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In the past decade, more students than ever entered medical school with the desire, if not the expectation, of participating in meaningful global health experiences. Schools must now weigh benefits to students of global experiences against burdens of students' learning experiences on institutions and individuals with whom schools partner. Most often, global health training is done as offsite immersion rotations in research or clinical settings. This article explores ethical dimensions of expanding global health offerings while respecting local partners' goals by focusing on the experience of the University of Pennsylvania's global health training programs.

As of May 2020, the global COVID-19 pandemic had reached 187 countries with more than 3.7 million confirmed cases and 263,000 deaths. While sub-Saharan Africa (SSA) has not been spared, the extent of disease is currently far less than in Europe or North America leading some to posit that climatic, genetic or other conditions will self-limit disease in this location. Nonethe­less, infections in tropical Africa continue to rise at an alarming pace with the potential to soon exceed health resource availability and to exhaust a health care workforce that is already grossly under supported and ill-equipped. This perspective outlines the context of COVID-19 disease in Africa with a focus on the distinctive challenges faced by African nations and a potential best path forward. Ethn Dis. 2020;30(4):693-694; doi:10.18865/ed.30.4.693

Objective The thymus serves as a critical site of T-lymphocyte ontogeny and selection. Thymic infection by HIV-1 is known to disrupt thymocyte maturation by both direct and indirect means; however, the mechanism behind these effects remains poorly defined. Macrophages represent one of the most important peripheral targets of HIV-1 infection, are resident in the thymic stroma, and play a central role in thymocyte maturation. Materials and Methods Studies presented here define three primary features and outcomes of thymic macrophages (TM) and HIV-1 infection: (1) The distinctive TM phenotype (surface markers and cytokine production measured by immunofluorescence, fluorescence-activated cell sorting, and reverse transcriptase polymerase chain reaction) relative to macrophages from other sources (blood [monocyte-derived macrophages] and bone marrow); (2) infection of TM by different HIV-1 subtypes (X4, R5, and X4/R5) measured by enzyme-linked immunosorbent assay and polymerase chain reaction; and (3) consequences of HIV-1 infection on cytokine production by TM measured by reverse transcriptase polymerase chain reaction. Results The results demonstrate that TM display a distinctive phenotype of HIV-1 receptors (CD4 lo , CXCR4 lo , CCR5 med , CCR3 hi ), chemokine production (macrophage inflammatory protein−1α + ; regulated on activation, normal T expressed and secreted + ; macrophage inflammatory protein−1b − ; stromal cell−derived factor -1 − ); and cytokine production (tumor necrosis factor−α + , interleukin-8 + , macrophage colony-stimulating factor + , interleukin-6 − ) relative to either monocyte-derived macrophages or bone marrow. TM were infected in vitro with R5 and X4/R5-tropic HIV-1 subtypes, and developed syncytia formation during long-term X4/R5 culture. In contrast, TM supported only transient replication of X4-tropic HIV-1. Lastly, infection of TM with HIV-1 abolished the production of all cytokines tested in long-term in vitro cultures. Conclusions Taken together, these results indicate that TM are a potential direct target of in situ HIV-1 infection, and that this infection may result in the disruption of macrophage functions that govern normal thymocyte maturation.

The ability to detect HIV-1 in tissues that are not readily amenable to biopsy greatly limits the diagnosis and control of HIV infection, and ultimately, our ability to understand HIV-induced disease pathology. In view of this, we explored the utility of diagnostically measuring HIV-1 infection using (31)P nuclear magnetic resonance ((31)P-NMR). (31)P-NMR enables the correlation of infection to changes in the concentration of specific intracellular metabolites, macromolecules and of bioenergetic parameters that are key to mammalian cell physiology. Examples include primary components of biological membranes such as phosphomonoester (PME) and phosphodiester (PDE) lipids. Using (31)P-NMR we found that changes in the ratio of PDE/PME in human cell lines and primary isolates were significantly altered following HIV-1 infection. Our findings showed that the ratio of cellular PDE/PME uniformly decreased 2.00-2.26 fold in HIV-1 infected cells. Using the altered PDE/PME ratio as a selection criterion, we next assessed HIV-1 infection in lymphocytes isolated from both HIV-1 seropositive and non-infected human subjects. A decreased PDE/PME ratio was characteristic of HIV-1 infection in each instance. These results demonstrate that changes in cellular phospholipids induced during HIV-1 infection may be used to uncover basic mechanisms of HIV-1 pathology, and potentially, may be extrapolated to explore the application of NMR analysis as a technique for imaging infected organs and tissues in situ.

Infection with murine leukemia virus (MLV) TR1.3 or the related molecular construct W102G causes severe neuropathology in vivo. Infection is causally linked to the development of extensive syncytia in brain capillary endothelial cells (BCEC). These viruses also induce cell fusion of murine cell lines, such as SC-1 and NIH 3T3, which are otherwise resistant to MLV-induced syncytium formation. Although the virulence of these viruses maps within the env gene, the mechanism of fusion enhancement is not fully determined. To this end, we examined the capacity of the syncytium-inducing (SI) TR1.3 and W102G MLVs to overcome the fusion inhibitory activity inherent in the full-length Env cytoplasmic tail. These studies showed that the TR1.3 and W102G Envs did not induce premature cleavage of p2E, nor did they override p2E fusion inhibition. Indeed, in the presence of mutations that disrupt p2E function, the TR1.3 and W102G Envs significantly increased the extent of cell fusion compared to that with the non-syncytium-inducing MLV FB29. Surprisingly, we also observed that TR1.3 and W102G Envs failed to elicit syncytium formation in these in vitro assays. Coexpression of gag-pol with env restored syncytium formation, and accordingly, mutations within gag-pol were used to examine the minimal functional requirements for the SI phenotype. The results indicate that both gag -dependent particle budding and cleavage of p2E are required to activate the SI phenotype of TR1.3 and W102G viruses. Collectively, these data suggest that the TR1.3 and W102G viruses induce cell fusion by the fusion-from-without pathway.

