Hepatitis B virus transgenic mouse model of chronic liver disease.

A model for hepatitis B virus-associated chronic liver disease has been made using cloned hepatitis B virus DNA as a transgene in a severe combined immunodeficient host. These mice consistently support virus gene expression and replication. After adoptive transfer of unprimed, syngeneic splenocytes, these mice cleared virus from liver and serum, and developed chronic liver disease. This model will permit identification of the host and virus contributions to chronic liver disease in the absence of tolerance.

Human- and mouse-inducible nitric oxide synthase promoters require activation of phosphatidylcholine-specific phospholipase C and NF-kappa B.

BACKGROUND: The production of nitric oxide by type II inducible nitric oxide synthase (type II NOS) gene is controlled at least in part by transcriptional activation. Although the murine and human type II NOS genes share significant sequence homology, they differ in the induction stimuli required for activation. MATERIALS AND METHODS: The A549 human and murine RAW 264.7 cell lines were cultured in the presence of inducers of the type II NOS gene and exposed to specific inhibitors of phosphatidyl choline-specific phospholipase C, NF-kappa B, and endocytosis, as well as to reagents that deplete stores of ATP or prevent the acidification of endosomes. The effect of these reagents on the induction of the type II NOS gene transcription, translation, and NO expression was studied using electromobility shift assays, Western blotting, and the detection of NO as nitrates, as appropriate. Additionally, the ability of the native human type II NOS NF-kappa B recognition sequence to bind NF-kappa B was compared with a concensus sequence and with a mutated oligomer. RESULTS: Type II NOS production by both human and mouse cells could be prevented by the addition of the specific inhibitor of phosphatidylcholine-specific phospholipase C, D609, and of agents that interfere with the activation of NF-kappa B. Both mouse and human cells also required acidic endosome formation and the production of 1,2-diacylglycerol for type II NOS expression. Additionally, the native human type II NOS NF-kappa B recognition sequence bound NF-kappa B with significantly less affinity than did the recognition sequence derived from the human immunoglobulin light-chain gene promoter. CONCLUSIONS: These experiments show that whereas mouse cells can be activated by lipopolysaccharide to produce nitric oxide, and human cells require activation by a mixture of cytokines to produce nitric oxide, the intracellular activation pathway following receptor binding of these heterologous stimuli is shared. Additionally, NF-kappa B activation is necessary but not sufficient for inducible nitric oxide synthase production in human cells, in contrast to murine cells in which it serves as a complete inducer.

Characterization and functional analysis of the human inducible nitric oxide synthase gene promoter.

BACKGROUND: Nitric oxide has a wide variety of homeostatic and pathological effects. Control of the production of nitric oxide by the inducible form of the enzyme resides in the 5' promoter region of the gene. Although control of the murine isoform has been investigated, little is known about the functional aspects of the human analog. MATERIALS AND METHODS: A 3.9-kb 5' nontranslated region of the human gene was cloned, sequenced, and several reporter constructs prepared. The promoter-reporter constructs were transfected into human or murine monocytoid cells and reporter expression quantified following cytokine activation of the cells. The production of nitric oxide was also monitored. RESULTS: Although a murine promoter-reporter functioned efficiently in both human and mouse cells, the human constructs functioned only in human cells. The activity of the mouse construct increased progressively with the addition of activating cytokines, but the human promoter-reporter did not. Although interleukin 1 beta drove expression of the human inducible nitric oxide synthase reporter, actual expression of nitric oxide required both interleukin 1 beta and interferon-gamma. CONCLUSIONS: The data indicate that despite the significant homology between the human and mouse inducible nitric oxide synthase promoter sequence, control of the two genes is quite different. In addition to being more efficient in promoter activity, the murine promoter responds increasingly to cytokines that are not effective for the human analog. It is also apparent that human inducible nitric oxide synthase is controlled at both the level of transcription and post-translationally.

Activation of the inducible form of nitric oxide synthase in the brains of patients with multiple sclerosis.

Nitric oxide (NO) has been implicated as a pathogenic mediator in a variety of central nervous system (CNS) disease states, including the animal model of multiple sclerosis (MS) and experimental allergic encephalomyelitis. We have examined post-mortem brain tissues collected from patients previously diagnosed with MS, as well as tissues collected from the brains of patients dying without neuropathies. Both Northern blot analysis and reverse transcriptase (RT)-driven in situ PCR (RT-in situ PCR) studies demonstrated that inducible NO synthase (iNOS) mRNA was present in the brain tissues from MS patients but was absent in equivalent tissues from normal controls. We have also performed experiments identifying the cell type responsible for iNOS expression by RT-in situ PCR in combination with immunohistochemistry. Concomitantly, we analyzed the tissues for the presence of the NO reaction product nitrotyrosine to demonstrate the presence of a protein nitrosylation adduct. We report here that iNOS mRNA was detectable in the brains of 100% of the CNS tissues from seven MS patients examined but in none of the three normal brains. RT-in situ PCR experiments also demonstrated the presence of iNOS mRNA in the cytoplasm of cells that also expressed the ligand recognized by the Ricinus communis agglutinin 1 (RCA-1), a monocyte/macrophage lineage marker. Additionally, specific labeling of cells was observed when brain tissues from MS patients were exposed to antisera reactive with nitrotyrosine residues but was significantly less plentiful in brain tissue from patients without CNS disease. These results demonstrate that iNOS, one of the enzymes responsible for the production of NO, is expressed at significant levels in the brains of patients with MS and may contribute to the pathology associated with the disease.

Expression of the rabies virus glycoprotein in transgenic tomatoes.

