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Many HIV-1-infected patients treated with protease inhibitors (PI) develop PI-resistant HIV-1 variants and rebounds in viremia, but their CD4+ T-cell counts often do not fall. We hypothesized that in these patients, T-cell counts remain elevated because PI-resistant virus spares intrathymic T-cell production. To test this, we studied recombinant HIV-1 clones containing wild-type or PI-resistant protease domains, as well as uncloned isolates from patients, in activated peripheral blood mononuclear cells, human thymic organ cultures and human thymus implants in SCID-hu Thy/Liv mice. In most cases, wild-type and PI-resistant HIV-1 isolates replicated to similar degrees in peripheral blood mononuclear cells. However, the replication of PI-resistant but not wild-type HIV-1 isolates was highly impaired in thymocytes. In addition, patients who had PI-resistant HIV-1 had abundant thymus tissue as assessed by computed tomography. We propose that the inability of PI-resistant HIV-1 to replicate efficiently in thymus contributes to the preservation of CD4+ T-cell counts in patients showing virologic rebound on PI therapy.

To better understand the relation of cytomegalovirus (CMV)-specific CD4+ T lymphocyte immunity and clinical outcome in AIDS-related CMV end-organ disease, 2 patient groups were prospectively studied: patients recently diagnosed with active CMV end-organ disease and survivors of CMV retinitis who had responded to highly active antiretroviral therapy and had quiescent retinitis when anti-CMV therapy was discontinued. Most patients with active CMV disease had negative CMV-specific CD4+ T lymphocyte responses at diagnosis, as measured by lymphoproliferation (7/7) or cytokine flow cytometry (3/5) assays. In contrast, all 10 subjects with quiescent retinitis and >150 absolute CD4+ T lymphocytes/microL whose anti-CMV therapy was discontinued during 6 months of follow-up had positive CMV-specific immune responses at least once by each assay. However, 6 of these 10 subjects also had negative CMV-specific immune responses > or =1 time. Such patients may be at risk for future CMV disease progression and should be closely monitored.

The size and composition of the CD4+ T-cell population is regulated by balanced proliferation of progenitor cells and death of mature progeny. After infection with the human immunodeficiency virus, this homeostasis is often disturbed and CD4+ T cells are instead depleted. Such depletion cannot result simply from accelerated destruction of mature CD4+ T cells - sources of T-cell production must also fail. Ironically, this failure may be precipitated by physiological mechanisms designed to maintain homeostasis in the face of accelerated T-cell loss.

Clinical histories are reported for 2 patients treated with highly active antiretroviral therapy (HAART) who experienced multiple relapses of cytomegalovirus (CMV) retinitis, despite suppression of human immunodeficiency virus type 1 (HIV-1) viremia and improvement in CD4+ T cell counts (to >400 cells/microL). CMV-specific CD4+ T cell immune reconstitution was measured directly, using cytokine flow cytometry, which revealed persistent deficits in CMV-specific CD4+ T cell responses in both patients. CMV-specific T cells constituted 0.14% and 0.05% of the total CD4+ T cell count in these patients, which is significantly lower than the percentages for 34 control subjects (0.6%-46%; CD4+ T cell count range, 7-1039 cells/microL; P=.019). Deficits in pathogen-specific immune responses may persist in some individuals, despite suppression of HIV-1 replication and substantial increases in circulating CD4+ T cells after HAART, and such deficits may be associated with significant morbidity from opportunistic infections.

Data from murine models of chronic viral infection suggest that CD4+ T-cell responses to viral pathogens are important in sustaining the number and/or function of CD8+ cytotoxic T-cell (CTL) effectors. In this study, we used cytokine flow cytometry (CFC), staining with HLA-A*0201-peptide tetramers, and peptide stimulation with epitopic peptides to study functional CD4+ and CD8+ T-cell responses to cytomegalovirus (CMV) in human subjects coinfected with CMV and the human immunodeficiency virus, type 1 (HIV-1). We show that strong CD4+ and CD8+ T-cell responses to CMV antigens are sustained over time in HIV-1-infected individuals. Those who maintain a strong CD4+ T-cell response to CMV are also likely to maintain higher frequencies of CD8+ T cells capable of binding to HLA-A*0201-CMV pp65 (A2-pp65) tetramers as well as responses to pp65 peptide stimulation with effector cytokine production. These data support the hypothesis that declines in frequencies of CD4+ T-cell responses to CMV are associated with an inability to sustain high levels of CMV-specific CD8+ T-cell responses in HIV-1-infected subjects. These declines may precede the onset of CMV-associated end organ disease.

We hypothesized that HIV-1-mediated T-cell loss might induce the production of factors that are capable of stimulating lymphocyte development and expansion. Here we perform cross-sectional (n = 168) and longitudinal (n = 11) analyses showing that increased circulating levels of interleukin (IL)-7 are strongly associated with CD4+ T lymphopenia in HIV-1 disease. Using immunohistochemistry with quantitative image analysis, we demonstrate that IL-7 is produced by dendritic-like cells within peripheral lymphoid tissues and that IL-7 production by these cells is greatly increased in lymphocyte-depleted tissues. We propose that IL-7 production increases as part of a homeostatic response to T-cell depletion.

