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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.)

Human cytomegalovirus (HCMV) is an opportunistic human herpesvirus that causes a sight-threatening retinitis in immunosuppressed patients, especially those with AIDS. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunodeficiency (MAIDS), we have been attempting to define with greater clarity the immunologic mechanisms that contribute to the progression of AIDS-related HCMV retinitis in the unique immunosuppressive setting of HIV infection. Toward this end, we provide herein a comprehensive assessment of immune response gene expression during the onset and development of MAIDS-related MCMV retinitis employing NanoString nCounter. In so doing, we analyzed and compared the intraocular expressions of 561 immune response genes within MCMV-infected eyes of groups of healthy mice, MCMV-infected mice with MAIDS of 4 weeks' (MAIDS-4) duration, and MCMV-infected eyes of mice with MAIDS of 10 weeks' (MAIDS-10) duration. These animal groups show a progression of retinal disease from absolute resistance to retinitis development in healthy mice to the development of classic full-thickness retinal necrosis in MAIDS-10 mice but through an intermediate stage of retinal disease development in MAIDS-4 mice. Our findings showed that increased susceptibility to MCMV retinitis during the progression of MAIDS is associated with robust upregulation or downregulation of a surprisingly large number of immune response genes that operate within several immune response pathways often unique to each animal group. Analysis of 14 additional immune response genes associated with programmed cell death pathways suggested involvement of necroptosis and pyroptosis during MAIDS-related MCMV retinitis pathogenesis. Use of the NanoString nCounter technology provided new and unexpected information on the immunopathogenesis of retinitis within MCMV-infected eyes of mice with retrovirus-induced immunosuppression. Our findings may provide new insights into the immunologic events that operate during the pathogenesis of AIDS-related HCMV retinitis., Competing Interests: The authors have declared that no competing interests exist.

The mechanisms that contribute to retinal tissue destruction during the onset and progression of AIDS-related human cytomegalovirus (HCMV) retinitis remain unclear. Evidence for the stimulation of multiple cell death pathways including apoptosis, necroptosis, and pyroptosis during the pathogenesis of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS) has been reported. Parthanatos is a caspase-independent cell death pathway mediated by rapid overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) and distinct from other cell death pathways. Using the MAIDS model of MCMV retinitis, studies were performed to test the hypothesis that intraocular MCMV infection of mice with MAIDS stimulates parthanatos-associated messenger RNAs (mRNAs) and proteins within the eye during the development of retinal necrosis that takes place by 10 days after MCMV infection. MCMV-infected eyes of MAIDS mice exhibited significant stimulation of PARP-1 mRNA and proteins at 3 days after infection but declined thereafter at 6 and 10 days after infection. Additional studies showed the intraocular stimulation of mRNAs or proteins before MCMV retinitis development for two additional participants in parthanatos, polymer of ADP-ribose and poly (ADP-ribose) glycohydrolase. These results provide new evidence for a role for parthanatos during MAIDS-related MCMV retinitis that may also extend to AIDS-related HCMV retinitis., (© 2019 Wiley Periodicals, Inc.)

AIDS-related human cytomegalovirus retinitis remains the leading cause of blindness among untreated HIV/AIDS patients worldwide. To study mechanisms of this disease, we used a clinically relevant animal model of murine cytomegalovirus (MCMV) retinitis with retrovirus-induced murine AIDS (MAIDS) that mimics the progression of AIDS in humans. We found in this model that MCMV infection significantly stimulates ocular suppressor of cytokine signaling 1 (SOCS1) and SOCS3, host proteins which hinder immune-related signaling by cytokines, including antiviral type I and type II interferons. The present study demonstrates that in the absence of retinal disease, systemic MCMV infection of mice without MAIDS, but not in mice with MAIDS, leads to mild stimulation of splenic SOCS1 mRNA. In sharp contrast, when MCMV is directly inoculated into the eyes of retinitis-susceptible MAIDS mice, high levels of intraocular SOCS1 and SOCS3 mRNA and protein are produced which are associated with significant intraocular upregulation of gamma interferon (IFN-γ) and interleukin-6 (IL-6) mRNA expression. We also show that infiltrating macrophages, granulocytes, and resident retinal cells are sources of intraocular SOCS1 and SOCS3 protein production during development of MAIDS-related MCMV retinitis, and SOCS1 and SOCS3 mRNA transcripts are detected in retinal areas histologically characteristic of MCMV retinitis. Furthermore, SOCS1 and SOCS3 are found in both MCMV-infected cells and uninfected cells, suggesting that these SOCS proteins are stimulated via a bystander mechanism during MCMV retinitis. Taken together, our findings suggest a role for MCMV-related stimulation of SOCS1 and SOCS3 in the progression of retinal disease during ocular, but not systemic, MCMV infection. IMPORTANCE Cytomegalovirus infection frequently causes blindness in untreated HIV/AIDS patients. This virus manipulates host cells to dysregulate immune functions and drive disease. Here, we use an animal model of this disease to demonstrate that cytomegalovirus infection within eyes during retinitis causes massive upregulation of immunosuppressive host proteins called SOCS. As viral overexpression of SOCS proteins exacerbates infection with other viruses, they may also enhance cytomegalovirus infection. Alternatively, the immunosuppressive effect of SOCS proteins may be protective against immunopathology during cytomegalovirus retinitis, and in such a case SOCS mimetics or overexpression treatment strategies might be used to combat this disease. The results of this work therefore provide crucial basic knowledge that contributes to our understanding of the mechanisms of AIDS-related cytomegalovirus retinitis and, together with future studies, may contribute to the development of novel therapeutic targets that could improve the treatment or management of this sight-threatening disease., (Copyright © 2018 American Society for Microbiology.)

The immunomodulatory effects of a dietary supplement of purple sweet potato extract (PSPE) in LP-BM5 murine leukemia virus (MuLV)-induced immune-deficient mice were investigated. Mice were divided into six groups: normal control, infected control (LP-BM5 MuLV infection), positive control (LP-BM5 MuLV infection+dietary supplement of red ginseng 300 mg/kg), purple sweet potato water extract (PSPWE) (LP-BM5 MuLV infection+dietary supplement of PSPE 300 mg/kg), PSP10EE (LP-BM5 MuLV infection+dietary supplement of 10% ethanol PSPE 300 mg/kg), and PSP80EE (LP-BM5 MuLV infection+dietary supplement of 80% ethanol PSPE 300 mg/kg). Dietary supplementation began on the day of LP-BM5 MuLV infection and continued for 12 weeks. Dietary supplementation of PSPE inhibited LP-BM5 MuLV-induced splenomegaly and lymphadenopathy and attenuated the suppression of T- and B-cell proliferation and T helper 1/T helper 2 cytokine imbalance in LP-BM5 MuLV-infected mice. Dietary supplement of PSPE increased the activity of the antioxidant enzymes, superoxide dismutase and glutathione peroxidase. The data suggest that PSPE may ameliorate immune dysfunction due to LP-BM5 MuLV infection by modulating antioxidant defense systems.

