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.

Myeloid-derived suppressor cells in murine AIDS inhibit B-cell responses in part via soluble mediators including reactive oxygen and nitrogen species, and TGF-ß.

Monocytic myeloid-derived suppressor cells (M-MDSCs) were increased during LP-BM5 retroviral infection, and were capable of suppressing not only T-cell, but also B-cell responses. In addition to previously demonstrating iNOS- and VISTA-dependent M-MDSC mechanisms, in this paper, we detail how M-MDSCs utilized soluble mediators, including the reactive oxygen and nitrogen species superoxide, peroxynitrite, and nitric oxide, and TGF-ß, to suppress B cells in a predominantly contact-independent manner. Suppression was independent of cysteine-depletion and hydrogen peroxide production. When two major mechanisms of suppression (iNOS and VISTA) were eliminated in double knockout mice, M-MDSCs from LP-BM5-infected mice were able to compensate using other, soluble mechanisms in order to maintain suppression of B cells. The IL-10 producing regulatory B-cell compartment was among the targets of M-MDSC-mediated suppression.

Glutathione Depletion Is Linked with Th2 Polarization in Mice with a Retrovirus-Induced Immunodeficiency Syndrome, Murine AIDS: Role of Proglutathione Molecules as Immunotherapeutics.

UNLABELLED: Injection of the LP-BM5 murine leukemia virus into mice causes murine AIDS, a disease characterized by many dysfunctions of immunocompetent cells. To establish whether the disease is characterized by glutathione imbalance, reduced glutathione (GSH) and cysteine were quantified in different organs. A marked redox imbalance, consisting of GSH and/or cysteine depletion, was found in the lymphoid organs, such as the spleen and lymph nodes. Moreover, a significant decrease in cysteine and GSH levels in the pancreas and brain, respectively, was measured at 5 weeks postinfection. The Th2 immune response was predominant at all times investigated, as revealed by the expression of Th1/Th2 cytokines. Furthermore, investigation of the activation status of peritoneal macrophages showed that the expression of genetic markers of alternative activation, namely, Fizz1, Ym1, and Arginase1, was induced. Conversely, expression of inducible nitric oxide synthase, a marker of classical activation of macrophages, was detected only when Th1 cytokines were expressed at high levels. In vitro studies revealed that during the very early phases of infection, GSH depletion and the downregulation of interleukin-12 (IL-12) p40 mRNA were correlated with the dose of LP-BM5 used to infect the macrophages. Treatment of LP-BM5-infected mice with N-(N-acetyl-l-cysteinyl)-S-acetylcysteamine (I-152), an N-acetyl-cysteine supplier, restored GSH/cysteine levels in the organs, reduced the expression of alternatively activated macrophage markers, and increased the level of gamma interferon production, while it decreased the levels of Th2 cytokines, such as IL-4 and IL-5. Our findings thus establish a link between GSH deficiency and Th1/Th2 disequilibrium in LP-BM5 infection and indicate that I-152 can be used to restore the GSH level and a balanced Th1/Th2 response in infected mice. IMPORTANCE: The first report of an association between Th2 polarization and alteration of the redox state in LP-BM5 infection is presented. Moreover, it provides evidence that LP-BM5 infection causes a decrease in the thiol content of peritoneal macrophages, which can influence IL-12 production. The restoration of GSH levels by GSH-replenishing molecules can represent a new therapeutic avenue to fight this retroviral infection, as it reestablishes the Th1/Th2 balance. Immunotherapy based on the use of pro-GSH molecules would permit LP-BM5 infection and probably all those viral infections characterized by GSH deficiency and a Th1/Th2 imbalance to be more effectively combated.

Morphine increases hippocampal viral load and suppresses frontal lobe CCL5 expression in the LP-BM5 AIDS model.

Chronic opiate abuse accelerates the development of cognitive deficits in human immunodeficiency virus (HIV)-1 patients. To investigate morphine's effects on viral infection of the central nervous system, we applied chronic morphine treatment to the LP-BM5 murine acquired immunodeficiency syndrome (MAIDS) model. LP-BM5 infection induces proinflammatory cytokine/chemokine production, correlating to increased blood-brain barrier permeability. Morphine treatment significantly increased LP-BM5 viral load in the hippocampus, but not in the frontal lobe. Morphine reduced the chemokine CCL5 to non-infected levels in the frontal lobe, but not in the hippocampus. These data indicate a region-specific mechanism for morphine's effects on virally-induced neurocognitive deficits.

