High-throughput chromatin accessibility profiling at single-cell resolution

Infection by human herpesvirus 8 (HHV-8) is associated with the development of Kaposi's sarcoma (KS). Since regression of KS can be achieved by treatment of the patients with alpha interferon (IFN-alpha), we analyzed the effects of IFN-alpha or anti-IFN-alpha antibodies (Ab) on HHV-8 latently infected primary effusion lymphoma-derived cell lines (BCBL-1 and BC-1) and on peripheral blood mononuclear cells (PBMC) from patients with all forms of KS and from at-risk subjects. IFN-alpha inhibited in a dose-dependent manner the amplification of HHV-8 DNA in BCBL-1 cells induced to lytic infection with tetradecanoyl phorbol acetate (TPA). This effect was associated with the inhibition of the expression of HHV-8 nut-1 and kaposin genes that are induced early and several hours, respectively, after TPA treatment. In addition, IFN-alpha inhibited virus production and/or release from BCBL-1 cells. Inhibition of nut-1 and kaposin genes by IFN-alpha was also observed in BC-1 cells induced with n-butyrate. Conversely, the addition of anti-IFN-alpha Ab to TPA-induced BCBL-1 cells resulted in a larger number of mature enveloped particles and in a more extensive cytopathic effect due to the neutralization of the endogenous IFN produced by these cells. IFN was also produced by cultured PBMC from HHV-8-infected individuals, and this was associated with a loss of viral DNA during culture. However, the addition of anti-IFN-alpha Ab or anti-type I IFN receptor Ab promoted the maintenance of HHV-8 DNA in these cells that was associated with the detection of the latency-associated kaposin RNA. Finally, the addition of IFN-alpha reduced the HHV-8 load in PBMC. Thus, IFN-alpha appears to have inhibitory effects on HHV-8 persistent infection of PBMC. These results suggest that, in addition to inhibiting the expression of angiogenic factors that are key to KS development, IFN-alpha may induce KS regression by reducing the HHV-8 load and/or inhibiting virus reactivation.

First Blood: Pre-Therapy BRAFV600E+ PBMC Identifies a Novel Clinical Risk Category for Patients with LCH

Single-Cell Analyses of Colon and Blood Reveal Distinct Immune Cell Signatures of Ulcerative Colitis and Crohn’s Disease

The pathogenesis of HIV-associated neurocognitive disorders is complex and multifactorial. It is hypothesized that the critical events initiating this condition occur outside the brain, particularly in the peripheral blood. Diagnoses of HIV-induced neurocognitive disorders largely rely on neuropsychometric assessments, which are not precise. Total HIV DNA in the peripheral blood mononuclear cells (PBMCs), quantified by PCR, correlate with disease progression, which is a promising biomarker to predict HAND. Numerous PCR assays for HIV DNA in cell compartments are prone to variation due to the lack of standardization and, therefore, their utility in predicting HAND produced different outcomes. This review evaluates the clinical relevance of total HIV DNA in circulating mononuclear cells using different published quantitative PCR (qPCR) protocols. The rationale is to shed light on the most appropriate assays and sample types used to accurately quantify HIV DNA load, which predicts severity of neurocognitive impairment. The role of monocytes as a vehicle for trafficking HIV into the CNS makes it the most suitable sample for determining a HAND associated reservoir. Studies have also shown significant associations between monocyte HIV DNA levels with markers of neurodamage. However, qPCR assays using PBMCs are cheaper and available commercially, thus could be beneficial in clinical settings. There is need, however, to standardise DNA extraction, normalisation and limit of detection.