Interferon regulatory factor (IRF) regulation of the type I interferon response has not been extensively explored in murine retroviral infections. IRF-3(-/-) and select IRF-3/7(-/-) mice were resistant to LP-BM5-induced pathogenesis. However, further analyses strongly suggested that resistance could be attributed to strain 129-specific contamination of the known retrovirus resistance gene Fv1. Therefore, caution should be taken when interpreting phenotypes observed in these knockout mice, as strain 129-derived genetic polymorphisms may explain observed differences.

B6 mice infected with LP-BM5 develop severe immunodeficiency (termed murine acquired immunodeficiency syndrome (MAIDS)) and peripheral neuropathy. To determine whether microglial CD40 is involved in LP-BM5-induced peripheral neuropathy, B6-CD40 knockout (KO) mice and B6-CD40 KO mice adoptively transferred either total leukocytes or B cells were examined for behavioral sensitivity, tissue viral loads, cytokine responses, and the development of MAIDS. All three CD40 KO groups developed MAIDS, the severity of which was correlated with peripheral cytokine responses. CD40 KO mice displayed significantly reduced mechanical hypersensitivity post-infection compared to wild-type mice regardless of cell transfer. These findings support microglial CD40 involvement in LP-BM5-induced peripheral neuropathy., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Interleukin-17 (IL-17), a pro-inflammatory cytokine produced by CD4+ Th17 cells, has been associated with the pathogenesis of several autoimmune diseases including uveitis. The fate of IL-17 during HIV/AIDS, however, remains unclear, and a possible role for IL-17 in the pathogenesis of AIDS-related diseases has not been investigated. Toward these ends, we performed studies using a well-established animal model of experimental murine cytomegalovirus (MCMV) retinitis that develops in C57/BL6 mice with retrovirus-induced immunosuppression (MAIDS). After establishing baseline levels for IL-17 production in whole splenic cells of healthy mice, we observed a significant increase in IL-17 mRNA levels in whole splenic cells of mice with MAIDS of 4-weeks (MAIDS-4), 8-weeks (MAIDS-8), and 10-weeks (MAIDS-10) duration. In contrast, enriched populations of splenic CD4+ T cells, splenic macrophages, and splenic neutrophils exhibited a reproducible decrease in levels of IL-17 mRNA during MAIDS progression. To explore a possible role for IL-17 during the pathogenesis of MAIDS-related MCMV retinitis, we first demonstrated constitutive IL-17 expression in retinal photoreceptor cells of uninfected eyes of healthy mice. Subsequent studies, however, revealed a significant decrease in intraocular levels of IL-17 mRNA and protein in MCMV-infected eyes of MAIDS-10 mice during retinitis development. That MCMV infection might cause a remarkable downregulation of IL-17 production was supported further by the finding that systemic MCMV infection of healthy, MAIDS-4, or MAIDS-10 mice also significantly decreased IL-17 mRNA production by splenic CD4+ T cells. Based on additional studies using IL-10 -/- mice infected systemically with MCMV and IL-10 -/- mice with MAIDS infected intraocularly with MCMV, we propose that MCMV infection downregulates IL-17 production via stimulation of suppressor of cytokine signaling (SOCS)-3 and interleukin-10., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

AIDS-related human cytomegalovirus (HCMV) retinitis remains a major ophthalmologic problem worldwide. Although this sight-threatening disease is well characterized clinically, many pathogenic issues remain unresolved, among them a basic understanding of the relative roles of cell death pathways during development of retinal tissue destruction. Using an established model of experimental murine cytomegalovirus (MCMV) retinitis in mice with retrovirus-induced immunosuppression (MAIDS), we initially investigated MCMV-infected eyes for evidence of apoptosis-associated molecules in mice with MAIDS of 4 weeks' (MAIDS-4) and 10 weeks' (MAIDS-10) duration, which were resistant and susceptible to retinal disease, respectively, but which harbored equivalent amounts of infectious MCMV. Whereas MCMV-infected eyes of MAIDS-4 mice showed little evidence of apoptosis-associated molecules, MCMV-infected eyes of MAIDS-10 mice showed significant amounts of tumor necrosis factor alpha (TNF-α), TNF receptors 1 and 2, active caspase 8, active caspase 3, TNF-related apoptosis-inducing ligand (TRAIL), TRAIL-R(DR5), Fas, and Fas ligand mRNAs and/or proteins, all detected at peak amounts prior to development of most severe retinal disease. Immunohistochemical staining showed macrophages, granulocytes (neutrophils), Müller cells, and microglial cells as TNF-α sources. Remarkably, quantification of apoptosis by terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL) assay suggested that apoptosis contributed minimally to retinal disease in MCMV-infected eyes of MAIDS-10 mice. Subsequent studies demonstrated that MCMV-infected eyes of MAIDS-10 mice, but not MAIDS-4 mice, showed evidence of significant increases in molecules associated with two additional cell death pathways, necroptosis (receptor-interacting protein 1 [RIP1] and RIP3 mRNAs) and pyroptosis (caspase 1, interleukin 1β [IL-1β], and IL-18 mRNAs). We conclude that apoptosis, necroptosis, and pyroptosis participate simultaneously during MAIDS-related MCMV retinitis, and all may play a role during AIDS-related HCMV retinitis.