Murine immunodeficiency virus-induced peripheral neuropathy and the associated cytokine responses.

Distal symmetrical polyneuropathy is the most common form of HIV infection-associated peripheral neuropathy and is often associated with pain. C57BL/6 (B6) mice infected with LP-BM5, a murine retroviral isolate, develop a severe immunodeficiency syndrome similar to that in humans infected with HIV-1, hence the term murine AIDS. We investigated the induction of peripheral neuropathy after LP-BM5 infection in B6 mice. Infected B6 mice, like HIV-infected humans, exhibited behavioral (increased sensitivity to mechanical and heat stimuli) and pathological (transient loss of intraepidermal nerve fibers) signs of peripheral neuropathy. The levels of viral gag RNA were significantly increased in all tissues tested, including spleen, paw skin, lumbar dorsal root ganglia, and lumbar spinal cord, postinfection (p.i.). Correlated with the development of peripheral neuropathy, the tissue levels of several cytokines, including IFN-γ, IL-1ß, IL-6, and IL-12, were significantly elevated p.i. These increases had cytokine-specific and tissue-specific profiles and kinetics. Further, treatment with the antiretroviral agent zidovudine either significantly reduced or completely reversed the aforementioned behavioral, pathologic, and cytokine changes p.i. These data suggest that LP-BM5 infection is a potential mouse model of HIV-associated distal symmetrical polyneuropathy that can be used for investigating the roles of various cytokines in infection-induced neuropathic pain. Further investigation of this model could give a better understanding of, and lead to more effective treatments for, HIV infection-associated painful peripheral neuropathy.

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.

Inhibition of premature death by isothiocyanates through immune restoration in LP-BM5 leukemia retrovirus-infected C57BL/6 mice.

The purpose of this study was to determine the effect of isothiocyanates (ITCs) in delaying the progression of the murine immunodeficiency virus to murine AIDS, resulting in increased life span. Furthermore, we investigated the role of ITCs in modulating immune dysfunction caused by LP-BM5 retrovirus infection. Among the tested ITCs, oral administration of sulforaphane (SUL), benzyl isothiocyante (BITC), and phenethyl isothiocyanate (PEITC) showed the inhibition of premature death caused by LP-BM5 retrovirus infection, while indolo[3,2-b] carbazole (ICZ) and indole-3-carbinol (I3C) did not delay the progress of the LP-BM5 retrovirus to murine AIDS. Inhibition of premature death by BITC, PEITC, and SUL could be explained by restoration of the immune system and down regulation of free radicals. Dysfunction of T and B cell mitogenesis caused by retrovirus infection in primary cultured splenocytes has been partially recovered with administration of BITC, PEITC, and SUL. There was a shift from imbalanced cytokine production (increased Th2 and decreased Th1 cell cytokine production) into balanced Th1/Th2 cell secretion of cytokines under administration of these ITCs during the development of murine AIDS. Hepatic vitamin E level was significantly restored by administration of these ITCs, in accordance with reduced hepatic lipid peroxidation levels. This study suggests that certain types of ITCs have beneficial effects in preventing premature death during progression to murine AIDS by restoration of immune dysfunction and removal of excessive free radicals, implying that selective usage of ITCs would be helpful in retarding the progression from HIV infection to AIDS.

Mice with disrupted type I protein kinase A anchoring in T cells resist retrovirus-induced immunodeficiency.

Type I protein kinase A (PKA) is targeted to the TCR-proximal signaling machinery by the A-kinase anchoring protein ezrin and negatively regulates T cell immune function through activation of the C-terminal Src kinase. RI anchoring disruptor (RIAD) is a high-affinity competitor peptide that specifically displaces type I PKA from A-kinase anchoring proteins. In this study, we disrupted type I PKA anchoring in peripheral T cells by expressing a soluble ezrin fragment with RIAD inserted in place of the endogenous A-kinase binding domain under the lck distal promoter in mice. Peripheral T cells from mice expressing the RIAD fusion protein (RIAD-transgenic mice) displayed augmented basal and TCR-activated signaling, enhanced T cell responsiveness assessed as IL-2 secretion, and reduced sensitivity to PGE(2)- and cAMP-mediated inhibition of T cell function. Hyperactivation of the cAMP-type I PKA pathway is involved in the T cell dysfunction of HIV infection, as well as murine AIDS, a disease model induced by infection of C57BL/6 mice with LP-BM5, a mixture of attenuated murine leukemia viruses. LP-BM5-infected RIAD-transgenic mice resist progression of murine AIDS and have improved viral control. This underscores the cAMP-type I PKA pathway in T cells as a putative target for therapeutic intervention in immunodeficiency diseases.

