used as adjunctive therapeutic agents to control/inhibit SIV/HIV infection.

Human T-cell leukemia virus type-I Tax induces the expression of CD83 on T cells.

BACKGROUND: CD83, a cell surface glycoprotein that is stably expressed on mature dendritic cells, can be transiently induced on other hematopoietic cell lineages upon cell activation. In contrast to the membrane form of CD83, soluble CD83 appears to be immunosuppressive. In an analysis of the phenotype of leukemic CD4(+) T cells from patients with adult T-cell leukemia (ATL), we found that a number of primary CD4(+) T cells became positive for cell surface CD83 after short-term culture, and that most of these CD83(+) CD4(+) T cells were positive for human T-cell leukemia virus type-I (HTLV-I) Tax (Tax1). We hypothesized that Tax1 is involved in the induction of CD83. RESULT: We found that CD83 was expressed selectively on Tax1-expressing human CD4(+) T cells in short-term cultured peripheral blood mononuclear cells (PBMCs) isolated from HTLV-I(+) donors, including ATL patients and HTLV-I carriers. HTLV-I-infected T cell lines expressing Tax1 also expressed cell surface CD83 and released soluble CD83. CD83 can be expressed in the JPX-9 cell line by cadmium-mediated Tax1 induction and in Jurkat cells or PBMCs by Tax1 introduction via infection with a recombinant adenovirus carrying the Tax1 gene. The CD83 promoter was activated by Tax1 in an NF-κB-dependent manner. Based on a previous report showing soluble CD83-mediated prostaglandin E2 (PGE2) production from human monocytes in vitro, we tested if PGE2 affected HTLV-I propagation, and found that PGE2 strongly stimulated expression of Tax1 and viral structural molecules. CONCLUSIONS: Our results suggest that HTLV-I induces CD83 expression on T cells via Tax1 -mediated NF-κB activation, which may promote HTLV-I infection in vivo.

Thymic B cells promote thymus-derived regulatory T cell development and proliferation.

Thymic CD4(+) FoxP3(+) regulatory T (Treg) cells are critical for the development of immunological tolerance and immune homeostasis and requires contributions of both thymic dendritic and epithelial cells. Although B cells have been reported to be present within the thymus, there has not hitherto been a definition of their role in immune cell development and, in particular, whether or how they contribute to the Treg cellular thymic compartment. Herein, using both phenotypic and functional approaches, we demonstrate that thymic B cells contribute to the maintenance of thymic Treg cells and, using an in vitro culture system, demonstrate that thymic B cells contribute to the size of the thymic Treg compartment via cell-cell MHC II contact and the involvement of two independent co-stimulatory pathways that include interactions between the CD40/CD80/CD86 co-stimulatory molecules. Our data also suggest that thymic B cells promote the generation of thymic Treg cell precursors (pre-Treg cells), but not the conversion of FoxP3(+) Treg cells from pre-Treg cells. In addition, thymic B cells directly promote the proliferation of thymic Treg cells that is MHC II contact dependent with a minimal if any role for co-stimulatory molecules including CD40/CD80/CD86. Both pathways are independent of TGFß. In conclusion, we rigorously define the critical role of thymic B cells in the development of thymic Treg cells from non-Treg to precursor stage and in the proliferation of mature thymic Treg cells.

Whole-body immunoPET reveals active SIV dynamics in viremic and antiretroviral therapy-treated macaques.

The detection of viral dynamics and localization in the context of controlled HIV infection remains a challenge and is limited to blood and biopsies. We developed a method to capture total-body simian immunodeficiency virus (SIV) replication using immunoPET (antibody-targeted positron emission tomography). The administration of a poly(ethylene glycol)-modified, (64)Cu-labeled SIV Gp120-specific antibody led to readily detectable signals in the gastrointestinal and respiratory tract, lymphoid tissues and reproductive organs of viremic monkeys. Viral signals were reduced in aviremic antiretroviral-treated monkeys but detectable in colon, select lymph nodes, small bowel, nasal turbinates, the genital tract and lung. In elite controllers, virus was detected primarily in foci in the small bowel, select lymphoid areas and the male reproductive tract, as confirmed by quantitative reverse-transcription PCR (qRT-PCR) and immunohistochemistry. This real-time, in vivo viral imaging method has broad applications to the study of immunodeficiency virus pathogenesis, drug and vaccine development, and the potential for clinical translation.