Human herpesvirus 6 (HHV-6) has been proposed as a potential cofactor in the progression of human immunodeficiency virus type 1 (HIV-1) disease. We used the SCID-hu Thy/Liv mouse model to evaluate the in vivo interactions between HHV-6 and HIV-1. Our results demonstrate that HHV-6 and HIV-1 can simultaneously replicate in the human thymus in vivo. In this model, however, the presence of one virus appears not to modify the replication or cytopathicity of the other.

The pathogenic differences between CXCR4 (X4)- and CCR5 (R5)-utilizing strains of HIV-1 may be predominantly due to differences in viral tropism, which in turn may be due to differential coreceptor utilization. We tested this hypothesis in the human thymus organ of the SCID-hu Thy/Liv mouse, using recombinants of NL4-3 that were isogenic except for Env coreceptor-binding determinants of the V1-V3 loops. Conversion of NL4-3 from an X4 to an R5 isolate was associated with altered tropism for cell subpopulations within the Thy/Liv organ (with a higher frequency of infection of thymic stromal cells, including macrophages), a slower rate of replication, and a lower level of cytopathicity. These observations underscore the causal relationships between tropism, coreceptor use, and cytopathicity in the human thymus in vivo.

Human thymocytes at several stages of maturation express Fas, yet resist apoptosis induction through its ligation. A proximal step in apoptotic signaling through Fas is implicated in this resistance, as these cells undergo normal levels of apoptosis induction after exposure to tumor necrosis factor-alpha. We studied the Fas receptors expressed in human thymocytes to search for mechanisms of receptor-mediated inhibition of Fas signaling in these cells. We describe here a unique, membrane-bound form of Fas receptor that contained a complete extracellular domain of Fas but that lacked a death domain due to alternative splicing of exon 7. This Fas decoy receptor (FDR) was shown to have nearly wild-type ability to bind native human Fas ligand and was expressed predominantly at the plasma membrane. Unlike soluble forms of Fas receptor, FDR dominantly inhibited apoptosis induction by Fas ligand in transfected human embryonic kidney cells. Titration of FDR in Fas-expressing cells suggests that FDR may operate through the formation of mixed receptor complexes. FDR also dominantly inhibited Fas-induced apoptosis in Jurkat T cells. In mixing experiments with wild-type Fas, FDR was capable of inhibiting death signaling at molar ratios less than 0.5, and this relative level of FDR:wild type message was observed in at least some thymocytes tested. The data suggest that Fas signal pathways in primary human cells may be regulated by expression of a membrane-bound decoy receptor, analogous to the regulation of tumor necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis by decoy receptors.

HIV-1 disease is associated with pathological effects on T-cell production, destruction, and distribution. Using the deuterated (2H) glucose method for endogenous labeling, we have analyzed host factors that influence T-cell turnover in HIV-1-uninfected and -infected humans. In untreated HIV-1 disease, the average half life of circulating T cells was diminished without compensatory increases in cell production. Within 12 weeks of the initiation of highly active antiretroviral therapy (HAART), the absolute production rates of circulating T cells increased, and normal half-lives and production rates were restored by 12-36 months. Interpatient heterogeneity in the absolute degree of turnover correlated with the relative proportion of naive- and memory/effector-phenotype T cells in each of the CD4+ and CD8+ populations. The half-lives of naive-phenotype T cells ranged from 116-365 days (fractional replacement rates of 0.19-0.60% per day), whereas memory/effector-phenotype T cells persisted with half-lives from 22-79 days (fractional replacement rates of 0.87-3.14% per day). Naive-phenotype T cells were more abundant, and the half-life of total T cells was prolonged in individuals with abundant thymic tissue, as assessed by computed tomography. Such interpatient variation in T-cell kinetics may be reflective of differences in functional immune reconstitution after treatment for HIV-1 disease.

Oral administration of 2'-deoxy-3'-oxa-4'-thiocytidine (BCH-10652), a nucleoside analog structurally similar to lamivudine (3TC), caused dose-dependent inhibition of viral replication in SCID-hu Thy/Liv mice infected with human immunodeficiency virus type 1 NL4-3 and with an NL4-3 clone containing the M184V mutation in reverse transcriptase that confers resistance to 3TC. These experiments demonstrate the utility of this mouse model for evaluating drug resistance and for performing direct comparisons between antiviral compounds in vivo.

Human immunodeficiency virus (HIV) infection is associated with progressive loss of circulating CD4+ lymphocytes. Treatment with highly active antiretroviral therapy (HAART) has led to increases in CD4+ T lymphocytes of naive (CD45RA+62L+) and memory (CD45R0+RA-) phenotypes. Thymic computerized tomography scans were obtained on 30 individuals with HIV disease to investigate the role of the thymus in cellular restoration after 48 weeks of HAART. Individuals with abundant thymic tissue had higher naive CD4+ T lymphocyte counts at weeks 2-24 after therapy than individuals with minimal thymic tissue. Individuals with abundant thymic tissue had significantly larger increases in naive CD4+ cells during the first 4 weeks of therapy. These individuals were also more likely to experience viral rebound despite comparable initial declines in plasma HIV-1 RNA. These findings suggest that there is a complex relationship among the thymus, viral replication, and lymphocyte restoration after application of HAART in HIV disease.

