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We recently reported that a chimeric simian/human immunodeficiency virus (SHIVKU-1) developed in our laboratory caused progressive depletion of CD4+ T lymphocytes and AIDS within 6 months of inoculation into pig-tailed macaques (M. nemestrina). None of the pig-tailed macaques showed productive SHIV infection in the central nervous system (CNS). In this report, we show that by further passage of the pathogenic virus in rhesus macaques [M. mulatta], we have derived a new strain of SHIV (SHIVKU-2) that has caused AIDS and productive CNS infection in 3 of 5 rhesus macaques infected with the virus. Productive replication of SHIV in the CNS was clearly shown by high infectivity titers and p27 protein levels in brain homogenates, and in 2 of the 3 rhesus macaques this was associated with disseminated, nodular, demyelinating lesions, including focal multinucleated giant cell reaction, largely confined to the white matter. These findings were reminiscent of HIV-1 associated neurological disease, and our immunohistochemical and in situ hybridization data indicated that the neuropathological lesions were associated with the presence of SHIV-specific viral antigens and nucleic acid respectively. However, the concomitant reactivation of opportunistic infections in these macaques suggested that such pathogens may have influenced the replication of SHIV in the CNS, or modified the neuropathological sequelae of SHIV infection in the rhesus species, but not in pig-tailed macaques. Our findings in the two species of macaques highlight the complexities of lentiviral neuropathogenesis, the precise mechanisms of which are still elusive.

By animal-to-animal passage in macaques we derived a pathogenic chimeric simian-human immunodeficiency virus (SHIV) that caused CD4+ T cell loss and AIDS in pigtail macaques and used it to inoculate 20 rhesus and pigtail macaques by the intravaginal and intravenous routes. On the basis of the outcome of infection and patterns of CD4+ T cell loss and viral load, disease was classified into four patterns: acute, subacute, chronic, and nonprogressive infection. During the study period, 15 of the 20 animals developed fatal disease, including AIDS, encephalitis, pneumonia, and severe anemia. Opportunistic pathogens identified in these animals included Pneumocystis, cytomegalovirus, Cryptosporidium, Toxoplasma, and Candida. No single parameter by itself predicted outcome, although a combination of low CD4+ T cell counts in blood, high plasma virus levels, and presence of autoantibodies to red blood cells reliably predicted a fatal outcome. Five animals (25%) died within 3 months of inoculation and constituted the group with acute disease, whereas the nine animals (45%) with subacute disease died between 3 and 8 months postinoculation. This 70% mortality within 8 months is significantly shorter than in HIV-1-infected human beings, of whom 70% develop fatal disease a decade after infection. SHIV infection in macaques provides a useful model with which to evaluate antiviral strategies, combining all the advantages of the SIVmac system, yet using a virus bearing the envelope gene of HIV-1.

Chimeric simian/human immunodeficiency virus (SHIV) consists of the env, vpu, tat, and rev genes of human immunodeficiency virus type 1 (HIV-1) on a background of simian immunodeficiency virus (SIV). We derived a SHIV that caused CD4+ cell loss and AIDS in pig-tailed macaques (S. V. Joag, Z. Li, L. Foresman, E. B. Stephens, L. J. Zhao, I. Adany, D. M. Pinson, H. M. McClure, and O. Narayan, J. Virol. 70:3189-3197, 1996) and used a cell-free stock of this virus (SHIV(KU-1)) to inoculate macaques by the intravaginal route. Macaques developed high virus burdens and severe loss of CD4+ cells within 1 month, even when inoculated with only a single animal infectious dose of the virus by the intravaginal route. The infection was characterized by a burst of virus replication that peaked during the first week following intravenous inoculation and a week later in the intravaginally inoculated animals. Intravaginally inoculated animals died within 6 months, with CD4+ counts of <30/microl in peripheral blood, anemia, weight loss, and opportunistic infections (malaria, toxoplasmosis, cryptosporidiosis, and Pneumocystis carinii pneumonia). To evaluate the kinetics of virus spread, we inoculated macaques intravaginally and euthanized them after 2, 4, 7, and 15 days postinoculation. In situ hybridization and immunocytochemistry revealed cells expressing viral RNA and protein in the vagina, uterus, and pelvic and mesenteric lymph nodes in the macaque euthanized on day 2. By day 4, virus-infected cells had disseminated to the spleen and thymus, and by day 15, global elimination of CD4+ T cells was in full progress. Kinetics of viral replication and CD4+ loss were similar in an animal inoculated with pathogenic SHIV orally. This provides a sexual-transmission model of human AIDS that can be used to study the pathogenesis of mucosal infection and to evaluate the efficacy of vaccines and drugs directed against HIV-1.

