Joseph Bryant

HIV-associated nephropathy in transgenic mice expressing HIV-1 genes

Transgenic mice were produced that bore copies of a defective HIV provirus. The transgenic offspring from three independently derived mouse lines manifested renal disease associated with proteinuria, a high mortality rate, and HIV-specific gene expression in the kidney. An early histopathological lesion in the kidney was focal glomerulosclerosis. Moribund animals had diffuse glomerulosclerosis with prominent microcystic tubular dilatation, tubular epithelial degeneration, and interstitial nephritis. Electron microscopy revealed ultrastructural features consistent with the glomerulosclerosis: effacement of the foot processes of visceral epithelium and an increase in mesangial cell matrix. Transgenic mice variably expressed 6-, 4.3-, and 2-kb HIV-specific RNAs and HIV-related polypeptides in several tissues including kidney. Immunocytostaining revealed the presence of HIV-related protein in the glomeruli of affected animals. Glomerulopathy in these transgenic mice and HIV-associated nephropathy in man have similar features.

The effects of preparations of human chorionic gonadotropin on AIDS- related Kaposi's sarcoma

BACKGROUND Kaposis sarcoma is the most common cancer in patients with the acquired immunodeficiency syndrome (AIDS). Recently, certain preparations of human chorionic gonadotropin (hCG) have been shown to inhibit the growth of Kaposis sarcoma cell lines in vitro and in immunodeficient mice. METHODS After in vitro evaluation of four commercially available hCG preparations, the most active product was evaluated in 36 patients with AIDS-related Kaposis sarcoma. In a phase 1-2 trial, 24 patients received intralesional injections of hCG three times a week for two weeks at doses of 250, 500, 1000, or 2000 IU (6 patients each). In each patient three nodular lesions were injected, two with the drug and one with diluent alone. In a double-blind trial, 12 additional patients were randomly assigned to receive intralesional injections of 2000 IU of hCG or diluent alone (6 patients each; two lesions per patient). At the conclusion of therapy, the lesions were measured, their gross appearance assessed, and biopsy specimens evaluated. RESULTS A.P.L. (Wyeth-Ayerst), which had the most in vitro activity against Kaposis sarcoma cell lines, was selected for the clinical investigation. Treatment with A.P.L. was well tolerated at all doses. In the cohorts given 250, 500, 1000, and 2000 IU, 1, 5, 5, and 10 of the 12 injected lesions responded, respectively (P=0.03 for trend). Complete tumor regression was observed in one lesion each at the 250-IU and 500-IU doses, in two lesions given the 1000-IU dose, and in five lesions given the 2000-IU dose. In the double-blind study, none of the 12 lesions in the six patients injected with diluent had responses, as compared with 10 of the 12 lesions in the six patients injected with hCG (P=0.015). Microscopical evidence of apoptosis was observed only in hCG-treated lesions. The percentage of cells that died increased in a dose-dependent manner (P<0.001). Serum levels of follicle-stimulating hormone (P=0.002) and luteinizing hormone (P=0.001) declined after the last injection of hCG, but there was no effect on these hormones in the diluent-treated patients. CONCLUSIONS The intralesional injection of hCG induces the regression of AIDS-related Kaposis sarcoma lesions in a dose-dependent manner. The response of these tumors appears to be mediated by the induction of apoptosis.

International NeuroAIDS: prospects of HIV-1 associated neurological complications.

ABSTRACTNeurological complications associated with HIV-1/AIDS are being recognized with a high frequency that parallels the increased number of AIDS cases. The early infiltration by HIV-1 into the nervous system can cause primary and/or secondary neurological complications. The most common neurocognitive disorder is AIDS Dementia Complex (ADC). In developing countries of Asia the three most opportunistic infections are tuberculosis (TB), cryptococcosis, and Pneumocystis carinii pneumonia. Therefore, it is expected that secondary neurological complications due to TB and cryptococcosis will be the most common cause of morbility and mortality in HIV-1/AIDS cases in China. Research of NeuroAIDS in China is necessary to understand the impact and the biology of HIV-1 in the nervous system. Future studies would include, the molecular epidemiology and the description of opportunistic infections associated to HIV-1; the neuropathological description of primary and secondary HIV-1 complications in different groups; the HIV-1 neurotropism and immune response studies for Chinas unique HIV-1 strains and recombinant forms derived from the nervous system, including experimental models such as the use of transgenic rats; and the study of potential resistant virus, primarily when the anti-retroviral therapy (ART) has not full access in the brain.

