Determination of HIV-1 coreceptor tropism using proviral DNA in women before and after viral suppression.

BACKGROUND: An HIV-1 tropism test is recommended prior to CCR5 antagonist administration to exclude patients harboring non-R5 virus from treatment with this class of antiretrovirals. HIV-1 tropism determination based on proviral DNA (pvDNA) may be useful in individuals with plasma viral RNA suppression. We developed a genotypic tropism assay for pvDNA and assessed its performance in a retrospective analysis of samples collected longitudinally. RESULTS: We randomly selected paired plasma/PBMC samples from the Women's Interagency HIV Study with plasma viral load ≥5,000 cp/mL at time 1 (T1), undetectable viral load maintained for ≥1 year and CD4+ >200 cells/µL at time 2 (T2). pvDNA was isolated from cryopreserved PBMCs. Sequences were analyzed in triplicate from 49/50 women, with tropism assigned using the geno2pheno (g2p) algorithm. The median time between T1 and T2 was 4.1 years. CXCR4-using virus was detected in 24% of the RNA samples and 33% of the pvDNA samples at T1, compared to 37% of the pvDNA samples at T2. Concordance between plasma RNA and pvDNA tropism was 88% at T1 and 80% at T2. The g2p scores for RNA (T1) vs DNA (T1, T2) were strongly correlated (Spearman rho: 0.85 (T1); 0.78 (T2)). In women with evidence of tropism switch at T2 (either R5 to non-R5 or non-R5 to R5), there was a correlation between change in tropism and time. Mean pvDNA viral load decreased by 0.4 log10 copies/106 cells between T1 and T2 (p < 0.0001), but this decrease was not significantly associated with tropism status. CONCLUSIONS: We demonstrated that pvDNA tropism in women with HIV-1 suppression is concordant with prior RNA tropism results, even after a median time of >4 years. pvDNA tropism testing may be useful to determine eligibility of patients with viral suppression to switch to a CCR5-antagonist based regimen as well as for research purposes.

A genotypic HIV-1 proviral DNA coreceptor tropism assay: characterization in viremic subjects.

BACKGROUND: HIV-1 coreceptor tropism testing is used to evaluate eligibility for CCR5 antagonist therapy. However, HIV-1 RNA-based tests are not suitable for virologically suppressed patients, therefore the use of proviral DNA tropism testing has been investigated. We describe a novel proviral DNA-based genotypic tropism assay and compare its performance to that of a sensitive HIV-1 RNA-based genotypic test. METHODS: Tropism was determined using HIV-1 plasma RNA and proviral DNA from 42 paired samples from patients with plasma viral loads ≥1000 HIV-1 RNA copies/mL. Proviral DNA sample types included whole blood, separated peripheral blood mononuclear cells resuspended in phosphate-buffered saline and peripheral blood mononuclear cells resuspended in spun plasma. The HIV-1 envelope V3 region was PCR-amplified, sequenced in triplicate, and analyzed for tropism with the geno2pheno algorithm using a 10% false-positive rate (FPR). RESULTS: Amplicons were obtained from proviral DNA and plasma RNA in 41/42 samples. Tropism predictions were highly concordant (93%-98%) between proviral DNA and plasma RNA, regardless of the proviral DNA isolation method. Non-R5 proviral DNA results were obtained for 100% of patients with detectable non-R5 plasma HIV-1 RNA results. Geno2pheno FPRs for proviral DNA and plasma RNA were highly correlated (Spearman rho = 0.86). CONCLUSIONS: Our findings demonstrate that proviral DNA tropism determinations from whole blood or peripheral blood mononuclear cells were highly concordant with plasma HIV-1 RNA tropism determinations. This assay may be useful for screening virologically suppressed patients for CCR5-antagonist eligibility and for research purposes.

Effectiveness of Topical Cyclosporin-A 0.1% Compared to Combined Topical Cyclosporin-A 0.1% with Topical Sodium Hyaluronate on Interleukin-6 Levels in the Tears of Patients with Dry Eye Disease.

INTRODUCTION: Pro-inflammatory cytokines are important contributors to dry eye disease (DED). The cytokine interleukin (IL)-6 has become a therapeutic target in several DED drug studies. This randomized controlled trial aimed to determine the effectiveness of topical cyclosporin-A 0.1% compared to the combination of topical cyclosporin-A 0.1% and sodium hyaluronate in reducing tear IL-6 levels in DED patients. METHODS: The participants were 20 patients, each with two eyes, who had moderate-to-severe DED. Before and after treatment, the clinical degree of DED was examined in each group, using ocular surface disease index (OSDI) scores, tear break-up time (TBUT), fluorescent tests, and Schirmer I tests. In addition, tear samples were taken to examine IL-6 levels through the ELISA method. The results were analyzed using the t-test, Wilcoxon test, and Mann-Whitney test. The correlation between tear IL-6 levels and the severity of DED was analyzed using the Spearman correlation test. RESULTS: The study showed a significantly lower tear IL-6 level, OSDI score, and degree of ocular staining after either topical cyclosporin-A 0.1% or a combination of topical cyclosporin-A 0.1% and sodium hyaluronate (all values p < 0.05). CONCLUSIONS: The combination therapy was superior in reducing tear IL-6 levels. In addition, a correlation existed between tear IL-6 levels and the severity of DED based on the TBUT, although it was weak and not statistically significant.

Human Ocular Thelaziasis: A Case Report from Indonesia.