We report here a new diagnostic approach to the direct detection of HIV in blood or other body fluids that is rapid, sensitive and potentially applicable in a point-of-care setting. The approach follows on the development of a novel BioNanoSensor (BNS) device that utilizes piezoelectric technology to detect the presence of the HIV surface glycoprotein gp120 in a nanoscale format. The detection range of the BNS device for the biomarker gp120 displayed a low-end sensitivity of 6.5×104 HIV viral particles/ml, while using a small fluid sample (5 µl) and with a reaction time of less then 30 seconds. Performance of this device indicated that the BNS has utility for direct detection of HIV particles prior to, and independent from, antibody formation. Accordingly, this device holds utility to monitor the status of HIV infection both early after exposure to virus as well as during chronic HIV infection. The BNS parameters of small sample volume, compact device size, and detection sensitivity indicate that the BNS is potentially useful in the point-of-care and/or home setting for monitoring decisions regarding HIV treatment on a real-time basis.

TR1.3 is a Friend murine leukemia virus (MLV) that induces selective syncytium induction (SI) of brain capillary endothelial cells (BCEC), intracerebral hemorrhage, and death. Syncytium induction by TR1.3 has been mapped to a single tryptophan-to-glycine conversion at position 102 of the envelope glycoprotein (Env102). The mechanism of SI by TR1.3 was examined here in comparison to the non-syncytium-inducing, nonpathogenic MLV FB29, which displays an identical BCEC tropism. Envelope protein expression and stability on both infected cells and viral particles were not statistically different for TR1.3 and FB29. However, affinity measurements derived using purified envelope receptor binding domain (RBD) revealed a reduction of >1 log in theKDof TR1.3 RBD relative to FB29 RBD. Whole-virus particles pseudotyped with TR1.3 Env similarly displayed a markedly reduced binding avidity compared to FB29-pseudotyped viral particles. Lastly, decreased receptor affinity of TR1.3 Env correlated with the failure to block superinfection following acute and chronic infection by TR1.3. These results definitively show that acquisition of a SI phenotype can be directly linked to amino acid changes in retroviral Env that decrease receptor affinity, thereby emphasizing the importance of events downstream of receptor binding in the cell fusion process and pathology.

Many viruses enter cells via an interaction of the viral envelope glycoprotein (Env) with receptor inducing fusion of viral and cellular membranes. These interactions are often evaluated in cell-cell fusion, gene-reporting systems with effector cells expressing Env and target cells expressing receptors. A common system utilizes vaccinia virus encoding T7 RNA polymerase (RNAP) in effector cells and a T7 promoted reporter plasmid in target cells. Fusion is quantified with expression of the reporter plasmid. However, direct activation of reporter plasmid from vaccinia virus can occur increasing background activity. We report here a modification of this assay in which T7 RNAP is expressed from a plasmid rather than vaccinia. This modification increased sensitivity with a ten-fold reduction in background. A novel dual T7/SP6 RNAP fusion assay was also developed to allow rapid screening for functional Env clones. Using these assays, we show that Envs from two CD4-independent HIV-2 isolates (VCP and ROD/B), which are able to fuse with chemokine receptor CXCR4 in a CD4-independent manner, are also able to fuse with alternative coreceptors GPR1 and GPR15 in the absence of CD4. The assay could also detect fusion of murine leukemia virus on target cells expressing the ecotropic MCAT-1 receptor showing its broad utility in other viral systems.

The thymus is considered to play an important role in the pathogenesis of Myasthenia gravis, an autoimmune disease characterized by antibody-mediated skeletal muscle weakness. However, its role is yet to be defined. The studies described herein summarize our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine (ACh) receptors is involved in the immunopathogenesis of this autoimmune disease. We review the work characterizing the expression of neuromuscular ACh receptors in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

Page e1 The remarkable advances in application of anti-retroviral therapy (ART) to treat HIVinfection has had a profound impact on the HIV epidemic as well as improved the quality and longevity of life for those who receive such treatment. Nonetheless, as individuals receiving ART live longer, they may develop chronic non-communicable disease at an increased incidence and/or severity when compared to non-infected and/or untreated individuals. Prime examples of this include cardiovascular and neurological disease, as well as metabolic syndrome and diabetes mellitus. Whether these growing disease burdens are identical in origin and outcome to disease in uninfected individuals, and whether ART impacts disease incidence and severity in addition to limiting HIV infection is often poorly distinguished. The data on type 2 diabetes mellitus (T2DM) provides a welcome case in point for this discussion.

