Joann M. Taylor

Genetic and Phenotypic Analyses of Human Immunodeficiency Virus Type 1 Escape from a Small-Molecule CCR5 Inhibitor

ABSTRACT We have described previously the generation of an escape variant of human immunodeficiency virus type 1 (HIV-1), under the selection pressure of AD101, a small molecule inhibitor that binds the CCR5 coreceptor (A. Trkola, S. E. Kuhmann, J. M. Strizki, E. Maxwell, T. Ketas, T. Morgan, P. Pugach, S. X. L. Wojcik, J. Tagat, A. Palani, S. Shapiro, J. W. Clader, S. McCombie, G. R. Reyes, B. M. Baroudy, and J. P. Moore, Proc. Natl. Acad. Sci. USA 99:395-400, 2002). The escape mutant, CC101.19, continued to use CCR5 for entry, but it was at least 20,000-fold more resistant to AD101 than the parental virus, CC1/85. We have now cloned the env genes from the the parental and escape mutant isolates and made chimeric infectious molecular clones that fully recapitulate the phenotypes of the corresponding isolates. Sequence analysis of the evolution of the escape mutants suggested that the most relevant changes were likely to be in the V3 loop of the gp120 glycoprotein. We therefore made a series of mutant viruses and found that full AD101 resistance was conferred by four amino acid changes in V3. Each change individually caused partial resistance when they were introduced into the V3 loop of a CC1/85 clone, but their impact was dependent on the gp120 context in which they were made. We assume that these amino acid changes alter how the HIV-1 Env complex interacts with CCR5. Perhaps unexpectedly, given the complete dependence of the escape mutant on CCR5 for entry, monomeric gp120 proteins expressed from clones of the fully resistant isolate failed to bind to CCR5 on the surface of L1.2-CCR5 cells under conditions where gp120 proteins from the parental virus and a partially AD101-resistant virus bound strongly. Hence, the full impact of the V3 substitutions may only be apparent at the level of the native Env complex.

Increased CCR5 Affinity and Reduced CCR5/CD4 Dependence of a Neurovirulent Primary Human Immunodeficiency Virus Type 1 Isolate

ABSTRACT Most human immunodeficiency virus type 1 (HIV-1) viruses in the brain use CCR5 as the principal coreceptor for entry into a cell. However, additional phenotypic characteristics are necessary for HIV-1 neurotropism. Furthermore, neurotropic strains are not necessarily neurovirulent. To better understand the determinants of HIV-1 neurovirulence, we isolated viruses from brain tissue samples from three AIDS patients with dementia and HIV-1 encephalitis and analyzed their ability to induce syncytia in monocyte-derived macrophages (MDM) and neuronal apoptosis in primary brain cultures. Two R5X4 viruses (MACS1-br and MACS1-spln) were highly fusogenic in MDM and induced neuronal apoptosis. The R5 viruses UK1-br and MACS2-br are both neurotropic. However, only UK1-br induced high levels of fusion in MDM and neuronal apoptosis. Full-length Env clones from UK1-br required lower CCR5 and CD4 levels than Env clones from MACS2-br to function efficiently in cell-to-cell fusion and single-round infection assays. UK1-br Envs also had a greater affinity for CCR5 than MACS2-br Envs in binding assays. Relatively high levels of UK1-br and MACS2-br Envs bound to CCR5 in the absence of soluble CD4. However, these Envs could not mediate CD4-independent infection, and MACS2-br Envs were unable to mediate fusion or infection in cells expressing low levels of CD4. The UK1-br virus was more resistant than MACS2-br to inhibition by the CCR5-targeted inhibitors TAK-779 and Sch-C. UK1-br was more sensitive than MACS2-br to neutralization by monoclonal antibodies (2F5 and immunoglobulin G1b12 [IgG1b12]) and CD4-IgG2. These results predict the presence of HIV-1 variants with increased CCR5 affinity and reduced dependence on CCR5 and CD4 in the brains of some AIDS patients with central nervous system disease and suggest that R5 variants with increased CCR5 affinity may represent a pathogenic viral phenotype contributing to the neurodegenerative manifestations of AIDS.