Hepatitis B virus (HBV) can persist in peripheral blood mononuclear cells (PBMCs) as an extrahepatic reservoir in treatment-naive patients; however, its persistence during long-term nucleos(t)ide analogue therapy with sustained serum viral suppression remains unclear. This study aimed to evaluate the frequency and predictors of HBV DNA detectability in PBMCs of chronic hepatitis B (CHB) patients following long-term nucleos(t)ide analogue therapy-induced sustained serum viral suppression. Eighty-eight CHB patients, on long-term tenofovir disoproxil fumarate ( n = 44) or entecavir ( n = 44) with greater than 2 years of sustained viral suppression, were enrolled. PBMCs were tested for HBV DNA by quantitative PCR. Clinical and laboratory data were analyzed to identify predictors of PBMC HBV DNA detection. Receiver operating characteristic analysis assessed the performance of posttreatment serum quantitative hepatitis B surface antigen (qHBsAg). HBV DNA was detected in PBMCs of eight (9.1%) patients, equally distributed between treatment groups. Pretreatment hepatitis B e-antigen (HBeAg) positivity predicted PBMC HBV DNA detection ( P = 0.036). PBMC HBV DNA-positive patients were older males with worse baseline liver chemistry and advanced fibrosis. Posttreatment qHBsAg was higher in PBMC HBV DNA-positive patients (139.5 vs. 21.5 IU/ml; P < 0.001) and strongly correlated with PBMC viral load ( r = 0.905, P = 0.002); qHBsAg greater than 89 IU/ml predicted PBMC HBV DNA detection with high accuracy. HBV DNA may persist in PBMCs despite long-term nucleos(t)ide analogue therapy-induced sustained viral suppression. Pretreatment HBeAg positivity and elevated posttreatment qHBsAg can help identify patients at risk, highlighting the need to monitor extrahepatic reservoirs in CHB management.

Hematopoietic Cell Cluster Identifier (HCCI): A Knowledgebase Computational Tool to Facilitate the Characterization of Hematopoietic Cell Populations in Single Cell RNA-Sequencing

Background and ObjectivesMultiple sclerosis (MS) is an immune-mediated demyelinating disease of the CNS characterized by a heterogeneous disease trajectory, highlighting the need for biomarkers to predict disease activity. Current disease-monitoring tools primarily reflect existing disease damage rather than impending activity. Peripheral blood mononuclear cells (PBMCs) are an ideal source of potential biomarkers due to their accessibility and their known role in MS pathology. Among PBMCs, myeloid cells are key players in MS pathogenic processes, yet they have not been as extensively studied than lymphocytes. The objective of our study was to identify indicators of MS disease activity through immune profiling.MethodsWe analyzed PBMCs using high-dimensional flow cytometry with a panel focusing on myeloid cells. We performed unsupervised clustering analyses to define a comprehensive immune landscape at a single-cell resolution. Supervised machine learning methods were used to extract immune features indicative of MS activity.ResultsWe analyzed PBMCs from 135 individuals with MS with retrospective longitudinal follow-up and 44 healthy controls (HCs). Among the individuals with MS, 53 were untreated and were compared with HCs. Using an elastic-net model, 20 immune features were identified as contributors to the classification of MS and HCs (receiver operating characteristic-AUC 0.8881). To explore associations between immune features and disease activity, we focused on individuals with relapsing-remitting MS (n = 106). We identified a subpopulation of classical monocytes (CMs) with high expression of human leukocyte antigen - DR isotype (HLA-DR) and positive for CD141 (HLA-DRhiCD141+) as a predictor of impending relapses over 2 years (hazard ratio [HR] 2.8, 95% CI 1.6–4.7) and disability worsening in patients with higher relapse activity. HLA-DRhiCD141+ CMs could be retrieved by manual gating using 9 parameters and were similarly indicative of 2-year relapse risk (HR 1.9, 95% CI 1.3–2.8), highlighting its potential as a practical, translational approach. Compared with the widely studied biomarker serum neurofilament light chain reflecting acute activity, HLA-DRhiCD141+ CMs provided a stronger prognostic value for impending relapse risk, suggesting different kinetics related to the underlying pathology.DiscussionOur findings suggest that the frequency of HLA-DRhiCD141+ CMs could serve as a valuable predictor of disease activity complementary to current clinical tools to guide evidence-based treatment decisions.