Anaplastic plasmacytomas: relationships to normal memory B cells and plasma cell neoplasms of immunodeficient and autoimmune mice.

Anaplastic plasmacytomas (APCTs) from NFS.V(+) congenic mice and pristane-induced plasmacytic PCTs from BALB/c mice were previously shown to be histologically and molecularly distinct subsets of plasma cell neoplasms (PCNs). Here we extended these comparisons, contrasting primary APCTs and PCTs by gene expression profiling in relation to the expression profiles of normal naïve, germinal centre, and memory B cells and plasma cells. We also sequenced immunoglobulin genes from APCT and APCT-derived cell lines and defined surface phenotypes and chromosomal features of the cell lines by flow cytometry and by spectral karyotyping and fluorescence in situ hybridization. The results indicate that APCTs share many features with normal memory cells and the plasma cell-related neoplasms (PLs) of FASL-deficient mice, suggesting that APCTs and PLs are related and that both derive from memory B cells. Published in 2010 by John Wiley & Sons, Ltd.

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.

Blockade of interferon-gamma-inducible protein-10 attenuates chronic experimental colitis by blocking cellular trafficking and protecting intestinal epithelial cells.

The role of chemokines, especially CXCL10/interferon-gamma-inducible protein 10 kDa (IP-10), a chemokine to attract CXCR3(+) T-helper 1-type CD4(+) T cells, is largely unknown in the pathophysiology of inflammatory bowel disease; ulcerative colitis and Crohn's disease. The authors have earlier shown that IP-10 neutralization protected mice from acute colitis by protecting crypt epithelial cells of the colon. To investigate the therapeutic effect of neutralization of IP-10 on chronic colitis, an anti-IP-10 antibody was injected into mice with newly established murine AIDS (MAIDS) colitis. Anti-IP-10 antibody treatment reduced the number of colon infiltrating cells when compared to those mice given a control antibody. The treatment made the length of the crypt of the colon greater than control antibody. The number of Ki67(+) proliferating epithelial cells was increased by the anti-IP-10 antibody treatment. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL)(+) apoptotic cells were observed in the epithelial cells of the luminal tops of crypts in control MAIDS colitis, whereas TUNEL(+) apoptotic epithelial cells were rarely observed with anti-IP-10 antibody treatment. In conclusion, blockade of IP-10 attenuated MAIDS colitis through blocking cellular trafficking and protecting intestinal epithelial cells, suggesting that IP-10 plays a key role in the development of inflammatory bowel disease as well as in chronic experimental colitis.

Dilated cardiomyopathy in retrovirally infected mice: a novel model for silent viral DCM?

Dilated cardiomyopathy (DCM) is a clinically relevant disease that can occur independently or secondary to other diseases such as HIV infection and AIDS. To study this latter process, we used a model in which mice are infected with the LP-BM5 murine AIDS (MAIDS) retrovirus. Cardiac function of control and infected mice was determined through the in vivo analysis of left ventricular pressure-volume loops. Furthermore, the role of myocarditis was investigated through immunohistochemistry for T-cell, B-cell, and macrophage cardiac infiltrates and Northern blot analysis for tumor necrosis factor (TNF)-alpha and inducible nitric oxide synthase (iNOS). End-systolic and end-diastolic volumes were significantly increased and ventricular stiffness was significantly decreased in infected mice, consistent with DCM; however, no staining for inflammatory cellular infiltrates or TNF-alpha and iNOS was seen. These data support the conclusion that the LP-BM5 HIV model virus causes DCM in the absence of chronic cardiac inflammation. These findings support MAIDS retroviral infection as a new model of idiopathic DCM in which myo-carditis does not occur.

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.

Murine cytomegalovirus retinitis during MAIDS: Susceptibility correlates with elevated intraocular levels of interleukin-4 mRNA.