We identified five human T-lymphoid cell lines (PB-1, Sez-4, C19PL, HUT 102B and ATL-2) which highly express CD4 in addition to CXCR4 and CCR5, In order to evaluate if these cells are infectabile by human immunodeficiency virus (HIV) and could be employed as a model in HIV research we exposed these cell lines to X4 (T-cell tropic) and R5 (macrophage tropic) and subsequently tried to correlate their infectability with (i) level of chemokine coreceptor (CXCR4 and CCR5) expression, (ii) coreceptor functionality (calcium flux, chemotaxis and phosphorylation of MAPK p42/44 and AKT) and (iii) endogenous expression and secretion of HIV-related chemokines which compete with the virus for binding to CXCR4 (SDF-1/CXCL12) or CCR5 (MIP-1β/ CCL4, MIP-1α/CCL3, RANTES/CCL5, MCP-2/CCL8, MCP-3/CCL7 and MCP-4/CCL13). We demonstrated that while PB-I cells are infectable by both X4 and R5 HIV, Sez-4, C91PL, HUT 102B and ATL-2 cells were infected by X4 HIV only. Moreover, we noticed that the susceptibility of these cells to HIV did not correspond either with the level of surface expression or with the functionality of CXCR4 or CCR5; however, it was modulated to some degree by the endogenously secreted HIV-related chemokines. Thus all five mature T-cell lines described here may provide useful new models for studying various aspects of HIV infection. In addition we demonstrate that the infectability of cells by HIV is modulated by so far unidentified intrinsic factors as well as some already known endogenously secreted chemokines. The identification of these factors may be important for developing new strategies to protect cells from HIV infection.

Objective The resistance of human bone marrow (BM) CD34 + cells to human immunodeficiency virus (HIV) infection is at this point not fully understood. Recently we reported that the chemokines MIP-1α, MIP-1β, and RANTES secreted by BM-derived CD34 + cells may compete with the macrophagotropic HIV (R5 HIV) strain for the CCR5 coreceptor. Materials and Methods In this study we extended our previous observations and examined various lympho-hematopoietic CD34 + cells isolated from thymus (Th), cord blood (CB), mobilized peripheral blood (mPB), and BM for the expression of β-chemokines binding to CCR5, i.e., MIP-1α, MIP-1β, RANTES, MCP-2, MCP-3, and MCP-4, and the α chemokine SDF-1 (binding to CXCR4) as these chemokines may compete with the R5 and X4 HIV strains, respectively, for entry into cells. Results We found that Th-, CB-, mPB-, and BM-derived CD34 + cells express mRNA transcripts for all the β -chemokines tested but not for SDF-1. Using sensitive ELISA assays we found that although MIP-1 α and MIP-1 β proteins were secreted by all the lympho-hematopoietic CD34 + cells tested, RANTES was detectable only in media conditioned by BM- and CB-derived CD34 + cells and not Th-derived cells. However, media conditioned by BM-, mPB- and Th-derived CD34 + cells protected the T lymphocytic cell line (PB-1) from infection by the R5 but not the X4 HIV strain. Conclusions Hence this study demonstrates that β-chemokines are secreted by lympho-hematopoietic CD34 + cells originating from various sources and that these endogenously secreted chemokines may limit entry of the R5 HIV strain into the cells by competing for the CCR5 coreceptor.

The aim of this study was to learn more about the role of the HIV-related chemokine-chemokine receptor axes in human hematopoiesis. To address this issue we phenotyped 35 selected hematopoietic cell lines for the expression of CD4, CXCR4 and CCR5. We next evaluated the functionality of these chemokine receptors by calcium flux and chemotaxis assays, and by the ability of SDF-1, MIP-1alpha, MIP-1beta and RANTES to influence the growth of the cells expressing CXCR4 and/or CCR5. Lastly, we examined whether human hematopoietic cell lines may secrete some HIV-related chemokines, and whether endogenously secreted chemokines might interfere with the infectability. of hematopoietic cells by X4 and R5 HIV strains. These results demonstrate that: (1) HIV-related receptors are widely expressed on human hematopoietic cell lines; (2) stimulation of CXCR4 by SDF-1 induces calcium flux and chemotaxis in several hematopoietic cell lines more efficiently than stimulation of CCR5 by receptor-specific beta-chemokines; (3) chemokines do not regulate proliferation of the hematopoietic cells; and finally (4) infectability of the hematopoietic cells by HIV-1 may be auto-modulated by endogenously secreted chemokines. These data shed more light on the role of HIV-related chemokine-chemokine receptors axes in human hematopoiesis and interaction of hematopoietic cells with HIV.

CD34+ cells are nonpermissive to infection by HIV strains X4 and R5, despite the fact that many CD34+ cells express high levels of the viral receptor protein CD4 and the coreceptor CXCR4 on their surface. In these cells, the co-receptor CCR5 protein, which, like CXCR4, is a chemokine receptor, is detected mainly intracellularly. We hypothesized that CD34+ cells secrete CCR5-binding chemokines and that these factors interfere with HIV R5 interactions with these cells, possibly by binding CCR5 or by inducing its internalization. We found that human CD34+ cells and CD34+KIT+ cells, which are enriched in myeloid progenitor cells, expressed and secreted the CCR5 ligands RANTES, MIP-1α, and MIP-1β and that IFN-γ stimulated expression of these chemokines. In contrast, SDF-1, a CXCR4 ligand, was not detectable in the CD34+KIT+ cells, even by RT-PCR. Conditioned media from CD34+ cell culture significantly protected the T lymphocyte cell line PB-1 from infection by R5 but not X4 strains of HIV. Interestingly, the secretion of endogenous chemokines decreased with the maturation of CD34+ cells, although ex vivo, expanded megakaryoblasts still secreted a significant amount of RANTES. Synthesis of CCR5-binding chemokines by human CD34+ cells and megakaryoblasts therefore largely determines the susceptibility of these cells to infection by R5 HIV strains. We postulate that therapeutic agents that induce the endogenous synthesis of chemokines in human hematopoietic cells may protect these cells from HIV infection. J. Clin. Invest. 104:1739–1749 (1999).