Background: APOBEC3 proteins are host factors that restrict infection by retroviruses like HIV, MMTV, and MLV and are variably expressed in hematopoietic and non-hematopoietic cells, such as macrophages, lymphocytes, dendritic, and epithelia cells. Previously, we showed that APOBEC3 expressed in mammary epithelia cells function to limit milk-borne transmission of the beta-retrovirus, mouse mammary tumor virus. In this present study, we used APOBEC3 knockout mice and their wild type counterpart to query the role of APOBEC3 in sexual transmission of LP-BM5 MLV - the etiological agent of murine AIDs (mAIDs)., Results: We show that mouse APOBEC3 is expressed in murine genital tract tissues and gametes and that genital tract tissue of APOBEC3-deficient mice are more susceptible to infection by LP-BM5 virus. APOBEC3 expressed in genital tract tissues most likely plays a role in decreasing virus transmission via the sexual route, since mice deficient in APOBEC3 gene have higher genitalia and seminal plasma virus load and sexually transmit the virus more efficiently to their partners compared to APOBEC3+ mice. Moreover, we show that female mice sexually infected with LP-BM5 virus transmit the virus to their off-spring in APOBEC3-dependent manner., Conclusion: Our data indicate that genital tissue intrinsic APOBEC3 restricts genital tract infection and limits sexual transmission of LP-BM5 virus.

The emergence of drug resistance threatens to limit the use of current anti-HIV-1 drugs and highlights the need to expand the number of treatment options available for HIV-1-infected individuals. Our previous studies demonstrated that two clinically approved drugs, decitabine and gemcitabine, potently inhibited HIV-1 replication in cell culture through a mechanism that is distinct from the mechanisms for the drugs currently used to treat HIV-1 infection. We further demonstrated that gemcitabine inhibited replication of a related retrovirus, murine leukemia virus (MuLV), in vivo using the MuLV-based LP-BM5/murine AIDS (MAIDS) mouse model at doses that were not toxic. Since decitabine and gemcitabine inhibited MuLV and HIV-1 replication with similar potency in cell culture, the current study examined the efficacy and toxicity of the drug combination using the MAIDS model. The data demonstrate that the drug combination inhibited disease progression, as detected by histopathology, viral loads, and spleen weights, at doses lower than those that would be required if the drugs were used individually. The combination of decitabine and gemcitabine exerted antiviral activity at doses that were not toxic. These findings indicate that the combination of decitabine and gemcitabine shows potent antiretroviral activity at nontoxic doses and should be further investigated for clinical relevance.

LP-BM5 retrovirus induces a complex disease featuring an acquired immunodeficiency syndrome termed murine AIDS (MAIDS) in susceptible strains of mice, such as C57BL/6 (B6). CD4 T helper effector cells are required for MAIDS induction and progression of viral pathogenesis. CD8 T cells are not needed for viral pathogenesis, but rather, are essential for protection from disease in resistant strains, such as BALB/c. We have discovered an immunodominant cytolytic T lymphocyte (CTL) epitope encoded in a previously unrecognized LP-BM5 retroviral alternative (+1 nucleotide [nt]) gag translational open reading frame. CTLs specific for this cryptic gag epitope are the basis of protection from LP-BM5-induced immunodeficiency in BALB/c mice, and the inability of B6 mice to mount an anti-gag CTL response appears critical to the initiation and progression of LP-BM5-induced MAIDS. However, uninfected B6 mice primed by LP-BM5-induced tumors can generate CTL responses to an LP-BM5 retrovirus infection-associated epitope(s) that is especially prevalent on such MAIDS tumor cells, indicating the potential to mount a protective CD8 T-cell response. Here, we utilized this LP-BM5 retrovirus-induced disease system to test whether modulation of normal immune down-regulatory mechanisms can alter retroviral pathogenesis. Thus, following in vivo depletion of CD4 T regulatory (Treg) cells and/or selective interruption of PD-1 negative signaling in the CD8 T-cell compartment, retroviral pathogenesis was significantly decreased, with the combined treatment of CD4 Treg cell depletion and PD-1 blockade working in a synergistic fashion to substantially reduce the induction of MAIDS.

Over the period of a year, colitis was observed in 44 mice raised in a conventional nonspecific pathogen-free colony, 41 of these having concomitant retrovirus-induced murine acquired immunodeficiency syndrome (MAIDS). The lesions varied from bacterial colonization to hyperplasia of colonic mucosa to severe, often fatal, ulceration. Citrobacter rodentium was isolated from the colon and/or liver of 2 mice with colitis. When C57BL/6 mice with or without MAIDS were given graded doses of the bacterium, only those with MAIDS developed colitis, and C rodentium was reisolated from their livers. Thus, mice with MAIDS can develop severe disease following opportunistic infection with an environmental contaminant of the colony that is nonpathogenic for normal adult mice.

Murine acquired immunodeficiency syndrome (MAIDS) is caused by exposure to murine leukemia virus and serves as a model to study human AIDS. In MAIDS-susceptible C57BL/6 mice, virus exposure leads to progressive immune deficiency, while resistant strains such as BALB/c recover from infection and develop protective immunity. The goal of this study was to identify early gene expression patterns that may be important in establishing this strain-specific differential response. Total RNA was isolated from spleens and pooled lymph nodes of both mouse strains at 3 and 7 days post virus infection. The complementary DNA generated from this RNA was hybridized to mouse oligonucleotide DNA microarrays using a strategy that controlled for inherent variability and highlighted only virus-induced changes. Fluorescent intensities were normalized and analyzed for statistically significant differential expression between strains across both time points and lymphoid organs. The majority of the resistance-associated genes was identified at day 3 post-infection and demonstrated the highest fold differences between strains, while more susceptibility-associated sequences were seen at 7 days post-infection. Among the most highly differentially expressed sequences seen at the earlier time point were genes related to protein metabolism, especially serine proteases. Differential patterns of chemokine-related genes were observed at the later time point. The overall pattern of expression suggests strain-specific differences in proteases and chemokines within secondary lymphoid organs shortly after infection influence the likelihood of disease progression.

Remnant proviral sequences in the genome resulting from the ancient germline infection of exogenous retroviruses are called endogenous retroviruses (ERVs). The transcriptional activation of human ERVs (HERVs) in the brain of patients with some neurologic diseases suggests that ERVs may participate in certain disease processes in the central nervous system. In this study, we identified putative murine ERVs (MuERVs) which are transcriptionally active in the brain and characterized their biological properties to better understand the ERVs' roles in the brain pathophysiology. The brain and selective non-nervous tissues (heart, muscle, adrenal gland, and salivary gland) of female C57BL/6J mice were subjected to RT-PCR analyses of MuERV expression by amplifying the 3'-end U3 regions and full-length/subgenomic transcripts. The expression patterns of the U3 regions and subgenomic transcripts in the brain were unique compared to the other tissues as well as the genomic MuERV profile. Two putative MuERVs (8,027 and 5,668 bp) were mapped on the mouse genome (chromosome 10, and chromosomes 4 and 8, respectively) using the MuERV U3 sequences, which were evidently expressed in the brain, as probes. Biological properties of these putative MuERVs, such as transcription potential, primer binding site, coding potential, integration age, recombination, and flanking host genes, were characterized. In particular, one of the two putative MuERV isolates had coding potentials for intact group specific antigen (gag), and truncated polymerase (pol) and envelope (env) polypeptides, while the other was defective for all three polypeptides. The findings from this study suggest that a specific group of MuERVs are constitutively expressed in the brain and they may participate in normal and pathogenic events pertaining to the brain through their replication gene products (e.g., gag and env polypeptides) as well as interactions with flanking host genes.