Thelaziasis is a parasitic disease caused by a nematode of genus Thelazia, which is rare in the world, including Indonesia. The definitive hosts for Thelazia are canids, felids, mustelids, and other mammals, while the vector is drosophila flies. Consequently, this study reported an uncommon occurrence of human ocular thelaziasis in Indonesia. Based on the patient's complaints and physical examination, we found a living worm that move actively in the anterior chamber; then documentation is carried out both during the examination at the polyclinic and in the operating room. The surgery was performed using topical anaesthesia, clear corneal incision, and removing worm through the main port. Morphological examination from the parasitology laboratory showed that the worm was Thelazia callipaeda species. Following this intervention, the patient was given an oral anti-helminthics drug, topical and oral antibiotics, topical steroid, and surgical treatment. There was no recurrence or appearance of any other symptoms reported in 2 months of follow-up.

Nutritional content and in vitro antioxidant potential of Garcinia atroviridis (Asam gelugor) leaves and fruits.

INTRODUCTION: The objective of this study was to determine antioxidant potential of Garcinia atroviridis leaves and fruits extracts in vitro. METHODS: Antioxidant activity was assessed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and ferric reducing antioxidant power (FRAP) assays. Total phenolic content (TPC) of the extracts was estimated as gallic acid equivalent by Folin-Ciocalteau method. Proximate analysis was determined based on the Association of Official Analytical Chemists (AOAC) procedures. RESULTS: Garcinia atroviridis leaves extracted at 100 degrees C/15 min demonstrated the highest TPC value (21.21 +/- 0.28 mg GAE/mg) and was significantly different (p < 0.05) from that of leaves extracted at 60 degrees C/6 h and 40 degrees C/12 h. On the other hand, the fruit extracted at 60 degrees C/6 h showed the highest TPC value (16.23 +/- 0.18 mg GAE/mg) (p < 0.05) compared to the fruit extracted at 40 degrees C/12 h and 100 degrees C/15 h respectively. The antioxidant activities of both samples were positively correlated with the TPC values based on DPPH-radical-scavenging activity and ferric reducing power estimation. Garcinia atroviridis leaf extract contained significantly higher proteins, carbohydrate and ash contents (2.16% +/- 0.08; 15.98% +/- 0.12 and 0.72% +/- 0.07 respectively) than its fruit extract (0.46% +/- 0.08, 8.64% +/- 0.06 and 0.15% +/- 0.06) respectively). The energy content was also found to be higher in the leaf (73.64% +/- 2.15) compared to the fruit (38.38% +/- 1.72) (p < 0.05). CONCLUSION: The findings indicate that G. atroviridis leaves and fruits have potential for use as a source of natural antioxidants and nutrients for therapeutic purposes against free radical mediated health conditions.

Alpha lipoic acid possess dual antioxidant and lipid lowering properties in atherosclerotic-induced New Zealand White rabbit.

There is accumulating data demonstrated hypercholesterolemia and oxidative stress play an important role in the development of atherosclerosis. In the present study, a protective activity of alpha-lipoic acid; a metabolic antioxidant in hypercholesterolemic-induced animals was investigated. Eighteen adult male New Zealand White (NZW) rabbit were segregated into three groups labelled as group K, AT and ALA (n=6). While group K was fed with normal chow and acted as a control, the rest fed with 100 g/head/day with 1% high cholesterol diet to induce hypercholesterolemia. 4.2 mg/body weight of alpha lipoic acid was supplemented daily to the ALA group. Drinking water was given ad-libitum. The study was designed for 10 weeks. Blood sampling was taken from the ear lobe vein at the beginning of the study, week 5 and week 10 and plasma was prepared for lipid profile estimation and microsomal lipid peroxidation index indicated with malondialdehyde (MDA) formation. Animals were sacrificed at the end of the study and the aortas were excised for intimal lesion analysis. The results showed a significant reduction of lipid peroxidation index indicated with low MDA level (p<0.05) in ALA group compared to that of the AT group. The blood total cholesterol (TCHOL) and low density lipoprotein (LDL) levels were found to be significantly low in ALA group compared to that of the AT group (p<0.05). Histomorphometric intimal lesion analysis of the aorta showing less of atheromatous plaque formation in alpha lipoic acid supplemented group (p<0.05) compared to that of AT group. These findings suggested that apart from its antioxidant activity, alpha lipoic acid may also posses a lipid lowering effect indicated with low plasma TCHOL and LDL levels and reduced the athero-lesion formation in rabbits fed a high cholesterol diet.

Biological Properties of Tinospora crispa (Akar Patawali) and Its Antiproliferative Activities on Selected Human Cancer Cell Lines.

The antioxidant and anti-proliferative activity of the aqueous crude extract of Tinospora crispa stem was investigated. The proximate composition of its stem and leaves was determined. Proximate analysis revealed that T. crispa contains - protein: leaves = 4.7%, stem = 1.2%; fat: leaves = 1.5%, stem = 0.43%; carbohydrate: leaves = 11.8%, stem = 19.4%; ash: leaves = 2.7%, stem = 1.1%; moisture: leaves = 79.3%, stem = 77.9%; fibre: leaves = 1.59%, stem = 0.65%; and energy: leaves = 1.59%, stem = 0.65%. The antioxidant activity of the extract prepared at various temperatures and incubation time was evaluated to determine the optimum extraction procedure. Based on DPPH and TBA tests, the preparation of the extract at 60oC for 6 hours was established as the best possible method as it demonstrated the highest inhibition percentage. The extract was tested against brine shrimp to evaluate its toxicity and no significant toxicity was recorded since the IC50 value was more than 1000 µg/ml. The extract produced moderate anti-proliferative activity on selected human cancer cell lines (IC50 MCF-7: 107 µg/ml, HeLa: 165 µg/ml, Caov-3: 100 µg/ml, and HepG2: 165 µg/ml). The findings from this study suggest that T. crispa has the potential to be a source of natural antioxidants and nutrients, besides having a moderate anti-proliferative effect on selected human cancer cell lines.