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Ande A.,University of Missouri - Kansas City | Sinha N.,University of Tennessee Health Science Center | Rao P.S.S.,University of Tennessee Health Science Center | McArthur C.P.,University of Missouri - Kansas City | And 5 more authors.
AIDS Research and Therapy

Background: Alcohol consumption is prevalent amongst HIV positive population. Importantly, chronic alcohol use is reported to exacerbate HIV pathogenesis. Although alcohol is known to increase oxidative stress, especially in the liver, there is no clinical evidence that alcohol increases oxidative stress in HIV positive patients. The mechanism by which alcohol increases oxidative stress in HIV positive patients is also unknown. Methods: To examine the effects of alcohol use on oxidative stress we recruited HIV+ patients who reported mild-to-moderate alcohol use. Strict inclusion and exclusion criteria were applied to reduce the effect of other therapeutic drugs metabolized via the hepatic system as well as the effect of co-morbidities such as active tuberculosis on the interaction between alcohol and HIV infection, respectively. Blood samples were collected from HIV-negative alcohol-users and HIV positive alcohol-users followed by collection of plasma and isolation and fractionation of monocytes from peripheral blood. We then determined oxidative DNA damage, glutathione level, alcohol level, transcriptional level of cytochrome P450 2E1 (CYP2E1) and several antioxidant enzymes, and plasma level of cytokines. Results: Compared to HIV-negative alcohol users, HIV-positive alcohol users demonstrated an increase in oxidative DNA damage in both plasma and CD14+ monocytes, as well as, a relative increase in oxidized/reduced glutathione (GSSG/GSH) in plasma samples. These results suggest an increase in oxidative stress in HIV-positive alcohol users compared with HIV-negative alcohol users. We also examined whether alcohol metabolism, perhaps by CYP2E1, and antioxidant enzymes are involved in alcohol-mediated increased oxidative stress in HIV-positive patients. The results showed a lower plasma alcohol level, which was associated with an increased level of CYP2E1 mRNA in monocytes, in HIV-positive alcohol users compared with HIV-negative alcohol users. Furthermore, the transcription of major antioxidants enzymes (catalase, SOD1, SOD2, GSTK1), and their transcription factor, Nrf2, were reduced in monocytes obtained from HIV positive alcohol users compared to the HIV-negative alcohol user group. However, no significant change in levels of five major cytokines/chemokines were observed between the two groups. Conclusions: The data suggests that alcohol increases oxidative stress in HIV+ patients, perhaps through CYP2E1- and antioxidant enzymes-mediated pathways. The enhanced oxidative stress is accompanied by a failure of cellular antioxidant mechanisms to maintain redox homeostasis. Overall, the enhanced oxidative stress in monocytes may exacerbate HIV pathogenesis in HIV positive alcohol users. © 2015 Ande et al. Source

Bristow C.L.,Cornell College | Babayeva M.A.,Cornell College | Modarresi R.,Cornell College | Mcarthur C.P.,University of Missouri - Kansas City | And 6 more authors.
Journal of Visualized Experiments

There is an urgent need for affordable CD4 enumeration to monitor HIV disease. CD4 enumeration is out of reach in resource-limited regions due to the time and temperature restrictions, technical sophistication, and cost of reagents, in particular monoclonal antibodies to measure CD4 on blood cells, the only currently acceptable method. A commonly used cost-saving and time-saving laboratory strategy is to calculate, rather than measure certain blood values. For example, LDL levels are calculated using the measured levels of total cholesterol, HDL, and triglycerides 1. Thus, identification of cell-free correlates that directly regulate the number of CD4 + T cells could provide an accurate method for calculating CD4 counts due to the physiological relevance of the correlates. The number of stem cells that enter blood and are destined to become circulating CD4 + T cells is determined by the chemokine CXCL12 and its receptor CXCR4 due to their influence on locomotion 2. The process of stem cell locomotion into blood is additionally regulated by cell surface human leukocyte elastase (HLE CS) and the HLE CS-reactive active α 1proteinase inhibitor (α 1PI, α 1antitrypsin, SerpinA1) 3. In HIV-1 disease, α 1PI is inactivated due to disease processes 4. In the early asymptomatic categories of HIV-1 disease, active α 1PI was found to be below normal in 100% of untreated HIV-1 patients (median=12 μM, and to achieve normal levels during the symptomatic categories 4, 5. This pattern has been attributed to immune inactivation, not to insufficient synthesis, proteolytic inactivation, or oxygenation. We observed that in HIV-1 subjects with >220 CD4 cells/μl, CD4 counts were correlated with serum levels of active α 1PI (r 2=0.93, p<0.0001, n=26) and inactive α 1PI (r 2=0.91, p<0.0001, n=26) 5. Administration of α 1PI to HIV-1 infected and uninfected subjects resulted in dramatic increases in CD4 counts suggesting α 1PI participates in regulating the number of CD4 + T cells in blood 3. With stimulation, whole saliva contains sufficient serous exudate (plasma containing proteinaceous material that passes through blood vessel walls into saliva) to allow measurement of active α 1PI and the correlation of this measurement is evidence that it is an accurate method for calculating CD4 counts. Briefly, sialogogues such as chewing gum or citric acid stimulate the exudation of serum into whole mouth saliva. After stimulating serum exudation, the activity of serum α 1PI in saliva is measured by its capacity to inhibit elastase activity. Porcine pancreatic elastase (PPE) is a readily available inexpensive source of elastase. PPE binds to α 1PI forming a one-to-one complex that prevents PPE from cleaving its specific substrates, one of which is the colorimetric peptide, succinyl-L-Ala-L-Ala-L-Ala-p-nitroanilide (SA 3NA). Incubating saliva with a saturating concentration of PPE for 10 min at room temperature allows the binding of PPE to all the active α 1PI in saliva. The resulting inhibition of PPE by active α 1PI can be measured by adding the PPE substrate SA 3NA. (Figure 1). Although CD4 counts are measured in terms of blood volume (CD4 cells/μl), the concentration of α 1PI in saliva is related to the concentration of serum in saliva, not to volume of saliva since volume can vary considerably during the day and person to person 6. However, virtually all the protein in saliva is due to serum content, and the protein content of saliva is measurable 7. Thus, active α 1PI in saliva is calculated as a ratio to saliva protein content and is termed the α 1PI Index. Results presented herein demonstrate that the α 1PI Index provides an accurate and precise physiologic method for calculating CD4 counts. © 2012 Journal of Visualized Experiments. Source

