Compartmentalized T cell profile in the lungs of patients with HIV-1-associated pulmonary Kaposi sarcoma.

ABSTRACT: Pulmonary Kaposi sarcoma (pKS) caused by Human herpesvirus 8 (HHV-8) is a devastating form of KS in patients with advanced acquired immunodeficiency syndrome (AIDS) and is associated with increased morbidity and mortality. Blood T cells play a central role in the response of HIV-1 and HHV-8. However, little information is available on T cells in the alveolar space of HIV-1-associated pKS patients.Therefore, we examined CD8+ and CD4+ T cells in the alveolar space in comparison with the blood of patients with pKS. We recruited 26 HIV-1 positive patients with KS, including 15 patients with pKS. Bronchoalveolar lavage (BAL) cells and blood mononuclear cells were analyzed for T cell memory phenotypes, surface markers associated with exhaustion, and intracellular cytokine staining (ICS) using flow cytometry. HIV-1 and HHV-8 viral loads were measured in plasma by quantitative PCR.BAL T cells showed reduced inflammatory capacities and significantly diminished polyfunctionality compared to blood T cells from patients with pKS. This was not accompanied by increased expression of exhaustion markers, such as TIM-3 and PD-1.More importantly, we found a negative correlation between the production of MIP1-ß and TNF-α in T cells in BAL and blood, indicating compartmentalised immune responses to pKS and accentuated chronic HIV-1/HHV-8 pathogenesis via T cells in the lungs of people with pKS.

Diagnostic Accuracy of Pan-Degenerate Amplification and Adaptation Assay for HIV-1 Drug Resistance Mutation Analysis in Low- and Middle-Income Countries.

HIV drug resistance (HIVDR) is a barrier to sustained virologic suppression in low- and middle-income countries (LMICs). Point mutation assays targeting priority drug resistance mutations (DRMs) are being evaluated to improve access to HIVDR testing. In a cross-sectional study (June 2018 to September 2019), we evaluated the diagnostic accuracy of a simple and rapid HIVDR assay (the pan-degenerate amplification and adaptation [PANDAA] assay targeting the mutations K65R, K103NS, M184VI, Y181C, and G190A) compared to Sanger sequencing and next-generation sequencing (NGS). Plasma samples from adolescents and young adults (aged 10 to 24 years) failing antiretroviral therapy (viral load, >1,000 copies/ml on 2 consecutive occasions 1 month apart) were analyzed. Sensitivity and specificity of the PANDAA assay were determined by a proprietary application designed by Aldatu Biosciences. Agreement between genotyping methods was evaluated using Cohen's kappa coefficient. One hundred fifty samples previously characterized by Sanger sequencing were evaluated using PANDAA. For all DRMs detected, PANDAA showed a sensitivity and specificity of 98% and 94%, respectively. For nucleotide reverse transcriptase inhibitor DRMs, sensitivity and specificity were 98% (95% confidence interval [CI], 92% to 100%) and 100% (94% to 100%), respectively. For non-nucleotide reverse transcriptase inhibitor DRMs, sensitivity and specificity were 100% (97% to 100%) and 76% (61% to 87%), respectively. PANDAA showed strong agreement with Sanger sequencing for K65R, K103NS, M184VI, and G190A (kappa > 0.85) and substantial agreement for Y181C (kappa = 0.720). Of the 21 false-positive samples genotyped by PANDAA, only 6 (29%) were identified as low-abundance variants by NGS. With the high sensitivity and specificity to detect major DRMs, PANDAA could represent a simple and rapid alternative HIVDR assay in LMICs.

Drug resistance and optimizing dolutegravir regimens for adolescents and young adults failing antiretroviral therapy.

OBJECTIVES: The integrase strand inhibitor dolutegravir (DTG) combined with tenofovir and lamivudine (TLD) is a single tablet regimen recommended for 1st, 2nd and 3rd-line public health antiretroviral therapy (ART). We determined drug resistance mutations (DRMs) and evaluated the predictive efficacy of a TLD containing regimen for viremic adolescents and young adults in Harare, Zimbabwe. METHODS: We sequenced plasma viral RNA from HIV-1-infected adolescents and young adults on 1st and 2nd-line ART with confirmed virologic failure (viral load >1000 copies/ml) and calculated total genotypic susceptibility scores to current 2nd, 3rd line and DTG regimens. RESULTS: A total of 160 participants were genotyped; 112 (70%) on 1st line and 48 (30%) on 2nd line, median (interquartile range) age 18 (15-19) and duration of ART (interquartile range) was 6 (4-8) years. Major DRMs were present in 94 and 67% of 1st and 2nd-line failures, respectively (P < 0.001). Dual class resistance to nucleotide reverse transcriptase inhibitors and nonnucleotide reverse transcriptase inhibitors was detected in 96 (60%) of 1st-line failures; protease inhibitor DRMs were detected in a minority (10%) of 2nd-line failures. A total genotypic susceptibility score of 2 or less may risk protease inhibitor or DTG monotherapy in 11 and 42% of 1st-line failures switching to 2nd-line protease inhibitor and TLD respectively. CONCLUSION: Among adolescents and young adults, current protease inhibitor-based 2nd-line therapies are poorly tolerated, more expensive and adherence is poor. In 1st-line failure, implementation of TLD for many adolescents and young adults on long-term ART may require additional active drug(s). Drug resistance surveillance and susceptibility scores may inform strategies for the implementation of TLD.