The P2X family of ATP receptors (P2XR) have been implicated in thymocyte death in vitro and in vivo. We characterized ATP-evoked Ca2+ transients and membrane currents in thymocytes to better understand the role of P2XR during thymocyte development. ATP4-, but not UTP or GTP, activated a sustained non-selective cation current in voltage-clamped CD4- CD8- and CD4+ CD8+ thymocytes that was reversed by apyrase, which hydrolyzes ATP, and by the P2XR antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS). The more selective P2XR agonist alphabeta-methylene ATP activated a smaller rapidly decaying current in both thymocyte populations. Reverse transcription-PCR results indicate that P2X1, P2X2, P2X6, and/or P2X7 are expressed in thymocytes. Finally, we used PPADS to examine the role of P2XR during thymocyte development in situ. PPADS-treated thymi yielded significantly more thymocytes (38%), due to a selective increase in CD4+ CD8+ cells. Together these data suggest that one or more PPADS-sensitive P2XR (P2X1, P2X2, P2X7) are involved in thymocyte apoptosis, and we propose more specifically a role associated with death by neglect.

Page e7 Despite remarkable advances in the prevention and treatment of HIV, over 35 million individuals currently live with active HIV infection and worldwide, there were nearly 2.1 million new cases of HIV in 2013.1,2 The benefits of rapid testing to determine HIV infection are well established the introduction of Highly active antiretroviral therapy (HAART) treatment within four hours of exposure has been shown to dramatically limit both virus spread and progression to AIDS.3 Equally critical is the need to continuously monitor virus load in individuals on HAART and/or other HIV therapies. With the expansion of HAART in countries with poor access to and/or quality of health care, individual variations in HAART effectiveness may be infrequently monitored. Negative outcomes of this well-intended expansion may be that individuals on HAART assume they are either virus-free or incapable of spreading infection whereas the opposite status would have potentially tragic outcomes. Lastly, as we look to the future and the implementation of HIV vaccines, the capacity to monitor virus directly, not just the presence of antibodies to HIV, is increasingly critical.

Replication of viruses within the thymic microenvironment may have a unique impact on viral persistence and pathology. The author's laboratory has studied thymic infection by both human and murine retroviruses. For human lentiviruses, such as HIV-1, the consequences of persistent thymic replication are frequently a severe disruption of the normal processes of thymopoiesis and potentially of progression to AIDS. Murine retroviruses, such as Gross murine leukemia virus, establish persistent infection with less cytopathic, but no less devastating effects. These include the alteration of immune recognition to retroviral antigens by the peripheral immune response, the thymic persistence of virus, and the establishment of viral-induced thymic leukemia. This article summarizes the analysis of both the common and distinctive means of pathology induced by these two retroviral families with particular attention on the influence and impact of the thymus as a unique site of virus replication.

The patch-clamp recording technique and RNApolymerase chain reaction were used to identify the voltage-dependent K channels expressed by murine fetal and adult CD4CD8 thymocytes. Two distinct currents, encoded by the genes Kv1.1 and Kv1.3 were identified based upon their biophysical and pharmacologic characteristics and confirmed with RNA-polymerase chain reaction. Peptide blockers of Kv1.1 and Kv1.3 gene products were also applied to a murine fetal thymic organ culture system to investigate the developmental role of these K channels. Dendrotoxin (DTX) and charybdotoxin (CTX), antagonists of Kv1.1 and Kv1.3 channels, respectively, decreased thymocyte yields in organ culture without affecting thymocyte viability. DTX-treated thymi contained 56 ± 8% (n = 8 experiments), and CTX-treated thymi contained 74 ± 4% (n = 7 experiments) as many thymocytes as untreated lobes. DTX and CTX also altered the developmental progression of thymocytes in fetal organ culture. These data provide the first evidence of Kv1.1 expression in a lymphoid cell and indicate that thymocyte voltage-dependent K channels are critical to thymocyte preclonal expansion and/or maturation.

TR1.3 is a Friend-related murine leukemia virus that has been shown to cause intracerebral hemorrhages and neurologic disease due to infection and subsequent cytopathology of cerebral vessel endothelium. A striking feature of this pathology is the formation of endothelial cell syncytia. The pathogenesis of this disease has now been mapped to a single amino acid substitution of tryptophan to glycine in the variable region of the envelope protein. This same mutation enabled TR1.3 to form syncytia and retard cell proliferation in vitro in the SC-1 mouse embryoblast line but did not affect the pH dependence of viral entry. These results demonstrate that subtle molecular changes in retroviral env genes can induce both syncytium formation and overt clinical disease.

To determine the impact of HIV-1 infection on thymocyte development, and the role of thymic infection on the pathogenesis of neonatal HIV-1 infection.The consequences of thymic infection by HIV-1 were examined by comparative histologic and molecular analyses of an asymptomatic, HIV-1-seropositive 3-day-old subject, versus age- and treatment-matched controls. The presence of replicating virus was established by in situ hybridization with specific molecular probes to HIV-1. The distribution of thymocyte subsets was determined by quantitative flow cytometry following staining with antibodies to CD4 and CD8 cell surface proteins.The results show clear evidence of severe thymic involution, HIV-1 infection of thymocytes, and selective depletion of thymocyte subpopulations. The consequences of HIV-1 infection were a marked depletion of CD3+CD4+ CD8hi and CD3+CD4+CD8- cells. The phenotype of the residual thymic lymphoid population was predominantly that of immature CD3-CD4-CD8- double negative and CD3+CD4+CD8lo cells.Changes in the distribution of thymocyte subsets suggests a role for thymic involvement in the pathogenesis of HIV-1 infection in neonates.

