HIV-1 Transgenic Mice Research Articles

Recruitment of renal tubular epithelial cells expressing verotoxin-1 (Stx1) receptors in HIV-1 transgenic mice with renal disease

Human immunodeficiency virus (HIV)-infected children are at risk of developing several renal parenchymal diseases, including hemolytic uremic syndrome (HUS). HUS is most frequently caused by infection with enteric Escherichia coli producing Shiga-like toxins (Stxs). In vitro studies have shown that cytokines known to be present at high systemic levels in HIV-1-infected children up-regulate the expression of the Stx glycolipid receptor (Gb3) in cultured endothelial cells. Thus, we studied whether HIV-1 or the HIV-associated "cytokine milieu" could modulate the expression of renal Stxs receptors in vivo. We used HIV-1 transgenic mice (HIV-Tg) expressing a deletion mutant of HIV-1 (pNL4-3). These mice develop renal disease similar to that of HIV-1-infected children. The expression of Gb3 was studied in renal sections from control and HIV-Tg mice by histochemistry, thin layer chromatography overlay studies, and high-pressure liquid chromatography. By histochemistry, we found a significant recruitment of renal tubular epithelial cells expressing Gb3 in HIV-Tg mice with nephropathy, whereas kidneys from control mice showed limited staining in renal tubules. Gb3 was not found in glomeruli of either control or HIV-Tg mice. Thin layer chromatography overlay studies with Stxs and high-pressure liquid chromatography studies confirmed the marked elevation of Gb3 in HIV-Tg kidneys with renal disease. These results suggest that the presence of HIV-associated nephropathy is associated with the recruitment of renal tubular epithelial cells expressing Stx1 receptors. The up-regulation of Stx1 receptors in HIV-diseased kidneys may increase the sensitivity of these cells to the cytotoxic effects of Stxs.

Captopril prevents nephropathy in HIV-transgenic mice.

Transgenic mice (T26) bearing the envelope, regulatory, and accessory genes of HIV- I develop renal disease resembling human HIV-associated nephropathy (HIVAN). Effects of vehicle (VEH) and the angiotensin-converting enzyme inhibitor captopril (CAP) were examined in wild-type (WT) or T26 mice treated from 7 to 100 d of age. Mortality was lower in CAP T26 mice (30 mg/kg: 8%; 100 mg/kg: 12%) than VEH T26 mice (52%). The urinary protein/creatinine ratio was increased in VEH T26 mice (19.5+/-7.60) versus WT mice (6.1+/-0.83), but not in low-dose (7.3+/-0.94) or high-dose (8.2+/-1.02) CAP T26 mice. Blood urea nitrogen was higher in VEH T26 mice (52+/-16.2 mg/dl) than VEH WT mice (24+/-0.8). Blood urea nitrogen was also elevated in CAP WT (high dose: 43+/-2.1 mg/dl) and T26 mice (high dose: 42+/-2.4 mg/dl). Glomerular injury was higher in VEH T26 mice (6.8+/-0.58) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 1.1+/-0.17; high dose: 0.7+/-0.13). Tubulointerstitial injury was also greater in VEH T26 mice (1.1+/-0.10) than VEH WT mice (0.2+/-0.08) or CAP T26 mice (low dose: 0.4+/-0.10; high dose: 0.3+/-0.10). These data validate recent nonrandomized studies of captopril in HIV-infected patients, and suggest that an angiotensin-converting enzyme substrate is an important mediator in HIVAN. A randomized placebo-controlled trial of captopril in HIVAN may be warranted.

Nephropathy in human immunodeficiency virus-1 transgenic mice is due to renal transgene expression.

HIV-associated nephropathy (HIVAN) is a progressive glomerular and tubular disease that is increasingly common in AIDS patients and one of the leading causes of end stage renal disease in African Americans. A major unresolved issue in the pathogenesis of HIVAN is whether the kidney disease is due to renal cell infection or a "bystander" phenomenon mediated by systemically dysregulated cytokines. To address this issue, we have used two different experimental approaches and an HIV-1 transgenic mouse line that develops a progressive renal disease histologically similar to HIVAN in humans. In the murine model, kidney tissue expresses the transgene and in heterozygous adults, renal disease develops shortly thereafter. We demonstrate by terminal deoxynucleotide transferase-mediated dUTP-biotin nick-end labeling assay that similar to the disease in humans, apoptosis of renal tubular epithelial cells is a component of the molecular pathogenesis. To determine whether apoptosis is due to transgene expression or environmental factors, we treated fetal kidney explants (normal and transgenic) with UV light to induce transgene expression. Apoptosis occurred in transgenic but not normal littermates after stimulation of transgene expression. To confirm a direct effect of HIV expression on the production of HIVAN, we transplanted kidneys between normal and transgenic mice. HIVAN developed in transgenic kidneys transplanted into nontransgenic littermates. Normal kidneys remained disease free when transplanted into transgenic littermates. Thus, the renal disease in the murine model is intrinsic to the kidney. Using two different experimental approaches, we demonstrate a direct effect of transgene expression on the development of HIVAN in the mouse. These studies suggest that in humans, a direct effect of HIV-1 expression is likely the essential cause of HIVAN, rather than an indirect effect of cytokine dysregulation.

