Effects of depot-medroxyprogesterone acetate on the immune microenvironment of the human cervix and endometrium: implications for HIV susceptibility.

Depot-medroxyprogesterone acetate is a commonly used injectable contraceptive that has been associated with an increased risk of HIV acquisition. This study compares effects of depot-medroxyprogesterone acetate on immune parameters from several upper reproductive tract compartments relevant to HIV-1 susceptibility in repetitive samples from 15 depot-medroxyprogesterone acetate users and 27 women not on hormonal contraceptives. Compared with samples from unexposed women in the mid-luteal phase, depot-medroxyprogesterone acetate use was associated with: increased endocervical concentrations of MCP1 and IFNalpha2; decreased endocervical concentrations of IL1beta and IL6; increased proportions of endometrial CD4+ and CD8+ cells expressing the activation marker HLADR; increased density of endometrial macrophages; and decreased density of endometrial regulatory T cells. Unlike previous reports with samples from the vagina, we did not observe increased expression of the HIV co-receptor CCR5 on CD4+ T cells in the endocervix or endometrium. Our results indicate important differences in anatomic compartments regarding mechanisms by which depot-medroxyprogesterone acetate could be associated with increased risk of HIV acquisition, including increased recruitment of macrophages to the endometrium, decreased levels of pro-inflammatory cytokines in the endocervix possibly leading to enhanced susceptibility to viral infection, and activation of endometrial T cells.

Predominance of weakly cytotoxic, T-betLowEomesNeg CD8+ T-cells in human gastrointestinal mucosa: implications for HIV infection.

The gastrointestinal mucosa is an important site of HIV acquisition, viral replication, and pathogenesis. Immune cells in mucosal tissues frequently differ in phenotype and function from their non-mucosal counterparts. Although perforin-mediated cytotoxicity as measured in blood is a recognized correlate of HIV immune control, its role in gastrointestinal tissues is unknown. We sought to elucidate the cytotoxic features of rectal mucosal CD8+ T-cells in HIV infected and uninfected subjects. Perforin expression and lytic capacity were significantly reduced in rectal CD8+ T-cells compared with their blood counterparts, regardless of HIV clinical status; granzyme B (GrzB) was reduced to a lesser extent. Mucosal perforin and GrzB expression were higher in participants not on antiretroviral therapy compared with those on therapy and controls. Reduction in perforin and GrzB was not explained by differences in memory/effector subsets. Expression of T-bet and Eomesodermin was significantly lower in gut CD8+ T-cells compared with blood, and in vitro neutralization of TGF-ß partially restored perforin expression in gut CD8+ T-cells. These findings suggest that rectal CD8+ T-cells are primarily non-cytotoxic, and phenotypically shaped by the tissue microenvironment. Further elucidation of rectal immune responses to HIV will inform the development of vaccines and immunotherapies targeted to mucosal tissues.

Mucosal T-cell responses to HIV: responding at the front lines.

Mucosal surfaces of the body serve as the major portal of entry for human immunodeficiency virus (HIV). These tissues also house a majority of the body's lymphocytes, including the CD4(+) T cells that are the major cellular target for HIV infection. Mucosal surfaces are defended by innate and adaptive immune mechanisms, including secreted antibodies and CD8(+) cytotoxic T cells (CTL). CTL in mucosal lymphoid tissues may serve to limit viral replication, decreasing the host's viral burden as well as reducing the likelihood of sexual transmission to a naïve host. This review summarizes recent literature on HIV-specific T-cell responses in mucosal tissues, with an emphasis on the gastrointestinal tract.

Isoquinolinesulphonamide derivatives inhibit transcriptional elongation of human immunodeficiency virus type 1 RNA in a promyelocytic model of latency.

