Progress toward a human CD4/CCR5 transgenic rat model for de novo infection by human immunodeficiency virus type 1.

The development of a permissive small animal model for the study of human immunodeficiency virus type (HIV)-1 pathogenesis and the testing of antiviral strategies has been hampered by the inability of HIV-1 to infect primary rodent cells productively. In this study, we explored transgenic rats expressing the HIV-1 receptor complex as a susceptible host. Rats transgenic for human CD4 (hCD4) and the human chemokine receptor CCR5 (hCCR5) were generated that express the transgenes in CD4(+) T lymphocytes, macrophages, and microglia. In ex vivo cultures, CD4(+) T lymphocytes, macrophages, and microglia from hCD4/hCCR5 transgenic rats were highly susceptible to infection by HIV-1 R5 viruses leading to expression of abundant levels of early HIV-1 gene products comparable to those found in human reference cultures. Primary rat macrophages and microglia, but not lymphocytes, from double-transgenic rats could be productively infected by various recombinant and primary R5 strains of HIV-1. Moreover, after systemic challenge with HIV-1, lymphatic organs from hCD4/hCCR5 transgenic rats contained episomal 2-long terminal repeat (LTR) circles, integrated provirus, and early viral gene products, demonstrating susceptibility to HIV-1 in vivo. Transgenic rats also displayed a low-level plasma viremia early in infection. Thus, transgenic rats expressing the appropriate human receptor complex are promising candidates for a small animal model of HIV-1 infection.

Haploinsufficiency identifies STAT5 as a modifier of IL-7-induced lymphomas.

The requirement for receptor components and the signalling effector, signal transducer and activator of transcription (STAT) 5A/5B, was assessed genetically in a lymphoma development model induced by interleukin-7 (IL-7). This growth factor for T- and B-cell progenitors and mature lymphocytes activates survival and proliferative pathways including Bcl-2, phosphatidylinositol-3 kinase and STAT5. Overexpression of IL-7 in vivo causes early mortality from lymphoma development. Mice overexpressing IL-7 that were heterozygous for the IL-7Ralpha subunit showed improved survival compared to wild-type mice. In addition, STAT5A/5B+/- compound heterozygous mice with one targeted allele each of STAT5A and STAT5B showed striking amelioration of IL-7-induced mortality and disease development. STAT5A/5B+/- compound heterozygous mice were otherwise normal in stem cell and lymphocyte development and cellularity. Lower STAT5 protein levels accompanied the reduction in STAT5A/5B copy number, which suggests that STAT5 haploinsufficiency is a modifier of IL-7 signal strength.

Injection of human primary effusion lymphoma cells or associated macrophages into severe combined immunodeficient mice causes murine lymphomas.

The pathogenesis of immunodeficiency-associated lymphoma is poorly understood. During the past several years, numerous lines of evidence implicating a multistep process of malignant transformation, also known as sequential pathogenesis, have emerged. Tumor-associated macrophage production of specific lymphostimulatory products has been demonstrated and hypothesized to be central to this process. While attempting to establish primary effusion lymphoma in severe combined immunodeficient (SCID) mice, we discovered a potential model of murine lymphomagenesis consistent with the sequential pathogenesis model. This pathogenesis-based model of lymphoma could significantly impact the current thinking about posttransplantation and other immunodeficiency-related lymphoproliferative disorders. Human primary effusion lymphoma-derived CD14+ cell-injected SCID mice developed aggressive murine large cell lymphomas. Tumor cell preparations containing CD14 cells or isolated CD14 cells induced lymphoma/lymphoproliferative diseases in 74% (20 of 27) of injected SCID mice. No tumors were induced by tumor-associated CD3 cells (0 of 4), normal human macrophages (0 of 13), or a murine macrophage cell line (0 of 10). Human macrophages were detected in tumor-bearing animals up to 6 months postinjection in association with the murine T-cell tumors but were not detected in controls or unaffected animals. These observations are consistent with the macrophage-initiated sequential pathogenesis model of disease (M. S. McGrath et al., Acquir. Immune Defic. Syndr., 8: 379-385, 1995; M. S. McGrath et al., Infectious Causes of Cancer: Targets for Intervention, pp. 231-242, Totowa, NJ: Humana Press, 2000).

Development of WF10, a novel macrophage-regulating agent.

WF10 represents a new class of drug involved in regulating macrophage function both in vitro and in vivo. In the US, WF10 is being evaluated in patients with advanced HIV infection as an adjunct to highly active antiretroviral therapy (HAART). To date, most therapeutic efforts to treat HIV infection have focused on inhibition of viral replication with the goal of decreasing viral load. The introduction of HAART was associated with a dramatic decline in AIDS-related mortality; however, recent indications suggest that the trend maybe changing. WF10, which contains chlorite as the active principle, causes profound changes in macrophage function and activation of gene expression, and appears to downregulate inappropriate immunological activation. The loss of T-cell function observed in HIV-infected patients likely requires the involvement of chronically activated macrophages. Therefore, the persistently activated macrophage represents a therapeutic target that is, unlike HIV, not highly mutable. With this target as a focus, WF10 is being developed for use in advanced HIV disease. WF10 is currently being studied in the US, Europe and Asia for treatment of late-stage HIV disease, as well as recurrent prostate cancer, late post-radiation cystitis, autoimmune disease and chronic active hepatitis C disease.