Relationship of stable integration of herpes simplex virus‐2 BglII N subfragmentXho2 to malignant transformation of human papillomavirus‐immortalized cervical keratinocytes

Transfection of the right end Xho2 subfragment of BglII N of herpes simplex virus‐2 (HSV‐2) into human genital keratinocytes immortalized by human papillomavirus (HPV) type 16 or 18 resulted in invasive and noninvasive indolent cystic squamous carcinomas when cells were injected into immunocompromised mice. Retention and expression of the right end portion of the BglII N fragment correlated with malignancy, as the corresponding HSV‐2 sequences were integrated and transcribed in the tumorigenic cell lines. HPV‐immortalized cells alone were not tumorigenic. In contrast, previous results have shown that using the entire BglII N region can malignantly transform HPV‐immortalized cells, although HSV2 DNA was not retained. Together, these observations localize the transforming activity of BglII N to Xho2 and suggest that the remaining sequences have an inhibitory effect on stable integration. The Xho2 sequence is 2480 bp long and contains an open reading frame (ORF) extending from nucleotides 559 to 1797. The ORF encodes a putative protein of 412‐aa with a m.w. of 42–43 kDa and is highly homologous to UL43 of HSV‐1. The correlation of tumorigenicity with stable integration and expression of Xho2 DNA in HPV‐immortalized cells indicates that HSV‐2 should be investigated further for a possible role in cervical cancer. Int. J. Cancer 76:865–871, 1998.© 1998 Wiley‐Liss, Inc.+ This article is a US Government work and, as such, is in the public domain in the United States of America.

Resistance of Streptococcus gordonii to Polymorphonuclear Leukocyte Killing Is a Potential Virulence Determinant of Infective Endocarditis

ABSTRACT Significant differences in virulence among seven representative Streptococcus gordonii strains were observed by using the rat model of infective endocarditis. Five strains, including S. gordonii DL1, caused severe disease, while the other two strains, including S. gordonii SK12, caused minimal or no disease. The differences in virulence were evident from the visible presence of streptococci in the vegetations present on the aortic valves of catheterized rats that were challenged with individual strains and also from the much greater recovery of rifampin-resistant S. gordonii DLl than of streptomycin-resistant S. gordonii SK12 from the hearts of animals coinfected with both organisms. Each S. gordonii strain aggregated with human platelets and bound to polymorphonuclear leukocytes (PMNs), as shown by the stimulation of PMN superoxide anion production. These interactions were reduced or abolished by pretreatment of the platelets or PMNs with sialidase, indicating that there was bacterial recognition of host sialic acid-containing receptors. Adhesin-mediated binding of each S. gordonii strain to PMNs also triggered phagocytosis. However, the subsequent PMN-dependent killing differed significantly for the seven strains. The five virulent strains included three strains that were not killed and two strains whose numbers were reduced by approximately 50%. In contrast, the level of killing of each avirulent strain under the same conditions was significantly greater and approached 90% of the bacteria added. Parallel studies performed with rat PMNs revealed comparable differences in the resistance or susceptibility of representative virulent and avirulent strains. Thus, the ability of S. gordonii to survive in PMNs following adhesin-mediated phagocytosis may be an important virulence determinant of infective endocarditis.

Effects of Vitamin A Deficiency and Opioids on Parvalbumin + Interneurons in the Hippocampus of the HIV-1 Transgenic Rat

