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Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18 following formation of gasdermin D (GSDMD)-mediated membrane pores. To determine the possible pathogenic contributions of pyroptosis toward development of full-thickness retinal necrosis during AIDS-related human cytomegalovirus retinitis, we performed a series of studies using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS). Initial investigations demonstrated significant transcription and translation of key pyroptosis-associated genes within the ocular compartments of MCMV-infected eyes of mice with MAIDS. Subsequent investigations compared MCMV-infected eyes of groups of wildtype MAIDS mice with MCMV-infected eyes of groups of caspase-1 -/- MAIDS mice, GSDMD -/- MAIDS mice, or IL-18 -/- MAIDS mice to explore a possible contribution of pyroptosis towards the pathogenesis of MAIDS-related MCMV retinitis. Histopathologic analysis revealed typical full-thickness retinal necrosis in 100% of MCMV-infected eyes of wildtype MAIDS mice. In sharp contrast, none (0%) of MCMV-infected eyes of MAIDS mice that were deficient in either caspase-1, GSDMD, or IL-18 developed full-thickness retinal necrosis but instead exhibited an atypical pattern of retinal disease characterized by thickening and proliferation of the retinal pigmented epithelium layer with relative sparing of the neurosensory retina. Surprisingly, MCMV-infected eyes of all groups of deficient MAIDS mice harbored equivalent intraocular amounts of infectious virus as seen in MCMV-infected eyes of groups of wildtype MAIDS mice despite failure to develop full-thickness retinal necrosis. We conclude that pyroptosis plays a significant role in the development of full-thickness retinal necrosis during the pathogenesis of MAIDS-related MCMV retinitis. This observation may extend to the pathogenesis of AIDS-related HCMV retinitis and other AIDS-related opportunistic virus infections., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Sjögren's syndrome (SjS)-like sialoadenitis and exocrine pancreatitis were induced in mice infected with LP-BM5 murine leukemia virus, which induces a severe immunodeficiency termed murine AIDS (MAIDS). All mice with MAIDS showed advancing cellular infiltration around the pancreatic ducts as well as systemic exocrinopathy. The primary target tissue of the pancreas was acinar cells, and the pancreatic islets were well preserved until a late phase of the disease. Immunofluorescence and flow cytometry demonstrated that CD4(+) T cells, Mac-1(+) cells, and B220(+) cells were major inflammatory components, and IFN-gamma and IL-10 were mainly detected on CD4(+) T and Mac-1(+) cells in the pancreas. Both Th1 and Th2 cells were found. TUNEL(+) apoptotic cells were mostly detected among pancreas-infiltrating cells. Fas ligand and TNF-alpha were also detected among pancreas-infiltrating cells, whereas Fas was rarely expressed in the pancreatic acinar cells. Thus, MAIDS mice could be valuable for analyzing the pathogenesis of autoimmune-related pancreatitis associated with SjS.

The results of the combined use of Fludarabine, an anticancer agent that may be able to target latently infected cells, and conventional antiretroviral therapy (AZT+DDI) in a murine model of AIDS, i.e., LP-BM5 infection, are reported. Eighty percent of infected mice, treated with four cycles of alternate administration of Fludarabine and AZT+DDI, showed undetectable levels of proviral DNA in lymph nodes. After 8 weeks of treatment interruption, the infected/treated animals, although still alive at a time when all untreated animals had succumbed to the infection, showed disease progression and reappearance of proviral DNA in lymph nodes. The retrospective analysis of proviral DNA content in spleen and bone marrow at the end of the fourth cycle of treatment revealed a low but detectable amount of BM5d proviral DNA. We thus concluded that the spleen and bone marrow may be less sensitive to lympholitic drugs and therefore act as viral reservoirs in LP-BM5 infection. This study suggests that optimized protocols of alternate administration of cytolytic and antiretroviral drugs may represent a useful strategy to eradicate retroviral infections.

To better understand the molecular signaling events leading to systemic inflammatory response syndrome (SIRS) and multiple organ failure (MOF), changes in gene expression profiles after burn injury were investigated by differential display. C57BLKS/J mice were subjected to 18% total body surface area (TBSA) full-thickness burn and various tissues were harvested at multiple time points after injury. Initial differential display revealed that retroviral transcripts similar to the envelope sequence of murine AIDS (MAIDS) virus were rapidly and transiently up-regulated after injury. Subsequent RT-PCR and DNA sequencing analyses confirmed the transient up-regulation of retroviral sequences similar to those of the MAIDS virus. In addition, the presence and induction of the subgenomic envelope transcripts of these MAIDS virus-related sequences, including a novel double spliced message, were identified after burn injury. These data suggest that the transcriptional efficiency of the integrated retroviral DNA and reactivation of defective MAIDS virus-related sequences may be affected by pathophysiological signals, such as burn injury. The elevated expression of these MAIDS virus-related retroviral sequences may affect the transcriptional activities of the flanking genes at the integration sites and may be a cause of altered local and systemic immune responses to burn-related stress.