We evaluated the effects of a reverse transcriptase inhibitor, 9-(2-phosphonylmethoxyethyl)adenine (PMEA), on simian immunodeficiency virus (SIV) infection in rhesus macaques (Macaca mulatta). Four macaques were given PMEA (20 mg/kg) subcutaneously on days 1 and 2 and inoculated with virus on day 2. Drug treatment was continued for 30 consecutive days, after which the virus burdens and course of infection were monitored for a further 6 months. Four control animals that did not receive PMEA all developed high virus burdens and two of the four developed clinical disease. In contrast, virus burdens remained low in three of the four macaques treated with PMEA and all four remained healthy. Our results show that suppression of virus replication early in infection can result in reduced virus burdens for a much longer period.

By animal-to-animal passage of simian/human immunodeficiency virus (SHIV) in pig-tailed macaques, we have developed a macaque model of human immunodeficiency virus type 1 (HIV-1) disease in humans. Passaging was begun with a chimeric virus containing the env gene of HIV-1 HXBc2 and the gag and pol genes of simian immunodeficiency virus SIVmac239. SHIV was passaged serially in cohorts of two macaques each, using bone marrow-to-bone marrow transfers at 5, 5, and 16 weeks for passages 2, 3, and 4, respectively. The fifth passage was done by using cell-free virus isolated from cerebrospinal fluid of a passage 4 macaque. The virus became more virulent with each passage. Virus replication was restricted in all three animals in passages 1 and 2 but not in five of the six animals in passages 3, 4, and 5. In these animals, intense virus replication in the lymphoid tissues resulted in almost total elimination of CD4+ T cells within weeks of inoculation, and three of these animals developed AIDS in less than 1 year. The more uniform virus-host interaction initiated by the cell-free virus in the passage 5 animals contrasted with a more variable pattern of disease initiated by infectious bone marrow cells during earlier passages. The virulent cell-free SHIV can now be used to screen the efficacy of vaccines directed against the envelope of HIV-1.

This study was designed to evaluate the effects of hypergastrinaemia induced via suppression of gastric acid by omeprazole on carcinogen-induced colon cancer in rats. The carcinogen methylazoxymethanol (MAM), 30 mg/kg, was administered intraperitoneally at 6-weekly intervals to Sprague-Dawley rats. Four weeks after the last MAM injection, the first daily dose of omeprazole, 40 mg/kg, was given by gastric gavage to one group of rats, and the rest were given buffered methylcellulose vehicle. After 10 weeks of daily omeprazole or vehicle, the rats were anaesthetized with ether, blood samples obtained, and animals sacrificed. Gastrin levels in serum from omeprazole-treated rats were elevated nearly six-fold. DNA and RNA levels in gastric mucosa were unchanged by omeprazole, but protein content was somewhat reduced. No biochemical or histological changes related to omeprazole treatment were observed in normal colon. The number of tumours, tumour volumes, and total tumour burden were not significantly different in colons of vehicle- or omeprazole-treated rats. Analysis by flow cytometry revealed that the S phase fraction was lower in tumour cells from omeprazole-treated animals; and that the frequency of DNA aneuploidy was also reduced. The results indicate that while omeprazole-induced suppression of stomach acid in rats elevates levels of gastrin in serum, it does not substantially alter the biochemical or cellular characteristics of carcinogen-induced colon tumours.

Drugs and other chemicals that have the potential to induce or exacerbate systemic autoimmune diseases in humans are of great concern. The aim of this study was to examine the immune-disregulating potential of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) by using the popliteal lymph node (PLN) assay. Chlorpromazine (CPZ) was used as a reference compound for 2 reasons: (a) CPZ is known to elicit a positive response in this assay, and (b) CPZ is a structural analogue of TCDD. Male Sprague-Dawley rats were injected subcutaneously with either TCDD or CPZ into the right hind footpad, whereas vehicle alone was injected into the contralateral footpad. Control rats were injected with vehicle in both hind footpads. Animals were sacrificed on day 7, and their PLNs were removed, weighed, and immersed in 10% formalin. The PLN weight index (the weight ratio of right PLN over left PLN) was significantly higher in both CPZ- and TCDD-treated rats than in controls. Histological examinations of PLNs in the CPZ- and TCDD-treated rats revealed similar morphological changes in both groups (e.g., mild follicular hyperplasia with no evidence of an acute inflammatory response). These results indicate that TCDD has the potential to induce or exacerbate autoimmune-like reactions. Results also suggest that drugs may be useful surrogates to study the mechanism of toxicity of environmental chemicals that cannot be administered to humans.