Some individuals infected with only R5 strains of human immunodeficiency virus type 1 progress to AIDS as quickly as individuals harboring X4 strains. We determined that three R5 viruses were much less pathogenic than an X4 virus in SCID-hu Thy/Liv mice, suggesting that R5 virus-mediated rapid disease progression is associated with host, not viral, factors.

We investigated the expression and function of Fas on human thymocytes prepared from fetal and pediatric tissue specimens and from SCID-hu Thy/Liv grafts. Unlike mouse thymocytes, human thymocytes exhibited a pattern of Fas expression skewed to immature cells, in that the highest expression was seen on double negative thymocytes and on intrathymic T progenitor cells. Fas expression was intermediate on double positive human thymocytes, and low or negative on mature single positive CD4 and CD8 medullary thymocytes. In spite of this relatively abundant surface expression, cross-linking of Fas with agonist mAb was incapable of triggering an apoptotic signal in human thymocytes. Apoptotic signaling was not enhanced by treatment with cycloheximide, nor by restoring a cosignaling milieu by addition of thymic stromal cells. Mouse thymocytes were induced to apoptosis by cross-linked recombinant soluble human Fas ligand both in vitro and in vivo, though human thymocytes were also resistant to this mode of receptor ligation. Membrane-bound Fas ligand also induced apoptotic death in murine thymocytes but not in human thymocytes. Human thymocytes were as sensitive as Jurkat cells, however, to apoptosis induced by TNF-alpha, suggesting that these cells have a signaling defect before activation of the earliest caspases. These data demonstrate a durable and specific resistance of human thymocytes to apoptosis induced through Fas receptor engagement, and reveal significant species-specific differences in the biology of thymocyte-programmed cell death.

The SCID-hu Thy/Liv mouse and human fetal thymic organ culture (HF-TOC) models have been used to explore the pathophysiologic mechanisms of HIV-1 infection in the thymus. We report here that HIV-1 infection of the SCID-hu Thy/Liv mouse leads to the induction of MHC class I (MHCI) expression on CD4+CD8+ (DP) thymocytes, which normally express low levels of MHCI. Induction of MHCI on DP thymocytes in HIV-1-infected Thy/Liv organs precedes their depletion and correlates with the pathogenic activity of the HIV-1 isolates. Both MHCI protein and mRNA are induced in thymocytes from HIV-1-infected Thy/Liv organs, indicating induction of MHCI gene expression. Indirect mechanisms are involved, because only a fraction (<10%) of the DP thymocytes were directly infected by HIV-1, although the majority of DP thymocytes are induced to express high levels of MHCI. We further demonstrate that IL-10 is induced in HIV-1-infected thymus organs. Similar HIV-1-mediated induction of MHCI expression was observed in HF-TOC assays. Exogenous IL-10 in HF-TOC induces MHCI expression on DP thymocytes. Therefore, HIV-1 infection of the thymus organ leads to induction of MHCI expression on immature thymocytes via indirect mechanisms involving IL-10. Overexpression of MHCI on DP thymocytes can interfere with thymocyte maturation and may contribute to HIV-1-induced thymocyte depletion.

The dynamic basis for T-cell depletion in late-stage HIV-1 disease remains controversial. Using a new, non-radioactive, endogenous labeling technique, we report direct measurements of circulating T-cell kinetics in normal and in HIV-1-infected humans. In healthy, HIV-1-seronegative subjects, CD4+ and CD8+ T cells had half-lives of 87 days and 77 days, respectively, with absolute production rates of 10 CD4+ T cells/microl per day and 6 CD8+ T cells/microl per day. In untreated HIV-1-infected subjects (with a mean CD4 level of 342 cells/microl), the half-life of each subpopulation was less than 1/3 as long as those of healthy, HIV-1-seronegative subjects but was not compensated by an increased absolute production rate of CD4+ T cells. After viral replication was suppressed by highly active antiretroviral therapy for 12 weeks, the production rates of circulating CD4+ and CD8+ T cells were considerably elevated; the kinetic basis of increased CD4 levels was greater production, not a longer half-life, of circulating cells. These direct measurements indicate that CD4+ T-cell lymphopenia is due to both a shortened survival time and a failure to increase the production of circulating CD4+ T cells. Our results focus attention on T-cell production systems in the pathogenesis of HIV-1 disease and the response to antiretroviral therapy.

CCR5-utilizing (R5) and CXCR4-utilizing (X4) strains of human immunodeficiency virus type 1 (HIV-1) have been studied intensively in vitro, but the pathologic correlates of such differential tropism in vivo remain incompletely defined. In this study, X4 and R5 strains of HIV-1 were compared for tropism and pathogenesis in SCID-hu Thy/Liv mice, an in vivo model of human thymopoiesis. The X4 strain NL4-3 replicates quickly and extensively in thymocytes in the cortex and medulla, causing significant depletion. In contrast, the R5 strain Ba-L initially infects stromal cells including macrophages in the thymic medulla, without any obvious pathologic consequence. After a period of 3 to 4 weeks, Ba-L infection slowly spreads through the thymocyte populations, occasionally culminating in thymocyte depletion after week 6 of infection. During the entire time of infection, Ba-L did not mutate into variants capable of utilizing CXCR4. Therefore, X4 strains are highly cytopathic after infection of the human thymus. In contrast, infection with R5 strains of HIV-1 can result in a two-phase process in vivo, involving apparently nonpathogenic replication in medullary stromal cells followed by cytopathic replication in thymocytes.