Clinical features and pathobiology of Ebolavirus infection.

There has clearly been a deluge of international press coverage of the recent outbreak of Ebolavirus in Africa and is partly related to the "fear factor" that comes across when one is confronted with the fact that once infected, not only is the speed of death in a majority of cases rapid but also the images of the cause of death such as bleeding from various orifices gruesome and frightening. The fact that it leads to infection and death of health care providers (10% during the current epidemic) and the visualization of protective gear worn by these individuals to contain such infection adds to this "fear factor". Finally, there is a clear perceived notion that such an agent can be utilized as a bioterrorism agent that adds to the apprehension. Thus, in efforts to gain an objective view of the growing threat Ebolavirus poses to the general public, it is important to provide some basic understanding for the lethality of Ebolavirus infection that is highlighted in Fig. 1. This virus infection first appears to disable the immune system (the very system needed to fight the infection) and subsequently disables the vascular system that leads to blood leakage (hemorrhage), hypotension, drop in blood pressure, followed by shock and death. The virus appears to sequentially infect dendritic cells disabling the interferon system (one of the major host anti-viral immune systems) then macrophages (that trigger the formation of blood clots, release of inflammatory proteins and nitric oxide damaging the lining of blood vessels leading to blood leakage) and finally endothelial cells that contribute to blood leakage. The virus also affects organs such as the liver (that dysregulates the formation of coagulation proteins), the adrenal gland (that destroys the ability of the patient to synthesize steroids and leads to circulation failure and disabling of regulators of blood pressure) and the gastro-intestinal tract (leading to diarrhea). The ability of the virus to disable such major mechanisms in the body facilitates the ability of the virus to replicate in an uncontrolled fashion leading to the rapidity by which the virus can cause lethality. Various laboratories have been working on defining such mechanisms utilizing in vitro culture systems, a variety of animal models including inbred strains of normal and select gene knock out mice, guinea pigs and nonhuman primates that have led to a better understanding of the potential mechanisms involved. There have also been some major advances made in the identification of therapies from the very simple (major supportive type of therapy), to the identification of a number of highly effective chemotherapeutic agents, a variety of highly effective preventive (demonstrating 100% effectiveness in nonhuman primate models) recombinant formulations (adenovirus based, VSV-based, rabies virus based), therapeutic candidate vaccines (cocktail of monoclonal antibodies such as ZMAPP) and alternate approaches (RNAi-based such as TKM-Ebola and antisense based such as AVI-7537) that show great promise and at an unprecedented rate of discovery that speaks well for the scientific research community at large.

Shotgun proteomics: identification of unique protein profiles of apoptotic bodies from biliary epithelial cells.

UNLABELLED: Shotgun proteomics is a powerful analytic method to characterize complex protein mixtures in combination with multidimensional liquid chromatography-tandem mass spectrometry (LC-MS/MS). We used this platform for proteomic characterization of apoptotic bodies in an effort to define the complex protein mixtures found in primary cultures of human intrahepatic biliary epithelial cells (HiBEC), human renal proximal tubular epithelial cells, human bronchial epithelial cells, isolated intrahepatic biliary epithelial cells from explanted primary biliary cirrhosis (PBC), and control liver using a total of 24 individual samples. Further, as additional controls and for purposes of comparison, proteomic signatures were also obtained from intact cells and apoptotic bodies. The data obtained from LC-MS/MS, combined with database searches and protein assembly algorithms, allowed us to address significant differences in protein spectral counts and identify unique pathways that may be a component of the induction of the signature inflammatory cytokine response against BECs, including the Notch signaling pathway, interleukin (IL)8, IL6, CXCR2, and integrin signaling. Indeed, there are 11 proteins that localize specifically to apoptotic bodies of HiBEC and eight proteins that were specifically absent in HiBEC apoptotic bodies. CONCLUSION: Proteomic analysis of BECs from PBC liver compared to normal liver are significantly different, suggesting that an immunological attack affects the repertoire of proteins expressed and that such cells should be thought of as living in an environment undergoing continuous selection secondary to an innate and adaptive immune response, reflecting an almost "Darwinian" bias.