Using the OM-10.1 promyelocytic model of inducible human immunodeficiency virus type 1 (HIV-1) infection, we tested a panel of known protein kinase inhibitors for an ability to block tumour necrosis factor-alpha-induced HIV-1 expression. Among the compounds tested, the broad-spectrum protein kinase inhibitor H-7 uniquely blocked HIV-1 expression at the level of viral transcription, but did not inhibit nuclear factor kappaB activation or function. In structure-activity analysis this inhibitory activity of H-7 on HIV-1 expression corresponded with the known structural requirements for the interaction of H-7 with the ATP-binding region of protein kinase C, suggesting that it was indeed related to the kinase inhibitory properties of H-7. The mechanism of H-7 transcriptional inhibition did not involve chromatin remodelling at the HIV-1 long terminal repeat promoter, as shown by nuc-1 disruption, and appeared to involve HIV-1 RNA elongation but not initiation. Therefore, H-7 and related isoquinolinesulphonamide analogues are most likely inhibiting a kinase target essential for HIV-1 transcriptional elongation whose identity may provide new therapeutic targets for intervention.

Casein kinase II is a selective target of HIV-1 transcriptional inhibitors.

The identification of cellular factors that are required to complete various steps of the HIV-1 life cycle may lead to the development of new therapeutics. One key step, transcription from the integrated provirus, is inhibited by members of two distinct classes of compounds, the flavonoids and the benzothiophenes, via an unknown mechanism, possibly involving a cellular factor. A marked specificity toward inhibiting HIV-1 transcription is evidenced by the ability of drug-treated cells to retain their proliferative and differentiation capabilities. In addition, the compounds do not impede the activation and function of the transcriptional factor NF-kappaB. Here we report on the identification of several cellular proteins that mediate the HIV-1 transcriptional inhibitory property of the flavonoid chrysin. Chemical and immunologic analyses identified these cellular proteins as the individual subunits of casein kinase II (CKII). Though structurally unrelated to chrysin, an HIV-1 inhibitory benzothiophene also bound selectively to CKII. Both chrysin and the benzothiophenes inhibited human recombinant CKII enzymatic activity and showed competitive kinetics with respect to ATP, analogous to the classic CKII inhibitor 5, 6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB). Moreover, DRB potently inhibited HIV-1 expression in chronically infected cells. CKII may regulate HIV-1 transcription by phosphorylating cellular proteins involved in HIV-1 transactivation that contain multiple CKII phosphorylation consensus sequences.

Inhibition of HIV activation in latently infected cells by flavonoid compounds.

Acute HIV-1 infection of H9 and C8166 cultures has been shown to be suppressed by certain flavonoids, and evidence for inhibition of HIV-1 protease, integrase, and reverse transcriptase by flavonoids also exists. The present aim was to determine whether flavonoids inhibit HIV-1 activation in models of latent infection. By screening flavonoids from six different classes, three structurally related compounds (chrysin, acacetin, and apigenin) were identified that inhibited HIV expression in TNF-alpha-treated OM-10.1 cultures. The three compounds had favorable potencies against HIV activation in relation to their growth inhibitory effects (therapeutic index 5-10). Chrysin also inhibited HIV expression in response to PMA in OM-10.1 cells, in ACH-2 cells stimulated with either TNF-alpha or PMA, and in 8E5 cultures. Furthermore, return to viral latency in OM-10.1 cells previously exposed to TNF-alpha occurred over a shorter time interval when chrysin was added. The inhibition of HIV activation was not dependent on preincubation with flavonoids relative to TNF, and was characterized by a lack of HIV RNA accumulation by Northern analysis. Gel-shift experiments revealed that NF-kappa B activation after TNF-alpha treatment was not inhibited by these agents, suggesting that some other critical factor(s) needed for viral transcription was being affected. These findings indicate that flavonoids inhibit HIV-1 activation via a novel mechanism, and that these agents are potential candidates for therapeutic strategies aimed at maintaining a cellular state of HIV-1 latency.

Compounds that target novel cellular components involved in HIV-1 transcription.