LP-BM5 murine leukemia virus-infected C57BL/6 mice develop profound immunodeficiency and B-cell lymphomas. The LP-BM5 complex contains a mixture of defective (BM5def) and replication-competent helper viruses among which BM5def is the primary causative agent of disease. The BM5def primary open reading frame (ORF1) encodes the single gag precursor protein (Pr60gag). Our lab has recently demonstrated that a novel immunodominant cytotoxic-T-lymphocyte (CTL) epitope (SYNTGRFPPL) is expressed from a +1-nucleotide translational open reading frame of BM5def during the course of normal retrovirus expression. The SYNTGRFPPL CTL epitope may be generated from either of two initiation methionines present, ORF2a or ORF2b, located downstream of the ORF1 initiation site. This study investigates the role(s) of the alternative ORF2-derived gag protein(s) of BM5def in viral pathogenesis. We have examined the disease-inducing capabilities of mutant viruses in which the translational potential of either the initiating ORF2a or ORF2b AUG has been disrupted. Although these mutated viruses are capable of wild-type ORF1 expression, they are unable to induce disease. Our data strongly suggest the existence of a novel ORF2 product(s) that is required for LP-BM5-induced pathogenesis and have potentially broad implications for other retroviral diseases.

The ribonucleotide reductase inhibitor hydroxyurea (HU) has demonstrated some benefit as a component of drug cocktails for the treatment of HIV-1 infection. However, HU is notoriously myelosuppressive and often administered only as salvage therapy to patients with late-stage disease, potentially exacerbating the bone marrow toxicity of HU. In this report we have compared the antiviral effects of HU and two novel RR inhibitors trimidox (3,4,5-trihydroxybenzamidoxime) and didox (3,4-dihydroxybenzohydroxamic acid) in combination with didanosine (2,3-didoxyinosine; ddI) in the LPBM5 MuLV retrovirus model (murine AIDS). We also evaluated the effects of these drug combinations on the hematopoietic tissues of LPBM5 MuLV-infected animals. The combination of RR inhibitors and ddI was extremely effective (DX>TX>HU) in inhibiting development of retrovirus-induced disease (splenomegaly, hypergammaglobulinemia, activated B-splenocytes and loss of splenic architecture). In addition, relative levels of proviral DNA were significantly lower in combination drug-treated animals compared to infected controls. Evaluation of femur cellularity, numbers of marrow-derived myeloid progenitor cells (CFU-GM and BFU-E) and peripheral blood indices revealed that TX and DX in combination with ddI were well-tolerated. However, treatment with HU and ddI induced moderate myelosuppression. These data demonstrate that RR inhibitors in combination with ddI provide significant protection against retroviral disease in murine AIDS. Moreover, the novel RR inhibitors TX and DX appear to be more effective and less myelosuppressive than HU when administered with ddI in this model.

MAIDS (murine AIDS) is caused by infection with the murine leukaemia retrovirus RadLV-Rs and is characterized by a severe immunodeficiency and T-cell anergy combined with a lymphoproliferative disease affecting both B- and T-cells. Hyperactivation of the cAMP-protein kinase A pathway is involved in the T-cell dysfunction of MAIDS and HIV by inhibiting T-cell activation through the T-cell receptor. In the present study, we show that MAIDS involves a strong and selective up-regulation of cyclo-oxygenase type 2 in the CD11b+ subpopulation of T- and B-cells of the lymph nodes, leading to increased levels of PGE2 (prostaglandin E2). PGE2 activates the cAMP pathway through G-protein-coupled receptors. Treatment with cyclo-oxygenase type 2 inhibitors reduces the level of PGE2 and thereby reverses the T-cell anergy, restores the T-cell immune function and ameliorates the lymphoproliferative disease.

A real-time PCR assay using SYBR Green I for quantification of provirus load in a murine model of AIDS (i.e., LP-BM5 infection) was developed and validated. In this method, data are normalized against the 18S rRNA gene. The method has a dynamic range of 8 logs and a sensitivity of one copy.

Murine acquired immunodeficiency disease (MAIDS) induced by LPBM5 MuLV is characterized by a late-stage lymphoma and hematopoietic cytopenias similar to those observed in human AIDS. The pathogenesis of MAIDS-related lymphoma/cytopenia is unknown but it has been postulated to involve a defective marrow microenvironment or stroma. The basic Fibroblast Growth Factor (bFGF) of stromal origin is an important stimulator for hematopoietic progenitors of several lineages. Long-term bone marrow cultures (LTBMCs) were established and pure stromal cell cultures were used for in vitro infection hematopoietic reconstitution studies. Reverse transcription-polymerase chain reaction (RT-PCR) was used to analyze bFGF gene expression in stromal cells derived from either viral-infected marrow or uninfected marrow. RT-PCR analysis showed a 40% reduction in the expression of bFGF transcript expression from viral-infected stromal cells, however, the levels of bek and flg bFGF receptors remained unchanged indicating virus-infection only inhibited bFGF gene expression in stromal cells. Viral infection was associated with a progressive decrease in bFGF transcript expression 35% of control at day 7, 50% of control at day 14 and 60% of control at day 21 compared to the mock-infected cultures. In addition, for bek and flg the transcript expression in, in vitro-infected primary cultures were comparable to the mock-infected cultures and remained essentially unchanged throughout culture period. Western blot analysis revealed viral-infected stromal cells produced a 45% decrease in bFGF protein production. Reduction of bFGF protein was confirmed by indirect immunofluorescent staining. We report MuLV infection reduces bFGF transcript expression but not its surface-receptors (bek and flg) in infected stromal cells. Impaired hematopoiesis consistently exhibited from MuLV-infected stromal cultures was restored by exogenous bFGF; therefore, bFGF was responsible in restoration of normal marrow stromal support function. These results suggest a role for bFGF deficiency in the pathogenesis of MAIDS-related marrow failure.