Human chorionic gonadotropin hormone prevents wasting syndrome and death in HIV-1 transgenic mice.

At birth, transgenic mice, homozygous for the HIV-1 provirus pNL4-3, deleted in gag/pol, are normal in appearance and weight. Within several days after birth, the pups develop a syndrome characterized by dry, scaly, hyperkeratotic skin, growth failure, and death. The possibility that the homozygous embryos are being protected during gestation by a maternal factor led us to treat the newborn animals with various pregnancy-related hormones including human chorionic gonadotropin (hCG), estrogen, progesterone, and dexamethasone. Treatment with hCG prevented death, led to normal growth, and markedly reduced skin lesions. In contrast to the skin of the untreated homozygous pups, which expressed high levels of HIV mRNA and proteins (i.e., gp120 and Nef), the skin of the hCG-treated pups showed a marked reduction in both HIV mRNA and proteins. Discontinuation of hCG resulted in the reappearance of HIV transcripts and proteins, skin lesions, and growth failure resulting in death. In addition, HIV transcripts and proteins were reduced significantly in heterozygous mothers during pregnancy, but reappeared after parturition. Similarly, hCG treatment resulted in a decrease of HIV proteins in the skin of nonpregnant heterozygous transgenic mice. These findings suggest that the inhibiting effect of hCG on HIV expression may be clinically useful in the treatment of HIV infections, and may be responsible, during pregnancy, for the low transmission of HIV from infected mothers to their offspring.

Models of HIV type 1 proviral gene expression in wild-type HIV and MLV/HIV transgenic mice.

Two proviral HIV transgenic mouse models, one bearing wild-type HIV proviral DNA and the other a modified provirus in which the viral LTRs contained the core enhancer of the Moloney murine leukemia virus (MLV), were compared. The MLV/HIV chimeric LTR, in which the MLV enhancer replaced the NF-kappa B-binding motifs, was transcriptionally active in human and murine cells in vitro and virus containing the chimeric LTR was replication competent in human cell cultures. Transgenic mice derived from microinjections of chimeric MLV/HIV proviral DNA transcribed HIV genes at a greater frequency and at higher levels than wild-type HIV proviral transgenic mice. MLV/HIV mice were also more apt to develop disease; wasting, periocular infections, and a degenerative myopathy characterized the most predominant phenotype. The tissue specificities of the wild-type and chimeric LTRs in transgenic mice were remarkably similar, but a significant difference was apparent in lymphoid cells. Basal level and LPS-inducible HIV gene expression occurred in peritoneal and bone marrow-derived macrophages from wild-type HIV transgenic mice. In contrast, HIV gene expression in macrophages from MLV/HIV mice was undetectable, even following LPS induction. However, cultured splenocytes from MLV/HIV mice supported HIV proviral gene transcription better than splenocytes from HIV mice, particularly after induction with LPS or anti-IgD antibody but not with concanavalin A. These data suggest that in transgenic mice, the HIV and MLV/HIV LTRs display a differential tropism for macrophages and B cells, respectively. HIV and MLV/HIV transgenic mice represent alternative models amenable to in vivo studies of HIV gene regulation in lymphoid cells, the induction of HIV-related disease and the evaluation of anti-HIV therapies.

Infection of human immunodeficiency virus 1 transgenic mice with Toxoplasma gondii stimulates proviral transcription in macrophages in vivo.

Human immunodeficiency virus (HIV) 1 transgenic mice expressing low or undetectable levels of viral mRNA in lymphoid tissue were infected with the intracellular protozoan Toxoplasma gondii. Exposure to this parasite resulted in an increase in HIV-1 transcript in lymph nodes, spleens, and lungs during the acute phase of infection and in the central nervous system during the chronic stage of disease. In vivo and ex vivo experiments identified macrophages as a major source of the induced HIV-1 transcripts. In contrast, T. gondii infection failed to stimulate HIV-1 transcription in tissues of two HIV-1 transgenic mouse strains harboring a HIV-1 proviral DNA in which the nuclear factor (NF) kappa B binding motifs from the viral long terminal repeats had been replaced with a duplicated Moloney murine leukemia virus core enhancer. A role for NF-kappaB in the activation of the HIV-1 by T. gondii was also suggested by the simultaneous induction of NF-kappaB binding activity and tumor necrosis factor alpha synthesis in transgenic mouse macrophages stimulated by exposure to parasite extracts. These results demonstrate the potential of an opportunistic pathogen to induce HIV-1 transcription in vivo and suggest a mechanism for the in vivo dissemination of HIV-1 by macrophages.