BACKGROUND: Therapeutic intervention designed to block expression of human immunodeficiency virus (HIV) at a cellular level may slow the clinical progression of HIV-1 disease. MATERIALS AND METHODS: Cellular models of latent (OM-10.1 and U1) and chronic (8E5) HIV infection were used to evaluate two benzothiophene derivatives, PD 121871 and PD 144795, for an ability to inhibit HIV activation and expression. RESULTS: The benzothiophene derivatives were effective at micromolar concentrations in preventing tumor necrosis factor alpha (TNF alpha)-induced HIV-1 expression in OM 10.1 and U1 cultures. These compounds inhibited the activation of HIV-1 transcription; however, this inhibition was selective in that another TNF alpha-induced response, the transcription of autocrine TNF alpha, was unaffected. Constitutive HIV-1 expression by chronically infected 8E5 cells was also significantly reduced when treated with these experimental compounds. In TNF alpha-treated OM-10.1 cultures, the inhibition of HIV-1 transcription by these compounds was not due to a block of nuclear factor-kappa B induction. The benzothiophene derivatives also inhibited HIV-1 activation by phorbol ester treatment of OM-10.1 promyelocytes, although no inhibition of cellular differentiation toward a macrophage-like phenotype was observed. Furthermore, these experimental compounds induced a state of HIV-1 latency in cytokine-activated OM-10.1 cultures even when maintained under constant TNF alpha stimulation. The benzothiophene derivatives did not inhibit the activity of the HIV-1 trans-activator, Tat, when evaluated in transient transfection assays. CONCLUSIONS: The benzothiophene derivatives appear to inhibit a critical cellular component, distinct from nuclear factor-kappa B, involved in HIV transcription and may serve to identify new therapeutic targets to restrict HIV expression.

Modulation of adriamycin accumulation and efflux by flavonoids in HCT-15 colon cells. Activation of P-glycoprotein as a putative mechanism.

Since P-glycoprotein (P-gp) in normal tissues may serve as a cellular defense mechanism against naturally occurring xenobiotics, we considered whether physiologically active components of commonly ingested plant foods could influence P-gp function. To examine this possibility, a series of flavonoids commonly found in plant foods was tested for their ability to modulate [14C]Adriamycin ([14C]ADR) accumulation and efflux in P-gp-expressing HCT-15 colon cells. Many flavonoids, in the micromolar range, inhibited the accumulation of [14C]ADR. Detailed experiments utilizing flavonoids with the greatest activity in reducing [14C]ADR accumulation, i.e. galangin, kaempferol, and quercetin, revealed that the efflux of [14C]ADR is increased markedly in the presence of these compounds. Flavonoid-induced stimulation of efflux was rapid and was blocked by the multidrug-resistant (MDR) reversal agents verapamil, vinblastine, and quinidine. The magnitude of flavonoid-stimulated efflux in sodium butyrate-treated cells with a 4-fold induction of P-gp protein was similar to that in uninduced cells. [3H]Azidopine photoaffinity labeling of P-gp in crude membrane preparations revealed mild to no competition for binding by flavonoids possessing either activity or inactivity in reducing ADR accumulation. Although flavonoid hydrophobicity was found to be unrelated to flavonoid activity in altering [14C]ADR accumulation, certain structural features were associated with enhancement or diminution of activity. Finally, the significance of flavonoid-related reduction of [14C]ADR accumulation was underscored in cell growth studies, showing partial protection by quercetin against ADR-induced growth inhibition. It is concluded that certain naturally occurring plant flavonoids may acutely upregulate the apparent activity of P-gp.

Anticonvulsant-induced changes in tissue manganese, zinc, copper, and iron concentrations in Wistar rats.