The mechanisms of endothelial cell damage that lead to cerebral hemorrhage are not completely understood. In this study, a cloned murine retrovirus, TR1.3, that uniformly induced stroke in neonatal BALB/c mice is described. Restriction digest mapping suggests that TR1.3 is part of the Friend murine leukemia virus (FMuLV) family. However, unlike mice exposed to other FMuLVs, mice infected with TR1.3 virus developed tremors and seizures within 8 to 18 days postinoculation. This was uniformly followed by paralysis and death within 1 to 2 days. Postmortem examination of TR1.3-inoculated mice revealed edematous brain tissue with large areas of intracerebral hemorrhage. Histologic analysis revealed prominent small vessel pathology including syncytium formation of endothelial cells. Immunohistochemical analysis of frozen brain sections using double fluorescence staining demonstrated that TR1.3 virus specifically infected small vessel endothelial cells. Although infection of vessel endothelial cells was detected in several organs, only brain endothelial cells displayed viral infection associated with hemorrhage. The primary determinant of TR1.3-induced neuropathogenicity was found to reside within a 3.0-kb fragment containing the 3' end of the pol gene, the env gene, and the U3 region of the long terminal repeat. The restricted tropism and acute pathogenicity of this cloned murine retrovirus provide a model for studying virus-induced stroke and for elucidating the mechanisms involved in syncytium formation by retroviruses in vivo.

The relative levels of phosphatidylinositol-3'-kinase (PI3K) activity were measured in interleukin-2 (IL-2)-dependent helper (HT-2) and cytolytic (CTLL-2) T-cell clones that had been stably transfected with expression plasmids encoding either the normal p56-Lck kinase or a mutant version of this kinase, p56-Lck(Y505F), that has constitutively high levels of kinase activity. Stimulation of untransfected T-cells or of transfected T-cells containing increased levels of normal p56-Lck resulted in an approximate doubling of the relative amounts of total cellular PI3K activity. In contrast, T-cells producing the activated version of p56-Lck contained levels of PI3K activity comparable to or slightly higher than those found IL-2-stimulated control cells, even in the absence of IL-2. Assessments of the relative levels of PI3K activity in immunoprecipitates prepared with the use of anti-phosphotyrosine-specific antibodies revealed constitutively high levels of anti-phosphotyrosine-immunoprecipitable PI3K activity in T-cells containing p56-Lck(Y505F), as opposed to T-cells containing normal p56-Lck where increases in anti-PY-immunoprecipitable PI3K activity were IL-2-inducible. IL-2 stimulation of T-cells containing the normal p56-Lck kinase resulted in marked increases in the relative amounts of PI3K activity and p85 that were coimmunoprecipitated when using anti-p56-Lck antibodies. In contrast, PI3K activity and the p85 subunit of PI3K could be coimmunoprecipitated from T-cells producing the activated p56-Lck(Y505F) kinase even in the absence of IL-2 stimulation, implying constitutive association of PI3K with the activated Lck kinase. Taken together with previous studies showing that IL-2 induces rapid increases in the activities of both p56-Lck and PI3K in T-cells, these findings suggest that p56-Lck lies immediately upstream of PI3K in a signal transduction cascade initiated by the binding of IL-2 to its specific receptor on T-lymphocytes.

The biological activity of interleukin-2 receptors (IL-2R) is dependent on the functional coupling of IL-2R beta molecules to intracellular enzymes such as protein tyrosine kinase. The serine-rich, S-domain within the cytosolic portion of IL-2R beta plays an essential role in transduction of the IL-2 proliferative signal. Cells bearing either wild type IL-2R beta (Baf alpha/beta) or deletions within the S-domain (Baf alpha/beta SD1) were used to evaluate the importance of the S-domain in linking ligand binding to protein tyrosine kinase induction. Multiparameter, side by side comparisons showed that only those cells that expressed wild type IL-2R beta responded to IL-2 by increased cellular proliferation, accumulation of the c-myc proto-oncogene, and activation of protein tyrosine kinase. Activation of protein tyrosine kinase was, in turn, linked to increased tyrosine phosphorylation and activation of phosphatidylinositol-3-kinase. These findings indicate that the S-domain of the IL-2R beta chain is an essential component in the signal transduction cascade that links IL-2 binding to tyrosine kinase and phosphatidylinositol-3-kinase activation.