HIV type 1 Nef perturbs eye lens development in transgenic mice.

HIV transgenic mice often display lens cataracts as a consequence of viral-specific expression of HIV gene products in the developing lens. Cataractous mouse lines encoding either HIV-1 proviral DNA, HIV delta Gag/Pol] proviral DNA, or the HIV-1 nef gene alone were examined to ascertain the effect of Nef on murine lens development. Ocular disease was characterized by a progressive architectural disorganization within the lens fiber cell compartment developing in 100% of HIV-positive mice in five reported transgenic lines. Late embryonic stage transgenic lenses featured a mild microphthalmia, pyknotic nuclei within the lens fiber department, ballooning lens fiber cells, and elongated lens epithelial cells. Increased DNA fragmentation was evident in transgenic embryonic lenses, suggesting that cell death occurred by apoptosis. As studied in HIV delta Gag/Pol] transgenic mice, HIV transcription was developmentally linked to alpha A- and alpha B-crystallin gene expression, preceded disease development (in E14.5-E16.5 embryos), and persisted for weeks after birth. HIV-1 Nef was the predominant HIV gene product detected in the lens fiber cells of this line and was expressed almost to the exclusion of other HIV gene products. Nef was implicated as a major determinant of disease because (1) cataracts developed in mice transgenic for Nef alone and (2) the expression of other HIV gene products in wild-type HIV provirus transgenic mice occurred without a concomitant change in lens pathology.

Evaluating Birth Frequencies and Embryonic Lethality in Transgenesis Following Pronuclear Injection of HIV DNA.

The likelihood that expression of a foreign gene in a mammalian cell is deleterious to viability is confronted whenever novel transgenic animals are made. A pathological response to transgene expression is even desired in transgenic mouse models of human disease. The derivation of HIV-transgenic mice in our laboratory using multiple recombinant forms of an HIV provirus has resulted in mixed success best explained by the variable toxicity of the different transgenes. Employing a standardized approach to pronuclear injections, experimental variation amongst recombinant HIV transgenes was documented in terms of the percentage of pregnancies following embryo transfer into pseudopregnant mice and the percentage of transplanted embryos leading to term births in these pregnant females (giving rise to an index of birth success, SI). Results compiled over 5 years suggested that the SI reflected transgene toxicity, in this case of HIV gene products early in embryogenesis. These observations have guided the design of productive transgenes for mouse models of HIV-related diseases and may be generally applicable in transgenesis. Copyright 1995 S. Karger AG, Basel

Study on transgenic mice growing into craniofacial anomaly

In order to elucidate the genetic effects on the growth and development of craniofacial regions, HIV-1 transgenic mice (TG mice) which exhibited the phenotype of anterior crossbites were investigated by various methods. Northern hybridization showed that the introduced gene was expressed from 14.5 days post coitum to adult. In morphological measurements using digital converter "GRADICON", significant differences between TG mice and wild type mice (WT mice) were found at 5 weeks of age (5W) for most measurement items. Especially at 7W, the results indicated remarkable reductions in maxilla and catch up growth in the mandible. On the other hand, the serum alkaline phosphatase activity of TG mice (3W, 5W, and 7W) was significantly higher than that of WT mice, and also the low level of inorganic phosphate was detected at 3W for TG mice. These results suggested that TG mice had a disorder of bone metabolism. Other TG mouse lines carrying the same DNA construct showed normal figures in the craniofacial regions, suggesting the possibility that the phenotype of anterior crossbites and the disorder of bone metabolism were not caused by functions of HIV-1 genome but by insertional mutation in craniofacial development.

BFGF and its low affinity receptors in the pathogenesis of HIV-associated nephropathy in transgenic mice

HIV-associated nephropathy is characterized by extensive tubulointerstitial disease with epithelial cell injury, microcystic proliferation, and tubular regeneration with glomerulosclerosis. To explore the role of bFGF as a mediator of HIV-induced interstitial disease, we utilized an HIV transgenic mouse model that manifests clinical and histological features observed in patients. In transgenic mice, simultaneous renal epithelial cell proliferation and injury were detected in vivo. In areas of microcystic proliferation, immunoreactive bFGF colocalized with extracellular matrix. Kidneys from transgenic mice had increased bFGF low affinity binding sites, particularly in the renal interstitium. In vitro, transgenic renal tubular epithelial cells proliferated more rapidly and generated tubular structures spontaneously, in marked contrast to nontransgenic renal cells where these pathologic features could be mimicked by exogenous bFGF. These studies suggest that renal bFGF and its receptors play an important role in the pathogenesis of HIV-associated nephropathy.