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 causative agent of murine AIDS (MAIDS) in C57BL/6 mice, is a defective murine leukemia virus (BM5d) that requires the replication-competent helper virus (BM5e). Since this animal model of immunodeficiency, which shows many similarities to human AIDS, is also used to test the efficacy and toxicity of antiretroviral drugs, a method that allows the quantitative detection of both viruses would be very useful also if hampered potentially by endogenous viral sequences usually present in mice. While BM5d alone could induce the disease, the effect of BM5e on the immune system of diseased mice is unclear. A specific and reliable one-step RT-PCR method was developed for the co-amplification, with the same efficiency, of BM5d or BM5e with ss-actin used as an internal standard. The standard curves produced with cloned cDNA sequences (ss-actin and BM5d or BM5e) assure that all samples are analyzed during the exponential phase of the reaction. Using this new assay which provided a dynamic range of at least four-log-unit, the ratio of initial absolute amounts of the virus and ss-actin RNA was determined, obtaining quantitative information on virus-specific cellular-transcript in the lymph nodes and spleen during the natural history of the disease and during therapeutic regimens.

Murine AIDS (MAIDS) develops in susceptible mouse strains after infection with the LP-BM5 murine leukemia virus (MuLV) complex that contains a mixture of defective (BM5def) and replication-competent viruses. While the BM5def virus is the causative agent in MAIDS, the replication-competent viruses in LP-BM5, including ecotropic MuLV (BM5eco), are required for BM5def propagation and thus function as helper viruses. We describe quantitative real-time RT-PCR assays for RNA encoded by the BM5def and BM5eco components of LP-BM5. The assays were used to standardize better the input doses of LP-BM5 viruses across viral preparations and to quantify BM5def and BM5eco gag RNA levels in spleen and blood cells from MAIDS-susceptible and -insusceptible infected mice. Spleens of MAIDS-susceptible infected mice harbored approximately similar levels of BM5def gag RNA as infected spleens of mice that are insusceptible to MAIDS due to lack of CD40. In contrast, the same infected spleens of CD40-deficient mice contained substantially higher (up to 10-fold) levels of BM5eco gag RNA compared with susceptible controls. Similar to that seen in spleen, infected blood of CD40-deficient mice contained similar levels of BM5def gag as susceptible strains, but increased levels (up to threefold) of BM5eco gag RNA. The assays described below can be used to characterize better the contributions of different functional viral components of the LP-BM5 mixture to the development of MAIDS.

In genetically susceptible strains, such as C57BL/6 (B6) mice, LP-BM5 causes murine AIDS (MAIDS). LP-BM5 is a complex mixture of murine leukemia viruses (MuLV) that includes replication competent ecotropic (BM5eco) and mink cell focus inducing (MCF), and replication defective (BM5d) MuLV. At present, for the BM5eco virus, sequence information on only the gag region is available. In this paper, we describe for the first time the sequencing of the entire BM5eco viral genome as well as analysis of homology with two other previously sequenced and well-characterized MuLVs, Emv-11 and Emv-2, the latter constituting the parental virus for BM5eco. We propose that the detailed sequence comparisons herein provide cogent evidence that BM5eco utilizes variations in cytotoxic T lymphocytes (CTL) epitopes as an immune escape mechanism. This CTL evasion mechanism may contribute substantially to the underlying prototypic susceptibility of B6 mice to LP-BM5-induced MAIDS.

Monocyte-macrophages play a central role in HIV-1 infection because they are among the first cells to be infected and because later they are important reservoirs for the virus. Thus, newly designed therapies should take into account the protection of this cell compartment. Herein, we report the results obtained in a murine AIDS model, by the addition to AZT+DDI of a system (GSH-loaded erythrocytes) able to protect macrophages against HIV-1 infection. Five groups of LP-BM5-infected mice were treated as follows: one group was treated by AZT, one group was treated by DDI, one group was treated by the combination of both, another by GSH-loaded erythrocytes, and finally, one by the combination of all three. After 10 weeks of infection the parameters of the disease were studied and the proviral DNA content in different organs and in macrophages of bone marrow and of the peritoneal cavity was quantified. The results obtained show that mice treated with AZT+DDI+GSH-loaded erythrocytes showed proviral DNA content in the brain and in macrophages of bone marrow that was significantly lower than in mice treated with AZT+DDI. This study may help developing strategies aimed at blocking HIV-1 replication in its reservoirs in the body.

Infection of genetically susceptible mice with the LP-BM5 mixture of murine leukemia viruses including an etiologic defective virus (BM5def) causes an immunodeficiency syndrome called murine AIDS (MAIDS). The disease is characterized by interactions between B cells and CD4(+) T cells resulting in polyclonal activation of both cell types. It is known that BM5def is expressed at highest levels in B cells and that B cells serve as viral APC. The CD19-CD21 complex and CD22 on the surface of B cells play critical roles as regulators of B cell responses to a variety of stimuli, influencing cell activation, differentiation, and survival. CD19 integrates positive signals induced by B cell receptor ligation by interacting with the protooncogene Vav, which leads to subsequent tyrosine phosphorylation of this molecule. In contrast, CD22 negatively regulates Vav phosphorylation. To analyze the role of CD19, CD21, Vav, and CD22 in MAIDS, we infected mice deficient in CD19, CD21 (CR2), Vav-1, or CD22 with LP-BM5 murine leukemia viruses. Infected CR2(-/-) mice developed MAIDS with a time course and severity indistinguishable from that of wild-type mice. In contrast, CD19 as well as Vav-1 deficiency restricted viral replication and suppressed the development of typical signs of MAIDS including splenomegaly, lymphadenopathy, and hypergammaglobulinemia. Finally, CD22 deficiency was found to accelerate MAIDS development. These results provide novel insights into the B cell signaling pathways required for normal induction and progression of MAIDS.

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.