Background/aims: Published studies suggest that hypergastrinemia stimulates growth of normal or malignant colon tissue. Other studies dispute these findings. This study was designed to test the hypothesis that hypergastrinemia enhances progression or invasiveness of colon cancer., Methods: Colonic carcinomas were induced in male Sprague-Dawley rats by six weekly intraperitoneal injections of methylazoxymethanol. Four weeks after the last injection of carcinogen, the animals were randomized into four treatment groups, including vehicle control, low- and high-dose omeprazole, and ranitidine. After 10 weeks of treatment, the animals were bled, stomach weights were recorded, and colon tumors were mapped, enumerated, measured, and scored histopathologically by Dukes' classification. Crypt and mucosal heights were determined in colonic mucosa unaffected by tumor., Results: Drug administration induced a sustained hypergastrinemia that did not enhance tumor burden or invasiveness or crypt height/mucosal height ratios. Ranitidine-treated rats consumed less food, weighed less, and developed fewer tumors. This group also had lower crypt and mucosal heights than rats in the vehicle- or omeprazole-treated rats., Conclusions: The results suggest that endogenous hypergastrinemia induced by these acid-suppressing drugs has no stimulatory effect on colon mucosal growth or progression or biological behavior of experimental rat colon cancer.

SIVmac239 replicates productivity in activated CD4+ T lymphocytes, but inefficiently in macrophages from rhesus macrophages. Inoculation of the virus into animals results in an acute, highly productive burst of virus replication in activated T lymphocytes in lymphoid tissues and infected cells invade the central nervous system (CNS). This phase lasts a few weeks and is eventually followed by development of immunosuppression of different degrees of severity, opportunistic infections, and tumors related to the loss of T lymphocytes. On rare occasions, infected immunosuppressed animals develop encephalitis and/or interstitial pneumonia, syndromes that are associated with selection of mutant viruses that replicate efficiently in macrophages of these tissues. Usually, however, brains of animals dying with AIDS caused by SIVmac239 appear histologically normal. Is the brain infected with virus? We report here on a macaque dying with AIDS, a neuroinvasive tumor and interstitial pneumonia associated with macrophage-tropic virus. Except for focal infiltration of tumor cells, the brain was normal histologically. We examined the virus and viral DNA from different tissues and found that lymphocytes but not macrophages from lymph nodes and spleen yielded virus, whereas macrophages but not lymphocytes from the lung produced virus. No virus was recovered from the brain but small amounts of viral p27 were present in the brain homogenate. Viral sequences were present in the brain as determined by PCR from tissue DNA. Comparison showed that the viral sequences in the brain closely resembled those from the spleen. Presumably, the virus caused a minimally productive infection detectable by production of small amounts of p27, but was not accompanied by any histopathological changes. It is unclear why the macrophage-tropic virus in the lung failed to 'take-off' in the brain of this animal. To determine whether this virus had encephalitic potential, we inoculated the lung homogenate containing cell-free, macrophage tropic virus into a young pigtail macaque, a species known to be sensitive to primate lentiviral infections. This animal developed severe encephalitis 10 weeks later. Virus from the brain was very similar to the inoculum virus, proving its encephalitic potential. Possible reasons for the differences in neurovirulence of this virus between the two animals remain speculative.

We inoculated four rhesus macaques with molecularly cloned simian immunodeficiency virus SIVmac239/17E env, a chimeric virus whose env gene was derived from the brain of an SIV-encephalitic macaque. Blood and lymphoid tissues had high frequencies of infected cells. The virus was neuroinvasive, but productive virus replication did not occur in the brain, and animals did not develop encephalitis.

