Isolation of Size Exclusion Chromatography Elution-Fractions of Coal and Petroleum-Derived Samples and Analysis by Laser Desorption Mass Spectrometry

High-mass species in a coal- and a petroleum-derived sample were investigated by LD-MS of eluent fractions collected from SEC. Results were compared with polymer-based calibrations of SEC elution times. It was observed that polymer- and PAH-based calibrations provide good mass estimates for material eluting in the resolved (later eluting) part of SEC chromatograms. At elution times shorter that 15 min (i.e., in the “excluded” region) there was significant deviation from the PS calibration for both the coal tar pitch and the petroleum-derived samples. In all cases, however, material eluting early during SEC was observed by LD-MS to have higher average masses compared to later-eluting material. The data appears conclusive. An upper limit to the detection of high-mass material by LD-MS appears to have been reached. When comparing mass estimates by LD-MS of SEC elution-fractions of material from the coal tar pitch and petroleum asphaltene, similar molecular mass ranges were found for fractions collected at similar elution times. This provides significant confirmation for the suitability of SEC as a technique for estimating molecular masses of complex hydrocarbon mixtures. The analytical approach presented in this paper provides a valid and useful basis for exploring relationships between molecular size, molecular mass, and structure for complex coal and petroleum-derived materials

Immune activation despite preserved CD4 T cells in perinatally HIV-infected children and adolescents

<div><p>Background</p><p>HIV disease progresses more rapidly in children than adults with mortality rates exceeding 50% by 2 years of age without antiretroviral therapy (ART) in sub-Saharan Africa. Recent World Health Organization (WHO) guidelines recommend universal treatment for all living persons with HIV, yet there is limited supporting evidence in pediatric populations. The objective of this study was to determine whether CD4 cell counts reflect immunological markers associated with disease progression in ART naïve perinatally-infected HIV+ children and adolescents and their response to ART.</p><p>Methods</p><p>PBMC and plasma samples were collected from 71 HIV negative and 132 HIV+ children (65 ART naïve and 67 on ART) between ages 1–19 years from Mombasa, Kenya. Untreated HIV+ subjects were sub-categorized by high or low CD4 T cell counts. Immune activation markers CD38, HLA-DR and Ki67 were analyzed by flow cytometry. Plasma soluble CD14 (sCD14) was quantified by ELISA.</p><p>Results</p><p>HIV-infected children and adolescents with preserved CD4 cell counts had depleted CD4 percentages and CD4:CD8 ratios, and high immune activation levels. ART initiation rapidly and persistently reversed T cell activation, but failed to normalize CD4:CD8 ratios and plasma sCD14 levels.</p><p>Conclusions</p><p>Diminished CD4 percentages and CD4:CD8 ratios along with profound immune activation occur independent of CD4 cell count thresholds in ART naïve HIV+ children and adolescents. Immediate ART initiation, as recommended in the most recent WHO guidelines may protect them from pathologic sequelae associated with persistent inflammation.</p></div

HIV disease progression in ART-CD4_hi children and adolescents.

<p>Comparison of the <b>(A)</b> CD4 percent and <b>(B)</b> HIV viral load in HIV-, ART-CD4_hi, ART- and ART+ children and adolescents. (C) Comparison of the Í38+DR+ CD8 T cells in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ children and adolescents. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05. (D) The percent of CD38+DR+ CD8 T cells vs. CD4 cell count in total lymphocytes in children and adolescents. P and <i>R</i>^<i>2</i> values are shown for a linear regression model. Shaded bar represents interquartile range of CD38+HLA-DR+ CD8 T cell frequencies in HIV-uninfected children.</p

Subject characteristics.

<p>Subject characteristics.</p

ART-CD4_hi children and adolescents exhibit markers of HIV progression associated with mortality.

<p>Comparison of the CD4:CD8 ratio in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ <b>(A)</b> children and <b>(B)</b> adolescents. <b>(C)</b> CD4:CD8 ratios are shown in subjects before antiretroviral treatment (T0), 5–7 months post-ART (T1), and 10–16 months post-ART (T2) and in comparison to HIV- children on the right. Plasma sCD14 levels in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ <b>(D)</b> children and <b>(E)</b> adolescents and <b>(F)</b> pre-and post-ART. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test and the Wilcoxon matched-pairs signed rank test (C and F). **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05.</p

Significant CD4 T cell activation in ART-CD4_hi children and adolescents.