Opioid use in HIV infection has been associated with an increased frequency of neurological disease and cognitive impairment and vitamin A deficiency has been linked to progressive HIV disease in drug users. In this report the potential effects of these factors, alone and in combination, on gamma amino butyric acid (GABA)-expression interneurons in hippocampus in the HIV-1 transgenic rat (TG) model were studied. TG and wild-type (WT) F344 Fisher rats deficient in vitamin A from birth were implanted either with a 37.5 mg morphine tablet or with a matching placebo and total numbers of neurons and of parvalbumin+ neurons were quantitated and parvalbumin expression was quantitated in the CA1 hippocampal region of the rats. These studies showed that total neuronal numbers were decreased in the TG versus WT Fisher rats and that this decrease was enhanced by the vitamin A deficient diet and by treatment with morphine. In contrast, there was no significant change noted in numbers of parvalbumin+ neurons. However, levels of parvalbumin expression were decreased for vitamin A deficient and morphine-treated WT rats as compared to WT rats on the normal diet and placebo-treated WT rats. For TG rats, parvalbumin expression was higher for vitamin A deficient TG rats treated with either placebo or morphine than for WT vitamin A deficient rats treated with placebo, and placebo treated vitamin A deficient TG rats showed higher expression than morphine treated vitamin A deficient rats. Expression was also higher for vitamin A deficient morphine-treated rats than for the corresponding WT rat groups and for vitamin A deficient TG rats treated with placebo. For the remaining groups, parvalbumin was similar for the TG and WT rats. These findings suggest that in hippocampus vitamin A deficiency and morphine can increase parvalbumin expression, perhaps as a manifestation of a stress response. Parvalbumin-expressing GABA-ergic interneurons regulate the primary neuronal output from hippocampus that is important for memory and behavior. Therefore, these studies suggest that vitamin A deficiency and morphine might have effects that may impact such outputs and thereby have lasting effects on cognitive status.

Role of T lymphocytes in renal disease in HIV-transgenic mice.

The pathogenesis of human immunodeficiency virus (HIV)-associated focal segmental glomerulosclerosis (FSGS) has remained obscure. It has been proposed that renal parenchymal cells may be infected with HIV-1. If such infection occurs, the target cells would be expected to express viral proteins and thus could be targets for cytotoxic T lymphocytes. We previously described mice transgenic for a gag-pol-deleted HIV-1 genome that developed FSGS. In the present study, we tested the requirement for functional T cells in the evolution of renal disease in this model. We bred the HIV-transgenic mice (T26) with athymic nude mice to produce athymic T26 mice. We confirmed by flow cytometry of peripheral blood, thymus, lymph node, and spleen that the athymic T26 mice lacked mature T cells. The athymic T26 mice developed renal disease characterized by FSGS, tubular atrophy and dilatation, and interstitial infiltrate that was qualitatively identical to that seen in the parental T26 mice. Quantitative assessment of the athymic T26 mouse kidneys showed that glomerulosclerosis, tubular injury, and interstitial infiltrate were less severe compared with the parental T26 mouse kidneys. Although T26 mouse kidneys had a mixed cellular infiltrate composed of CD4 cells, CD8 cells, and macrophages, interstitial infiltrates within the athymic T26 mouse kidneys included macrophages but lacked both CD4 and CD8 cells. The renal expression of the HIV transgene was 1. 7-fold greater in T26 mice compared with athymic T26 mice. We conclude that mature T cells are not absolutely required for the development of HIV-associated nephropathy in transgenic mice but that, in their absence, renal disease is significantly milder. These data suggest that T-cell-mediated cytotoxicity directed against renal cells expressing virally encoded proteins is not an essential feature of renal pathogenesis in this model.

Targeting of CDK9 with indirubin 3'-monoxime safely and durably reduces HIV viremia in chronically infected humanized mice.

Successful propagation of HIV in the human host requires entry into a permissive cell, reverse transcription of viral RNA, integration into the human genome, transcription of the integrated provirus, and assembly/release of new virus particles. Currently, there are antiretrovirals against each of these viral steps, except for provirus transcription. An inhibitor of HIV transcription could both increase potency of treatment and suppress drug-resistant strains. Cellular cyclin-dependent kinase 9 (CDK9) serves as a cofactor for the HIV Tat protein and is required for effective transcription of the provirus. Previous studies have shown that the CDK9 inhibitor Indirubin 3’-monoxime (IM) inhibits HIV transcription in vitro and in short-term in vivo studies of HIV acute infection in humanized mice (PBMC-NSG model), suggesting a therapeutic potential. The objective of this study is to evaluate the toxicity, pharmacokinetics and long-term antiviral activity of IM during chronic HIV infection in humanized mice (HSC-NSG model). We show that IM concentrations above EC50 values are rapidly achieved and sustained for > 3 h in plasma, and that non-toxic concentrations durably reduce HIV RNA levels. In addition, IM enhanced the antiviral activity of antiretrovirals from the reverse transcriptase, protease and integrase inhibitor classes in in vitro infectivity assays. In summary, IM may enhance current antiretroviral treatments and could help achieve a “functional cure” in HIV patients by preventing expression of proviruses.