Background: Murine acquired immunodeficiency syndrome (MAIDS) is characterized by generalized lymphoproliferation and progressive immunodeficiency. It is induced by a mixture of two replication-competent murine leukemia viruses (MuLV) and a disease-causing, replication-incompetent defective MuLV. Infection leads to specific phenotypic and functional alterations of lymphocytes in lymphoid organs., Methods: We analyzed phenotypic, virological and functional parameters in the blood of mice infected with MAIDS virus., Results: Disease progression correlated with increasing viremia, a loss of mitogen responsiveness of T lymphocytes, and the appearance of CD4+ Thy1- T lymphocytes. At >9 weeks after infection, the distribution of leukocyte cell populations became very heterogeneous, and late-stage leukemic events were observed in 5 of 23 mice., Conclusions: Virus titers, mitogen responsiveness and the presence of CD4+ Thy1- T lymphocytes can efficiently be monitored in the blood and serve as diagnostic parameters to monitor disease progression. Acute leukemic events occurring at the terminal stage could be responsible for the death of at least some of the mice with MAIDS., (Copyright 2002 S. Karger AG, Basel)

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.

The immunodeficiency syndrome murine AIDS (MAIDS), caused by the BM5 retrovirus preparation, involves the activation, division, and subsequent anergy of the entire CD4(+) T cell population as well as extensive B cell hyperproliferation and hypergammaglobulinemia, resulting in splenomegaly and lymphadenopathy, followed many weeks later by death. The development of MAIDS requires CD4(+) T cells and MHC class II expression by the infected host, supporting a role for T-B interaction in disease development or progression. To explore this possibility, we examined development of MAIDS in mice deficient in CD4 (CD4 knockout), in which T-B interactions are compromised. We find that in CD4 knockout hosts, BM5 causes T cell immunodeficiency in the remaining T cells but has only a limited ability to induce B cell phenotypic changes, hyperproliferation, hypergammaglobulinemia, or splenomegaly. There is also delayed death of infected mice. This implies that CD4 dependent T-B interaction is needed to induce the B cell aspects of disease and supports a multistep mechanism of disease in which B cell changes follow and are caused by CD4(+) T cell effects.

Genetically susceptible C57BL/6 (B6) mice that are infected with the LP-BM5 isolate of murine retroviruses develop profound splenomegaly, lymphadenopathy, hypergammaglobulinemia, terminal B-cell lymphomas, and an immunodeficiency state bearing many similarities to the pathologies seen in AIDS. Because of these similarities, this syndrome has been called murine AIDS (MAIDS). We have previously shown that CD154 (CD40 ligand)-CD40 molecular interactions are required both for the initiation and progression of MAIDS. Thus, in vivo anti-CD154 monoclonal antibody (MAb) treatment inhibited MAIDS symptoms in LP-BM5-infected wild-type mice when either a short course of anti-CD154 MAb treatment was started on the day of infection or a course was initiated 3 to 4 weeks after LP-BM5 administration, after disease was established. Here, we further characterize this required CD154-CD40 interaction by a series of adoptive transfer experiments designed to elucidate which cellular subsets must express CD154 or CD40 for LP-BM5 to induce MAIDS. Specifically with regard to CD154 expression, MAIDS-insusceptible B6 nude mice reconstituted with highly purified CD4+ T cells from wild-type, but not from CD154 knockout, B6 donors displayed clear MAIDS after LP-BM5 infection. In contrast, nude B6 recipients that received CD8+ T cells from wild-type B6 donors did not develop MAIDS after LP-BM5 infection. B6 CD40 knockout mice, which are also relatively resistant to LP-BM5-induced MAIDS, became susceptible to LP-BM5-induced disease after reconstitution with highly purified wild-type B cells but not after receiving purified wild-type dendritic cells (DC) or a combined CD40+ population composed of DC and macrophages obtained from B6 SCID mouse donors. Based on these and other experiments, we thus conclude that the cellular basis for the requirement for CD154-CD40 interactions for MAIDS induction and progression can be accounted for by CD154 expression on CD4+ T cells and CD40 expression on B cells.

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.)

Coxsackievirus initiates myocarditis especially in the immunologically deficient or immature. To test whether Coxsackievirus B3 (CVB3) induced pronounced cardiomyopathy during severe immune dysfunction of murine AIDS, female C57BL/6 mice were infected with LP-BM5 retrovirus and superinfected with CVB3. Some were also injected daily with cocaine hydrochloride in 0.9% saline solution (30 mg/kg) intraperitoneally, because cocaine also suppresses cellular immune response. Heart tissue was analyzed histopathologically. Mice experiencing concurrent retrovirus and Coxsackievirus infection had a high degree of cardiac lesions consistent with myopathy compared with findings in uninfected animals (p <.05). Cocaine injection during murine retrovirus infection greatly exacerbated the pathogenesis of Coxsackievirus infection. C57BL/6 mice, essentially resistant to Coxsackievirus-induced cardiomyopathy, became susceptible during the immune dysfunction in murine AIDS. This suggests that retrovirus infection causes conditions favoring Coxsackie-induced cardiac lesions. Interleukin (IL)-2 and IL-4 expression by splenocytes from the dually infected retrovirus and Coxsackievirus group showed no significant differences when the animals were also cocaine treated. However tumor necrosis factor TNF-alpha production was significantly decreased in dually infected retrovirus + Coxsackievirus mice treated with cocaine, compared with findings in various controls (p <.05).

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.)

Idiopathic interstitial pneumonitis (IP), characterized by lymphocytic infiltration of the lung and pulmonary dysfunction, is a major noninfectious complication of human immunodeficiency virus (HIV) infection. The role of the CD4+ and CD8+ T cell populations and INF-gamma in the development of IP were analyzed using a murine model of retroviral-associated IP. Infected mice depleted of CD8+ T cells developed IP similarly to untreated infected mice, suggesting that the CD8+ T cell population does not play a role in IP. Furthermore, depletion of CD8+ T cells did not alter the level of viral RNA in lungs, suggesting that cytotoxic T cells may not serve a role in controlling virus burden in lungs. In contrast, depletion of CD4+ T cells in infected mice prevented the development of IP and inhibited inflammatory cytokine expression, suggesting that CD4+ T cells are important for the development of IP. IFN-gamma -/- mice infected with virus for 10 weeks developed IP, although the severity of lymphocytic infiltration was substantially reduced compared to infected wild-type mice. The data suggest that persistent viral antigen in the lung may drive a CD4+ T cell-mediated immune response, resulting in the chronic production of IFN-gamma which amplifies a chronic inflammatory response in the lung resulting in tissue injury.