The problem of Alzheimer's disease (AD) exemplifies the challenges of dealing with a broad range of aging-related chronic disorders that require long-term, labor-intensive, and expensive care. As the baby boom generation ages and brain diseases become more prevalent, the need to confront the pending health care crisis is more urgent than ever before. Indeed, there is now a critical need to expand significantly the national effort to solve the problem of AD, with special focus on prevention. The Campaign to Prevent Alzheimer's Disease by 2020 (PAD2020) aims to create a new paradigm for planning and supporting the organization of worldwide cooperative research networks to develop new technologies for early detection and treatments of aging-related memory and motor impairments. PAD 2020 is developing an implementation plan to justify (1) increasing the federal budget for research, (2) developing novel national resources to discover new interventions for memory and motor disorders, and (3) creating innovative and streamlined decision-making processes for selecting and supporting new ideas. Since 1978 the National Institute on Aging or National Institute of Health (NIH) established an extensive national network of AD research facilities at academic institutions including AD Centers (ADCs), Consortium to Establish a Registry for AD, AD Cooperative Study (ADCS), AD Drug Discovery Program, National Alzheimer's Coordinating Center, National Cell Repository for AD, and AD Neuroimaging Initiative. However, despite the success of these programs and their critical contributions, they are no longer adequate to meet the challenges presented by AD. PAD 2020 is designed to address these changes by improving the efficiency and effectiveness of these programs. For example, the ADCs (P30s and P50s) can be enhanced by converting some into Comprehensive Alzheimer's Disease Centers (CADCs) to support not only research, but also by being demonstration projects on care/treatment, clinical trials, and education as well as by seamlessly integrating multisite collaborative studies (ADCS, AD Neuroimaging Initiative, Patient Registries, Clinical Data Banks, etc) into a cohesive structure that further enhances the original mission of the National Institute on Aging ADCs. Regional CADCs offer greater efficiency and cost savings while serving as coordinating hubs of existing ADCs, thereby offering greater economies of scale and programmatic integration. The CADCs also broaden the scope of ADC activities to include research on interventions, diagnosis, imaging, prevention trials, and other longitudinal studies that require long-term support. Thus, CADCs can address the urgent need to identify subjects at high risk of AD for prevention trials and very early in the course of AD for clinical trials of disease modification. The enhanced CADCs will allow more flexibility among ADCs by supporting collaborative linkages with other institutions and drawing on a wider expertise from different locations. This perspective article describes the University of Pennsylvania (Penn) CADC Model as an illustrative example of how an existing ADC can be converted into a CADC by better utilization of Penn academic resources to address the wide range of problems concerning AD. The intent of this position paper is to stimulate thinking and foster the development of other or alternative models for a systematic approach to the study of dementia and movement disorders.

Inhibitory effects of human immunodeficiency virus (HIV) on T lymphocyte function have been linked to perturbation of signaling through the T cell antigen receptor-CD3 complex. Comparative biochemical analyses of signaling responses were performed in T cells that were either uninfected or chronically infected with the HIV-1/IIIB strain. Stimulation with antibodies to CD3 triggered both Ca2+ accumulation and phosphoinositide hydrolysis responses that were equivalent in uninfected and infected cells. Treatment with anti-CD3 or with phorbol diester also stimulated serine phosphorylation of CD4 molecules in uninfected T cells. However, phosphorylation of CD4 was not observed after anti-CD3 treatment in HIV-infected T cells despite normal phosphorylation responses to phorbol diester. Identical results were obtained using a T cell line that was infected with an env (gp160/120-) HIV-1 defective variant. These studies indicate that infection with HIV-1 inhibits the activation of protein kinase associated with the T cell receptor-CD3 complex by a mechanism which is independent of viral env protein components.

Emergency care research (ECR) does not fit neatly into the traditional National Institutes of Health (NIH) funding model, because emergency research involves undifferentiated disease presentations involving multiple disciplines and time-sensitive interventions. A task force of emergency care researchers and other stakeholders was convened to discuss the present and future state of clinical research networks. Integration of ECR with the Clinical Translational and Science Award (CTSA) program through a multidisciplinary emergency care research network (ECRN) would obviate the duplication of research efforts by disease-specific or institute-specific multicenter networks and reduce startup and maintenance costs. Strategies to enhance integration must include the training of emergency physician investigators in biostatistical and epidemiologic methods, as well as educating collaborative investigators in emergency care-related methodologies. Thus, an ECRN would be of great benefit to CTSA awardees and applicants and should be considered a priority.

Glycosylated phosphatidylinositol (gly-Pl) molecules have been implicated as precursors for insulin-sensitive second messengers (1–4) and lipid-anchored membrane proteins (5–9). The relationship between the diverse functions of these lipids and their predicted structural heterogeneity within gly-Pl subtypes was examined in human T lymphocytes. Four subtypes of gly-Pl molecules were identified in T lymphocytes after separation over high-performance thin-layer chromatography by sensitivity to Pl-specific phospholipase C and nitrous acid. Antibody probes of the glycan domain of gly-Pl were developed and used to assess the partial sensitivity of gly-Pl to insulin action. This analysis showed that the effects of insulin are linked to differential utilization of only two of the four gly-Pl subtypes in T lymphocytes. Polar fragments of this reaction were identified in extracellular supernatants from insulin-treated cells. The biological significance of insulin-dependent gly-Pl hydrolysis was demonstrated by insulin and inositol phosphoglycan regulation of glucose metabolism in intact lymphocytes. These results support the hypothesis that multifunctional roles of gly-Pl are served by discrete gly-Pl populations and that metabolites of gly-Pl subsets participate as signaling elements in insulin action.

The molecular mechanism of signal transduction through the interleukin 2 (IL-2) receptor remains an enigma. Glycosylphosphatidylinositol (GPI) lipids were investigated as one component of this process. IL-2 stimulated the rapid (30 sec) loss of greater than 50% of GPI in the IL-2-dependent T-cell line CTLL-2. Half-maximal GPI loss was detected at 40 pM IL-2, coincident with the EC50 (20 pM) for IL-2-induced proliferation of this cell line. This effect was specifically inhibited by antibodies that bind either IL-2 or the IL-2 receptor. The loss of GPI was mirrored by the accumulation of both polar inositolphosphoglycan (IPG) and diacylglycerol lipid fragments within cells. Increases in lipids were initially restricted to myristoyl diacylglycerol but were followed by the accumulation of myristoyl phosphatidic acid. These results are indicative of IL-2-dependent hydrolysis of GPI in T cells. The biological relevance of this hydrolysis was demonstrated by synergism of purified IPG with IL-2 in T-cell proliferation responses. The inclusion of IPG (0.1 microM) in determinations of IL-2-dependent CTLL-2 growth shifted the EC50 from 20 to 7 pM IL-2. IPG had no effect on either the number or affinity of IL-2 receptors; therefore, half-maximal CTLL-2 proliferation was obtained at less than 10% IL-2 receptor occupancy. These results demonstrate that GPI lipids are an important component of the biological response to IL-2.