Human epileptics have been reported to have low blood manganese (Mn) concentrations in comparison to nonepileptics, an observation that is important because Mn deficiency can increase seizure susceptibility in experimental animals. Factors that have been suggested to contribute to the low blood Mn levels in epileptics include anticonvulsant use, seizure-induced tissue redistribution of Mn, and genetics; in the present study, the first of these possibilities was tested. Wistar rats were fed semipurified diets containing diphenylhydantoin ([DPH] 3 g/kg diet), phenobarbital ([PB] 2 g/kg diet), or primidone ([PRIM] 3 g/kg diet) for 7 weeks, at which time they were killed and tissues collected and analyzed for Mn, zinc (Zn), copper (Cu), and iron (Fe) concentrations. In comparison to pair-fed rats, DPH- and PRIM-fed rats had significantly elevated liver Mn concentrations, while Mn concentrations in blood, brain, heart, and kidney were unaffected by anticonvulsant exposure. Changes in the concentrations of Zn, Cu, and Fe in specific tissues were also found. Overall, these findings suggest that the anticonvulsants tested do not lead to significant derangements in the metabolism of Mn.

Manganese and epilepsy: brain glutamine synthetase and liver arginase activities in genetically epilepsy prone and chronically seizured rats.

Low blood manganese (Mn2+) concentration is associated with epilepsy in humans and rats. The low Mn2+ concentration is attributed by some investigators to the seizure activity associated with the epilepsy, whereas others propose that the low Mn2+ concentration may be secondary to genetic mechanisms underlying the epilepsy. To begin to differentiate between these possibilities, Mn(2+)-binding enzymes of liver and brain (i.e., arginase and glutamine synthetase, respectively) were assayed in rats exposed to chronically induced seizures and in genetically epilepsy-prone rats (GEPRs). Chronic seizures caused a decrease in whole blood Mn2+ levels but did not affect brain Mn2+ concentrations. Arginase activity was increased in livers of rats with chronic seizure as compared with controls, but this difference was eliminated when Mn2+ was added to the assay. Brain glutamine synthetase activity was unaffected by chronic seizures, but the activity of this enzyme was significantly lower in GEPR brain than in control brain. Liver arginase activity tended to be lower in GEPRs, although the difference was not statistically significant. These data indicate that seizures affect liver arginase activity through changes in liver Mn2+ concentration, but GEPRs show abnormalities in Mn(2+)-dependent enzymes apparently independent of seizure activity.

The influence of manganese supplementation on seizure onset and severity, and brain monoamines in the genetically epilepsy prone rat.

Human and experimental animal studies suggest a relationship between low Mn status and seizures. The genetically epilepsy prone rat (GEPR), which has low tissue Mn levels, was studied in the context of Mn supplementation. Manganese was provided at 45 micrograms/g diet (control) or 1000 micrograms/g diet (supplemented) to dams during pregnancy and lactation, then to the offspring after weaning. Offspring were tested for seizure susceptibility as young adults; tissue trace elements, brain monoamines and brain glutamine synthetase activity were measured as endpoint biochemical indices. Supplementation, although developmentally encompassing and highly effective in elevating tissue Mn levels, had no effect on seizure latency or severity. Similarly, brain monoamine concentrations and glutamine synthetase activities were resistant to Mn supplementation. Notably, the GEPR was confirmed to have low whole brain glutamine synthetase activity. These findings suggest that seizure activity in the GEPR does not stem from an increased nutritional/metabolic need for Mn.

Manganese + 2 exhibits dynamic binding to multiple ligands in human plasma.

Plasma from fasted adult male subjects was labeled in vitro with 54MnCl2 and then fractionated using several techniques. Molecular sieve chromatography showed that the major 54Mn-containing peak had a very low molecular weight (VLMW), although four other significant peaks, one of which corresponded to the mass of transferrin (Tf), were also observed. The 54Mn content of the Tf peak increased with increasing incubation time in vitro, suggesting the oxidation of Mn+2 to Mn+3 before its association with Tf. This time-dependent effect was verified using affinity chromatography consisting of immobilized anti-Tf. Electrophoretic analyses of plasma yielded equivocal results, indicating a limited value of this method for investigating plasma manganese localization. The above findings are discussed in the context of factors that influence the oxidation and metabolism of Mn2+ in human plasma.

Copper, zinc, manganese, and magnesium status and complications of diabetes mellitus.