<p>(A) Comparison of frequencies of the CD38+HLA-DR+ CD4 T cells in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ children and adolescents. Percentages of (B) CD38+ and (C) Ki67+ cells within in memory CD4 T cells in HIV-, ART-CD4_hi, ART-CD4_lo and ART+ children and adolescents. To identify memory populations, CD4 T cell were first gated on CD45RO+ CD4 T cells. Bars represent median values with IQRs. P values were calculated using the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test. **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05.</p

Antiretroviral therapy lowers immune activation rapidly and persistently.

<p>Comparisons of <b>(A)</b> the CD4 percentages and <b>(B-E)</b> frequencies of the following IA markers in paired longitudinal samples pre-ART (T0), 5–7 months post-ART (T1), and 10–16 months post-ART (T2): CD38+HLA-DR+ <b>(B)</b> CD8 and <b>(C)</b> CD4 T cells and <b>(D)</b> CD38+ <b>(E)</b> and Ki67+ memory CD4 T cells. Right graphs show comparison between IA markers in HIV- and the prospective cohort pre- and post-ART. Bars represent median values with IQRs. P values were calculated using the paired Wilcoxon matched-pairs signed rank test (left graphs) and the Kruskal-Wallis test corrected for multiple comparisons by controlling the false discovery rate with the Benjamini, Krieger, and Yekutieli test (right graphs). **** p<0.0001; *** p<0.001; ** p<0.01; * p<0.05.</p

HIV-1 Variants and Drug Resistance in Pregnant Women from Bata (Equatorial Guinea): 2012-2013

<div><p>Objectives</p><p>This is the first study describing drug resistance mutations (DRM) and HIV-1 variants among infected pregnant women in Equatorial Guinea (GQ), a country with high (6.2%) and increasing HIV prevalence.</p><p>Methods</p><p>Dried blood spots (DBS) were collected from November 2012 to December 2013 from 69 HIV-1 infected women participating in a prevention of mother-to-child transmission program in the Hospital Regional of Bata and Primary Health Care Centre <i>María Rafols</i>, Bata, GQ. The transmitted (TDR) or acquired (ADR) antiretroviral drug resistance mutations at partial <i>pol</i> sequence among <i>naive</i> or antiretroviral therapy (ART)-exposed women were defined following WHO or IAS USA 2015 lists, respectively. HIV-1 variants were identified by phylogenetic analyses.</p><p>Results</p><p>A total of 38 of 69 HIV-1 specimens were successfully amplified and sequenced. Thirty (79%) belonged to ART-experienced women: 15 exposed to nucleoside reverse transcriptase inhibitors (NRTI) monotherapy, and 15 to combined ART (cART) as first regimen including two NRTI and one non-NRTI (NNRTI) or one protease inhibitor (PI). The TDR rate was only found for PI (3.4%). The ADR rate was 37.5% for NNRTI, 8.7% for NRTI and absent for PI or NRTI+NNRTI. HIV-1 group M non-B variants caused most (97.4%) infections, mainly (78.9%) recombinants: CRF02_AG (55.2%), CRF22_A101 (10.5%), subtype C (10.5%), unique recombinants (5.3%), and A3, D, F2, G, CRF06_cpx and CRF11_cpx (2.6% each).</p><p>Conclusions</p><p>The high rate of ADR to retrotranscriptase inhibitors (mainly to NNRTIs) observed among pretreated pregnant women reinforces the importance of systematic DRM monitoring in GQ to reduce HIV-1 resistance transmission and to optimize first and second-line ART regimens when DRM are present.</p></div

HIV-1 group M variants reported in patients from GQ during 1997–2013.

<p>HIV-1 group M variants reported in patients from GQ during 1997–2013.</p

Epidemiological and virological features of the HIV-1 infected study population.

<p>Epidemiological and virological features of the HIV-1 infected study population.</p