The addition of interleukin 2 (IL2) to the IL2-dependent murine cytotoxic T cell line CTTL-2 induced increased tyrosine phosphorylation of a protein with a molecular weight of 80,000 and, to a lesser extent, proteins with molecular weights of 130,000, 100,000, and 69,000. To correlate the stimulation of tyrosine phosphorylation with increased tyrosine kinase activity, cell-free phosphorylation assays were performed. Phosphotyrosine-containing proteins were purified from detergent-solubilized cell lysates by immunoprecipitation with anti-phosphotyrosine antibodies. The level of tyrosine kinase activity was determined by incorporation of [gamma-32P]ATP into the exogenous substrate histone H2B. IL2 treatment of cells increased H2B phosphorylation 10-fold when compared with nonstimulated cells. Phosphorylation was first detected after 2.5 min of incubation with physiologically relevant (100 pM) IL2 doses. To examine if tyrosine kinase activity was resident within the IL2 receptor complex, cell-free phosphorylation assays were performed with ligand-receptor complexes following cross-linking with IL2 and purification by immunoabsorption with an anti-IL2 antibody. Tyrosine kinase activity was found specifically associated with the IL2 receptor complex. These results indicate that the IL2 receptor complex contains a tyrosine kinase activity that is induced by IL2 binding and suggest that components of the complex may be a substrate of this activity.

Neonatal exposure to Gross murine leukemia virus results in a profound inhibition of the virus-specific T and B cell responses of adult animals. Animals exposed to virus as neonates exhibit a marked depression in virus-specific T cell function as measured by the virtual absence of in vivo delayed type hypersensitivity responses and in vitro proliferative responses to virally infected stimulator cells. Further, serum obtained from neonatally treated mice failed to either immunoprecipitate viral proteins or neutralize virus in an in vitro plaque assay, suggesting the concurrent induction of a state of B cell hyporesponsiveness. The specificity of this effect at the levels of both T and B cells was demonstrated by the ability of neonatally treated mice to respond normally after adult challenge with either irrelevant reovirus or influenza virus. The replication of Gross virus within both stromal and lymphocytic compartments of the neonatal thymus suggests that thymic education plays a key role in the induction of immunologic nonresponsiveness to viruses.

The thymus is thought to play an important role in the pathogenesis of myasthenia gravis (MG), an autoimmune disease characterized by skeletal muscle weakness. However, its role remains a mystery. The studies described represent our efforts to determine how intrathymic expression of the neuromuscular type of acetylcholine receptors (nAChRs) is involved in the immunopathogenesis of MG. We review our work characterizing the expression of the alpha subunit of nAChR (nAChRalpha) in the thymus and advance a new hypothesis that examines the intrathymic expression of this autoantigen in disease pathogenesis.

There is a large body of circumstantial evidence highlighting a primary role of the thymus in the pathogenesis of MG. Nevertheless, the etiologic link remains to be forged. We are reexamining the hypothesis that AChR expressed in the thymus drives the pathogenic autoimmune response. To this end, we have established a model of intrathymic inflammation that is localized to the thymic medulla and demonstrated that this inflammatory process promotes the nonspecific entry of peripheral CD4+ T cells into the thymus. Using this model, we are in the process of determining whether (a) AChR-reactive CD4+ T cell homing to the thymus is augmented by a concurrent intrathymic inflammatory response to an unrelated antigen, and (b) AChR-reactive T cell immigrants undergo activation following their engagement of autoantigen in this inflammatory milieu, provide help for the production of anti-AChR antibodies by immigrant autoreactive B cells, and thereby promote the development of a myasthenic syndrome.

Objective: Under some circumstances the HIV virus may infect cells that do not express receptors essential to HIV-entry. We hypothesized that platelet- and megakaryocyte-derived microparticles (MP) could play a role in such infections. MP are circular membrane fragments shed from the surface of eukaryotic cells. After adhesion to target cells, MP may transfer membrane-associated proteins to these cells. We found that peripheral blood platelet- (PMP) and megakaryocyte-derived MP (MegaMP) that highly express CXCR4 may transfer this receptor from the surface of platelets or megakaryocytes to the surface of CXCR4-null cells. Design: Since this mechanism could potentially allow CD4+/CXCR4-null cells to become infected by T-tropic HIV, we incubated several human CD4+/CXCR4-null cells such as normal erythroblasts, glioblastomas U87, MAGI and hematopoietic cell lines UT-7, HEL and TF-1 with PMP or MegaMP. We found that these cells became CXCR4+. We next exposed these cells to X4-HIV (IIIB) and evaluated their susceptibility to infection by PCR, ELISA, and morphological analysis. Results: We observed in all instances that after CD4+/CXCR4-null cell lines ‘acquired’ CXCR4 from PMP or MegaMP, they could became infected by X4 HIV. Conclusions: We postulate that both PMP and MegaMP may play a novel and important role in spreading HIV-1 infection by transferring the CXCR4 co-receptor to CD4+/CXCR4-null cells.