OBJECTIVE: To evaluate copper, zinc, manganese, magnesium, and other indices of peroxidative status in diabetic and nondiabetic human subjects. RESEARCH DESIGN AND METHODS: Convenience sample of 57 insulin-dependent or non-insulin-dependent diabetic subjects recruited from the diabetes clinic of the University of California, Davis, Medical Center and 28 nondiabetic subjects recruited from the staffs of the Departments of Internal Medicine and Nutrition. Individuals conducting laboratory analyses were blind to subject group. A fasting blood sample was collected from all subjects and appropriately processed for future analyses. A 24-h urine collection was obtained in a subset of subjects. RESULTS: Hyperzincuria and hypermagnesuria were evident in diabetic subjects compared with control subjects. There were no differences in plasma magnesium or whole-blood manganese between groups. Plasma copper was higher and plasma zinc was lower in diabetic than in control subjects. When data were viewed with respect to specific diabetes-associated complications, diabetic subjects with retinopathy, hypertension, or microvascular disease had higher plasma copper concentrations compared with both diabetic subjects without complications and with control subjects. There were no significant differences between control and diabetic subjects in erythrocyte copper-zinc superoxide dismutase activity or whole-blood glutathione peroxidase or glutathione reductase activities. Plasma peroxide concentrations were higher in diabetic than control subjects. CONCLUSIONS: Diabetes can alter copper, zinc, magnesium, and lipid peroxidation status. Perturbations in mineral metabolism are more pronounced in diabetic populations with specific complications. It is not known whether differences in trace element status are a consequence of diabetes, or alternatively, whether they contribute to the expression of the disease.

Genetically epilepsy-prone rats are characterized by altered tissue trace element concentrations.

Since trace element abnormalities have been found in the human epileptic population, trace element concentrations were determined in blood and tissues of genetically epilepsy-prone rats both exposed to an unexposed to seizure-inducing stimuli and in genetically related epilepsy-resistant rats. Half of the epilepsy-prone group were exposed to seizure-inducing sound twice daily for 3 weeks. Food intake and weight gain were monitored for each animal. Genetically epilepsy prone rats with induced seizures consumed significantly less food and gained less weight than did the epilepsy resistant group. Seizure prone rats without seizures consumed the same amount of food as the resistant rats but gained less weight than the resistant strain but more than the seizure-induced animals. Epilepsy-prone animals had significantly altered trace element concentrations in tissues as compared with the resistant animals independent of seizure induction. Brain and liver iron, liver copper, and brain and heart manganese levels were all significantly lower in the seizure-prone rats as compared with the seizure-resistant rats. In the seizure-prone rats, induction of seizures resulted in an increase in brain and heart zinc levels and a decrease in whole blood manganese levels. These results demonstrate that both genetic factors relevant to susceptibility to seizures and the seizures themselves are associated with changes in trace element concentrations.

Effect of kainate-induced seizures on tissue trace element concentrations in the rat.

It has been shown that epileptics have lower mean blood concentration of manganese than do controls but the cause of this abnormality has not been determined. In order to investigate the effects of seizures on manganese distribution in the body, rats were treated with kainic acid to produce spontaneous seizures which were quantitated for number and severity. Manganese, zinc, copper and iron concentrations were determined in blood, brain, liver, heart and kidney. Kainate-treated animals ate more food but gained less weight than controls. Liver and kidney manganese concentrations were significantly higher in kainate-treated animals than in controls. Blood manganese concentration showed a significant negative correlation with seizure index while heart manganese concentration showed a significant positive correlation with seizure index. None of the other trace elements showed a significant correlation between trace element concentration and seizure index in any of the tissues, although iron concentration was lower in brain and copper concentration was lower in kidney of kainate-treated animals than in their appropriate controls. These data show that manganese concentrations are generally elevated in tissues of kainate-treated animals. This increased manganese concentration may be related to the increased energy demand of these animals.