HIV-1 disease is often associated with CD4+ T lymphopenia as well as quantitative reductions in naive CD8+ T cells and cytopenias involving nonlymphoid hemopoietic lineages. Studies in HIV-1-infected humans as well as in animal models of lenti-virus disease indicate that these effects may be secondary to infection and destruction of multilineage and lineage-restricted hemopoietic progenitor cells. To define the stages of T cell differentiation that might be susceptible to HIV-1, we performed flow cytometric analysis of the surface expression of CXCR4 and CCR5 on T cells and their progenitors from fetal tissue, cord blood, SCID-hu Thy/Liv mice, and adult peripheral blood. We found that CXCR4 is expressed at low levels on hemopoietic progenitors in the bone marrow, is highly expressed on immature (CD3-CD4+CD8-) T cell progenitors in the thymus, and then is down-regulated during thymocyte differentiation. As thymocytes leave the thymus and enter the peripheral circulation, the expression of CXCR4 is again up-regulated. In contrast, CCR5 is undetectable on most hemopoietic progenitors in the bone marrow and on intrathymic T progenitor cells. It is up-regulated when thymocytes coexpress CD4 and CD8, then down-regulated either in the thymus (CD4+ cells) or during exit from the thymus (CD8+ cells). These results indicate that discrete, lineage-related populations of T cell progenitors may vary widely in their potential to respond to chemokines and to be infected by HIV-1, and that T lymphoid differentiation is particularly vulnerable to CXCR4-using viruses.

Recent studies of subjects infected with human immunodeficiency virus (HIV-1) have produced conflicting results about the extent of reconstitution possible in the CD4+ lymphocyte repertoire after highly active antiretroviral therapy (HAART). The effect of HAART on the incidence of opportunistic infections will probably depend on reconstitution of antigen-specific CD4+ lymphocyte responses to important pathogens, including cytomegalovirus (CMV), the leading cause of blindness in AIDS. Several studies have demonstrated an important role for CD4+ lymphocytes in controlling CMV replication in vitro and in clinical studies. It is now possible to quantitate antigen-specific CD4+ lymphocyte responses by flow cytometry. Using this method, we studied CMV-specific CD4+ lymphocyte responses in individuals infected with HIV-1 with and without a history of active CMV-associated end organ disease (EOD), and in those with quiescent CMV EOD after ganciclovir therapy and HAART. The presence of active CMV-associated EOD strongly correlated with loss of CMV-specific lymphocyte responses (P = 0.0004). In contrast, patients with no history of CMV-associated EOD and most patients with quiescent EOD after HAART demonstrated strong CMV-specific CD4+ lymphocyte responses. These data indicate that the loss of CMV-specific CD4+ lymphocyte responses in individuals infected with HIV-1 who have active CMV EOD may be restored after ganciclovir therapy and HAART, which provides evidence for functional immune reconstitution to an important pathogen.

Viral replication was inhibited in a dose-dependent manner after administration of the phosphorothioate oligonucleotide TTGGGGTT (ISIS 5320) to human immunodeficiency virus type 1 (HIV-1)-infected SCID-hu Thy/Liv mice. Potent in vivo antiviral activity was observed against the T-cell-tropic molecular clone NL4-3; the agent was found to have weak activity against one primary HIV-1 isolate, and the agent was inactive against a second primary isolate.

The thymus in adults infected with the HIV-1 is generally thought to be inactive, both because of age-related involution and viral destruction. We have revisited the question of thymic function in adults, using chest-computed tomography (CT) to measure thymic tissue in HIV-1-seropositive (n = 99) or HIV-1-seronegative (n = 32) subjects, and correlating these results with the level of circulating CD4(+) and CD8(+) T cells that are phenotypically described as naive thymic emigrants. Abundant thymic tissue was detectable in many (47/99) HIV-1-seropositive adults, aged 20-59. Independent of age, radiographic demonstration of thymic tissue was significantly associated with both a higher CD4(+) T cell count (P = 0.02) and a higher percentage and absolute number of circulating naive (CD45RA+CD62L+) CD4(+) T cells (P < 0.04). The prevalence of an abundant thymus was especially high in younger HIV-1-seropositive adults ( 40 yr) regardless of CD4 count (P = 0.03). These studies suggest that the thymus is functional in some but not all adults with HIV-1 disease.