Control of radiation-induced pneumopathy and lung fibrosis by angiotensin-converting enzyme inhibitors and an angiotensin II type 1 receptor blocker

Purpose : This report summarizes our experiences on the protective effect of angiotensin-converting enzyme (ACE) inhibitors, especially captopril and an angiotensin II type 1 receptor blocker on radiation-induced pulmonary injury. Method : In the first series of experiments, adult male Sprague Dawley rats were given a single dose of either 20 or 30 Gy of gamma rays to a 35 cm 2 right hemithorax port, whilst shielding the left, contralateral, lung. Perfusion scans and autopsies were performed at intervals up to 12 months post-radiation. Three different ACE inhibitors, penicillamine and pentoxifylline were given as radiation protectors and their activity compared. A model of irradiation for total bone marrow transplant (BMT) was used for the second group of experiments. Male WAC/Rij/MCW rats received total-body irradiation and a regimen of cyclophosphamide (CTX) in preparation for bone marrow transplant. The modifiers were two ACE inhibitors, captopril and enalapril, and L-158,809, an angiotensin II (A II) type 1 receptor blocker. All drugs were administered in the rats‚ drinking water and all were well-tolerated. Results : In the irradiated rats, pulmonary damage progressed from the presence of blebs and detachment from basement membranes of endothelial cells a few days after injury, to severe arteritis and interstitial collagen deposition at 3 months, and then on to severe pneumonitis and extensive pulmonary fibrosis at 6 months. Marked increase of hydroxyproline was also found in the lungs at 6 months. These morphological changes were associated with significant decrease of ACE and plasminogen activator activity (PLA) and a marked increase of prostaglandins (PG12) and thromboxane (Txa2), substances considered as indicators of endothelial pulmonary damage. ACE inhibitors captopril, CL 24817, enalapril and CGS 13945 prevented the markers of endothelial dysfunction. Captopril and CL 24817, which contain a sulphydryl (-SH) radical in their moiety and the AII type 1 receptor blocker, L-158,809, were the most efficient in protecting the lung parenchyma from the inflammatory response and subsequent fibrosis. Penicillamine, an SH-containing compound with weak ACE inhibitory activity was also a strong antifibrotic agent but showed only modest anti-inflammatory properties. Additionally, in the irradiated rats, captopril also reduced the incidence of squamous cell skin carcinomas and subcutaneous sarcomas consequent to the highest doses of radiation. Conclusion : ACE inhibitors and one AII type 1 receptor blocker were effective in protecting lungs from radiation-induced pneumonitis and the development of lung fibrosis in two models of rat radiation injury. In the first series of experiments (unilateral irradiation), those ACE inhibitors containing a sulphydryl radical were more effective than those without it. This observation led to the question of whether this protective effect is related to inhibition of AII synthesis or rather to some of the collateral pharmacologic properties of these drugs, such as anti-oxidation or protease inhibition. The AII receptor blocker, however, was shown to be equally effective, if not better, in its antifibrotic capacity than any ACE inhibitor with or without an SH radical, reaffirming the role of AII in modulation of collagen synthesis.

Genetic and Functional Analysis of Full-Length Human Immunodeficiency Virus Type 1 env Genes Derived from Brain and Blood of Patients with AIDS