Keratoconus: I. Biochemical studies.

The present study analyses collagenous and non-collagenous components from age-matched normal and keratoconus corneas. Intact keratoconus corneas showed decreased collagen, total protein, and hydroxylysine levels, with normal reducible collagen cross-linking. Non-collagenous fractions were isolated from corneas with a 4 M guanidine procedure. As demonstrated by PAGE-silver stain, the keratoconus cornea guanidine extracts had a 75 kDa band that was absent in normal cornea guanidine extracts. In addition, there was a markedly increased level of protein, uronic acid and neutral hexose in keratoconus extracts as compared with controls. Our Western blot studies showed increased affinity for the castor-bean agglutinin (RCA120, specific for terminal galactose) in the keratoconus extracts as compared with normals. These data suggest the presence of an abnormal noncollagenous component in keratoconus corneas.

Changes in response to ascorbic acid administered orally to rat pups: lung collagen, elastin and protein synthesis.

Rat pups were supplemented orally with high doses of L-ascorbic acid (AA) or D-isoascorbic acid (IA) throughout suckling. The regulation of AA in the lung and its relationship to collagen and elastin deposition were examined. Based on known responses of smooth muscle cells and fibroblasts in culture to high concentrations of AA, it was hypothesized that a markedly elevated intake of AA should increase net collagen deposition, but decrease net elastin deposition in the neonatal rat lung. In two experiments, groups of rat pups were gavaged daily with AA (in saline), in amounts corresponding to 0.1, 1 or 2% of the total consumed milk solids. As controls, pups were gavaged with IA (2% of the milk solids) or saline. The treatments were initiated 2 d postpartum and continued for 19 or 23 d. Compared to the saline-gavaged pups. AA and IA were elevated (twofold) in serum and lung at d 11, but not at d 25. Urinary excretion represented a major route for elimination of excess AA and IA. With respect to collagen and elastin accumulation, only elastin consistently was altered (10-20% decrease) in groups supplemented with AA or IA at the termination of experiments. The rodent appears to defend against elevation of AA concentration in the lung. Consequently, the putative effects of AA on the net deposition of lung collagen and elastin in vivo are less obvious than effects reported by others from in vitro studies.

Ascorbic acid turnover in the mouse following acute ozone exposure.

Swiss Webster mice were continuously exposed to an atmosphere containing 1.5 ppm ozone (O3) for 5 days. Control mice breathed filtered air. Immediately following the exposure period each mouse was then injected with [1-14C] ascorbic acid. The rate of disappearance of [1-14C] ascorbic acid and the levels of total ascorbic acid were determined in serum, lung, liver, and the remaining carcass over a 9-day period. O3 exposure caused transient decreases in the ascorbic acid levels in liver and serum, whereas lung ascorbic acid levels increased. The apparent biological half-life of ascorbate in carcasses (minus lung and liver) from O3-exposed mice, was significantly prolonged. Significant changes were also observed in the net flux of ascorbic acid in all tissues examined. The results indicated that the change in ascorbic acid levels in a given tissue appeared to reflect changes in the rate of ascorbic acid degradation, its mobilization or tissue compartmentalization, rather than increased synthesis in response to O3 exposure.

Altered succinate dehydrogenase activity of basal ganglia following to mesotelencephalic dopaminergic projection.

Unilateral damage to the mesotelencephalic dopamine-containing projection of rats by intracerebral 6-hydroxydopamine injection, alters the staining of the tricarboxylic acid cycle enzyme succinate dehydrogenase in the basal ganglia. Relative to the intact hemisphere, the neostriatum ipsilateral to the damage shows diminished enzyme staining, while the ipsilateral globus pallidus, entopeduncular nucleus and substantia nigra pars reticulata all show enhanced deposition of reaction product. These asymmetries are detectable in some animals by 3 days after surgery and increase in magnitude at longer postoperative survival times. The alterations in succinate dehydrogenase staining appear to be related to the severity of the sensorimotor impairments resulting from the injury.