It is still uncertain whether multilineage hematopoietic progenitor cells are affected by human immunodeficiency virus-1 (HIV-1) infection in vivo. The SCID-hu Thy/Liv model is permissive of long-term multilineage human hematopoiesis, including T lymphopoiesis. This model was used to investigate the effects of HIV-1 infection on early hematopoietic progenitor function. We found that both lineage-restricted and multilineage hematopoietic progenitors were depleted from grafts infected with either a molecular clone or a primary isolate of HIV-1. Depletion of hematopoietic progenitors (including CD34(+) cells, colony-forming units in methylcellulose, and long-term culture-initiating cells) occurred several days before the onset of thymocyte depletion, indicating that the subsequent rapid decline in thymocyte numbers was due at least in part to loss of thymocyte progenitors. HIV-1 proviral genomes were not detected at high frequency in hematopoietic cells earlier than the intrathymic T-progenitor cell stage, despite the depletion of such cells in infected grafts. Proviral genomes were also not detected in colonies derived from progenitor cells from infected grafts. These data demonstrate that HIV-1 infection interrupts both lineage-restricted and multilineage hematopoiesis in vivo and suggest that depletion of early hematopoietic progenitor cells occurs in the absence of direct viral infection.

The thymus involutes over time and is generally considered to be non-functional in adults. If this is the case, T-cell depletion in adult HIV-1 disease would in part be due to insufficient intrathymic production. This review summarizes information relevant to the structure and function of the thymus under normal conditions and in the setting of infection with HIV-1. The speculation is raised: could residual thymic reserve be present in some adults and be inducible during pathophysiological states of T-cell depletion? If so, the contribution of thymopoiesis during adult HIV-1 disease may be more significant than otherwise assumed., (Copyright 1997 Academic Press Limited.)

Infection by HIV-1 has been associated with perturbations in the TCR V beta repertoire, suggesting the involvement of a superantigen. Among the hallmarks of superantigens is the capacity to delete T cells bearing specific TCR V beta families in the developing thymus. To verify the presence of a superantigen in HIV-1, we analyzed the SCID-hu Thy/Liv TCR V beta repertoire within CD4+CD8+, CD4+CD8-, or CD4-CD8+ thymocytes subsets by flow cytometry using a panel of Abs recognizing about 60% of the TCR repertoire following injection of SEB or infection by two different HIV-1 isolates. Seven days following SEB injection, thymocyte subsets bearing TCR V beta 3, V beta 12, V beta 17, and V beta 20, but not V beta 5 or V beta 8 , were deleted relative to mock-injected mice. In contrast, no changes were observed in the TCR V beta repertoire in CD4+CD8+, CD4+CD8-, or CD4-CD8+ thymocyte subsets after infection with HIV-1. The T cell depletion caused by HIV-1 infection is most likely not mediated by an HIV-encoded superantigen.

Pathogenic organisms are frequently attenuated after long-term culture in vitro. The mechanisms of the attenuation process are not clear, but probably involve mutations of functions required for replication and pathogenicity in vivo. To identify these functions, a direct comparison must be made between attenuated genomes and those that remain pathogenic in vivo. In this study, we used the heterochimeric SCID-hu Thy/Liv mouse as an in vivo model to define human immunodeficiency virus type 1 (HIV-1) determinants which are uniquely required for replication in vivo. The Lai/IIIB isolate and its associated infectious molecular clones (e.g., HXB2) were found to infect T cell lines but failed to replicate in the SCID-hu Thy/Liv model. When a lab worker was accidentally infected by Lai/IIIB, however, HIV-1 was isolated only from infection of primary PBMC, and not from infection of T cell lines. We hypothesized that the lab worker was exposed to a heterogeneous viral stock which had been attenuated by passage in immortalized T cell lines. Either a rare family member from this stock was selected for in vivo replication or, alternatively, an attenuated genotype dominant in vitro may have reverted to become more infectious in vivo. To address this hypothesis, we have used the SCID-hu Thy/Liv model to study the replication of HXB2 and of HXB2 recombinant viruses with HIV-1 fragments isolated from the infected lab worker. HXB2 showed no or very low levels of replication in the Thy/Liv organ. Replacement of its subgenomic fragment encoding the envelope gene with a corresponding fragment from the lab worker isolate generated a recombinant virus (HXB2/LW) which replicated actively in SCID-hu mice. The NEF mutation in the HXB2 genome is still present in HXB2/LW. Thus, the LW sequences encode HIV-1 determinants which enhance HIV replication in vivo in a NEF-independent mechanism. The specific determinants have been mapped to the V1-V3 regions of the HIV-1 genome. Six unique mutations in the V3 loop region of HXB2/LW have been identified which contribute to the increased replication in vivo.

The SCID-hu mouse was designed to serve as a preclinical model for the analysis of human physiology and pathophysiology. In distinction to other immunodeficient mouse models, SCID-hu mice are made upon implantation of intact human organ systems rather than dispersed cell populations. Two constructs, the SCID-hu Thy/Liv and the SCID-hu Bone models, have been developed to reproduce the differentiation and function of human hematopoietic progenitor cells within the human thymus and bone marrow microenvironments, respectively. This review focuses on several applications of these models: definition of multilineage and lineage-restricted human hematopoietic progenitor cells and evaluation of HIV disease and its treatment.

