Rabbit cells expressing human CD4 and human CCR5 are highly permissive for human immunodeficiency virus type 1 infection.

To evaluate the feasibility of using transgenic rabbits expressing CCR5 and CD4 as a small-animal model of human immunodeficiency virus type 1 (HIV) disease, we examined whether the expression of the human chemokine receptor (CCR5) and human CD4 would render a rabbit cell line (SIRC) permissive to HIV replication. Histologically, SIRC cells expressing CD4 and CCR5 formed multinucleated cells (syncytia) upon exposure to BaL, a macrophagetropic strain of HIV that uses CCR5 for cell entry. Intracellular viral capsid p24 staining showed abundant viral gene expression in BaL-infected SIRC cells expressing CD4 and CCR5. In contrast, neither SIRC cells expressing CD4 alone nor murine 3T3 cells expressing CCR5 and CD4 exhibited significant expression of p24. These stably transfected rabbit cells were also highly permissive for the production of virions upon infection by two other CCR5-dependent strains (JR-CSF and YU-2) but not by a CXCR4-dependent strain (NL4-3). The functional integrity of these virions was demonstrated by the successful infection of human peripheral blood mononuclear cells (PBMC) with viral stocks prepared from these transfected rabbit cells. Furthermore, primary rabbit PBMC were found to be permissive for production of infectious virions after circumventing the cellular entry step. These results suggest that a transgenic rabbit model for the study of HIV disease may be feasible.

Replication of HIV type 1 in rabbit cell lines is not limited by deficiencies in tat, rev, or long terminal repeat function.

HIV-1 infection has been documented in rabbits, but infection proceeds slowly in this species. Human and rabbit cell lines were compared in order to identify barriers to efficient HIV-1 infection of rabbit cells. A direct comparison of human and rabbit CD4 as receptor for HIV-1 indicated that the rabbit CD4 homolog did not function well even when expressed by human cells. Examination of viral RNA production indicated that the major HIV transcripts were produced in HIV-infected rabbit cells, but were present at levels significantly lower than those found for human cells. Ability of HIV-1 LTRs to direct protein expression in human and rabbit cells was compared using gene constructs with the chloramphenicol acetyltransferase (cat) gene flanked by HIV-1 LTRs. Chloramphenicol acetyltransferase protein expression was equivalent in rabbit and human cell lines transfected with the HIV-1/CAT constructs and cotransfections with the HIV-1 tat gene led to similar increases in CAT expression. Subsequent transfections with an infectious molecular HIV clone yielded approximately equal levels of HIV protein expression in rabbit and human cell lines, suggesting that major barriers to virus production in rabbit lines exist at steps prior to transcription of the viral genome. Because HTLV-I replicates with high efficiency in rabbit cells, a chimeric virus clone was constructed consisting of the 5' portion of HIV-1 through the nef coding sequence followed by the 3' HTLV-I LTR. Transfection of most rabbit cell lines with the chimera produced levels of p24gag protein higher than those transfected with the parent HIV-1 clone. By contrast, the unmodified HIV clone replicated more efficiently in all human cell lines tested.

HIV-1 mediates rapid apoptosis of lymphocytes from human CD4 transgenic but not normal rabbits.

Normal rabbit lymphocytes can be infected with HIV-1 although infection is much less efficient than in human lymphocytes. When peripheral blood mononuclear cells (PBMC) from rabbits transgenic for human CD4 (HuCD4) were exposed to HIV-1, enhanced infection and a rapid depletion of lymphocytes were observed. Cell death in the infected cultures occurred via apoptosis, but no similar effect was seen in nontransgenic rabbit PBMC cultures. Induction of apoptosis in HuCD4-expressing cells required virus replication; heat-inactivated virus or recombinant viral proteins had no effect on cell viability. Expression of the Fas antigen was increased in HIV-1-infected CD4+ rabbit lymphocytes. Characterization of the infected PBMC cultures revealed that apoptosis occurs both in HuCD4+ and HuCD4- cells, indicating that bystander cells are killed. These data define a requirement for HuCD4 in initiation, but not the spread, of HIV-1-induced apoptosis in rabbit PBMC and provide a model to probe mechanisms leading to lymphocyte depletion in HIV-1 infection.

Developmental and tissue-specific expression of human CD4 in transgenic rabbits.