Expression of the diffuse B-cell lymphoma family molecule SWAP-70 in human B-cell neoplasms: immunohistochemical study of 86 cases.

SWAP-70 is a recently discovered member of the Dbl (diffuse B-cell lymphoma) family of signal transduction molecules that is abundantly expressed in B cells. SWAP-70 mediates lipid second-messenger signals to the cytoskeletal-organizing GTPase Rac, functioning as a guanine-nucleotide exchange factor. SWAP-70 is strongly expressed in germinal center B cells, with low-level expression in resting B-cells. Expression of SWAP-70 in neoplastic B cells has not been described. We report the immunohistochemical expression of SWAP-70 in 86 B-cell neoplasms. SWAP-70 was strongly expressed in 59 of the 86 cases: 2 of 10 (20%) precursor B-cell lymphoblastic leukemias, 2 of 2 (100%) precursor B-cell lymphoblastic lymphomas, 2 of 4 (50%) mantle cell lymphomas, 7 of 9 (78%) Burkitt lymphomas, 9 of 9 (100%) diffuse large B-cell lymphomas, 8 of 8 (100%) follicular lymphomas, 6 of 6 (100%) nodular lymphocyte predominant Hodgkin lymphomas, 0 of 8 (0%) classic Hodgkin lymphomas, 12 of 13 (92%) chronic lymphocytic leukemias, 3 of 3 (100%) nodal marginal zone lymphomas, 5 of 5 (100%) extranodal marginal zone lymphomas, 1 of 2 (50%) splenic marginal zone lymphomas, 2 of 3 (66%) hairy cell leukemias, and 0 of 4 (0%) plasma cell neoplasms. All 4 T-cell lymphomas were nonreactive for SWAP-70: 0 of 3 peripheral T-cell lymphomas and 0 of 1 anaplastic large cell lymphoma. These results suggest that a spectrum of neoplastic B cells maintains activation of this signal transduction pathway. This is the first report of the expression of a Dbl family molecule in human lymphoma and leukemia tissues.

Productive infection of plasmacytoid dendritic cells with human immunodeficiency virus type 1 is triggered by CD40 ligation.

Immature plasmacytoid dendritic cells are the principal alpha interferon-producing cells (IPC), responsible for primary antiviral immunity. IPC express surface molecules CD4, CCR5, and CXCR4, which are known coreceptors required for human immunodeficiency virus (HIV) infection. Here we show that IPC are susceptible to and replicate HIV type 1 (HIV-1). Importantly, viral replication is triggered upon activation of IPC with CD40 ligand, a signal physiologically delivered by CD4 T cells. Immunohistochemical staining of tonsil from HIV-infected individuals reveals HIV p24(+) IPC, consistent with in vivo infection of these cells. IPC exposed in vitro to HIV produce alpha interferon, which partially inhibits viral replication. Nevertheless, IPC efficiently transmit HIV-1 to CD4 T-cells, and such transmission is also augmented by CD40 ligand activation. IPC produce RANTES/CCL5 and MIP-1alpha/CCL3 when exposed to HIV in vitro. IPC also induce naïve CD4 T cells to proliferate and would therefore preferentially infect these cells. These results indicate that IPC may play an important role in the dissemination of HIV.

HDAC7, a thymus-specific class II histone deacetylase, regulates Nur77 transcription and TCR-mediated apoptosis.

We report that HDAC7, a class II histone deacetylase, is highly expressed in CD4(+)CD8(+) double-positive thymocytes. HDAC7 inhibits the expression of Nur77, an orphan receptor involved in apoptosis and negative selection, via the transcription factor MEF2D. HDAC7 is exported from the nucleus during T cell receptor activation, leading to Nur77 expression. A triple HDAC7 mutant (S155A, S318A, S448A) is not exported from the nucleus in response to TCR activation and suppresses TCR-mediated apoptosis. Conversely, a fusion of HDAC7 to the transcriptional activator VP16 activates Nur77 expression. Inhibition of HDAC7 expression by RNA interference causes increased apoptosis in response to TCR activation. These observations define HDAC7 as a regulator of Nur77 and apoptosis in developing thymocytes.

Loss of tolerance and autoimmunity affecting multiple organs in STAT5A/5B-deficient mice.