Mortality from measles is caused mostly by secondary infections associated with the depression of cellular immunity. The mechanism of immune suppression and the role of virus strain differences on the immune system are incompletely understood. SCID-hu mice were used to determine the effects of virulent, wild-type (Chicago-1) and avirulent, vaccine (Moraten) strains of measles virus (MV) on the human thymus in vivo. Chicago-1 replicated rapidly, with a 100-fold decrease in numbers of thymocytes, whereas Moraten replicated slowly, without significant thymocyte death. Productive MV infection occurred not in thymocytes but in thymic epithelial and myelomonocytic cells. Wild-type MV infection of thymic stromata leads to induction of thymocyte apoptosis and may contribute to a long-term alteration of immune responses. The extent of thymic disruption reflects the virulence of the virus, and therefore the SCID-hu mouse may serve as the first small animal model for the study of MV pathogenesis.

SID 791, a bicyclam inhibiting human immunodeficiency virus (HIV) replication in vitro by blocking virus entry into cells, is an effective inhibitor of virus production and of depletion of human CD4+ T cells in HIV type 1-infected SCID-hu Thy/Liv mice. Steady levels of 100 ng of SID 791 or higher per ml in plasma resulted in statistically significant inhibition of p24 antigen formation. Daily injections of SID 791 caused a dose-dependent decrease in viremia, and this inhibition could be potentiated by coadministration of zidovudine or didanose. The present study suggests that SID 791 alone or in combination with licensed antiviral agents may decrease the virus load in HIV-infected patients and, by extension, that the infectious cell entry step is a valid target for antiviral chemotherapy of HIV disease. The SCID-hu Thy/Liv model in effect provides a rapid means of assessing the potential of compounds with novel modes of antiviral action, as well as the potential of antiviral drug combinations.

We have developed standardized procedures and practices for infection of SCID-hu Thy/Liv mice with human immunodeficiency virus type 1 for the prophylactic administration of antiviral compounds and for evaluation of the antiviral effect in vivo. Endpoint analyses included quantitation of viral load by intracellular p24 enzyme-linked immunosorbent assay, DNA PCR for the presence of proviral genomes, flow cytometry to measure the representation of CD4+ and CD8+ cells, and cocultivation for the isolation of virus. Efficacy tests in this model are demonstrated with the nucleoside analogs zidovudine and dideoxyinosine and with the nonnucleoside reverse transcriptase inhibitor nevirapine. This small-animal model should be particularly useful in the preclinical prioritization of lead compounds within a common chemical class, in the evaluation of alternative in vivo dosing regimens, and in the determination of appropriate combination therapy in vivo.

The development of effective therapies for the treatment of AIDS would be facilitated by a better understanding of HIV pathogenesis in vivo. While some aspects of pathogenesis may be assessed by standard tissue culture assays, in vivo animal models may provide clues to other aspects of HIV-mediated progression toward AIDS. Current animal models include primate models for the study of simian immunodeficiency virus (SIV) and HIV, SCID-hu and hu-PBL SCID mouse models for the study of HIV, and feline models for the study of feline immunodeficiency virus (FIV). In general these models are costly and labor intensive. We have developed a simple human fetal thymic organ culture (TOC) system that is permissive for HIV infection and that exhibits pathology similar to that observed in vivo. A key feature of this system is the time-dependent destruction of thymocytes typified by the preferential loss of CD4-expressing cells. HIV-mediated thymocyte destruction occurs by a process involving programmed cell death. We have infected TOC with a panel of HIV isolates and found that the resulting viral replicative and pathogenic profiles are similar to those seen in the SCID-hu Thy/Liv mouse, yet different from profiles observed in standard PHA-blast tissue culture assays. In addition, we find that TOC may be used to assess efficacy of antiviral agents such as AZT (3'-azido-3'-deoxythymidine) and ddI (2',3'-dideoxyinosine) in blocking both viral replication and virus-induced pathology. These results indicate that this model is amenable to the systematic manipulation, analysis, and characterization of a variety of HIV virus isolates and antiviral therapies.

Direct and indirect cytopathic mechanisms have been proposed to account for the loss of CD4+ T cells after infection with human immunodeficiency virus type 1 (HIV-1). We report here that HIV-1 infection of the human thymus in vivo results in thymocyte depletion by at least two different mechanisms. Thymocytes within multiple stages of differentiation are induced to die of apoptosis; most of these cells are uninfected. Additionally, thymopoiesis is interrupted by direct infection and destruction of intrathymic CD3-CD4+CD8- progenitor cells. These mechanisms are differentially induced by distinct isolates of HIV-1.

Clinical deterioration in human immunodeficiency virus type 1 (HIV-1) disease is associated with an increased viral burden in the peripheral blood and a loss of circulating CD4+ T cells. HIV-1 isolates obtained prior to this stage of disease often have a "slow-low," non-syncytium-inducing (NSI) phenotype, whereas those obtained afterwards are often characterized as "rapid-high" and syncytium inducing (SI). Paired NSI and SI isolates from two different patients were inoculated into the human thymus implants of SCID-hu mice. The two slow-low, NSI isolates replicated to minimal levels in the grafts and did not induce thymocyte depletion. In contrast, the two SI isolates from the same patients showed high levels of viral replication and induced a marked degree of thymocyte depletion, accompanied by evidence of programmed cell death. These observations reveal a correlation between the replicative and cytopathic patterns of HIV-1 isolates in vitro and in the SCID-hu mouse in vivo and provide direct evidence that the biological phenotype of HIV-1 switch may be a causal and not a derivative correlate of HIV-1 disease progression.

