Advancing Behavioral HIV Prevention: Adapting an Evidence-Based Intervention for People Living with HIV and Alcohol Use Disorders.

Alcohol use disorders (AUDs) are highly prevalent among people living with HIV/AIDS (PLWHA) and are associated with increased HIV risk behaviors, suboptimal treatment adherence, and greater risk for disease progression. We used the ADAPT-ITT strategy to adapt an evidence-based intervention (EBI), the Holistic Health Recovery Program (HHRP+), that focuses on secondary HIV prevention and antiretroviral therapy (ART) adherence and apply it to PLWHA with problematic drinking. Focus groups (FGs) were conducted with PLWHA who consume alcohol and with treatment providers at the largest HIV primary care clinic in New Orleans, LA. Overall themes that emerged from the FGs included the following: (1) negative mood states contribute to heavy alcohol consumption in PLWHA; (2) high levels of psychosocial stress, paired with few adaptive coping strategies, perpetuate the use of harmful alcohol consumption in PLWHA; (3) local cultural norms are related to the permissiveness and pervasiveness of drinking and contribute to heavy alcohol use; (4) healthcare providers unanimously stated that outpatient options for AUD intervention are scarce, (5) misperceptions about the relationships between alcohol and HIV are common; (6) PLWHA are interested in learning about alcohol's impact on ART and HIV disease progression. These data were used to design the adapted EBI.

Modulated photoacoustic spectroscopy study of an artificial tanning on human skin induced by dihydroxyacetone.

A modulated photoacoustic spectroscopy study on the effect of dihydroxyacetone, commonly used for artificial tan, is presented. The study was carried out in the presence and absence of dimethylisosorbide (a solvent for dihydroxyacetone) on fresh human skin, obtained from the breast region of recent autopsy cases (two females), at a frequency of 25 Hz, which enabled us to study the effect at a depth of 30 microm in the stratum corneum and beneath. By monitoring the photoacoustic signal intensity with time in the region of 300-400 nm, which is a specific region for melanin pigment, it is demonstrated that dihydroxyacetone in combination with dimethylisosorbide enhances the process of tanning. Dihydroxyacetone also has an effect on the amino acids and nucleic acids which is bad for the skin.

[13C]aminopyrine and [13C]caffeine breath test: influence of gender, cigarette smoking and oral contraceptives intake.

The [13C]aminopyrine breath test ([13C]ABT) measures the global activity of cytochrome P450 in vivo and is a sensitive indicator of liver metabolic dysfunction. The present study aims to determine whether gender and cigarette smoking influence the results of [13C]ABT as well as to confirm the effect of oral contraceptive steroids (OCS) intake on this metabolic test. Hundred and ten healthy subjects, including men and women, smoker and non-smoker, women taking OCS or not, were phenotyped for CYP1A2 using the [13C]caffeine breath test and underwent a [13C]ABT. Both tests showed large inter-individual variations in accordance with that of CYP450 liver content. [13C]ABT was sensitive enough to point out a significant induction or inhibition related to cigarette smoking habits or OCS. The combined effect of smoking and OCS resulted in an overall unchanged metabolic activity. Consequently, the impact of the studied conditions on the [13C]ABT parameters must be considered by clinicians or clinical investigators.

Mechanisms of wound reepithelialization: hints from a tissue-engineered reconstructed skin to long-standing questions.

Wound closure of epithelial tissues must occur efficiently to restore rapidly their barrier function. We have developed a tissue-engineered wound-healing model composed of human skin keratinocytes and fibroblasts to better understand the mechanisms of reepithelialization. It allowed us to quantify the reepithelialization rate, which was significantly accelerated in the presence of fibrin or platelet-rich plasma. The reepithelialization of these 6 mm excisional wounds required the contribution of keratinocyte proliferation, migration, stratification, and differentiation. The epidermis regenerated progressively from the surrounding wound margins. After 3 days, the neoepidermis showed a complete spectrum of changes. Near the wound margin, the differentiation of the neoepidermis (keratins 1/10, filaggrin, and loricrin) and regeneration of the dermoepidermal junction (laminin 5 and collagen IV) were more advanced than toward the wound center, where the proliferative index was significantly increased. The spatial distribution of keratinocytes distinguished by particular features suggests two complementary mechanisms of reepithelialization: 1) the passive displacement of the superficial layers near the wound margin that would rapidly regenerate a barrier function and 2) the crawling of keratinocytes over each other at the tip of the progressing neoepidermis. Therefore, this study brings a new perspective to long-standing questions concerning wound reepithelialization.