Lymphocyte-tropic (L-tropic) SIVmac predictably causes immunosuppression and AIDS in rhesus macaques. SIV encephalitis, on the other hand, is caused mainly by macrophage-tropic (M-tropic) SIVmac. We have previously described the derivation of M-tropic, neuroadapted SIVmac from molecularly cloned, L-tropic SIVmac239. In this report we show that inoculation of four macaques with neuroadapted virus resulted in L-tropic SIVmac-related diseases in all four but neurological disease in only two of the four animals. Because cocultivation of infected macrophages with CD4+ lymphocytes results in production of tumor necrosis factor alpha and interleukin-6, we asked whether infiltration of supernatant fluids containing these cytokines into the brains of macaques infected with neuroadapted virus would enhance the development of neurological disease. These procedures failed to promote productive virus replication in the brain. Thus, although different degrees of immunosuppression and AIDS could be induced predictably with L-tropic virus, induction of neurological disease was not predictable even when animals were inoculated with neuroadapted M-tropic virus and inflammatory cytokines were infiltrated into the brains of these animals.

We studied 15 macaques inoculated with SIV and identified three phases of infection. Phase 1 was characterized by activated lymphocytes in blood and infected cells in the CSF. In phase 2, activated cells were not detected but virus was recovered from mitogen-stimulated PBMC, while in phase 3, virus was recovered from mitogen-stimulated PBMC only after depletion of CD8+ lymphocytes, indicating effective control of the virus in peripheral blood. Early development of phase 3 status correlated with a longer period of clinical normalcy.

Transforming growth factor-beta (TGF-beta) is known phenomenologically as a negative regulator of several functions of mouse bone marrow macrophages. The studies reported here extend this list by showing that TGF-beta can suppress cytolytic activity of mouse bone marrow culture-derived macrophages that already have become activated by IFN-gamma and LPS for tumor cell killing, as well as confirm that this cytokine can interfere with the induction of activation. Suppression was caused by a shift in the dose response curve for IFN-gamma rather than absolute inhibition; the 50% effective dose for IFN-gamma was increased approximately fourfold by treatment with TGF-beta. TGF-beta also decreased the absolute number of IFN-gamma R on the surfaces of pretreated macrophages by approximately 30 to 35%, without altering the affinity with which IFN-gamma bound. The increased concentration of IFN-gamma needed to produce the higher level of receptor occupancy explained the observed shift in the IFN-gamma dose response curve. These results suggest that TGF-beta mediates its negative regulatory effects on macrophage activation by interfering with coupling of the IFN-gamma R to the pathways that induce and maintain macrophage activation for tumor cell killing. Such effects are consistent with the view that TGF-beta is a negative regulator of macrophage activation for tumor cell killing. Because of this fact, neoplastic cells that secrete this cytokine may have a distinct survival advantage.

To facilitate investigation of its physical and functional properties, 11 monoclonal antibodies (mAbs) and a goat polyclonal IgG specific for the mouse interferon- (IFN-gamma) receptor were characterized and their potential uses studied. Eight of the mAbs interacted with epitopes on the extracellular domain of the receptor, two interacted with epitopes on the intracellular domain, and one interacted with an epitope that could not be localized definitively to either region. Of the 11 mAbs, the majority (8) were IgGs, 2 were IgMs, and 1 was an IgA. Relative avidities of the seven that could be determined ranged from 333 to 0.002 microM-1. Both the polyclonal goat IgG and mAb GR-20 (the latter specific for an epitope in the binding site for IFN-gamma) blocked binding of the ligand and, as expected, prevented induction by IFN-gamma of priming of macrophages for tumor cell killing. None of the other mAbs had an effect despite the fact that GR-22 partially (greater than 50%) blocked binding of IFN-gamma. Neither the polyclonal IgG nor any of the mAbs had an agonist effect. The relative usefulness of the antibodies for immunoprecipitation, immunoblotting, immunoassay, and cell staining with and without prior fixation is described. The results of immunocytochemical staining directly confirmed that the majority of immunologically reactive receptor protein expressed by cells is intracellular. To facilitate use by other investigators, the hybridomas that produce these mAbs will be offered to the American Type Culture Collection.

The activation-associated markers, p47b and p71/73, have been recognized as minor proteins in peritoneal and bone marrow culture-derived macrophages activated for tumor cell killing. Proteins with identical characteristics of migration on 2-dimensional gels and comparable Cleveland peptide maps are described here in macrophage cell lines that could be activated for tumor cell killing (J774A.1, RAW 264, UNC-2). Macrophage cell lines that could not be activated (P388D1 and PU5-1.8) did not express the markers. The expression of these markers in activatable macrophage cell lines strengthens their association with the activation process and provides a bulk source of the proteins for purification studies.