We have engineered tomato plants (Lycopersicon esculentum Mill var. UC82b) to express a gene for the glycoprotein (G-protein), which coats the outer surface of the rabies virus. The recombinant constructs contained the G-protein gene from the ERA strain of rabies virus, including the signal peptide, under the control of the 35S promoter of cauliflower mosaic virus. Plants were transformed by Agrobacterium tumefaciens-mediated transformation of cotyledons and tissue culture on selective media. PCR confirmed the presence of the G-protein gene in plants surviving selection. Northern blot analysis indicated that RNA of the appropriate molecular weight was produced in both leaves and fruit of the transgenic plants. The recombinant G-protein was immunoprecipitated and detected by Western blot from leaves and fruit using different antisera. The G-protein expressed in tomato appeared as two distinct bands with apparent molecular mass of 62 and 60 kDa as compared to the 66 kDa observed for G-protein from virus grown in BHK cells. Electron microscopy of leaf tissue using immunogold-labeling and antisera specific for rabies G-protein showed localization of the G-protein to the Golgi bodies, vesicles, plasmalemma and cell walls of vascular parenchyma cells. In light of our previous demonstration that orally administered rabies G-protein from the same ERA strain elicits protective immunity in animals, these transgenic plants should provide a valuable tool for the development of edible oral vaccines.

The human immunodeficiency virus type 1 (HIV-1) vif protein is located in the cytoplasm of infected cells and its effect on viral replication is equivalent in HIV-2.

The human immunodeficiency virus type 1 (HIV-1) vif gene (viral infectivity gene) plays an important role in viral replication in vitro. We demonstrated that the Vif protein is membrane associated in HIV-1-infected cells and have investigated the role in viral replication of the equivalent gene in HIV-2. We constructed an HIV-2 vif minus mutant and studied its virulence and cellular tropism in vitro. Parallel experiments were also performed with an HIV-1 vif mutant to ascertain whether the two distantly related HIV-2 and HIV-1 genes might exert the same effect on viral replication. The results indicated that both HIV-1 and HIV-2 vif minus cell-free infection was not impaired when the SupT-1 cell line was used. However, differential degrees of impairment in viral replication were observed when other cell lines were used (Molt-3, U-937). Nevertheless, when viral production could not be detected, rescue experiments by coculture with the permissive cell line SupT-1 were generally positive, indicating that the viruses were still present in the inoculated cells. In contrast, when primary human cells (peripheral blood mononuclear cells and purified macrophages) were infected with HIV-1 and HIV-2 vif minus viruses no productive infection was observed and generally no virus was rescued by cocultivation. Thus, like in HIV-1, the vif gene of HIV-2 is crucial for viral infectivity in primary cells and might represent an attractive target for therapy.

Lessons from caprine and ovine retrovirus infections.

Two lentiviruses that infect sheep and goats have been shown to be closely related to the human immunodeficiency virus (HIV). These ungulate lentiviruses cause a spectrum of diseases, including arthritis in their natural hosts. The molecular and cellular biology of these viruses as well as possible pathogenic mechanisms is compared to HIV-1 in order to identify common features and significant differences between the human and animals pathogens.

Augmentation of lymphocyte and macrophage proliferation by caprine arthritis-encephalitis virus contributes to the development of progressive arthritis.

Infection by the lentivirus, caprine arthritis-encephalitis virus (CAEV), may lead to an intermittent arthritis due to the presence of lymphocytes and macrophages in synovial membranes. We have observed that the presence of CAEV increases the division of lymphocytes and macrophages. In association, antigen-induced arthritis is more severe in goats concurrently infected by CAEV than in noninfected goats. From these observations we propose that infection by this lentivirus increases reactivity of lymphocytes and macrophages to immune and nonimmune stimuli, leading to the increased likelihood of progressive arthritis in infected animals.

Contribution of various host factors to resistance to experimentally induced bacterial endotoxemia in calves.

The reactions of 15 calves to IV administered bacterial lipopolysaccharide was investigated and was correlated with the capacity of 2 host defense mechanisms. The calves had a wide range of changes in clinical response, total WBC count, plasma lactate dehydrogenase (LD) activity, reaction to intradermally inoculated lipid A, and rectal temperature response. The early rectal temperature response of an individual calf was correlated with plasma LD activity, indicating a relationship between cell damage and fever. The concentration of antibody against lipid A at the time of lipopolysaccharide inoculation was negatively correlated with the rectal temperature changes recorded during endotoxemia, suggesting a protective ability of antibody. The capacity of a plasma inhibitor of lipopolysaccharide-mediated clotting of limulus amebocyte lysate was correlated with increases in plasma LD activity. Therefore, antibody to lipid A probably is involved in protection of cattle during endotoxemia, but the plasma lipopolysaccharide inhibitor actually may potentiate lipopolysaccharide toxicity.

Lentivirus infection augments concurrent antigen-induced arthritis.

Experimental antigen-induced arthritis was compared in normal goats and goats infected with caprine arthritis-encephalitis virus. Although acute arthritis was the same in infected and uninfected animals, the disease lasted 16 weeks longer in the caprine arthritis-encephalitis virus-infected goats. Our findings suggest that the arthritis caused by this virus is due to events other than, or in addition to, the immune reaction to viral antigens.

Stimulation and killing of bovine mononuclear leukocytes by bacterial lipopolysaccharide (endotoxin).

Bovine adherent mononuclear leukocytes were incubated with bacterial lipopolysaccharides (LPS) in vitro, and these cells produced a factor that increased the blastogenic reaction of mouse thymocytes to concanavalin A. This factor most resembles interleukin 1. The LPS were also cytotoxic for bovine adherent mononuclear leukocytes in a dose-and time-dependent manner. Cytotoxicosis was determined by the release of cytoplasmic lactic dehydrogenase. This cytotoxicosis was blocked by treating the cells with corticosteroids. Variation in the reaction to LPS occurred in cells collected from the same cow on different days and from cells collected from different cows.