One of the major complications of HIV infection is the development of interstitial pneumonitis (IP). IP is characterized by lymphocytic infiltration of the lung and may lead to respiratory failure in some cases. The etiology of IP is unknown although it is likely the result of an antiviral or autoimmune response occurring in the lung. To determine the role of viral replication in the development of IP, AZT was evaluated for the ability to inhibit development of lung inflammation in a murine model of retrovirus-associated IP. Mice were infected with LP-BM5 retrovirus, which induces murine AIDS. Infected mice develop IP by 4 weeks postinfection characterized by infiltration of the lung with activated T cells, B cells, and macrophages. Virus could be detected in the lungs of these mice by 2 weeks postinfection and persisted throughout the course of disease. To determine if reduction in viral load affected the disease process, infected mice were treated with AZT for varying periods postinfection and analyzed for the development of IP. Treatment with AZT resulted in a treatment time-dependent reduction of viral RNA in the lungs of infected mice compared to untreated infected mice. The reduction of viral burden in the lungs correlated with a reduction in the severity of IP and decreased production of the proinflammatory cytokines interleukin (IL)-1 beta and interferon (IFN)-gamma. These results suggest that continuous viral replication in the lung contributes to the pathogenesis of IP.

Macrophages play a key role in AIDS pathogenesis and thus controlling infectivity and viral replication in these cells is a key issue in any antiretroviral therapy. In the present study, using a murine model of AIDS, we evaluated new therapeutic approaches specifically designed for the protection of macrophages. Based on previous observations, we took advantage of the unique ability of autologous erythrocytes to deliver drugs selectively to macrophages. The antiviral drugs selected were a new homodimer of AZT (AZTp2AZT) and reduced glutathione (GSH). The addition of an oral drug for the protection of lymphocytes (i.e., AZT) was also investigated. C57BL/6 mice infected with the retroviral complex LP-BM5 were treated with GSH-loaded erythrocytes, GSH-loaded erythrocytes plus oral AZT, or GSH/AZTp2AZT-loaded erythrocytes plus oral AZT. The treatments including AZT and erythrocytes loaded with GSH alone or with GSH plus AZTp2AZT provided similar results and were most effective in inhibiting the progression of MAIDS; they reduced splenomegaly, lymphadenopathy, and hypergammaglobulinemia by about 70%, 90% and 83%, respectively, when compared with infected animals at 10 weeks postinfection. Evaluation of BM5d proviral DNA content in infected organs revealed that both treatments were able to almost completely protect most infected animals. They were also able to normalize the blood lymphocyte phenotype and to restore the responses of T and B cells to mitogens significantly. Treatment with GSH-loaded erythrocytes alone did not provide significant results for most parameters investigated, but a marked reduction in proviral DNA content was obtained in infected organs, including the brain. The results reported in this paper confirm the important role of macrophages in retroviral infection and moreover prove that erythrocytes, by selectively protecting these cells, strongly affect MAIDS progression. Furthermore, the combination of GSH- or GSH/AZTp2AZT-loaded erythrocytes with an oral nucleoside analogue (AZT) for the protection of lymphocytes provides additive responses in all the parameters investigated.

Murine acquired immunodeficiency syndrome is a lymphoproliferative disease with a concurrently developing immunodeficiency. The disease is induced after injection of supernatant of a chronically infected cell line that releases a mixture of two replication competent virus classes and a replication incompetent virus species responsible for pathogenicity. An immunochemical detection assay for virus infected foci on cell monolayers has been developed. This assay allows quantification of all three types of virus.

Female C57BL/6 mice infected with the LP-BM5 leukaemia retrovirus developed murine acquired immune-deficiency syndrome (AIDS). Dehydroepiandrosterone (DHEA) and melatonin (MLT) modify immune dysfunction and prevent lipid peroxidation. We investigated whether DHEA and MLT could prevent immune dysfunction, excessive lipid peroxidation, and tissue vitamin E loss induced by retrovirus infection. Retrovirus infection inhibited the release of T helper 1 (Th1) cytokines, stimulated secretion of Th2 cytokines, increased hepatic lipid peroxidation, and induced vitamin E deficiency. Treatment with DHEA or MLT alone, as well as together, largely prevented the reduction of B- and T-cell proliferation as well as of Th1 cytokine secretion caused by retrovirus infection. Supplementation also suppressed the elevated production of Th2 cytokines stimulated by retrovirus infection. DHEA and MLT simultaneously reduced hepatic lipid peroxidation and prevented vitamin E loss. The use of DHEA plus MLT was more effective in preventing retrovirus-induced immune dysfunction than either DHEA or MLT alone. These results suggest that supplementation with DHEA and MLT may prevent cytokine dysregulation, lipid oxidation and tissue vitamin E loss induced by retrovirus infection. Similarly, hormone supplementation also modified immune function and increased tissue vitamin E levels in uninfected mice.

The pathogenicities of the murine AIDS (MAIDS) virus complex (LP-BM5) and ecotropic helper virus (BM5eco) isolated from the complex to BALB/c nude mice were studied to elucidate the possible role of replication-competent helper virus in inducing the monoclonal outgrowth of lymphoid cells. Neither LP-BM5 nor BM5eco was pathogenic in adult BALB/c nude mice. However, B-cell lymphoma developed with a very high frequency when either virus was inoculated into newborn BALB/c nude (nu/nu) mice. The cells from the B-cell lymphoma were easily transplanted into nude mice. These results suggested that ecotropic helper virus in the MAIDS virus complex plays an important role in inducing the monoclonal outgrowth of lymphoid cells under immunodeficient conditions caused by defective virus.

Mice infected with murine acquired immunodeficiency syndrome (MAIDS) virus developed lymphoadenopathy and profound immunodeficiency. Concomitantly the expression of endogenous mammary tumor virus (MTV) mRNA increased significantly, especially for the 1.7-kb 3' open reading frame (ORF) mRNA encoding MTV superantigen. B cell lines that are established from MAIDS mice and exhibit superantigen activity also express a high level of 1.7-kb endogenous MTV and mRNA. Infection of a B cell tumor line in vitro with retrovirus containing the cloned MAIDS virus gene induced superantigen activity and this cell line also expressed the 1.7-kb superantigen coding MTV 3' ORF mRNA. These results strongly suggest a link between MAIDS virus infection and the induction of endogenous superantigen activity. This may play an important role in the pathogenesis of the MAIDS virus.

Murine AIDS in C57BL/6 mice is caused by a unique mixture of murine leukemia viruses. We report the use of a competitive PCR to detect and quantitate BM5d proviral DNA. This assay allowed discrimination among endogenous wild-type murine retroviruses and BM5d sequences. Furthermore, the method was subsequently used to evaluate the amount of BM5d in infected mice and in infected AZT (zidovudine)-treated mice, providing an effective way to quantitatively evaluate drug efficacy in the murine AIDS model.