Canadian valsartan study in patients with mild-to-moderate hypertension.

OBJECTIVE: This study was designed mainly to establish the rates of response to valsartan 80mg once daily (qd) and to valsartan 160mg qd given to non-responders to 80mg qd, as well as to determine the safety of valsartan and the blood pressure control achieved using valsartan over a period of 24 h or more, using ambulatory blood pressure monitoring (ABPM) devices. METHODS: This was a single-blind, single-arm, multicenter study involving 256 out-patients with mild-to-moderate essential hypertension. After previous antihypertensive treatments had been 'washed out', the patients were entered into a 2-week placebo run-in period to confirm the diagnosis of mild-to-moderate hypertension. Patients who, at the end of the placebo run-in period, had a mean sitting diastolic blood pressure of between 95 and 115mmHg inclusive received valsartan 80mg qd for 4 weeks. Non-responders (those not demonstrating a diastolic blood pressure of less than 90mmHg or a decrease in diastolic blood pressure of 10mmHg or more compared with baseline) received valsartan 160mg qd for another 4 weeks. In selected centers, patients who agreed also had their blood pressure monitored for 24 h, provided their blood pressure was shown to be controlled. Of these patients, half skipped one dose of valsartan and were monitored for an additional 24h period. RESULTS: The rate of response to valsartan 80mg was 45.4%, and of those not responding to this dose, 36.3% responded to valsartan 160mg. The response rate to one or other dose was 63.2%. The ambulatory blood pressure data support a consistent reduction of blood pressure with valsartan over a 24h period and for up to 32 h after dosing in those who missed a dose. The overall incidence of adverse experiences per person-year, treatment related or otherwise, was 6.3 and 10.6 for the valsartan and placebo study periods respectively. CONCLUSION: Antihypertensive treatment with valsartan for 8 weeks produced a significant decrease in diastolic blood pressure in hypertensive patients. In addition, the drug may be safely administered, and the results of 24 h/48 h ambulatory monitoring demonstrate that valsartan is a true once-a-day antihypertensive.

Multidrug resistance in brain tumors: roles of the blood-brain barrier.

Malignant brain tumors and brain metastases present a formidable clinical challenge against which no significant advances have been made over the last decade. Multidrug resistance (MDR) is one of the main factors in the failure of chemotherapy against central nervous system tumors. The MDR1 gene encoding P-glycoprotein (P-gp), a drug efflux pump which plays a significant role in modulating MDR in a wide variety of human cancers, is highly expressed in the blood-brain barrier (BBB). The BBB controls central nervous system exposure to many endogenous and exogenous substances. The exact molecular mechanisms by which the BBB is involved in the resistance of brain tumors to chemotherapy remain to be identified. The purpose of this review is to summarize reports demonstrating that P-gp, one of the most phenotypically important markers of the BBB, is present in primary brain tumors and thus plays a crucial role in their clinical resistance to chemotherapy.

Association of cyclophilin A with renal brush border membranes: redistribution by cyclosporine A.

BACKGROUND: Administration of the immunosuppressive agent cyclosporine A (CsA) is associated with nephrotoxicity. The main target for CsA, cyclophilin A (CypA), was found in high levels in epithelial cells of renal proximal tubules. In the present study, CypA was immunodetected and characterized following CsA treatment in subcellular fractions of renal cortex. METHOD: The renal content and distribution of CypA was evaluated in untreated rats and in rats treated with a subcutaneous injection of CsA (10 mg. kg-1. day-1) for 10 days. RESULTS: In untreated rats, membrane-bound CypA represents 0.25% of total brush border membrane (BBM) proteins, similar to the proportion found in the soluble fraction. High ionic strength treatment was unable to extract CypA from BBMs, whereas alkaline treatment (Na2CO2, pH 11) and detergent 3 - [(3 - cholamidopropyl) - dimethyl - ammonio] - 1 - propanesulfate (CHAPS) released it from BBMs. These results indicate that CypA is associated with renal BBMs, and that hydrophobic interactions are involved in this association. The CypA distribution was strongly modified in both BBMs and the soluble fraction after CsA treatment, but its affinity for CsA estimated by photoaffinity labeling was unaffected. The CypA expression level decreased by 45% in BBMs, while it increased by 33% in the soluble fraction, compared with control rats. CypA remained associated with the membranes following in vitro incubation of renal BBMs with CsA. However, incubation of CypA with one of its substrates released CypA from renal BBMs. CONCLUSIONS: These experiments suggest that renal BBMs contain a significant amount of CypA and chronic exposure to CsA, and acute exposure to one of CypA substrates may modify its subcellular distribution.