Murine autoimmune cholangitis requires two hits: cytotoxic KLRG1(+) CD8 effector cells and defective T regulatory cells.

Primary biliary cirrhosis (PBC) is an enigmatic disease mediated by autoimmune destruction of cholangiocytes in hepatic bile ducts. The early immunological events leading to PBC are poorly understood; clinical signs of disease occur very late in the pathological process. We have used our unique murine model of PBC in dominant-negative TGF-ß receptor type II transgenic mice to delineate critical early immunopathological pathways, and previously showed that dnTGFßRII CD8 T cells transfer biliary disease. Herein we report significantly increased numbers of hepatic dnTGFßRII terminally differentiated (KLRG1(+)) CD8 T cells, a CD8 subset previously shown to be enriched in antigen specific cells during hepatic immune response to viral infections. We performed bone marrow chimera studies to assess whether dnTGFßRII CD8 mediated disease was cell intrinsic or extrinsic. Unexpectedly, mixed (dnTGFßRII and B6) bone marrow chimeric (BMC) mice were protected from biliary disease compared to dnTGFßRII single bone marrow chimerics. To define the protective B6 cell subset, we performed adoptive transfer studies, which showed that co-transfer of B6 Tregs prevented dnTGFßRII CD8 T cell mediated cholangitis. Treg mediated disease protection was associated with significantly decreased numbers of hepatic KLRG1(+) CD8 T cells. In contrast, co-transfer of dnTGFßRII Tregs offered no protection, and dnTGFßRII Treg cells were functionally defective in suppressing effector CD8 T cells in vitro compared to wild type B6 Tregs. In vitro cholangiocyte cytotoxicity assays demonstrated significantly increased numbers of cytotoxic hepatic dnTGFßRII KLRG1(+) CD8 cells compared to B6. Protection from disease by B6 Tregs was associated with elimination of hepatic dnTGFßRII CD8 mediated cholangiocyte cytotoxicity. These results emphasize that autoimmune cholangitis requires defects in both the T effector and regulatory compartments, and that an intrinsic T cell effector defect is not sufficient to mediate autoimmune biliary disease in the setting of intact immune regulation. These results have important implications for understanding the early pathogenesis of human PBC.

Elimination of human T cell leukemia virus type-1-infected cells by neutralizing and antibody-dependent cellular cytotoxicity-inducing antibodies against human t cell leukemia virus type-1 envelope gp46.

Human T cell leukemia virus type-1 (HTLV-1) is prevalent worldwide with foci of high prevalence. However, to date no effective vaccine or drug against HTLV-1 infection has been developed. In efforts to define the role of antibodies in the control of HTLV-1 infection, we capitalized on the use of our previously defined anti-gp46 neutralizing monoclonal antibody (mAb) (clone LAT-27) and high titers of human anti-HTLV-1 IgG purified from HAM/TSP patients (HAM-IgG). LAT-27 and HAM-IgG completely blocked syncytium formation and T cell immortalization mediated by HTLV-1 in vitro. The addition of these antibodies to cultures of CD8(+) T cell-depleted peripheral blood mononuclear cells (PBMCs) from HAM/TSP patients at the initiation of culture not only decreased the numbers of Tax-expressing cells and the production of HTLV-1 p24 but also inhibited the spontaneous immortalization of T cells. Coculture of in vitro-HTLV-1-immortalized T cell lines with autologous PBMCs in the presence of LAT-27 or HAM-IgG, but not an F(ab')2 fragment of LAT-27 or nonneutralizing anti-gp46 mAbs, resulted in depletion of HTLV-1-infected cells. A 24-h (51)Cr release assay showed the presence of significant antibody-dependent cellular cytotoxicity (ADCC) activity in LAT-27 and HAM-IgG, but not F(ab')2 of LAT-27, resulting in the depletion of HTLV-1-infected T cells by autologous PBMCs. The depletion of natural killer (NK) cells from the effector PBMCs reduced this ADCC activity. Altogether, the present data demonstrate that the neutralizing and ADCC-inducing activities of anti-HTLV-1 antibodies are capable of reducing infection and eliminating HTLV-1-infected cells in the presence of autologous PBMCs.