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.

A new antiretroviral drug (azidothymidine homodinucleotide, AZTp2AZT), designed for the protection of macrophages against retroviral infection, was evaluated in a murine retrovirus-induced immunodeficiency model of AIDS (MAIDS) alone and in combination with oral azidothymidine (AZT). C57BL/6 mice were infected with the retroviral complex LP-BM5 and treated for 3 months by weekly administrations of 15 nmol of AZTp2AZT encapsulated into autologous erythrocytes for macrophage protection. AZTp2AZT treatment was found to reduce lymphoadenopathy (48%), splenomegaly (26%), and BM5d proviral DNA content in lymph nodes, spleen, and brain of 37%, 40%, and 36%, respectively, compared with untreated animals. AZT administration in drinking water (0.25 mg/ml) was more effective than administration of AZTp2AZT encapsulated into erythrocytes in reducing lymphoadenopathy, splenomegaly, gammaglobulinemia, and proviral DNA content in lymph nodes, but it caused a reduction in erythrocyte count and hematocrit levels. Although combined treatments do not provide additive responses in the several parameters investigated, they were found to be much more effective in reducing the proviral DNA content in brain (67%) than were monotherapies. Furthermore, no apparent signs of hematotoxicity were observed. Thus, macrophage delivery of antiviral drugs may contribute to brain protection from retroviral infections by mechanisms other than those exerted by oral AZT administration.

Heterosexual transmission of a murine leukemia virus mixture named LP-BM5 MuLV, which is known as the murine AIDS virus, was investigated. Our results indicated that the heterosexual transmission of LP-BM5 MuLV occurs in both directions with high frequency and that the frequencies of virus transmission in the cervix and penis are higher than those in other genital organs. The results suggested that infection by LP-BM5 MuLV via heterosexual transmission may initially take place at particular retrovirus-sensitive sites (cells) in the genital organs.

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.

Experiments were performed to explore the ability of murine cytomegalovirus (MCMV) to disseminate to the eye following intravenous inoculation and to cause infection of ocular tissues and necrotizing retinitis in C57BL/6 mice with retrovirus-induced immunodeficiency syndrome (MAIDS). Although infectious virus could be detected in whole eye homogenates of mice with MAIDS at 10 days after intravenous MCMV inoculation, a recombinant MCMV (RM461) that carries an MCMV IE1 promoter-LacZ insert was used as a tracer virus to confirm direct infection of ocular tissues. Evidence for MCMV replication (determined by RM461-induced expression of beta-galactosidase) was consistently observed in the nonpigmented epithelium of the ciliary body of the eyes of MAIDS animals at 14 days after infection. In sharp contrast, however, the neurosensory retina was spared and necrotizing retinitis failed to develop. These findings demonstrate that systemic MCMV infection of mice with MAIDS results in ocular MCMV infection without development of ocular MCMV disease. Conversion of occult subclinical MCMV infection of ocular tissues to overt clinical MCMV retinitis may require as yet unidentified cofactor(s). Identification of these cofactors could lead to more innovative therapeutic approaches for prevention and/or treatment of CMV retinitis in patients with AIDS.

Lithium gamma linolenic acid (Li-GLA), was evaluated for its possible role as an antiviral agent. Li-GLA 15 micrograms ml-1 was administered to both normal and LP-BM5 MuLV retroviral infected murine bone marrow cultures. After 2 weeks of treatment, numbers of progenitors being produced by infected/treated cultures were reduced to some 10% that of normal cultures. In the remaining 4 weeks, numbers of CFU-GM and BFU-E hematopoietic progenitors returned within normal range. The efficacy of Li-GLA in relieving retroviral hematopoietic bone marrow suppression correlates to a reduction in interleukin-4 (IL-4) secretion, normally elevated in association with LP-BMP5 infection. These data indicate that this reduction in bone marrow suppression of LP-BMP5 infected cells may be due to a killing of infected cells by the Li-GLA, rather than stimulating hematopoiesis as with other lithium compounds. To conclude this may indicate the possible dual effect of administration of LiGLA to virally infected individuals in reducing viral titre and to lower the toxicities associated with long term drug therapy.

Ribonucleotide reductase inhibitors (RRIs) have been recently shown to inhibit retroviral replication. We examined a new series of RRIs, 3,4-dihydroxybenzohydroxamic acid (Didox) and 3,4,5-trihydroxybenzohydroxamidoxime (Trimidox) for their ability to alter disease progression in murine acquired immunodeficiency syndrome (MAIDS), both alone and in combination with 2',3'-dideoxyinosine (ddI). MAIDS disease was induced by inoculation of female C57BL/6 mice with the LP-BM5 murine leukemia virus (MuLV) and disease progression characterized by extensive peripheral lymphadenopathy and splenomegaly. Efficacy of treatment with these drugs was based upon their ability to influence survival and disease pathophysiology by monitoring the development of splenomegaly. Toxicity was determined by changes in body weight, total peripheral white blood cell count and hematocrit. Didox or trimidox monotherapy was associated with increased survival and decreased disease pathophysiology, with no apparent toxicity. Combined with ddI, their ability to reduce development of viral induced splenomegaly was enhanced compared to trimidox, didox or ddI alone. These results demonstrate RRIs have potent activity in reversing the disease manifestations characteristic of MAIDS. Further studies are warranted to determine human clinical efficacy.

The murine AIDS (MAIDS) virus has a unique sequence in its gag p12 region. A transcript which hybridizes with this sequence is expressed in normal C57BL/6 mice. This transcript has been proposed to be the origin of the MAIDS virus, since the virus was originally isolated from radiation-induced leukemic C57BL/6 mice. The transcript, designated Edv, was molecularly cloned and sequenced. Compared with the nucleotide sequence of the helper LP-BM5 ecotropic virus, the pathogenic defective MAIDS virus has 16-bp deletions and a 1-bp insertion in the 5' and 3' regions of the gag p12 sequence, respectively, and the Edv transcript contains only a 3-bp deletion. Therefore, the amino acid sequence of the gag p12 region of the MAIDS virus is less homologous to that of the helper virus and Edv transcript due to the frameshift mutations. This suggested that the MAIDS virus was generated by such frameshift mutations in the gag p12 region during recombination between the helper virus and the Edv or a related sequence.