ABSTRACT The genetic evolution of human immunodeficiency virus type 1 (HIV-1) in the brain is distinct from that in lymphoid tissues, indicating tissue-specific compartmentalization of the virus. Few primary HIV-1 envelope glycoproteins (Envs) from uncultured brain tissues have been biologically well characterized. In this study, we analyzed 37 full-length env genes from uncultured brain biopsy and blood samples from four patients with AIDS. Phylogenetic analysis of intrapatient sequence sets showed distinct clustering of brain relative to blood env sequences. However, no brain-specific signature sequence was identified. Furthermore, there was no significant difference in the number or positions of N-linked glycosylation sites between brain and blood env sequences. The patterns of coreceptor usage were heterogeneous, with no clear distinction between brain and blood env clones. Nine Envs used CCR5 as a coreceptor, one used CXCR4, and two used both CCR5 and CXCR4 in cell-to-cell fusion assays. Eight Envs could also use CCR3, CCR8, GPR15, STRL33, Apj, and/or GPR1, but these coreceptors did not play a major role in virus entry into microglia. Recognition of epitopes by the 2F5, T30, AG10H9, F105, 17b, and C11 monoclonal antibodies varied among env clones, reflecting genetic and conformational heterogeneity. Envs from two patients contained 28 to 32 N-glycosylation sites in gp120, compared to around 25 in lab strains and well-characterized primary isolates. These results suggest that HIV-1 Envs in brain cannot be distinguished from those in blood on the basis of coreceptor usage or the number or positions of N-glycosylation sites, indicating that other properties underlie neurotropism. The study also demonstrates characteristics of primary HIV-1 Envs from uncultured tissues and implies that Env variants that are glycosylated more extensively than lab strains and well-characterized primary isolates should be considered during development of vaccines and neutralizing antibodies.

The HIV Env variant N283 enhances macrophage tropism and is associated with brain infection and dementia

HIV infects tissue macrophages and brain microglia, which express lower levels of CD4 and CCR5 than CD4+ T cells in peripheral blood. Mechanisms that enhance HIV tropism for macrophages in the CNS and other tissues are not well understood. Here, we identify an HIV envelope glycoprotein (Env) variant in the CD4-binding site of gp120, Asn 283 (N283), that is present at a high frequency in brain tissues from AIDS patients with HIV-associated dementia (HAD). N283 increases gp120 affinity for CD4 by decreasing the gp120-CD4 dissociation rate, enhancing the capacity of HIV Envs to use low levels of CD4 for virus entry and increasing viral replication in macrophages and microglia. Structural modeling suggests that the enhanced ability of Envs with N283 to use low levels of CD4 is due to a hydrogen bond formed with Gln 40 of CD4. N283 is significantly more frequent in brain-derived Envs from HAD patients (41%; n = 330) compared with non-HAD patients (8%; n = 151; P < 0.001). These findings suggest that the macrophage-tropic HIV Env variant N283 is associated with brain infection and dementia in vivo, representing an example of a HIV variant associated with a specific AIDS-related complication.

Captopril modulates hormone receptor concentration and inhibits proliferation of human mammary ductal carcinoma cells in culture.

The present study evaluated the effect of the angiotensin converting enzyme (ACE) inhibitor captopril on estrogen (ER) and progesterone (PR) receptor concentration and on proliferation in two lines of human mammary ductal carcinoma cells in culture: T-47D (ER+/PR+) and Hs578T (ER−/PR−). The incorporation of [3H]thymidine, validated by cell count, served as an index of proliferation. Compared to control cells, T-47D cells incubated for 48 hrs in 1, 2, or 5 mM captopril (but not in 0.5 mM) exhibited a reduction in ER from 130 ± 6 to 32 ± 32 fmol/mg cytosolic protein, and an increase in PR from 1780 ± 120 to 2740 ± 400 fmol/mg protein (p < 0.05). Western analysis confirmed these drug-induced changes in the concentration of immunoreactive receptor proteins. Captopril also induced the appearance of low but detectable PR in the Hs578T cells at concentrations as low as 50 µM. Captopril inhibited the incorporation of [3H]thymidine by both cell types during a 48 hr incubation, although Hs578T cells were 2–3 times more resistant than were T–47D cells. This cytostatic effect of captopril was not due to cytotoxicity as indicated by 51Cr release, and was not accompanied by significant changes in cell cycle distribution as determined by flow cytometry. The incorporation of [3H]uridine (RNA synthesis) and [14C]alanine (protein synthesis) also were inhibited by captopril, suggesting a general antimetabolic effect of the drug in the ductal carcinoma cells. These are novel actions of a common antihypertensive agent. In contrast, the nonthiol ACE inhibitor lisinopril, and penicillamine, a thiol compound with virtually no ACE inhibitory activity, had no effect on any of these endpoints.