A major obstacle to understanding AIDS is the lack of a suitable small animal model for studying HIV-1 infection and the subsequent development of AIDS, and for testing diagnostic, therapeutic, and preventive modalities. Our goal is to produce a rabbit model for the study of AIDS. Here we report on the generation of transgenic rabbits that express the human CD4 (hCD4) gene. The transgene, which contains the coding region for hCD4 and approximately 23 kb of sequence upstream of the translation start site, was used previously to direct hcD4 expression on the surface of CD4+ T cells of transgenic mice (Gillespie et al., 1993: Mol Cell Biol 13:2952-2958). The hCD4 transgene was detected in five males and two females derived from the microinjection in five males and two females derived from the microinjection of 271 rabbit embryos. Both hCD4 RNA and protein were expressed in peripheral blood lymphocytes (PBLs) from all five males but neither of the females. Human CD4 was expressed on PBLs from F1 offspring of all founder males. T-cell subset analysis revealed that hCD4 expression was restricted to rabbit CD4 (rCD4) expressing lymphocytes; mature rCD4- rCD8+ lymphocytes did not express hCD4. In preliminary studies, PBLs from hCD4 transgenic rabbits produced greater amounts of HIV-1 p24 core protein following HIV-1 infection in vitro than HIV-1 p24 antigen in nontransgenic rabbit infected cultures. These results extend to rabbits our previous observation that this transgene contains the sequence elements required for high-level expression in the appropriate cells of transgenic mice. Furthermore, these and previous studies demonstrating that expression of hCD4 protein enhances HIV-1 infection of rabbit T cells in vitro, coupled with reports that normal, nontransgenic rabbits are susceptible to HIV-1 infection, suggests that the hCD4 transgenic rabbits described herein will have an increased susceptibility to HIV-1 infection. In vivo HIV-1 infection studies with these rabbits are under way.

Selection of rabbit CD4- CD8- T cell receptor-gamma/delta cells by in vitro transformation with human T lymphotropic virus-I.

In vitro transformation of rabbit peripheral blood mononuclear cells (PBMC) with human T lymphotropic virus-I (HTLV)-infected human or rabbit cells resulted in CD4- CD8- cell lines, some of which caused acute leukemia when injected into rabbits. Structural analyses of the proviruses from cell lines with diverse pathogenic effects provided no clear correlation with lethality. The rabbit lines were provisionally designated T cells because they express interleukin 2R (IL-2R) and CD5 and lack surface immunoglobulin, but none express functional T cell receptor (TCR) alpha or beta transcripts. A more detailed characterization of the HTLV-I-infected cells was required to determine cell lineage and its potential influence on pathogenic consequences. Probes for rabbit TCR gamma and delta genes were derived and used to detect gamma and delta TCR RNA transcripts, identifying the in vitro transformed lines as gamma/delta T cells. CD4+ and CD8+ lines were derived from PBMC of HTLV-I-infected rabbits and CD4+ TCR-alpha/beta HTLV-I lines were derived from rabbit thymus, eliminating the possibility that the HTLV-I isolates used here transform only CD4- CD8- TCR-gamma/delta cells. The percentage of gamma/delta cells in rabbit PBMC is relatively high (23% in adult rabbits); this with diminution of CD4+ and CD8+ cells in IL-2-supplemented PBMC or thymocyte cultures may account for selection of rabbit HTLV-I-infected gamma/delta T cell lines in vitro. The availability of well-characterized T cell lines with diverse in vivo effects in the rabbit HTLV-I disease model allows evaluation of roles played by cell type in HTLV-I-mediated disease.

CD4 and its role in infection of rabbit cell lines by human immunodeficiency virus type 1.

Human CD4 (HuCD4) is the principal receptor for human immunodeficiency virus type 1 (HIV-1) in human cell infection. Susceptibility of rabbit cell lines to infection with HIV-1 raised questions concerning whether a CD4 homolog serves as HIV-1 receptor on rabbit cells. Sequence comparisons of rabbit CD4 (RbCD4) cloned from a rabbit thymus cDNA library showed that 6 of the 18 residues implicated in HIV-1 binding by CD4 differ between the human and rabbit proteins. No correlation between RbCD4 expression by rabbit cell lines and their ability to support HIV-1 infection was seen. Transfection of RbCD4-negative, HTLV-I-transformed cell lines with HuCD4 significantly enhanced HIV-1 infectivity, suggesting that these lines lack a receptor present on other RbCD4-negative lines that produce high levels of p24 in their native state. Inhibition of HIV-1 infection with soluble HuCD4 was demonstrated for all rabbit lines tested, but complete inhibition was obtained only with a rabbit T-cell line expressing RbCD4 and with HuCD4 transfectants. The results suggest that HIV-1 infection of the RbCD4-positive line proceeds through a receptor similar to HuCD4 but that an additional receptor or receptors may serve this purpose in RbCD4-negative lines.

Autoradiographic study of the distribution of bound radioactivity in the respiratory tract of Syrian hamsters given N-[3H]nitrosodiethylamine.

