Impact of HIV-1 Resistance-associated Mutations on Susceptibility to Doravirine: Analysis of Real-world Clinical Isolates

Background: Clinical management of human immunodeficiency virus type-1 (HIV-1) infection may be negatively impacted by either acquired or transmitted drug resistance. Here, we aim to extend our understanding of the impact of resistance-associated mutations (RAMs) on the susceptibility of clinical isolates to the non-nucleoside reverse transcriptase inhibitor (NNRTI) doravirine. Methods: Clinical isolates from people living with HIV-1 undergoing routine testing for susceptibility to doravirine and other approved NNRTIs (etravirine, rilpivirine, efavirenz, and nevirapine) were collected from August 2018 to August 2019. Susceptibility in the presence/absence of NNRTI and nucleos(t)ide reverse transcriptase inhibitor (NRTI) mutations was determined using cut-offs for relative fold-change in inhibition (ratio of the 50% inhibitory concentration [IC 50 ] of patient virus compared with the IC 50 of a wild-type reference strain). Biological cut-offs of 3- to 15-fold-change were investigated for doravirine, with pre-established cut-offs used for the other NNRTIs. Results: Of 4,070 clinical isolates, 42.9% had ≥1 NNRTI RAM. More isolates were susceptible to doravirine (92.5–96.7%) than to etravirine (91.5%), rilpivirine (89.5%), efavirenz (81.5%), or nevirapine (77.5%). Based on a 3-fold cut-off, doravirine susceptibility was retained in 44.7–65.8% of isolates resistant to another NNRTI and 28.5% of isolates resistant to all other tested NNRTIs. The presence of NRTI RAMs including thymidine analog mutations was associated with doravirine hypersusceptibility in some isolates, particularly in the absence of NNRTI RAMs. Conclusion: These results support the favorable resistance profile of doravirine, and are of particular importance given the challenge posed by both acquired and transmitted resistance.

Changes in Testicular Biometry, Steroid Hormones and Receptor Expression in the Peripubertal Period of Indigenous Tenyi-vo Male Pigs of North-eastern Himalayan Region in India

Present study was conducted to characterize the testicular changes in the peripubertal period in Tenyi-vo, a miniature size pig of North-eastern Himalayan (NEH) region of India. A total of twenty-four male pigs were randomly selected and categorised for castration at different age groups, G1 (Days 30-45), G2 (Days 60-65), G3 (Days 80-100) and G4 (Days 150-160), n=6 each category. Paired testes and epididymis were used for the assessment of biometry, cauda epididymal spermiogram, testicular histology and relative expression of the androgen receptor (AR), estrogen receptors (ERα and ERβ), aromatase (CYP19A1), and insulin like growth factor-1β receptor (IGF-1R) in qPCR. Plasma testosterone (T), estradiol (E2), tri-iodothyronine (T3), thyroxine (T4), and cortisol concentrations were estimated on the day of castration in each group of male using commercial ELISA kits. In pigs of G2, a greater testicular weight, volume, epididymis weight was observed relative to G1. The presence of live spermatozoa at 1240.9±304.2×106/mLconcentration with 0.65% proximal droplets was recorded as early as day 60. The concentration of T increased steadily over the age of G1 to G4 and a significantly higher concentration was observed in G4 relative to the other categories. Among the transcripts analysed in the testis, the relative fold change of AR was 10.8 fold in G2, which was subsequently reduced in G3 and then down-regulated in G4. CYP19A1 was abundantly expressed in the testis and the fold change ranged from 41-54 fold, although it did not differ significantly from 60-150 days of age. Further, the presence of well-developed seminiferous tubules was evident in the Tenyi-vo male from day 60 onward with a body weight as low as 4.28 kg. The study concluded that the male of Tenyi-vo pig attained puberty at the earliest age of 60 days.