Cytostatic properties of some angiotensin I converting enzyme inhibitors and of angiotensin II type I receptor antagonists.

Angiotensin converting enzyme (ACE) inhibitors and angiotensin II (AII) type 1 receptor antagonists have strong cytostatic properties on in vitro cultures of many normal and neoplastic cells. They are effective, in particular, in reducing the growth of human lung fibroblasts, renal canine epithelial cells, bovine adrenal endothelial cells, simian T lymphocytes, and of neoplastic cell lines derived from human neuroblastomas, a ductal pancreatic carcinoma of the Syrian hamsters, human salivary glands adenocarcinomas, and two lines of human breast adenocarcinomas. ACE inhibitors are also effective in protecting lungs, kidneys and bladders from the development of nephropathy, pneumopathy, cystitis, and eventually fibrosis in different models of organ-induced damage such as exposure to radiation, chronic hypoxia, administration of the alkaloid monocrotaline or bladder ligation. ACE inhibitors and AII type 1 receptor antagonists are also effective in reducing excessive vascular neoformation in a model of injury to the cornea of rats and rabbits, and in controlling the excessive angiogenesis observed in the Solt-Farber model of experimentally induced hepatoma, in methylcholantrene or radiation-induced fibrosarcomas, in radiation-induced squamous cell carcinomas and in the MA-16 viral-induced mammary carcinoma of the mouse. Captopril was, in addition, effective in controlling tumor growth in a case of Kaposis sarcoma in humans. The inhibition of AII synthesis and/or its blockade by AII receptors is likely to be an important mechanism for this cytostatic action. The mitogenic effect of AII is well established and a reduction of AII synthesis may well explain cell and neoplasm delayed growth. Moreover, AII regulates and enhances the activity of several growth factors including transforming growth factor B (TGFB) and smooth muscle actin (SMA); and many of these factors are reduced in tissues of animals treated with ACE inhibitors and AII type 1 receptor antagonists. These processes seem to be particularly relevant in the control of fibroblast growth and in the control of the ensuing fibrosis. The ACE inhibitors containing a sulphydril (SH) or thiol radical in their moiety (Captopril and CL242817) seemed to be more effective in controlling fibrosis and the growth of some neoplastic cells than those ACE inhibitors without this thiol radical in their structure, even if the second group of these drugs show in vitro a stronger inhibitory effect on converting enzyme activity. Pharmacologically it is known that ACE inhibitors containing a thiol radical also have antioxidant properties and they are efficient in controlling metalloproteinase action. However, although these additional properties are pharmacologically relevant, the blockade of AII synthesis plays an essential role in the cytostatic activity of these two categories of drugs. These observations underline that in addition to the beneficial effect of these drugs on the cardiovascular system, new potential applications are opening for their wider deployment.

Calflagin inhibition prolongs host survival and suppresses parasitemia in Trypanosoma brucei infection.

ABSTRACT African trypanosomes express a family of dually acylated, EF-hand calcium-binding proteins called the calflagins. These proteins associate with lipid raft microdomains in the flagellar membrane, where they putatively function as calcium signaling proteins. Here we show that these proteins bind calcium with high affinity and that their expression is regulated during the life cycle stage of the parasite, with protein levels approximately 10-fold higher in the mammalian bloodstream form than in the insect vector procyclic stage. We also demonstrate a role for the calflagins in mammalian infection, as inhibition of the entire calflagin family by RNA interference dramatically increased host survival and attenuated parasitemia in a mouse model of sleeping sickness. In contrast to infection with parental wild-type parasites, which demonstrated an unremitting parasitemia and death within 6 to 10 days, infection with calflagin-depleted parasites demonstrated prolonged survival associated with a sudden decrease in parasitemia at approximately 8 days postinfection. Subsequent relapsing and remitting waves of parasitemia thereafter were associated with alternate expression of the variant surface glycoprotein, suggesting that initial clearance was antigen specific. Interestingly, despite the notable in vivo phenotype and flagellar localization of the calflagins, in vitro analysis of the calflagin-deficient parasites demonstrated normal proliferation, flagellar motility, and morphology. Further analysis of the kinetics of surface antibody clearance also did not demonstrate a deficit in the calflagin-deficient parasites; thus, the molecular basis for the altered course of infection is independent of an effect on parasite cell cycle progression, motility, or degradation of surface-bound antibodies.