Adult Syrian golden hamsters received a single intragastric dose of N-[3H]nitrosodiethylamine. Their tracheas, extrapulmonary stem bronchi, and lungs were processed for high-resolution light-microscopic autoradiography to monitor the distribution of bound radioactivity. In the trachea and extrapulmonary stem bronchi, mucous cells contained the most bound radioactivity, while in the lobar and segmental bronchi and bronchioles, Clara cells were the major site of binding. In conjunction with earlier conducted studies on the pathogenesis of N-[3H]nitrosodiethylamine-induced respiratory tract tumors, these findings indicate that metabolic competence and a preexisting capacity for proliferation are important factors in determining the target cell types of this compound.

Autoradiography in fetal golden hamsters treated with tritiated diethylnitrosamine.

Tritiated diethylnitrosamine was administered to female Syrian golden hamsters on each of the last 4 days (days 12-15) of pregnancy. The distribution of bound radioactivity was monitored by light microscopic autoradiography of fetal tracheas and livers, the placentas, and the maternal livers. In the trachea, the fetal target organ, bound radioactivity was restricted to the respiratory epithelium, where diethylnitrosamine-induced tracheal tumors arise. Mucous cells and nonciliated stem cells were identified as the principal sites of binding; other cell types within the tracheal epithelium contained only small amounts of bound radioactivity. The level of binding observed in the fetal trachea increased steadily from day 12 to day 15, which correlated well with the levels of differentiation of this tissue during this period. This observation also agrees with the previously reported observation that tumor incidence increases from 40 to 95% in Syrian golden hamsters between days 12 and 15.

Pathogenesis of toxic and neoplastic renal lesions induced by the flame retardant tris (2,3-dibromopropyl)phosphate in F344 rats, and development of colonic adenomas after prolonged oral administration.

Male F344 rats, 4 weeks old, were given tris (2,3-dibromopropyl)phosphate (TBP) via stomach tube at a dosage level of 100 mg. per kg. of body weight per day. Treatment was given 5 days per week and continued for 4 or 52 weeks. Representative treated and control animals were killed at predetermined time intervals and the histomorphology and ultrastructure of the kidneys investigated. Twenty-four hours after the first TBP treatment, the epithelial cells at the corticomedullary junction developed increased nucleus to cytoplasm ratios, cytomegaly, and nuclear vacuolization and pleomorphism. These changes increased in severity to a toxic tubular nephrosis as treatment continued, and extended to the peripheral cortex by 52 weeks. Electron microscopy showed loss of microvilli and polarity in the epithelium of the proximal convoluted tubules. The development of adenocarcinomas from hyperplastic areas at these sites were studied. At the ultrastructural level, the cytoplasm of the neoplastic cells was poorly differentiated, and in many areas, the surfaces of the cells were covered by microvilli. In one group of rats, treatment was terminated after 4 weeks. In these animals, a gradual but incomplete restoration of the tubular epithelia to nearly normal morphology was observed. Nuclear changes persisted after cytoplasmic abnormalities had disappeared. At 52 weeks, three of five surviving rats given TBP throughout the experiment were found to have polypoid adenomas of the descending colon.

Electron microscopic autoradiography of the pancreas in the hamster treated with tritiated N-nitroso-2,6-dimethylmorpholine.

Syrian golden hamsters were given a single dose of [3H]-N-nitroso-2,6-dimethylmorpholine and killed 8 hr later. The pancreas was processed for electron microscopic autoradiography to detect binding of radioactivity to cellular constituents. The pancreatic acinar cells and duct epithelia were found to be labeled, while islet cells, centroacinar cells, and all nonepithelial elements were not. Acinar cells active in secreting zymogen had a high concentration of grains over the zymogen granules and the rough endoplasmic reticulum. Their nonsecreting counterparts contained abundant bound material in the nuclei and rough endoplasmic reticulum. Labeling was lower in the duct epithelia than in acinar cells, with the majority of grains associated with the heterochromatin. Our findings suggest that the acinar cells are the principle site of metabolic activation in this organ.

A morphometric study of the pulmonary Clara cell in normal and nitrosoheptamethyleneimine-treated European hamsters.

Previous studies have identified the bronchial Clara cell as the progenitor of the lung tumors induced in European hamsters by nitrosoheptamethyleneimine (NHMI). Using stereological methods, we compared the ultrastructure of Clara cells from untreated animals and lung tumor cells, induced by NHMI, in the European hamster. The composition and volume of the cytoplasm was significantly altered in the tumor cells whereas the nuclei did not show any measurable changes in size or structure when compared with the controls.