Relationship between chromosomal aberrations and gene expressions in the p53 pathway in chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a neoplasm characterized by excessive accumulation of B lymphocytes in the peripheral blood, bone marrow and lymph nodes. We assessed the expressions of 22 genes in the p53 pathway in 30 CLL patients and 15 healthy subjects by a RT2 Profiler PCR (polymerase chain reaction) Array technique and their relation to cytogenetic aberrations detected by fluorescent in situ hybridization (FISH). Our Student’s t-test results indicated that ATM, ATR, BAX, CASP9, CDK4, CDKN2A, CHEK1, CHEK2, E2F3, MCL1, MDM2, MDM4, PCNA, RB1, P53 and BCL2 genes were statistically significant (p <0.001). For six genes (APAF1, CDKN1A, E2F1, GADD45A, PTEN and PTX3) were not statistically significant. The ATM, ATR, BAX, CASP9, CDK4, CDKN1A, CDKN2A, CHEK1, CHEK2, MDM2, MDM4, PCNA, RB1, P53, E2F1, GADD45A and BCL2 genes were found to be upregulated by the 2-ᐃᐃCt (relative fold change in gene expression) method. The highest up-regulation was detected in CDKN2A and BCL2 genes, 10.22- and 8.51-fold, respectively. On the other hand, the PTX3 gene with a fold regulation of 1.84 was found to the highest downregulation. Overall, the CDNK2A BCL2 and PTX3 genes are related to the mechanism of the disease in the p53 pathway and may be an important predictor of the prognosis of the disease. The BCL2 gene may be associated with increased risk of developing CLL. We suggest that the PTX3 gene may be considered as a marker associated with CLL disease. The CDKN2A gene expression seems to play a protective role in CLL.

reconCNV: interactive visualization of copy number data from high-throughput sequencing

Summary Copy number variation (CNV) is an important category of unbalanced structural rearrangement. While methods for detecting CNV in high-throughput targeted sequencing have become increasingly sophisticated, dedicated tools for interactive and dynamic visualization of CNV from these data are still lacking. We describe reconCNV, a tool that produces an interactive and annotated web-based dashboard for viewing and summarizing CNVs detected in next-generation sequencing (NGS) data. reconCNV is designed to work with delimited result files from most NGS CNV callers with minor adjustments to the configuration file. The reconCNV output is an HTML file that is viewable on any modern web browser, requires no backend server, and can be readily appended to existing analysis pipelines. In addition to a standard CNV track for visualizing relative fold change and absolute copy number, the tool includes an auxiliary variant allele fraction track for visualizing underlying allelic imbalance and loss of heterozygosity. A feature to mask assay-specific technical artifacts and a direct HTML link out to the UCSC Genome Browser are also included to augment the reviewer experience. By providing a light-weight plugin for interactive visualization to existing NGS CNV pipelines, reconCNV can facilitate efficient NGS CNV visualization and interpretation in both research and clinical settings. Availability and implementation The source code and documentation including a tutorial can be accessed at https://github.com/rghu/reconCNV as well as a Docker image at https://hub.docker.com/repository/docker/raghuc1990/reconcnv. Supplementary information Supplementary data are available at Bioinformatics online.

Piperine from Black Pepper Decreased the Expression of Intercellular Adhesion Molecule-1 in Macrophages

Background: Macrophages are the main players involved in inflammation. Intercellular adhesion molecule-1 (ICAM-1) facilitates macrophage polarization prior to extravasation into inflamed tissue. Piperine a natural product derived from black pepper possess useful biological and pharmacological activities. In current study, the possible anti-inflammatory effect of piperine on the expression of ICAM-1 on J774.1 murine macrophage cell line was investigated. Methods: Lipopolysaccharide (LPS)-stimulated J774.1 cells were cultured in the presence of different concentrations of piperine to examine the changes in ICAM-1 expression by real-time PCR and flow cytometry. Results: We found that piperine decreased ICAM-1 gene expression level from 2.4 ± 0.25 RFC (relative fold change) in LPS-only treated cells to 0.85 ± 0.525 RFC at 1μg/ml (p<0.05), 0.43 ± 0.27 RFC at 10μg/ml (p<0.01), and 0.26 ± 0.25 RFC at 20μg/ml (p<0.01). In flow cytometry, piperine at all concentrations significantly decreased ICAM-1 surface expressions (P<0.05). The geometric mean fluorescence intensity (g-MFI) in LPS-only treated cells (792 ± 57.3) decreased to 482±70 gMFI at 20 µg/ml piperine. Conclusion: According to the results of this study, by decreasing the expression of ICAM-1, piperine is suggested as a candidate to reduce inflammation and has the potential for therapeutic benefits for immune-mediated diseases.