Inhibition of P-glycoprotein by cyclosporin A analogues and metabolites.

The interaction between P-glycoprotein (P-gp) from membranes isolated from multidrug-resistant Chinese hamster ovary cells and cyclosporin A (CsA) analogues and its metabolites was characterized. Screening of these latter as chemosensitizers was performed using three different assays: (i) vinblastine uptake, (ii) photoaffinity labeling by [125I]iodoaryl azidoprazosin, and (iii) P-gp ATPase activity. Oxidation of the hydroxyl group at position I of CsA (200-096), CsG (215-834), or CsD (PSC-833) increased their inhibition of P-gp. CsA analogues (208-032, 208-183) modified at position 11 retained their ability to inhibit P-gp while analogues modified at position 2 (CsC and CsD) lost their efficiency. The inhibitions induced by metabolites of CsA were also compared to those obtained with CsG metabolites. From all the molecules tested, PSC-833 and 280-446 peptolide were the strongest inhibitors. Our results indicate that modifications of CsA analogues at position 1 and 2 are critical for their interaction with P-gp and that CsA metabolites retain a portion of the inhibitory activity of the parent drug.

Expression of heat shock proteins in mouse skin during wound healing.

Wound healing conditions generate a stressful environment for the cells involved in the regeneration process and are therefore postulated to influence the expression of heat shock proteins (Hsps). We have examined the expression of four Hsps (Hsp27, Hsp60, Hsp70 and Hsp90) and a keratin (keratin 6) by immunohistochemistry during cutaneous wound repair from Day 1 to Day 21 after wounding in the mouse. Hsps were constitutively expressed in normal mouse epidermis and their patterns of expression were modified during the healing process. The changes were not directly linked to the time course of the healing process but rather were dependent on the location of cells in the regenerating epidermis. In the thickened epidermis, Hsp60 was induced in basal and low suprabasal cells, Hsp70 showed a reduced expression, and Hsp90 and Hsp27 preserved a suprabasal pattern with an induction in basal and low suprabasal cells. All Hsps had a uniform pattern of expression in the migrating epithelial tongue. These observations suggest that the expression of Hsps in the neoepidermis is related to the proliferation, the migration, and the differentiation states of keratinocytes within the wound.

Identification of the cyclosporin-binding site in P-glycoprotein.

The binding site of cyclosporin A to P-glycoprotein was characterized by using a multidrug-resistant Chinese hamster ovary cell line. P-glycoprotein photolabeled with diazirine-cyclosporin A analogue was purified by a two-step process involving continuous elution electrophoresis followed by wheat germ agglutinin-agarose precipitation. The cyclosporin A covalently bound to P-glycoprotein and to subsequent proteolytic fragments was detected by Western blot analysis using a monoclonal antibody against cyclosporin A. Proteolytic digestion of purified P-glycoprotein by V8 generated a major fragment of 15 kDa photolabeled by cyclosporin A, while proteolysis of P-glycoprotein photolabeled by [125I]-iodoaryl azidoprazosin generated a major fragment of 7 kDa. Limited proteolysis of cyclosporin A-photolabeled P-glycoprotein with trypsin indicated that the major binding site for cyclosporin A was in the C-terminal half of the protein. This cyclosporin A binding site was further characterized with chemical agents (N-chlorosuccinimide, cyanogen bromide, and 2-nitro-5-thiocyanobenzoate). These three chemical agents established a proteolytic profile of P-glycoprotein for fragments photolabeled with cyclosporin A and for fragments that contained the C494 and C219 epitopes. The smallest fragments generated by these chemical agents include the transmembrane domains (TMs) 10, 11, and 12 of P-glycoprotein. When the fragments generated by these chemical agents are aligned, the region that binds cyclosporin A is reduced to the 953-1007 residues. These combined results suggest that the major binding site of cyclosporin A occurs between the end of TM 11 and the end of TM 12.