PURPOSE: To determine if murine cytomegalovirus (MCMV)-infected eyes of mice with murine acquired immunodeficiency syndrome (MAIDS) that are destined to develop MCMV retinitis display elevated intraocular levels of interleukin-4 (IL-4) mRNA when compared with uninfected eyes of mice with MAIDS and unmanipulated, uninfected, eyes of normal healthy mice. METHODS: Groups of C57BL/6 mice with MAIDS and normal C57BL/6 mice were infected uniocularly with MCMV by subretinal MCMV injection. IL-4 levels in individual spleens collected five days later from groups of MAIDS mice and normal mice were assessed by quantitative ELISA. MCMV-infected eyes and uninfected contralateral eyes from another group of mice with MAIDS were also collected at five days postinfection and individually subjected to competitive RT-PCR assay and real-time RT-PCR assay for detection and quantification of IL-4 mRNA. Unmanipulated eyes from normal C57BL/6 mice served as controls. RESULTS: IL-4 mRNA was detected at a level of 9.7 +/- 3.4 pg mRNA per 1000 ng total RNA in 100% of MCMV-infected eyes of mice with MAIDS by competitive RT-PCR assay, but could not be detected in any of the uninfected eyes of MAIDS mice. In comparison, the more sensitive technique of real-time RT-PCR assay detected copies of IL-4 cDNA in both MCMV-infected eyes and uninfected eyes of MAIDS mice. MCMV-infected eyes showed a 16-fold increase in the number of IL-4 cDNA copies when compared with uninfected eyes. Neither technique detected IL-4 mRNA in unmanipulated eyes of normal mice. As expected, spleen cells from mice with MAIDS expressed significantly greater levels of IL-4 when compared with spleen cells from normal mice. CONCLUSIONS: MCMV-infected mice with MAIDS exhibited an expected preferential activation of Th2 cells as determined by increased levels of IL-4 in spleen cells when compared with spleen cells of normal mice. MCMV-infected eyes destined to develop retinitis during MAIDS also showed increased levels of detectable IL-4 mRNA when compared with uninfected eyes of mice with MAIDS. It is therefore possible that IL-4 synthesis by Th2 CD4+ T cells during retrovirus-induced immunosuppression serves to inhibit the perforin cytotoxic pathway that subsequently allows susceptibility to MCMV retinitis during MAIDS.

Murine retrovirus infection and the effect of chronic alcohol consumption: proteomic analysis of cardiac protein expression.

AIMS: The cardiovascular complications of acquired immunodeficiency syndrome (AIDS) are serious, including the occurrence of pathological heart conditions such as cardiomyopathy. Chronic alcohol consumption accentuates the severity of AIDS and may contribute to the development of cardiomyopathy. The aim of this work was to use a proteomics approach to investigate global alterations in protein expression in a mouse model of AIDS in the presence or absence of chronic alcohol consumption. METHODS: Cardiac proteins were separated by two-dimensional polyacrylamide gel electrophoresis and quantitative computer analysis was used to evaluate the resulting two-dimensional protein profiles. Proteins that were differentially expressed in the hearts of mice from the different experimental groups were identified by peptide mass finger-printing by matrix-assisted laser desorption/ionization mass spectrometry. RESULTS: A number of specific proteins were observed to be differentially expressed in the mouse heart due to the effect of ethanol feeding alone. Differentially expressed proteins were also observed that were due to viral infection alone. Ethanol feeding and viral infection appeared to have similar effects on the expression of a number of proteins. A total of 24 proteins were altered by infection alone. Of these 24 proteins, eight were affected by alcohol, with six alterations being ameliorated and two being exacerbated by alcohol. Two of these proteins have been identified as the 27 kDa heat-shock protein and mitochondrial long-chain acyl-CoA thioesterase 1. CONCLUSIONS: These results suggest that chronic alcohol consumption may exacerbate the effects of viral infection on the heart by lowering the stress response leading to de-protection and further cytotoxic effects.

Supplementation with Lactobacillus reuteri or L. acidophilus reduced intestinal shedding of cryptosporidium parvum oocysts in immunodeficient C57BL/6 mice.