Dose-Response Effects of Radiation on the Permeability of Endothelial Cells in Culture

Increased permeability is an early and universal response of the vasculature to radiation injury, yet the biological basis of this reaction is poorly understood. The present study determined the time course and the dose-response relationship of radiation-induced hyperpermeability in cultured bovine pulmonary artery endothelial (BPAE) cells. BPAE cells were grown to a confluent monolayer on microcarrier beads, and column chromatography methods were used to evaluate permeability to two low molecular weight compounds: sodium fluorescein (NaFlsc, mol. wt. = 342) and cyanocobalamin (B12, mol. wt. = 1355). This is a novel in vitro model to study mechanisms and modifiers of radiation-induced permeability of endothelial cells under flow conditions using nonradioactive tracers. Cell-covered beads were exposed to a single dose of 10 Gy Of 137Cs gamma rays and placed in the column, and permeability was measured every 30 min for 3 h. There was a time-dependent increase in permeability to both tracers, reaching significance by 2 h. Increased permeability was accompanied by perturbations in F-actin distribution in the BPAE cells as determined by rhodamine-phalloidin fluorescence microscopy. Neither catalase nor captopril ameliorated this hyperpermeability, but dibutyryl cAMP partially prevented it. At 3 h after 0, 1, 2, 5 and 10 Gy irradiation, permeability values of 11.8 +/- 2.1, 13.9 +/- 2.2, 20.9 +/- 3.6, 24.8 +/- 2.8 and 27.2 +/- 3.3 (10(-5) cm/s, +/- SEM), respectively, were observed using NaFlsc. The increase was significant (P < 0.05) at 2 Gy or higher. Permeability to B12 was significantly elevated after 5 or 10 Gy. These results suggest that permeability of endothelial cells to low molecular weight solutes increases within 3 h after therapeutic doses of radiation, and that cAMP ameliorates this response.

Heat-Killed Trypanosoma cruzi Induces Acute Cardiac Damage and Polyantigenic Autoimmunity

Chagas heart disease, caused by the protozoan parasite Trypanosoma cruzi, is a potentially fatal cardiomyopathy often associated with cardiac autoimmunity. T. cruzi infection induces the development of autoimmunity to a number of antigens via molecular mimicry and other mechanisms, but the genesis and pathogenic potential of this autoimmune response has not been fully elucidated. To determine whether exposure to T. cruzi antigens alone in the absence of active infection is sufficient to induce autoimmunity, we immunized A/J mice with heat-killed T. cruzi (HKTC) emulsified in complete Freunds adjuvant, and compared the resulting immune response to that induced by infection with live T. cruzi. We found that HKTC immunization is capable of inducing acute cardiac damage, as evidenced by elevated serum cardiac troponin I, and that this damage is associated with the generation of polyantigenic humoral and cell-mediated autoimmunity with similar antigen specificity to that induced by infection with T. cruzi. However, while significant and preferential production of Th1 and Th17-associated cytokines, accompanied by myocarditis, develops in T. cruzi-infected mice, HKTC-immunized mice produce lower levels of these cytokines, do not develop Th1-skewed immunity, and lack tissue inflammation. These results demonstrate that exposure to parasite antigen alone is sufficient to induce autoimmunity and cardiac damage, yet additional immune factors, including a dominant Th1/Th17 immune response, are likely required to induce cardiac inflammation.