A rat monoclonal IgG2a antibody, GR-20, has been produced against the receptor for mouse interferon-gamma (MuIFN-gamma). Comparison of competitive binding studies performed with either 125I-labeled GR-20 or recombinant (r) MuIFN-gamma, as well as a variety of other studies, suggested that the epitope recognized by the monoclonal antibody (mAb) is in the domain of the receptor that binds ligand. The binding of GR-20 to cells of the monomyelocytic line WEHI-3 was of high affinity (1-2 nM). Approximately 20,000 binding sites were found per cell, a value that is in close agreement with the number of MuIFN-gamma receptors quantified on cells of the same type by ligand binding studies. The mAb also bound to a variety of other mouse cells, suggesting that the same epitope is shared by receptors for MuIFN-gamma, regardless of cell type. The epitope was not detected on two human cell types that were tested, while cells of a rat cell line shown to be minimally responsive to rMuIFN-gamma gave equivocal binding results when they were interacted with GR-20. Binding of the mAb to the receptor did not mimic the effects of ligand. In fact, the opposite was true: binding blocked the induction of three biological effects of MuIFN-gamma, including priming of macrophages for tumor cell killing, upregulation of the expression of class II major histocompatibility antigens (Ia) on the same cell type, and induction of antiviral activity in L cells. Following intravenous injection, initial removal of GR-20 was precipitous, followed after 1 h by a phase which was more gradual, resulting in 5-10% of biologically active mAb remaining in the circulation after 24 h. Such retention should make this mAb useful in a variety of studies in vivo.

To evaluate lesions of ammonia exposure and Mycoplasma pulmonis infection in gnotobiotic F344/N rats, groups of rats were inoculated with M. pulmonis, exposed to 76 micrograms/liter (100 parts per million) ammonia, or both, or served as controls. Six rats from each group were killed 3, 5, 7, and 9 days after inoculation and their respiratory organs prepared for histologic examination. Lesions were assessed by subjective scoring and computerized morphometry, and results obtained by the two methods were compared to assess agreement of results. Lesions were limited to the nasal passages. Ammonia exposure resulted in hyperplastic and degenerative changes in the anterior nasal epithelium. Lesions of mycoplasmosis were more severe in ammonia-exposed than unexposed rats, but qualitative differences in lesions were not apparent. Results of the two methods agreed in assessment of submucosal inflammatory cell accumulation. Epithelial lesions were difficult to evaluate by both methods in infected, ammonia-exposed rats. In evaluation of lesions of ammonia exposure alone, agreement was adequate. Both methods indicated that exudates were significantly greater in ammonia-exposed infected rats than in non-exposed infected rats. Thus, scoring and morphometry provided similar assessments of differences in lesion severity among the treatment groups.

During exacerbation of respiratory mycoplasmosis in rats by environmental ammonia, numbers of Mycoplasma pulmonis organisms in the respiratory tract are increased. To test whether or not exposure of respiratory epithelium to ammonia in vitro promotes growth of the organism, rat tracheal organ cultures were treated with 50 mM ammonium chloride, inoculated with M. pulmonis, and quantitatively cultured. After 48 hours, treated tracheas harbored almost 10 times more M. pulmonis colony-forming units than control tracheas. Cellular lesions in the epithelium of treated tracheas resembled those in the nasal passages of rats exposed to gaseous ammonia. To determine whether or not growth-modifying factors were released from tracheal epithelium exposed to ammonium chloride, M. pulmonis growth was assessed in medium collected from ammonium chloride-treated and control tracheas. Growth in medium from treated tracheas was greater than that in medium from untreated tracheas.

Two weeks after daily topical application of hexachlorophene, a 4-week-old female kitten developed cardiovascular collapse, corneal ulcers, trembling, lethargy, and weakness. The kitten was euthanatized. At necropsy, the tissues appeared macroscopically normal; however, microscopic examination of tissue specimens indicated status spongiosis, astrocytosis, and microgliosis of the cerebral and cerebellar white matter and corticospinal tracts. Neuronal cell bodies forming the affected white matter were intact, indicating that demyelination may have been the cause of the lesions. The neurologic lesions were considered compatible with those of hexachlorophene-induced toxicosis.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.