The strain and developmental parameters that control susceptibility to murine leukemia virus (MuLV)-induced intracerebral hemorrhages and infarction were studied using the endothelial cell tropic MuLV TR1.3. Inoculated animals displayed an absolute age dependence on the development of intracerebral vascular disease; however, other genetic determinants affected the timing and magnitude of susceptibility to neurologic disease. BALB/c mice were susceptible to neurologic disease only when inoculated prior to Day 4 postpartum. In contrast, Swiss/NIH and C3H/HeN mice consistently showed a less virulent phenotype and were only susceptible when infected prior to Day 3 postpartum. These studies demonstrate that susceptibility to TR1.3 murine leukemia virus-induced neurologic disease is regulated by age- and strain-dependent factors encoded within cerebral endothelial cells.

Glycosylated phosphatidylinositol (GPI) lipids are a structurally and functionally diverse molecular family. One of the most interesting and controversial aspects of GPI function is their ability to participate in signaling mechanisms or to directly function as second messengers of biological receptors. For example, while there is little dispute that subsets of GPI molecules are hydrolyzed following receptor ligation, there is no consensus as to the subsequent roles of intact GPI molecules or their cleavage products. The importance of these observations is underscored by two facts; many GPI anchored proteins participate in the regulation of cell proliferation, and several hormones metabolize GPI forms that are not linked to proteins. The purpose of this review is to outline the major structural and biological features of GPI molecules as they relate to their role in cellular signaling.

Tyrosine phosphorylation is thought to be one of the earliest steps in antigenic activation of T cells. Three nonreceptor tyrosine kinases, p56lck, p60fyn, and ZAP-70, are known to be involved in T cell receptor (TCR) signaling, albeit their functional roles appear to be different. Whereas p60fyn and ZAP-70 are functionally associated with the T cell antigen receptor, p56lck is essential for TCR signaling without being directly coupled to the TCR. We have studied a mutant variant of the Jurkat T cell line (J32–3.2), in which basal activities of p56lck and p60fyn are 2- to 2.5-fold reduced relative to those in its parental line (J32) while basal activity of ZAP-70 remains unchanged, and compared responses of J32–3.2 and J32 to TCR stimulation. We have demonstrated that tyrosine phosphorylation following CD3 cross-linking in J32–3.2 cells was extremely short-lived and thus insufficient for the induction of subsequent physiological responses. This was at least partially due to the diminished tyrosine kinase activity in these cells. A decrease in the activity of src-related kinases was caused primarily by their lower expression, whereas expression of ZAP-70 was unchanged but its response to CD3 crosslinking was diminished, correlating with the deficient tyrosine phosphorylation of the CD3 -chain, recently observed in J32–3.2. These data are consistent with the idea that sir-related kinases phosphorylate the -chain, which in turn recruits ZAP-70 required to sustain the signal. J. Leukoc. Biol. 55: 289–298; 1994.

The signalling mechanisms of the IL-2R remained an enigma until recent years. We now know that IL-2R are coupled to their own unique signalling pathways that complement rather than duplicate TCR signalling. The IL-2R beta- and gamma-chains are essential for signal coupling. Sequence comparisons indicate that portions of the extracellular and cytoplasmic domains of IL2R beta gamma molecules are homologous to several hematopoietic growth factor receptors, including erythropoietin. This may indicate that these receptors utilize common or related molecules in ligand binding or signal transduction. The cytoplasmic domain of the IL-2R beta-chain contains distinctive serine (S) and acidic (A) rich regions that participate in signalling. The overall scheme of IL-2R signalling is similar to other eukaryotic growth factors in that ligand binding activates a complicated and branching series of enzymatic steps that utilize protein tyrosine kinase (PTK) activation as a central component. More recent reports indicate that IL-2R are linked to additional membrane and cytosolic signalling molecules, including glycosylated phosphatidylinositol (GPI), phosphatidylinositol-3-kinase (PI3K), p74c-raf and p21ras. The regulation of these factors and their importance in IL-2 induced growth and differentiation awaits further study.

The biological effects of interleukin-2 (IL-2) were examined in T lymphocytes. IL-2 binding induced the rapid activation of diacylglycerol kinase in both cytosolic and membrane subfractions. This enzyme utilized diacylglycerol from multiple endogenous and exogenous sources for the synthesis of phosphatidic acid. Phosphatidic acid was, in turn, shown to stimulate the accumulation of c-myc mRNA and augment cellular proliferation when added to IL-2-dependent cell lines. These results link previous observations of IL-2 and glycosylphosphatidylinositol-dependent diacylglycerol production to phosphatidic acid accumulation, and suggest that diacylglycerol kinase activation is part of an intricate IL-2 signaling cascade that utilizes phosphatidic acid as an effector molecule.

Protooncogenes are the normal forms of cellular genes that when altered in their expression or coding sequences can contribute to neoplastic transformation. As these genes often are important for normal cellular growth control, we explored the possibility that protein kinases encoded by particular protooncogenes could participate in signal transduction pathways regulated by the T cell growth factor, interleukin-2 (IL-2). In this review we summarize our findings to date regarding Raf-1, a serine/threonine-specific kinase that becomes phosphorylated on tyrosine residues and enzymatically activated in response to IL-2 stimulation. In addition, we describe our investigations of Lck and Lyn, two closely related protein tyrosine kinases of the src gene family that physically associate with the IL-2 receptor complex and whose activities are regulated by IL-2 in at least some T cells and B cells, respectively., This work was supported in part by NIH Grant CA-54957.