Effects and metabolism of fumarate in the perfused rat heart. A 13C mass isotopomer study.

The cardioprotective effects of fumarate have been linked to its metabolism to succinate through both oxidative and reductive pathways. To date, the relative contribution of these pathways is a subject of controversy. To address this question, we designed a protocol with 13C substrates and took advantage of 13C isotopomer analysis by gas chromatography-mass spectrometry. Rat hearts were perfused with 11 mM glucose, 1 mM lactate, 0.2 mM pyruvate, 0.2 mM [1-13C]octanoate, and 0.04 or 0.4 mM [U-13C4]fumarate. On reoxygenation after 40 min of severe hypoxia, hearts perfused with 0.4 mM fumarate showed a better recovery of contractile function and released less lactate dehydrogenase (an index of cellular necrosis) than those perfused with 0.04 mM fumarate. The 13C data showed that, in hypoxic hearts, fumarate conversion to succinate occurred only through reduction, although it accounted for only 16% of total succinate release. Most of the succinate was formed through the oxidation of alpha-ketoglutarate or its precursors (50 +/- 5%) and by another yet-unidentified pathway (34 +/- 4%). These data show that, in a model of hypoxia-reoxygenation, the cardioprotective effects of fumarate were associated with its predominant metabolism to succinate through the reductive pathway.

Isotopomer analysis of citric acid cycle and gluconeogenesis in rat liver. Reversibility of isocitrate dehydrogenase and involvement of ATP-citrate lyase in gluconeogenesis.

We conducted an extensive mass isotopomer analysis of citric acid cycle and gluconeogenic metabolites isolated from livers of overnight fasted rats perfused with 4 mM glucose, 0.2 mM octanoate, 1 mM [U-13C3]lactate, and 0.2 mM [U-13C3]pyruvate, in the anterograde or retrograde mode. In both perfusion modes, two distinct isotopomer patterns were observed: (i) those of phosphoenolpyruvate, glucose, malate, and aspartate and (ii) those of citrate, alpha-ketoglutarate, glutamate, and glutamine. Key citric acid cycle parameters and, hence, rates of gluconeogenesis, calculated (Lee, W.-N.P. (1989) J. Biol. Chem. 264, 13002-13004 and Lee, W.-N.P. (1993) J. Biol. Chem. 268, 25522-25526) from our mass isotopomer data did not only vary, but lead to conclusions inconsistent with Lee's citric acid cycle model. Compared to lactate and pyruvate uptake, which sets an upper limit to glucose production, rates of gluconeogenesis calculated (i) with the phosphoenolpyruvate and citrate data were similar, but those calculated (ii) with the glutamate data amounted to only 60%, which is unlikely. All these conclusions are independent of the perfusion modes. We provide evidence that the following processes contribute to the observed labeling discrepancy: (i) the reversibility of the isocitrate dehydrogenase reaction and (ii) an active citrate cleavage pathway for the transfer of the oxaloacetate carbon skeleton from mitochondria to the cytosol. Also, a good fit of our labeling data was obtained with a model of citric acid cycle and gluconeogenesis which we developed to incorporate the above reactions (Fernandez, C.A., and Des Rosiers, C. (1995) J. Biol. Chem. 270, 10037-10042). The following conclusions can be drawn from the calculated reaction rates: (i) about half of the lactate conversion to glucose occurs via the citrate cleavage pathway, (ii) the flux through the reversal of the isocitrate dehydrogenase reaction is almost as fast as that through the citrate synthase reaction, and (iii) the flux through citrate synthase and alpha-ketoglutarate dehydrogenase is 1.6- and 3.2-fold that through pyruvate carboxylase, respectively.

Assay of blood and tissue oxaloacetate and alpha-ketoglutarate by isotope dilution gas chromatography-mass spectrometry.