Growth Failure and AIDS-like Cachexia Syndrome in HIV-1 Transgenic Mice

The mechanisms which predispose to growth failure in infants and children infected with immunodeficiency virus type-1 (HIV-1) are not fully understood. The contributions of viral replication and CD4+ T cell depletion to growth failure in an HIV-1 transgenic mouse model were investigated. Mice homozygous for the transgene, a gag-pol deletion mutant of the HIV-1 provirus pNL4-3, exhibited marked cachexia, growth retardation, lymphoproliferation with a reduction in the percentage of CD4+ T cells but an increase in the absolute number of splenic CD4+ and CD8+ T cells, thymic hypoplasia, and early death. Despite the absence of T cells, athymic nude mice, homozygous for the HIV transgene, displayed comparable growth failure. The results indicate that AIDS-like cachexia may be produced by expression of viral envelope or accessory genes, need not be accompanied by absolute depletion of CD4+ T cells, and may occur independent of T cell function.

Nephropathy in HIV-transgenic mice.

Nephropathy in HIV-transgenic mice.

Immunization with recombinant gp160 prolongs the survival of HIV-1 transgenic mice.

A strain of mouse transgenic for the env gene of the HIV-1 virus was used to study the immunogenicity of a gp160-derived vaccine (the protein encoded by the HIV env gene) and its effect on disease progression. Untreated transgenic mice frequently developed a rapidly progressive renal disease similar to that affecting approximately 10% of HIV-infected humans. When transgenic mice were immunized with recombinant purified gp160, their edema, proteinuria, and serum BUN levels were substantially reduced and their survival prolonged (p < 0.01). The increased longevity of immunized transgenic mice correlated with the production of IgG antibodies reactive with gp160.

Cutaneous Disorders and Viral Gene Expression in HIV-1 Transgenic Mice

Patients infected with HIV-1 experience several hyperproliferative skin disorders, including seborrheic dermatitis, ichthyosis, and psoriasis. Transgenic mice carrying a subgenomic HIV-1 proviral construct lacking the gag and pol genes were found to develop proliferative epidermal lesions, manifested as diffuse epidermal hyperplasia in homozygous transgenic mice and benign papillomas in heterozygous transgenic mice. Nonpapillomatous skin from both homozygotes and heterozygotes expressed viral RNA, and the viral envelope protein gp120 was localized to the suprabasal keratinocyte. Papillomas contained increased amounts of both viral mRNA and envelope glycoprotein. Exposure of transgenic mice to doses of ultraviolet B (UV-B) irradiation that induced cutaneous injury increased viral gene expression and resulted in the development of papillomas within 14-21 days. Cutaneous injury induced by phenol and liquid nitrogen had similar effects. These data support a role for HIV-1 gene products in the pathogenesis of proliferative epidermal disorders associated with HIV-1 infection. Further, they suggest that the process of wound repair increases HIV-1 gene expression in this transgenic mouse model.

HIV-1 expression during early mammalian development.

Our long-term objective is to determine the consequences of HIV-1 expression in the mammalian fetus using the mouse as a model system. This study describes HIV gene expression in micro-injected and electroporated embryos during the pre-implantation period. Procedures were adopted to create a non-infectious HIV system to assay HIV long terminal repeat (LTR) activation in a non-human mammal. Two constructs (RSV promoter-HIV tat gene; HIV LTR-lac Z gene) were co-micro-injected (10 micrograms/ml each) into pronuclei of fertilized eggs or one nucleus of the two-cell stage and expression assessed after 2-5 days. The techniques of electroporation and micro-injection were compared for their ability to deliver the constructs expressed subsequently. We found that HIV-1 expression, as monitored by beta-galactosidase production, can occur as early as the eight-cell to blastocyst stage. The intensity of HIV expression varied from high to low; the proportion of the embryo expressing HIV varied from half to only two cells. The material was too limited to allow a determination of integration. The expression rate of the HIV-LTR was low, averaging slightly over 1%. The data demonstrate that the HIV-LTR can be activated in early mammalian development, even before implantation. This information is valuable to those studying HIV-transgenic mice, to those interested in factors governing HIV expression, and to those concerned about pathologies accompanying developmental expression of HIV.