Determining the utility of the 60 min cortisol measurement in the short synacthen test.

OBJECTIVE: Despite the widespread use of the short synacthen test (SST), there remains no clear consensus on sampling times for the measurement of serum cortisol that best determines adrenal reserve. We set out to establish whether there is any value in measuring serum cortisol at 60 min following administration of synacthen. DESIGN: Retrospective data analysis of 500 SST results measuring 0, 30 and 60 min cortisol levels after administration of 250 µg of synacthen at 2 large urban National Health Teaching Hospitals in the UK. PATIENTS AND MEASUREMENTS: Individuals thought to have primary or secondary adrenal insufficiency given 250 µg of synacthen. MEASUREMENTS: Serum cortisol levels measured at 0, 30 and 60 min, looking to see how many people who had adrenal insufficiency at the 30 min sample but in whom the 60 min sample showed adequate adrenal reserve. RESULTS: The results from 384 people were analysed. A total of 276 had normal responses at 30 min and also at 60 min. A sum of 33 individuals had 'insufficient' (i.e., <550 nmol/l) 30 min cortisol levels, rising to ≥ 550 nmol/l at the 60 min test. All 75 individuals who were insufficient at 60 min were also insufficient at 30 min. No individuals passed (≥550 nmol/l) at 30 min and then failed (<550 nmol/l) at 60 min. CONCLUSIONS: These results suggest that a significant proportion of people undergoing a SST may be inappropriately diagnosed as having adrenal insufficiency if the 60 min sample is not measured. We suggest that the 60 min sample is measured in all individuals having a SST to prevent unnecessary over-diagnosis of adrenal insufficiency.

Monoclonal pathogenic antibodies to the thyroid-stimulating hormone receptor in Graves' disease with potent thyroid-stimulating activity but differential blocking activity activate multiple signaling pathways.

The thyroid target Ag for disease-inducing autoantibodies in Graves' disease is the receptor for thyroid-stimulating hormone (TSH), but little is known about the molecular basis of this pathogenic Ab response. We describe the characteristics of two high- affinity mAbs developed from an experimental murine model of hyperthyroid Graves' disease that exhibit potent thyroid-stimulating activity. Nanogram concentrations of the IgG mAbs KSAb1 and KSAb2 and their Fab induce full stimulation of the TSH receptor that is matched by the ligand TSH and, thus, act as full agonists for the receptor. However, KSAb1 and KSAb2 display differential activities in their ability to block TSH-mediated stimulation of the receptor, indicating subtle differences in their biological properties. In displacement studies, IgG and Fabs of KSAb1 and KSAb2 compete with Graves' disease autoantibodies as well as thyroid-blocking Abs present in some hypothyroid patients, indicating a close relationship between these autoimmune determinants on the receptor. In passive transfer studies, single injections of microgram quantities of KSAb1 or KSAb2 IgG led to rapid elevation of serum thyroxine and a hyperthyroid state that was maintained for a number of days. The thyroid glands showed evidence of cell necrosis, but there was no accompanying mononuclear cell infiltrate. In studying their receptor activation pathways, both KSAb1 and KSAb2 provoked phosphorylation of the intracellular ERK1/2 pathway in primary thyrocytes, indicating that multiple signaling pathways may participate in the pathogenesis of Graves' disease. In summary, our findings emphasize the similarities of the experimental mouse model in reproducing the human disorder and provide improved means for characterizing the molecular basis of this pathogenic response.

Dynamics of thyroid-stimulating and -blocking antibodies to the thyrotropin receptor in a murine model of Graves' disease.

Graves' disease is characterized by the presence of autoantibodies to the TSH receptor (TSHR). There are multiple antibodies to the TSHR, with thyroid-stimulating antibodies (TSAbs) and TSH-stimulating blocking antibodies (TSBAbs), which in patients can fluctuate over time, resulting in changes in disease activity. Recently, animal models of Graves' disease have been developed, but it is not known whether the induced TSAbs and TSBAbs change spontaneously with time to influence disease. We used fibroblasts expressing major histocompatability complex (MHC) class II and human TSHR murine model to study anti-TSHR antibody patterns in serial bleeds of 23 animals. Anti-TSHR antibody responses were first detectable after 7-8 wk of first immunization. Moreover, the pattern of the TSAbs or TSBAbs was selected early in the response. The majority of the animals showed anti-TSHR antibodies that were either TSAb or TSBAb responses and were maintained throughout the course of 17-24 wk of the experiment. Remarkably, a proportion of mice (13%) displayed presence of antibodies with both TSAbs and TSBAbs, which appeared to cycle over time and thus mimic the fluctuations described in some hyperthyroid patients. Analyses of the linear epitopes to TSHR by peptide scanning showed that there was no early restricted epitope response. Thus, despite using an inbred strain, the initial response appears to target different regions of the receptor in different animals. Our data show that anti-TSHR antibody epitopes in the model display heterogeneity in TSHR epitopes, which vary in individual animals as well as in their regulation.