Inflammatory Phenotypes Predict Changes in Arterial Stiffness Following Antiretroviral Therapy Initiation

Background Inflammation drives vascular dysfunction in HIV, but in low-income settings causes of inflammation are multiple, and include infectious and environmental factors. We hypothesized that patients with advanced immunosuppression could be stratified into inflammatory phenotypes that predicted changes in vascular dysfunction on ART. Methods We recruited Malawian adults with CD4 &lt;100 cells/μL 2 weeks after starting ART in the REALITY trial (NCT01825031). Carotid femoral pulse-wave velocity (cfPWV) measured arterial stiffness 2, 12, 24, and 42 weeks post–ART initiation. Plasma inflammation markers were measured by electrochemiluminescence at weeks 2 and 42. Hierarchical clustering on principal components identified inflammatory clusters. Results 211 participants with HIV grouped into 3 inflammatory clusters representing 51 (24%; cluster-1), 153 (73%; cluster-2), and 7 (3%; cluster-3) individuals. Cluster-1 showed markedly higher CD4 and CD8 T-cell expression of HLADR and PD-1 versus cluster-2 and cluster-3 (all P &lt; .0001). Although small, cluster-3 had significantly higher levels of cytokines reflecting inflammation (IL-6, IFN-γ, IP-10, IL-1RA, IL-10), chemotaxis (IL-8), systemic and vascular inflammation (CRP, ICAM-1, VCAM-1), and SAA (all P &lt; .001). In mixed-effects models, cfPWV changes over time were similar for cluster-2 versus cluster-1 (relative fold-change, 0.99; 95% CI, .86–1.14; P = .91), but greater in cluster-3 versus cluster-1 (relative fold-change, 1.45; 95% CI, 1.01–2.09; P = .045). Conclusions Two inflammatory clusters were identified: one defined by high T-cell PD-1 expression and another by a hyperinflamed profile and increases in cfPWV on ART. Further clinical characterization of inflammatory phenotypes could help target vascular dysfunction interventions to those at highest risk.

Abstract 075: Impaired Hypothalamic PVN G-alpha i 2 Signal Transduction Attenuates Endogenous Sympathoinhibitory PVN GABAergic Tone to Evoke the Development of Salt-sensitive Hypertension

Aim: We hypothesize hypothalamic PVN Gαi 2 proteins, which are up regulated during high salt intake, facilitate sympathoinhibition and blood pressure regulation via mediating GABA B receptor signal transduction to maintain salt-resistance in the Sprague Dawley (SD) rat. Methods: Groups of male salt-resistant SD rats received a bilateral PVN infusion of a scrambled (SCR) or Gαi 2 oligodeoxynucleotide (ODN-300ng/side/day) and a normal 0.6% (NS) or high 4% NaCl (HS) diet for 7-days. On day-7 24h Na + balance was assessed - in sub-groups MAP, plasma norepinephrine (NE) content, PVN GAD67 [marker of GABAergic expression] and vGLUT2 [marker of glutamatergic expression] immunoreactivity, and the cardiovascular responses to bilateral administration of the GABA B receptor antagonist (CGP52432; 3nmol/side) and the ionotropic glutamate receptor antagonist (kynurenate; 1.4 μg/side) was assessed (N=4/gp/study). Results: In control SCR ODN PVN infused SD rats HS intake suppressed plasma NE (plasma NE [nmol/L] SCR NS 76±7 vs HS 43±6, P<0.05) and evoked increased GAD67, but not vGLUT2, immunoreactivity (Medial Parvocellluar (MP) GAD67 [relative fold change (normalized to DAPI) 8.4±1.1) in all PVN parvocellular sub-regions without impacting MAP or sodium balance. Following ODN-mediated PVN Gαi 2 down-regulation HS intake caused salt-sensitive hypertension (MAP [mmHg] Gαi 2 ODN NS 128±3, HS 148±4, P<0.05), failed to increase GAD67 expression and increased PVN vGLUT2 immunoreactivity in multiple PVN parvocellular sub-regions (MP vGLUT2 [relative fold change (normalized to DAPI) 3.2±0.8). In separate groups of rats bilateral PVN GABA B antagonism increased MAP in control SCR but not Gαi 2 ODN infused rats maintained on a HS intake (peak ΔMAP [mmHg] SCR ODN HS +17±3, Gαi 2 ODN HS +2±2). In contrast, bilateral PVN glutamate antagonism did not alter MAP in control SCR ODN infused rats but decreased MAP in hypertensive Gαi 2 ODN infused SD rats on a HS intake (peak ΔMAP [mmHg] SCR HS +3±3, Gαi 2 ODN HS -13±2). Conclusion: These data suggest impaired Gαi 2 signaling attenuates sympathoinhibitory PVN GPCR-mediated GABAergic tone (i.e., modulates GPCR coupled GABA B responses) and enhances sympathoexcitatory glutamatergic tone to evoke salt-sensitive hypertension in the SD rat.

