Factors associated with elevated ALT in an international HIV/HBV co-infected cohort on long-term HAART

Background

Previous studies have demonstrated that hepatitis B virus (HBV) infection increases the risk for ALT elevations in HIV-HBV co-infected patients during the first year of HAART; however, there is limited data on the prevalence of ALT elevations with prolonged HAART in this patient group.

Methods/Principal findings

To identify factors associated with ALT elevations in an HIV-HBV co-infected cohort receiving prolonged HAART, data from 143 co-infected patients on HAART enrolled in an international HIV-HBV co-infected cohort where ALT measurements were obtained every 6 months was analysed. A person-visit analysis was used to determine frequency of ALT elevation (≥2.5×ULN) at each visit. Factors associated with ALT elevation were determined using multivariate logistic regression with generalized estimating equations to account for correlated data. The median time on HAART at the end of follow-up was 5.6 years (range 0.4–13.3) years. During follow-up, median ALT was 36 U/L with 10.6% of person-visits classified as having ALT elevation. Most ALT elevations were grade 2 (86.5%), with only 13.5% of all ALT elevations grade 3 or higher. Univariate associations with ALT elevation (p<0.05) included history of AIDS, HBV DNA ≥2,000 IU/ml, HBeAg positive, study visit CD4 <200 cells/ml and nadir CD4 <200 cells/ml. In the multivariate analysis, only study visit CD4 <200 cells/ml (OR 2.07, 95%CI 1.04–4.11, p = 0.04) and HBeAg positive status (OR 2.22, 95%CI 1.03–4.79, p = 0.04) were independently associated with ALT elevation.

Conclusions

In this HIV-HBV co-infected cohort, elevated ALT after >1 year of HAART was uncommon, and severe ALT elevations were rare. HIV-HBV co-infected patients on long-term HAART who are either HBeAg positive or have a CD4 count of <200 cells/ml are at increased risk for ALT elevations.     

Quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

A method is described for the quantitative determination of peptides using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. Known limitations imposed by crystal heterogeneity, peptide ionization differences, data handling, and protein quantification with MALDI-TOF mass spectrometry are addressed in this method with a "seed crystal" protocol for analyte-matrix formation, the use of internal protein standards, and a software package called maldi_quant. The seed crystal protocol, a new variation of the fast-evaporation method, minimizes crystal heterogeneity and allows for consistent collection of protein spectra. The software maldi_quant permits rapid and automated analysis of peak intensity data, normalization of peak intensities to internal standards, and peak intensity deconvolution and estimation for vicinal peaks. Using insulin proteins in a background of other unrelated peptides, this method shows an overall coefficient of variance of 4.4%, and a quantitative working range of 0.58-37.5 ng bovine insulin per spot. Coupling of this methodology to powerful analytical procedures such as immunoprecipitation is likely to lead to the rapid and reliable quantification of biologically relevant proteins and their closely related variants.     

Heterocyclic analogues of squamocin as inhibitors of mitochondrial complex I. On the role of the terminal lactone of annonaceous acetogenins

Heterocyclic analogues of squamocin have been semisynthesized by condensation reactions between squamocin-derived alpha-keto esters and heterodinucleophiles. The strong complex I inhibitory potency of squamocin-benzimidazole, a hybrid derivative, illustrates for the first time the functional analogy existing between the terminal butenolide of annonaceous acetogenins and heteroaromatic substructures of classic inhibitors of the enzyme. This finding supports the categorization of this atypical group of inhibitors as antagonists of the ubiquinone substrates. In addition, competition experiments of squamocin-benzimidazole versus squamocin and rolliniastatin-2 suggest that the binding of this hybrid inhibitor is responsible for a negative allosteric effect at the level of the first ubiquinone-binding site (A site) of mitochondrial complex I. This result supports the existence of a large cooperatively regulated inhibitor/ubiquinone-binding pocket located within the catalytic core of the enzyme, consisting of the association of the previously defined affinity sites A and B.     

Virologically suppressed HIV patients show activation of NK cells and persistent innate immune activation

FcRγ is an ITAM-containing adaptor required for CD16 signaling and function in NK cells. We have previously shown that NK cells from HIV patients receiving combination antiretroviral therapy (cART) have decreased FcRγ expression, but the factors causing this are unknown. We conducted a cross-sectional study of cART-naive viremic patients (ART(-)), virologically suppressed patients receiving cART (ART(+)), and HIV-uninfected controls. CD8(+) T cells were activated, as assessed by CD38(+)HLA-DR(+) expression, in ART(-) patients (p < 0.0001), which was significantly reduced in ART(+) patients (p = 0.0005). In contrast, CD38(+)HLA-DR(+) NK cells were elevated in ART(-) patients (p = 0.0001) but did not decrease in ART(+) patients (p = 0.88). NK cells from both ART(-) and ART(+) patients showed high levels of spontaneous degranulation in ex vivo whole blood assays as well as decreased CD16 expression (p = 0.0001 and p = 0.0025, respectively), FcRγ mRNA (p < 0.0001 for both groups), FcRγ protein expression (p = 0.0016 and p < 0.0001, respectively), and CD16-dependent Syk phosphorylation (p = 0.0001 and p = 0.003, respectively). HIV-infected subjects showed alterations in NK activation, degranulation, CD16 expression and signaling, and elevated plasma markers of inflammation and macrophage activation, that is, neopterin and sCD14, which remained elevated in ART(+) patients. Alterations in NK cell measures did not correlate with viral load or CD4 counts. These data show that in HIV patients who achieve viral suppression following cART, NK cell activation persists. This suggests that NK cells respond to factors different from those driving T cell activation, but which are associated with inflammation in HIV patients.     

Role of the C8 gem-dimethyl group of bryostatin 1 on its unique pattern of biological activity

The role of the C(8) gem-dimethyl group in the A-ring of bryostatin 1 has been examined through chemical synthesis and biological evaluation of a new analogue. Assays for biological function using U937, K562, and MV4-11 cells as well as the profiles for downregulation of PKC isozymes revealed that the presence of this group is not a critical determinant for the unique pattern of biological activity of bryostatin.