The effect of L. acidophilus supplementation to reduce fecal shedding of Cryptosporidium parvum oocysts was compared to L. reuteri using C57BL/6 female mice immunosuppressed by murine leukemia virus (strain LP-BM5) inoculation. After 12 weeks post LP-BM5 inoculation, 15 immunosuppressed mice each were randomly assinged to one of the following treatment groups: historical control (group A), LP-BM5 control (group B), C. parvum (group C), L. reuteri plus C. parvum (group D) or L. acidophilus plus C. parvum (group E). Mice were pre-fed the L. reuteri or L. acidophilus bacteria strains daily for 13 days, challenged with C. parvum oocysts and thereafter fed the specified Lactobacillus regimens daily during the experimental period. Animals supplemented with L. reuteri shed fewer (p<0.05) oocysts on day-7 post C. parvum challenge compared to controls. Mice supplemented with L. acidophilus also shed fewer (p<0.05) oocysts on days 7 and 14 post-challenge compared to controls. Overall, Lactobacillus supplementation reduced C. parvum shedding in the feces but failed to suppress the production of T-helper type 2 cytokines [interleukin-4 (IL-4), IL-8)] which are associated with immunosuppression. Additionally, Lactobacillus supplementation did not restore T-helper type 1 cytokines (interleukin-2 (IL-2) and gamma interferon (IFN-gamma), which are required for recovery from parasitic infections. Altered T-helper types 1 and 2 cytokine production as a consequence of immunodysfunction permitted the development of persistent cryptosporidiosis while mice with intact immune system were refractory to infection with C. parvum. Reduction in shedding of oocysts observed in the Lactobacillus supplemented mice during deminished IL-2 and IFN-gamma production may be mediated by factors released into the intestinal lumen by the Lactobacillus and possibly other host cellular mechanisms. These observations suggest that L. reuteri or L. acidophilus can reduce C. parvum parasite burdens in the intestinal epithelium during cryptosporidiosis and may serve potential benefits as probiotics for host resistance to intestinal parasitic infections. L. acidophilus was more efficacious in reducing fecal shedding than L. reuteri and therefore may also have implication in the therapy of cryptosporidiosis during immunosuppressive states including human AIDS.

Activation of the ubiquitin proteolytic system in murine acquired immunodeficiency syndrome affects IkappaBalpha turnover.

Murine acquired immunodeficiency syndrome (MAIDS) is a complex immunopathology caused by a defective murine leukemia virus (LP-BM5) that mainly targets B-lymphocytes. Lymphadenophathy, splenomegaly, hypergammaglobulinemia and progressive immunodeficiency are prominent features of MAIDS. Previously, we showed that the ubiquitin proteolytic system was upregulated in infected lymph nodes [Crinelli, R., Fraternale, A., Casabianca, A. & Magnani, M. (1997) Eur. J. Biochem. 247, 91-97]. In this report, we demonstrate that increased 26S proteasome activity is responsible for accelerated turnover of the IkappaBalpha inhibitor in lymph node extracts derived from animals with MAIDS. The molecular mechanisms mediating IkappaBalpha proteolysis involved constitutive phosphorylation of IkappaBalpha at Ser32 and Ser36 and subsequent ubiquitination, suggesting persistent activation of an NF-kappaB inducing pathway. Interestingly, enhanced IkappaBalpha degradation did not result in enhanced NF-kappaB DNA binding activity, but rather in a different subunit composition. The modulation of NF-kappaB/IkappaB system may affect multiple immunoregulatory pathways and may in part explain the mechanisms leading to the profound immune dysregulation involved in MAIDS pathogenesis.

Role of virus replication in a murine model of AIDS-associated interstitial pneumonitis.

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.

Extracellular glutamate levels are chronically elevated in the brains of LP-BM5-infected mice: a mechanism of retrovirus-induced encephalopathy.

Mice infected with the LP-BM5 leukemia retrovirus mixture develop a progressive immunodeficiency with associated behavioral, histological, and neurochemical alterations consistent with glutamatergic hyperactivation. To gain insight into the contribution of excitatory amino acids to the neurodegeneration observed in these mice, their concentrations were measured in the CSF and striatal microdialysates. Glutamate concentrations were significantly elevated in CSF but not plasma as early as 4 weeks postinoculation. Steady-state glutamate levels in striatal microdialysates were increased threefold and could be reduced 40% by application of L-alpha-aminoadipate, an inhibitor of microglial glutamate transport. Stimulation of infected mice with KCl/L-trans-2,4-pyrrolidine dicarboxylate further increased glutamate levels 170-270% above those evoked in control mice. Tetrodotoxin suppressed the depolarization-evoked increase in glutamate by 88% in control mice, but it had only negligible effects in 40% of infected mice. Analysis of glutamate transport and catabolism suggests that abnormal astrocytic function does not contribute to the increase in basal extracellular glutamate levels. These findings are the first direct evidence that infection with an immunodeficiency-inducing retrovirus leads to a chronic elevation of extracellular free glutamate levels in the brain, which contributes to the neurodegenerative and cognitive deficits observed in these mice.