The severe combined immunodeficient C.B.-17 scid/scid (SCID) mouse has been widely used to study the normal processes of murine lymphoid differentiation. To create an in vivo model of the human hematolymphoid system, this mouse strain has been engrafted with human organ systems (the SCID-hu mouse) or with human peripheral blood mononuclear cells (the hu-PBL-SCID mouse). These mouse models have now been characterized and used to analyze human infectious diseases, hematopoiesis, malignancies and vaccines.

Human immunodeficiency virus type 1 (HIV-1) infection is highly specific for its human host. In order to study HIV-1 infection of the human nervous system, we have established a small animal model in which second-trimester (11-17.5 weeks) human fetal brain or neural retina is transplanted into the anterior chamber of the eye of immunosuppressed adult rats (Epstein et al., 1992; Cvetkovich et al., 1992), and more recently in immunodeficient (SCID) mice. The human xenografts survive for many months, vascularize and form a blood-brain barrier. Immunohistochemistry with PGP 9.5 identified neuronal cell bodies and neuritic processes. Electron microscopy revealed axonal growth cones and synaptic junctions. Infection of these xenografts with cell-free HIV-1 proved difficult, however co-engraftment with HIV-1-infected human monocytes resulted in characteristic pathological changes, including the formation of syncytial giant cells, neuronal loss, and astroglial proliferation, supporting the hypothesis that these cells can mediate neurotoxicity. In other studies, xenografts of human fetal retinal tissue were readily infected with cell-free human cytomegalovirus (HCMV) strain AD169. These grafts contained cells with intracytoplasmic and intranuclear inclusions typical of HCMV infection. Productive infection within these grafts was demonstrated by the presence of immediate early, and late (capsid) HCMV antigens, by recovery of HCMV on human fibroblast cultures, and by serial passage of virus to additional retinal xenografts (DiLoreto et al., 1994).(ABSTRACT TRUNCATED AT 250 WORDS)

We have developed techniques by which normal functional elements of human bone marrow can be implanted into immunodeficient C.B-17 scid/scid (SCID) mice. Afterward, long-term multilineage human hematopoiesis is sustained in vivo. We evaluated the effect of irradiation on the function of human bone marrow with this in vivo model. After whole-body X irradiation of the engrafted animals, it was determined that the D0 value of human committed progenitor cells within the human marrow was 1.00 +/- 0.09 (SEM) Gy for granulocyte-macrophage colony-forming units (CFU-GM) and 0.74 +/- 0.12 Gy for erythroid burst-forming units (BFU-E). The effects of irradiation on the hematopoietic elements were reduced when the radioprotective agent WR-2721 was administered prior to irradiation. After low-dose irradiation, recovery of human myelopoiesis was accelerated by treatment with human granulocyte colony-stimulating factor (G-CSF). This small animal model may prove amenable for the analysis of the risk of the exposure of humans to radiation as well as for the development of new modalities for the prevention and treatment of radiation-induced hematopoietic damage.

Human immunodeficiency virus (HIV) disease is typified by declining CD4+ T lymphocyte counts in the peripheral circulation, a loss which may be secondary to accelerated destruction, to suppressed differentiation, and/or to sequestration of circulating cells into tissue spaces. As it is hard to distinguish between these possibilities in human subjects, the pathogenic mechanisms associated with HIV infection are unclear. In particular, little is known about the events that occur within infected lymphoid organs in which most CD4 T lymphocytes mature and function. To obtain a better description of HIV pathogenesis in vivo, we have implanted human haematolymphoid organs into the immunodeficient SCID mouse to create the SCID-hu mouse. We have previously shown that these organ systems promote long-term multilineage human haematopoiesis and are permissive for infection with HIV. Here we report that human thymopoiesis is suppressed by HIV infection, thereby precluding regeneration of the peripheral T-cell compartment.

Animal models of human cytomegalovirus (CMV) infections have not been available to study pathogenesis or to evaluate antiviral drugs. Severe combined immunodeficient mice implanted with human fetal tissues (SCID-hu) were found to support CMV replication and may provide a model for this species-specific virus. When conjoint implants of human fetal thymus and liver were inoculated with a low-passage-number isolate of CMV, strain Toledo, consistent high-level viral replication was detected 5, 12, 15, 28, and 35 days after inoculation and virus replication continued for up to 9 months. Other human tissue implants, including lung and colon, were also found to support viral growth but with greater variability in levels and for a shorter duration. As expected, the species specificity of human CMV was preserved in this model such that virus was detected in the human conjoint thymus/liver implant but not in surrounding mouse tissues. The majority of virus-infected cells were localized in the thymic medulla rather than cortical region of the implant and immunofluorescence analysis identified epithelial cells rather than any hematopoietic cell population as the principal hosts for viral replication. Finally, treatment of infected animals with ganciclovir reduced viral replication, thereby demonstrating the value of this system for evaluating antiviral therapies. This animal model opens the way for a range of investigations not previously possible with human CMV.