We previously showed that certain tyrphostin derivatives, known as protein tyrosine kinase inhibitors, also act as topoisomerase I-specific antagonists and inhibit Moloney murine leukemia virus replication in vitro in acutely and chronically infected cells. However, an accurate portrayal of retroviral-induced disease cannot rely exclusively on extrapolations from in vitro data. Therefore, experiments with animal models are essential for evaluating the efficacy of a specific drug in vivo. In this study, we examined the effect of tyrphostin AG-1387 on murine AIDS (MAIDS) development in C57BL/6J mice injected with the LP-BM5 virus mixture. A single dose of tyrphostin, administered together with or 24 h post virus inoculation, decreased the development of MAIDS symptoms as measured by spleen and lymph node weight, the T-cell response to concanavalin A (con A), and spleen architecture. Furthermore, weekly treatment with tyrphostins totally abolished MAIDS symptoms and prevented the viral infection of the spleen cells as measured by the absence of viral RNA and the restoration of T-cell function in these spleens. These results implicate that prolonged treatment with tyrphostins is needed for the prevention of MAIDS development in infected mice and suggest that it may be applied as a legitimate remedy for the treatment of retroviral-induced diseases., (Copyright 2000 Academic Press.)

Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus complex that contains causative defective, and ecotropic helper, retroviruses. We previously demonstrated that the MAIDS-resistant H-2(d) strains BALB/cByJ and C57BL/KsJ generate MHC class I (K(d)) restricted virus-specific CD8(+) cytolytic T lymphocytes (CTLs) that lyse cells expressing either defective or ecotropic gag proteins. In contrast, the congenic BALB.B and closely related C57BL/6J MAIDS-susceptible H-2(b) strains were unable to serve as a source of gag-specific CTLs (Schwarz and Green, 1994), suggesting that anti-gag CTLs might provide a basis for resistance to MAIDS. Although its susceptibility to MAIDS was unknown, the (BALB/c x C57BL/6J) F(1) (CBY6F(1)) strain could also produce H-2(d)-, but not H-2(b)-, restricted, anti-gag CTLs (Schwarz and Green, 1994). Because of this correlation between anti-gag CTLs and resistance to MAIDS, it was important to provide more direct evidence in support of CTL-mediated protection and to determine both the fine specificity of CByB6F(1) anti-gag CTLs, in comparison with the resistant C57BL/Ks and BALB/c strains, and the susceptibility of this F(1) strain to LP-BM5-induced MAIDS. We report here that no symptoms of MAIDS were observed in CBY6F(1) (H-2(dxb)) mice. For F(2) mice, in contrast to the high susceptibility of H-2(b/b) mice, 77% of H-2(d/d) and 81% of H-2(b/d) F(2) mice did not exhibit MAIDS after LP-BM5 infection. These results are in contrast to other published studies that concluded that susceptibility, rather than resistance, is dominant in F(1) (resistant x susceptible or susceptible x resistant) mice. We also show that CBY6F(1) anti-gag CTLs exhibit a fine specificity shared by the MAIDS-resistant BALB/c and C57BL/Ks strains, that is, the immunodominant gag epitope, SYNTGRFPPL, encoded by an alternative open reading frame. Together with our direct demonstration here that in vivo monoclonal antibody (mAb) depletion of CD8(+) T cells converts genetically resistant mice to MAIDS susceptibility, these data on the ability to mount anti-ORF2/SYNTGRFPPL, gag-specific CTL responses strongly suggest that CTLs are a primary factor in determining MAIDS resistance. Accordingly, given the K(d)-restricted nature of the CTLs, the main genetic determinant of resistance appeared to be the codominant expression of the resistant H-2(d) haplotype. Interestingly, however, 19% of H-2(d/b) and 23% of the H-2(d/d) F(2) mice had at least one clinical aspect of MAIDS, suggesting that a non-MHC genetic determinant(s) can negatively influence T-cell protection and thus disease outcome, (Copyright 2000 Academic Press.)