Rationale: Changes in peripheral blood cell populations have been observed, but not detailed, at single-cell resolution in idiopathic pulmonary fibrosis (IPF). Objectives: We sought to provide an atlas of the changes in the peripheral immune system in stable and progressive IPF. Methods: Peripheral blood mononuclear cells (PBMCs) from patients with IPF and control subjects were profiled using 10× chromium 5' single-cell RNA sequencing. Flow cytometry was used for validation. Protein concentrations of regulatory T cells (Tregs) and monocyte chemoattractants were measured in plasma and lung homogenates from patients with IPF and control subjects. Measurements and Main Results: Thirty-eight PBMC samples from 25 patients with IPF and 13 matched control subjects yielded 149,564 cells that segregated into 23 subpopulations. Classical monocytes were increased in patients with progressive and stable IPF compared with control subjects (32.1%, 25.2%, and 17.9%, respectively; P < 0.05). Total lymphocytes were decreased in patients with IPF versus control subjects and in progressive versus stable IPF (52.6% vs. 62.6%, P = 0.035). Tregs were increased in progressive versus stable IPF (1.8% vs. 1.1% of all PBMCs, P = 0.007), although not different than controls, and may be associated with decreased survival (P = 0.009 in Kaplan-Meier analysis; and P = 0.069 after adjusting for age, sex, and baseline FVC). Flow cytometry analysis confirmed this finding in an independent cohort of patients with IPF. The fraction of Tregs out of all T cells was also increased in two cohorts of lung single-cell RNA sequencing. CCL22 and CCL18, ligands for CCR4 and CCR8 Treg chemotaxis receptors, were increased in IPF. Conclusions: The single-cell atlas of the peripheral immune system in IPF reveals an outcome-predictive increase in classical monocytes and Tregs, as well as evidence for a lung-blood immune recruitment axis involving CCL7 (for classical monocytes) and CCL18/CCL22 (for Tregs).

In these studies, we designed and investigated the potential anticancer activity of five iron(II) cyclopentadienyl complexes bearing different phosphine and phosphite ligands. All complexes were characterized with spectroscopic analysis viz. NMR, FT–IR, ESI–MS, UV–Vis, fluorescence, XRD (for four complexes) and elemental analyses. For biological studies, we used three types of cells—normal peripheral blood mononuclear (PBM) cells, leukemic HL-60 cells and non-small-cell lung cancer A549 cells. We evaluated cell viability and DNA damage after cell incubation with these complexes. We observed that all iron(II) complexes were more cytotoxic for HL-60 cells than for A549 cells. The complex CpFe(CO)(P(OPh)3)(η1-N-maleimidato) 3b was the most cytotoxic with IC50 = 9.09 µM in HL-60 cells, IC50 = 19.16 µM in A549 and IC50 = 5.80 µM in PBM cells. The complex CpFe(CO)(P(Fu)3)(η1-N-maleimidato) 2b was cytotoxic only for both cancer cell lines, with IC50 = 10.03 µM in HL-60 cells and IC50 = 73.54 µM in A549 cells. We also found the genotoxic potential of the complex 2b in both types of cancer cells. However, the complex CpFe(CO)2(η1-N-maleimidato) 1 which we studied previously, was much more genotoxic than complex 2b, especially for A549 cells. The plasmid relaxation assay showed that iron(II) complexes do not induce strand breaks in fully paired ds-DNA. The DNA titration experiment showed no intercalation of complex 2b into DNA. Molecular docking revealed however that complexes CpFe(CO)(PPh3) (η1-N-maleimidato) 2a, 2b, 3b and CpFe(CO)(P(OiPr)3)(η1-N-maleimidato) 3c have the greatest potential to bind to mismatched DNA. Our studies demonstrated that the iron(II) complex 1 and 2b are the most interesting compounds in terms of selective cytotoxic action against cancer cells. However, the cellular mechanism of their anticancer activity requires further research.

Interrelationships among selenium status, antioxidant capacity and oxidative stress in the horse

Genomic Sequencing by Template Purification: Principles and Mapping of Protein-Bound and Single-Stranded Sequences in Vivo

The synthesis of a novel acetoxymethyltriazene designed to be a prodrug of multiple inhibitors of the epidermal growth factor receptor (EGFR) and a methyldiazonium species is described. Studies with each of the expected metabolites demonstrated significant EGFR tyrosine kinase inhibitory activities and the released methyldiazonium was trapped with p-nitrobenzylpyridine. Their ability to damage genomic DNA in whole cells was demonstrated by using the single cell microelectrophoresis (comet) assay. The results suggest that this approach may well represent a novel drug combination strategy involving single molecules masking multiple bioactive agents.