STAT5 has previously been reported to be dispensable for the maintenance of tolerance in vivo. However, in examining hemopoiesis in mice lacking both isoforms of STAT5, STAT5A, and STAT5B, we noted that a subset of these mice demonstrated dramatic alterations in several bone marrow progenitor populations concomitant with lymphocytic infiltration of the bone marrow. In addition, cellular infiltration affecting the colon, liver, and kidney was observed in these mice. Survival analysis revealed that STAT5A/5B(-/-) mice exhibited early death. The increased mortality and the pathology affecting multiple organs observed in these mice were abrogated on the recombination-activating gene 1(-/-) background. In light of the similarities between STAT5A/5B-deficient mice and mice unable to signal through the IL-2R, we hypothesized that the tolerizing role of STAT5A/5B was triggered via activation of the IL-2R. In agreement with this, we found that IL-2Rbeta chain-deficient mice exhibited similar hemopoietic abnormalities. Because IL-2 signaling is thought to contribute to tolerance through maintenance of a CD4(+)CD25(+) regulatory T cell population, we examined these cells and observed a numerical reduction in STAT5A/5B(-/-) mice along with a higher rate of apoptosis. These data provide strong evidence for a requirement for STAT5 in the maintenance of tolerance in vivo.

Increased HLA-DR expression on peripheral blood monocytes in subsets of subjects with primary HIV infection is associated with elevated CD4 T-cell apoptosis and CD4 T-cell depletion.

Whereas T-cell activation parameters of HIV disease have been extensively studied, the activation status of circulating monocytes has received less attention. Sixty-one subjects with primary HIV infection were evaluated by fluorescent-activated cell sorter (FACS) analysis at baseline (pretreatment) for CD4 T-cell count, CD4 T-cell apoptosis, and immune activation. A subset of 15 subjects with marked elevated (3 standard deviations above normal) monocyte DR expression had significantly reduced CD4 T-cell counts at baseline (p <.01) when compared with 46 subjects without monocyte activation. Ten subjects who presented with elevated levels of both CD14/DR, and CD4/CD38, had higher CD4 T-cell apoptosis (p <.001), and lower CD4 T-cell counts (p <.001) and higher baseline plasma HIV RNA (p <.01) than 21 subjects without elevated CD14/DR and CD4/CD38 coexpression. Fifty subjects were subsequently evaluated for immune cell activation over 24 weeks postinitiation of highly active antiretroviral therapy (HAART). A subgroup of 5 subjects who had persistent CD14/DR activation showed continuous depression of CD4 T-cell counts persisting for up to 2 years. The CD4 T-cell counts of this subgroup were significantly lower, at all time points, in comparison to 35 subjects who lacked any persistent expression of monocyte or CD4 T-cell activation (at 24 weeks, p <.002). We conclude that monocyte activation as defined by elevation of CD14/DR expression correlates to CD4 T-cell depletion in primary HIV infection, and is predictive of a poor CD4 T-cell response to HAART in a subset of patients.

HIV-related neuropathology, 1985 to 1999: rising prevalence of HIV encephalopathy in the era of highly active antiretroviral therapy.

Postmortem neuropathologic reports for a consecutive series of 436 HIV-seropositive patients who died between 1985 and 1999 were matched with clinical data for 371 of them. Cases were divided into four groups depending on the date of death. The chosen time periods reflected the type of antiretroviral therapy available: before 1987 (before zidovudine); 1987-1992, the period of monotherapy (nucleoside analog reverse transcriptase inhibitors [NRTIs]); 1993-1995, the era of the use of dual NRTI combinations; and 1996-1999, the era of highly active antiretroviral therapy (HAART) containing protease inhibitors. Fifty-seven percent of our cases in this group had been prescribed HAART. In our study population, accessibility to the latest antiretroviral therapy was widespread. The total number of HIV autopsies declined after the advent of combination therapy. The prevalence of opportunistic infections-cytomegalovirus, toxoplasmosis, cryptococcosis, and central nervous system lymphoma-decreased over time. Cerebral tuberculosis, aspergillosis, herpes, and progressive multifocal leukoencephalopathy showed a downward trend, but the numbers were too low for statistical analyses. The incidence of HIV encephalopathy increased over time (p =.014). The rising prevalence of HIV encephalopathy at time of death may reflect a longer survival time after initial HIV infection in the HAART era. Although combination therapies decrease overall mortality and prevalence of CNS opportunistic infections, these therapies may be less active in preventing direct HIV-1 effects on the brain.

Increased virus replication and virulence after serial passage of human immunodeficiency virus type 2 in baboons.

Similar to human immunodeficiency virus type 1 (HIV-1) infection of humans, the natural history of HIV-2 infection in baboons (Papio cynocephalus) is a slow and chronic disease that generally takes several years before an AIDS-like condition develops. To shorten the amount of time to the development of disease, we performed five serial passages of HIV-2(UC2) in baboons by using blood and bone marrow samples during the acute phase of infection when viral loads were at high levels. After these serial passages, virus levels in plasma, peripheral blood mononuclear cells (PBMC) and lymphatic tissues in the acutely infected baboons were increased. Within 1 year of the HIV-2 infection, all of the inoculated baboons showed specific signs of AIDS-related disease progression within the lymphatic tissues, such as vascular proliferation and lymphoid depletion. The HIV-2(UC2) recovered after four serial passages showed increased kinetics of viral replication in baboon PBMC and cytopathicity. This study suggests that the HIV-2 isolate recovered after several serial passages in baboons will be useful in future studies of AIDS pathogenesis and vaccine development by using this animal model.