Mice inoculated with the murine AIDS (MAIDS)-defective virus develop severe B and T cell dysfunctions. The primary event in the development of this disease is the infection and polyclonal expansion of the target cells of this defective virus, which have been reported to belong to the B cell lineage. To further study the central role that these cells play in the development of MAIDS, we attempted to establish MAIDS-defective virus-infected B cell lines in vitro. We succeeded in establishing two cell lines, SD1 and CSTB5, from the enlarged organs of C57BL/6 mice inoculated with helper-free stocks of the MAIDS-defective virus. Both cell lines are not transplantable in syngeneic C57BL/6 mice or in nude or CD8-/- mice and are apparently not malignant. They both belong to the B lineage, as their immunoglobulin (Ig) genes, but not the T cell receptor (TcR) beta locus, are rearranged, suggesting that they are relatively mature B cells. However, analysis of cell surface marker expression by FACS revealed a surface phenotype similar to that of pre-B cells (MHC I+, MHC II+, B7.2+, sIgM-, sIgG-, kappa-, B220-, CD5-, Thy1.2-, TcR-, CD3-, CD4-, CD8-, Mac-1-, 33D1-). Additionally, the CSTB5 cells express CD40 and the SD1 cells express CD43. Both cell lines contain the MAIDS-defective provirus and express the expected 4.2-kb viral RNA and the corresponding Pr60gag protein. The CSTB5 cells are nonproducer, while the SD1 cell line produces what appears to be an endogenous MuLV. The phenotype of these cell lines is very similar to what is known about the target B cells of this virus in vivo. These new established cell lines are likely to be useful in elucidating the mechanism(s) by which the MAIDS-defective virus causes its target B cells to proliferate and induce T cell anergy in infected animals.

In genetically susceptible C57BL/6 mice the LP-BM5 isolate of murine retroviruses causes profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, and an immunodeficiency syndrome bearing many similarities to the pathologies seen in AIDS. Because of these similarities, which also include terminal B cell lymphoma formation, this syndrome has been called murine AIDS or MAIDS. Prompted by previous reports showing that the onset of MAIDS is dependent on the presence of both CD4+ T and B cells, we have previously shown that anti-gp39/CD40 ligand mAb (anti-CD40L mAb) treatment of LP-BM5-infected mice is effective in inhibiting the induction of MAIDS when a short course of anti-CD40L mAb treatment was started on the same day as LP-BM5 administration. The success of anti-CD40L mAb therapy, as indicated by a much reduced degree of splenomegaly, hypergammaglobulinemia, and mitogen and allogeneic CTL unresponsiveness, demonstrated that CD40L/CD40 interactions were critical to the establishment of MAIDS. Here we extend these findings through the use of delayed anti-CD40L mAb treatment of mice, beginning 3-4 weeks after LP-BM5 infection, by showing that interruption of CD40L/CD40 interactions also interferes with the progression of MAIDS. About 60% of LP-BM5-preinfected mice were affected by delayed anti-CD40L mAb treatment, with substantially reduced spleen weights and serum hypergammaglobulinemia and normal or greatly restored proliferative responses to Con A stimulation and CTL responses to allogeneic stimulation. The other LP-BM5-infected mice that did not respond to anti-CD40L therapy were found to have made antibodies to the anti-CD40L mAb. Thus, in a majority of mice anti-CD40L mAb therapy was very effective in interfering with MAIDS pathogenesis well after the establishment of the virus infection and MAIDS symptomatology, indicating that CD40L/CD40 interactions are crucial to the maintenance and progression of the disease, as well as its initiation.

Mice inoculated with replication-competent stocks of the murine acquired immunodeficiency syndrome (MAIDS) virus are severely immunocompromised and proned to the development of T- and B-cell lymphomas. We have studied the development of T-cell lymphomas in C57BL/6 and RF/J mice inoculated with helper-free stocks of the MAIDS defective virus. We observed the expansion of T cell clones (detected by TCR gene rearrangements and by transplantation) only rarely in diseased C57BL/6 mice and slightly more frequently in RF/J mice. We succeeded in establishing four transplantable T cell tumors and malignant cell lines. The three cell lines from RF/J mice were immature T-cells (Thy-1+, CD3-, CD4+, CD8+, Mac-1+), while the line from the C57BL/6 mouse had the phenotype of mature T-cells (Thy-1+, CD3+, CD4+, CD8-). All lines were virus-producers despite the fact that helper-free stocks of the virus were inoculated. These helper MuLVs most likely originated from endogenous MuLV sequences. Also, the defective viral genome was clearly detectable in one cell line and was rearranged in two other lines. These established cell lines may be useful to determine whether they share some of the characteristics of the anergic T-cells in vivo and to study the role of the MAIDS defective virus in T cell transformation.

Blood cytopenia is a common feature in HIV infection, occurring in up to 70% of patients with AIDS. Since at present it is not clear to what extent this is intrinsic to HIV infection or due to opportunistic infections and antiretroviral agents we have investigated the long-term effects of conventional and new antiviral drugs on the bone marrow of normal and immunodeficient mice. The results show that azidothymidine (AZT), dideoxycytidine (DDC) and dideoxycytidine 5'-triphosphate (DDCTP) alone or in combination are all effective in inhibiting the expression of the retroviral protein Pr60gag in bone marrow cells. However, DDCTP was the most effective in preventing bone marrow cytopenia. Combined treatments of AZT plus DDCTP result in a reduction in erythroid precursors compared to that resulting from DDCTP administration, while DDC plus DDCTP results in a differential cell count similar to that found in uninfected mice. Thus, the bone marrow in murine AIDS may prove useful as a model for therapy of retroviral infections and for treating blood cytopenias.