There are several forms of human immunodeficiency virus type 1 (HIV-1) DNA in peripheral blood T cells and lymph nodes in untreated HIV-1-infected individuals and in patients whose plasma HIV-1 RNA levels are suppressed by long-term combination antiretroviral therapy. However, it remains to be established whether the concentration of HIV-1 DNA in cells predicts the clinical outcome of HIV-1 infection. In this report, we measured the concentration of HIV-1 DNA forms which has undergone the second template switch (STS DNA) and 2-long-terminal-repeat DNA circles in peripheral blood mononuclear cell (PBMC) samples. To do this, we used molecular-beacon-based real-time PCR assays and studied 130 patients with hemophilia in the Multicenter Hemophilia Cohort Study. We assessed the influence of baseline HIV-1 STS DNA levels on the progression of HIV-1 disease in the absence of combination antiretroviral therapy by Kaplan-Meier and Cox regression analysis. Among the patients who progressed to AIDS, the median levels (interquartile ranges) of STS HIV-1 DNA in PBMC were significantly higher than those of patients who remained AIDS free during the 16 years of follow-up (1,017 [235 to 6,059] and 286 [31 to 732] copies per 10(6) PBMC, respectively; P < 0.0001). Rates of progression to death and development of AIDS varied significantly (log rank P < 0.001) by quartile distribution of HIV-1 STS DNA levels. After adjustment for age at seroconversion, baseline CD4(+) T-cell counts, plasma viral load, and T-cell-receptor excision circles, the relative hazards (RH) of death and AIDS were significantly increased with higher HIV-1 STS DNA levels (adjusted RH, 1.84 [95% confidence interval (CI), 1.30 to 2.59] and 2.62 [95% CI, 1.75 to 3.93] per 10-fold increase per 10(6) PBMC, respectively). HIV-1 STS DNA levels in each individual remained steady in longitudinal PBMC samples during 16 years of follow-up. Our findings show that the concentration of HIV-1 STS DNA in PBMC complements the HIV-1 RNA load in plasma in predicting the clinical outcome of HIV-1 disease. This parameter may have important implications for understanding the virological response to combination antiretroviral therapy.

The presence of human immunodeficiency virus type 1 (HIV-1) proviral DNA in peripheral blood mononuclear cells (PBMC) of three groups (group 1, more than 500 CD4+ T cells per microliter; group 2, between 200 and 499 CD4+ T cells per microliter; group 3, fewer than 200 CD4+ T cells per microliter) of HIV-1-infected patients, in different stages of the disease, was determined by using a newly developed flow cytometry analysis of the product of in situ PCR assay and compared with other markers of viral replication (HIV-1 p24 antigenemia and viral isolation). Results showed varied percentages of HIV-1-infected PBMC, ranging from 0.6 to 20%. Patients with more than 500 CD4+ T cells per microliter showed the lowest percentage of HIV-1-infected PBMC (2.1 +/- 1.7), compared with patients with CD4+ T-cell counts of between 200 and 499 per microliter (6.5% +/- 4.1%; P < 0.001) and patients with fewer than 200 CD4+ T cells per microliter (4.9% +/- 4.7%; P < 0.05). The difference in the percentage of HIV-1-infected PBMC between group 2 and group 3 patients may in part reflect the loss of CD4+ T lymphocytes in more advanced stages of the disease. However, the results clearly indicate a striking coincidence between the fall of the CD4+ T-cell count below 400/microliter and the sharp increase in PBMC virus loading and p24 antigenemia. Since the procedure is relatively easy to perform, it could be used to monitor the evolution of HIV-1 infection and may prove a useful adjunct in tailoring therapeutic strategies.

Saliva and peripheral blood mononuclear cells from three patients, two with lymphoproliferative disorders and one suffering from multiple sclerosis, were examined for the presence of human herpesvirus-6 (HHV-6) genome by using the polymerase chain reaction and Southern blot analysis. The search for anti-HHV-6 antibodies, carried out in the sera of the same cases by an immunofluorescence assay, was negative in two cases at the lowest dilution used (1:40). These three patients had a high number of HHV-6 specific sequences in uncultured peripheral blood mononuclear cells, which are thought to be a normal site of viral latency although, in healthy individuals, the infected cells are extremely rare. In order to gain some insight into the state of the viral genome in this latent HHV-6 infection, we used pulsed field gel electrophoresis to separate HHV-6 DNA directly from HHV-6 (strain GS) infected HSB-2 cells and from the peripheral blood mononuclear cells of these three patients. Our study showed the presence of intact viral genome, of the expected length of 170 kb, persisting as free extrachromosomal element in the HSB-2 cells but not in patients' peripheral blood mononuclear cells. On the other hand, in strong contrast with the results obtained in infected HSB-2 DNA, the restriction analysis of the three patients' peripheral blood mononuclear cell DNA showed fragments of molecular weight constantly higher than the 170 kb segment, indicating that the viral sequences are linked to high molecular weight cellular DNA.(ABSTRACT TRUNCATED AT 250 WORDS)