Work in the SCID-hu system has progressed significantly over the past 3-4 years. Reliable SCID-hu constructs can now be created for the evaluation of long-term multilineage human hematopoiesis, human immune function, and HIV infection of human hematolymphoid organs in vivo. Given these constructs, a variety of therapeutically important modalities can now be discovered and developed with a relevant in vivo model, prior to the time that they are moved into the clinic. With further progress in this and other preclinical models, useful therapies for important human disease states will hopefully be forthcoming at an even faster pace.

Severe combined immunodeficiency (SCID) mice can be transplanted successfully with human fetal liver and thymus (SCID-hu mice). Precursor cells derived from the fetal liver differentiate in the thymus and migrate into the blood as mature T cells. In the present paper, the peripheral T cell compartment of such mice was studied. Peripheral WBC were activated by PHA and cultured in the presence of irradiated human feeder cells. The resultant cell population consisted exclusively of human CD1- CD2+ CD3+ CD7+ T lymphocytes; up to 4% of the T cells expressed the TCR gamma delta, whereas 95 to 100% were TCR alpha beta +. The CD4bright (42 to 66%) and CD8bright (30 to 54%) populations coexpressed variable but low levels of CD8 and CD4, respectively. The T cell cultures from the SCID-hu mice did not display reactivity towards the autologous human EBV-transformed B cell lines (B-LCL). On the other hand, these human T cells proliferated and were cytotoxic against allogeneic human B-LCL. T cell clones were established from cultured SCID-hu T cells. All T cell clones were TCR alpha beta + CD3+ CD2+; 61% of the clones were CD4+ CD8-, 27% were CD8+ CD4-, 11% were CD8+ CD4lo, and 2% were CD4+ CD8lo. None of these clones recognized the autologous B-LCL established from the fetal human donor. Fourteen of 100 T cell clones had specific alloreactivity, as tested on a panel of five B-LCL. Of these 14, two CD8+ CD4lo and two CD8+ CD4- clones were cytotoxic and did not proliferate in response to specific stimulator cells. Furthermore, two CD4+ CD8lo and eight CD4+ CD8- clones proliferated specifically in response to alloantigens. In conclusion, the peripheral human T cells of SCID-hu animals are functional and their TCR repertoire is polyclonal, alloreactive, and devoid of self-reactive cells. Therefore, the SCID-hu mouse can be a suitable model for the study of alloreactivity and allotolerance in vivo, as well as for the study of negative selection in the human thymus.

SCID-hu mice are heterochimeric animals that are constructed by transplanting human fetal thymus (Thy), liver (Liv), and/or lymph nodes into congenitally immunodeficient C.B-17 scid/scid (SCID) mice. Sensitive and specific two-color flow cytometric assays were used to evaluate human lymphocytes from peripheral blood of SCID-hu mice. Kinetic studies presented in this report show long term T lymphopoiesis in SCID-hu mice. Approximately one-half of SCID-hu mice constructed with Thy and Liv tissue develop detectable levels of circulating human T cells by 4 mo after transplantation. The average level of circulating human cells in SCID-hu mice is generally less than 2% of the total lymphoid cells in the peripheral blood of these mice. Some SCID-hu mice with as high as 13% human lymphocytes, however, have been detected. Nearly all human cells in the peripheral blood of SCID-hu mice are CD3+ cells that express TCR-alpha beta. The percentages of gamma delta+, CD4+, CD8+, CD25+, CD69+, and Leu-8+ cells among CD45+ cells in SCID-hu blood are similar to the levels found in adult peripheral blood. On average, 74% of SCID-hu T cells express CD45RA and 18% express CD29. Functional studies demonstrate that cells from SCID-hu Thy/Liv grafts or human T cells from SCID-hu peripheral blood are functionally competent to respond to mitogens or allogeneic human cells in vitro. They are similar to fetal thymocytes or adult T cells, respectively, in these responses. These studies demonstrate that the SCID-hu mouse is a useful model for the analysis of human immune differentiation and function in vivo.

The SCID-hu mouse is a small animal in which human hematolymphoid organs can be engrafted and maintained in vivo. In this study, parameters are described for reproducible infection of SCID-hu mice after i.v. inoculation. Infection was found to be dependent upon the time after inoculation, the virus isolate, the titer of virus, and the human target organ implanted into the mouse. Ten to 14 days after the i.v. administration of HIV isolates derived freshly from patients (e.g., JR-CSF, JR-FL, SM), 100% of engrafted human lymph nodes in SCID-hu mice were infected; greater than 95% of these animals were also viremic. Implants of human thymus or connective tissue, as well as the endogenous murine hematolymphoid organs, were not infected. As demonstrated by a combination of in situ hybridization and immunohistochemistry, both T-lymphoid and myelomonocytic lineage cells were infected in this system. HIV isolates that have been adapted to growth in vitro (e.g., HTLV-IIIb) were not infectious. When either 3'-azido-3'-deoxythymidine (AZT) or 2',3'-dideoxyinosine (ddIno) was administered to SCID-hu mice before HIV infection, the animals were protected in dose ranges similar to those used in man. This animal model may now be used as an efficient intermediate step between the lab and the clinic to study the infectious process in vivo and to best select efficacious antiviral compounds against HIV.