Jubulis J.,Johns Hopkins Medical Institutions | Jubulis J.,Tufts University | Dionne K.,Johns Hopkins Medical Institutions | Osterhout G.,Johns Hopkins Medical Institutions | And 6 more authors.
International Journal of Tuberculosis and Lung Disease

OBJECTIVE : To determine the resistance of Mycobacterium tuberculosis to first- And second-line agents in adult pulmonary tuberculosis (TB) patients in Cameroon using a novel phenotypic assay. SETTING : Samples were collected from TB patients at Bamenda Hospital in Bamenda, Cameroon. DESIGN : Samples were collected consecutively from adult pulmonary TB patients over a 2-month period. TREK SensititreTM MYCOTB panels were used to perform phenotypic drug susceptibility testing (DST). Susceptibility/resistance was determined by comparing minimum inhibitory concentrations to standard critical concentrations established for first- And second-line antituberculosis drugs. RESULTS : Of 103 sputum samples processed, growth on Löwenstein-Jensen media was confirmed in 78 samples, 65 of which were suitable for DST. Thirtynine strains (60%) were susceptible to all first- And second-line drugs. Five strains (8%) were categorized as multidrug-resistant TB. Two strains (3%) were classified as pre-extensively drug-resistant TB. Of those isolates susceptible to first-line drugs, 20% were resistant to at least one second-line drug. CONCLUSION: Antimicrobial resistance may be higher than assumed in TB strains in Cameroon, especially with regard to second-line drugs. There remains a need for rapid, comprehensive DST. © 2015 The Union. Source

Ande A.,University of Missouri - Kansas City | McArthur C.,University of Missouri - Kansas City | Ayuk L.,Regional Hospital | Awasom C.,Regional Hospital | And 9 more authors.

Mild-to-moderate tobacco smoking is highly prevalent in HIV-infected individuals, and is known to exacerbate HIV pathogenesis. The objective of this study was to determine the specific effects of mild-to-moderate smoking on viral load, cytokine production, and oxidative stress and cytochrome P450 (CYP) pathways in HIV-infected individuals who have not yet received antiretroviral therapy (ART). Thirty-two human subjects were recruited and assigned to four different cohorts as follows: a) HIV negative non-smokers, b) HIV positive non-smokers, c) HIV negative mild-to-moderate smokers, and d) HIV positive mild-tomoderate smokers. Patients were recruited in Cameroon, Africa using strict selection criteria to exclude patients not yet eligible for ART and not receiving conventional or traditional medications. Those with active tuberculosis, hepatitis B or with a history of substance abuse were also excluded. Our results showed an increase in the viral load in the plasma of HIV positive patients who were mild-to-moderate smokers compared to individuals who did not smoke. Furthermore, although we did not observe significant changes in the levels of most pro-inflammatory cytokines, the cytokine IL-8 and MCP-1 showed a significant decrease in the plasma of HIV-infected patients and smokers compared with HIV negative non-smokers. Importantly, HIV-infected individuals and smokers showed a significant increase in oxidative stress compared with HIV negative non-smoker subjects in both plasma and monocytes. To examine the possible pathways involved in increased oxidative stress and viral load, we determined the mRNA levels of several antioxidant and cytochrome P450 enzymes in monocytes. The results showed that the levels of most antioxidants are unaltered, suggesting their inability to counter oxidative stress. While CYP2A6 was induced in smokers, CYP3A4 was induced in HIV and HIV positive smokers compared with HIV negative non-smokers. Overall, the findings suggest a possible association of oxidative stress and perhaps CYP pathway with smoking-mediated increased viral load in HIV positive individuals. © 2015 Ande et al. Source

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