Men Show Significantly Higher T Lymphocyte Mediated Immune Responses Against H-Y Than Multiparous Women: Results from a Cross-Sectional Analysis in 114 Volunteer Blood Donors and Implications for Immunotherapy

Background: Immune responses against Y-chromosomal encoded epitopes are well known as a driving force for both strong graft-versus-host (GvH) and graft-versus-leukemia (GvL) effects in female-into-male allogeneic hematopoietic stem cell transplantations (HSCT). Female donors with a history of pregnancies with male infants are likely to carry T lymphocyte based immune responses against minor histocompatibility antigens derived from the Y chromosome (H-Y antigens). A feto-maternal cell transfer and consecutive immunization of the mother forms the likely basis of this phenomenon. Interestingly, male individuals may carry T lymphocyte based immune responses against H-Y as well. So far, little is known about their frequency, their origin and their functionality particularly in light of allogeneic immunotherapy. Therefore, we screened a group of volunteer blood donors including men and women for H-Y responses. This group has been previously reported for immune responses against tumor-associated antigens (Lutz M et al, ASH 2012). Material and Methods:After obtaining local ethical approval and written informed consent 114 HLA-A*02:01-positive healthy volunteer blood donors were enrolled in this scientific study including 38 nulligravidous women with a median age of 28 (21–53) years, 38 primi- or multiparous women with a median age of 46 (28–63) years and 38 men with a median age of 41 (21–56) years, respectively. Peripheral blood was drawn and peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation. CD8+ T lymphocytes were isolated and stimulated with irradiated T2 cells (ATCC CRL-1992) which were pulsed with two different concentrations (0.1 and 10 µM) of the HLA-A*02:01-restricted minor antigen H-Y (FIDSYICQV) to distinguish between low- and high-avidity immune responses. After extraction of total RNA, Interferon gamma (IFNγ) mRNA expression was analyzed by quantitative reverse transcriptase polymerase chain reaction (RT-qPCR). IFNγ mRNA expression was normalized to CD8 mRNA levels and expressed as relative fold change compared to the irrelevant melanoma antigen Glycoprotein 100 (gp100). A two-fold change or more as compared to gp100 was considered as a positive result. Results:Significant immune responses were detected in a small fraction of both women and men using two different peptide concentrations. Interestingly, women with a history of pregnancy did not show higher immune responses than those with no (known) history of pregnancy for both peptide concentrations. However, men showed the highest frequency of positive immune responses for both peptide concentrations. For the higher peptide concentration men showed significantly higher immune responses than nulligravidous (P < 0.05) or primi-/multiparous women (P < 0.01) or all women (P < 0.01). All results of this analysis are depicted in Figure 1. The horizontal line represents the cut-off for positive immune responses (relative fold change of 2.0). Conclusions: The results in women suggest that their immune responses – if not boosted by a further pregnancy or immunotransfer to an allogeneic individual – are short-lived. The detection of H-Y responses in nulligravidous women could be a result of unknown pregnancies in the past. We expected the highest frequency of significant immune responses against H-Y in primi- or multiparous women. However, to our surprise men showed the highest frequency and had significantly higher levels than women. This finding is in line with prior data where we found men to carry frequent immune responses against tumor-associated antigens such as WT1. At the end H-Y may serve as an auto-antigen to men suggesting that continuous gonadal expression of Y-chromosomal encoded antigens maintains these low-avidity autoimmune responses as previously described for cancer/testis antigens (Lutz M et al, ASH 2012). The transfer of these immune responses in a male-to-male HSCT may therefore contribute preferably to the GvL effect and thus be beneficial. Disclosures No relevant conflicts of interest to declare.