Atypical cytomegalovirus retinal disease in pyroptosis-deficient mice with murine acquired immunodeficiency syndrome.

Pyroptosis is a caspase-dependent programmed cell death pathway that initiates and sustains inflammation through release of pro-inflammatory cytokines interleukin (IL)-1ß 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.

Transcriptional analysis of immune response genes during pathogenesis of cytomegalovirus retinitis in mice with murine acquired immunodeficiency syndrome.

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.

Parthanatos-associated proteins are stimulated intraocularly during development of experimental murine cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression.

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.

Suppressor of Cytokine Signaling 1 (SOCS1) and SOCS3 Are Stimulated within the Eye during Experimental Murine Cytomegalovirus Retinitis in Mice with Retrovirus-Induced Immunosuppression.

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.

Immunomodulatory and Antioxidant Effects of Purple Sweet Potato Extract in LP-BM5 Murine Leukemia Virus-Induced Murine Acquired Immune Deficiency Syndrome.

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.

Use of IRF-3 and/or IRF-7 knockout mice to study viral pathogenesis: lessons from a murine retrovirus-induced AIDS model.

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.

Involvement of microglial CD40 in murine retrovirus-induced peripheral neuropathy.

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.

Murine cytomegalovirus downregulates interleukin-17 in mice with retrovirus-induced immunosuppression that are susceptible to experimental cytomegalovirus retinitis.

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.

Evidence for multiple cell death pathways during development of experimental cytomegalovirus retinitis in mice with retrovirus-induced immunosuppression: apoptosis, necroptosis, and pyroptosis.

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 1ß [IL-1ß], 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.

A novel role for APOBEC3: susceptibility to sexual transmission of murine acquired immunodeficiency virus (mAIDS) is aggravated in APOBEC3 deficient mice.

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.

Activity of a novel combined antiretroviral therapy of gemcitabine and decitabine in a mouse model for HIV-1.

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.

Immunotherapy of murine retrovirus-induced acquired immunodeficiency by CD4 T regulatory cell depletion and PD-1 blockade.

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.

Citrobacter-induced colitis in mice with murine acquired immunodeficiency syndrome.

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.

MAIDS resistance-associated gene expression patterns in secondary lymphoid organs.

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.

Identification of putative endogenous retroviruses actively transcribed in the brain.

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.

Role of a cytotoxic-T-lymphocyte epitope-defined, alternative gag open reading frame in the pathogenesis of a murine retrovirus-induced immunodeficiency syndrome.

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.

Combination of inhibitors of lymphocyte activation (hydroxyurea, trimidox, and didox) and reverse transcriptase (didanosine) suppresses development of murine retrovirus-induced lymphoproliferative disease.

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.

Cyclo-oxygenase type 2-dependent prostaglandin E2 secretion is involved in retrovirus-induced T-cell dysfunction in mice.

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.

Development of a real-time PCR assay using SYBR Green I for provirus load quantification in a murine model of AIDS.

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.

Basic fibroblast growth factor (bFGF) and its receptor expression (bek and flg) In bone marrow stroma of murine AIDS.

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.