The assay of oxaloacetate and alpha-ketoglutarate in biological samples is complicated by their chemical instability and low concentrations. We present a quantitative assay for physiological concentrations of these metabolites by isotope dilution gas chromatography-mass spectrometry. Samples are spiked with the corresponding internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate prior to their treatment with hydroxylamine. After ethyl acetate extraction and evaporation of the organic phases, the oximes are converted to t-butyldimethylsilyl ethers and analyzed by selected ion monitoring gas chromatography-mass spectrometry of the [M-57]+ ion in electron impact. Although the internal standards of [U-13C4]oxaloacetate and [U-13C5] alpha-ketoglutarate are not commercially available, they can easily be synthesized in 30 min by reacting [1,2,3,6-13C4]citrate with citrate lyase, and L-[U-13C5]glutamate with pyruvate and glutamate-pyruvate transaminase, respectively. Because of their chemical instability, the internal standards are prepared on the day of the analysis. A stock solution of [1,2,3,6-13C4]citrate is prepared from L-[U-13C4]aspartate using citrate synthase and glutamate-oxaloacetate transaminase and then purified and kept frozen until required. The detection limit of the method is 0.05 nmol in a given sample. The method was applied to measurements of oxaloacetate and alpha-ketoglutarate in human blood and rat liver.

Intercellular adhesion molecule-1 contributes to pulmonary oxygen toxicity in mice: role of leukocytes revised.

In immature or injured lungs, impaired alveolar gas exchange forces the use of elevated levels of inhaled oxygen to maintain life. But, at high concentrations oxygen induces lung injury, edema, and bronchopulmonary dysplasia, probably by stimulating the generation of reactive oxygen radicals and subsequent neutrophil infiltration. In addition to regulating neutrophil diapedesis, intercellular adhesion molecule-1 (ICAM-1) expression is marked on inflamed alveolar epithelium, suggesting a role for ICAM-1 in oxygen-induced, neutrophil-mediated parenchymal damage. To test this, we evaluated the rat anti-mouse ICAM-1 monoclonal antibody YN1/1.7 in 2 protocols of oxygen-induced toxicity in adult, male Balb-c mice: greater than or equal to 95% O2 for 84 hr and greater than or equal to 95% O2 for 60 hr followed by 48 hr at 21% (ambient) O2. YN1/1.7 treatment partially attenuated the neutrophil infiltration, lung damage (lavage lactate dehydrogenase [LDH] activity) and dysfunction (reductions in respiratory system compliance [Crs] and diffusion capacity of the lungs for carbon monoxide [DLCO] in the 84 hr exposure protocol. In the milder 60 hr exposure protocol, YN1/1.7 completely blocked the oxygen-induced lung dysfunction (reductions in Crs and DLco). These results confirm the contribution of leukocytes in the pathogenesis of pulmonary oxygen toxicity and indicate that antagonism of ICAM-1 may provide a therapeutic approach to reducing hyperoxic lung injury and dysfunction.

BI-L-239, a 5-lipoxygenase inhibitor, blocks inhaled antigen-induced airway hyperresponsiveness in conscious guinea pigs.

Male Hartley guinea pigs were actively sensitized to ovalbumin (OA). Respiratory system resistance (Rrs) was measured by forced oscillations superimposed on tidal breathing. Airway responsiveness (inhaled methacholine PC100) was determined three days prior and three days after (day 10) three alternate day inhalations of OA. Airway cell composition was assessed on day 10 by lung lavage. Three groups (n = 5-6) were studied: A) vehicle challenged, B) OA challenged/placebo treated, C) OA challenged/BI-L-239 (2,6-dimethyl-4-[2-(4-fluorophenyl)ethenyl]phenol) treated (10 x 0.75 mg/actuation, 10 minutes prior to each OA challenge). Animals were treated with pyrilamine and indomethacin (10 mg/kg i.p.) 30 minutes prior to each OA challenge. OA induced acute increases in Rrs of 143 +/- 29%, 238 +/- 73% and 102 +/- 43% in placebo and 86 +/- 34%, 45 +/- 35% (p, 0.05 vs. placebo) and 102 +/- 31% in BI-L-239 treated. OA induced a significant (p less than 0.05) increase in airway leukocytes in placebo (487 +/- 36 to 1615 +/- 421 x 10(3)/ml) but not BI-L-239 treated (to 881 +/- 155 x 10(3)/ml) and decrease in methacholine PC100 in placebo (1.487 +/- 0.49 to 0.39 +/- 0.18 mg/ml) but not BI-L-239 treated (0.99 +/- 34 to 1.04 +/- 0.39 mg/ml). We conclude that BI-L-239 attenuates the airway constriction, inflammation and hyperresponsiveness induced by repeated antigen inhalations in conscious guinea pigs.