ObjectiveAlthough the risk of morbidity and mortality of children and adolescents was lower during the COVID-19 pandemic, it appears that their mental health was strongly impacted. The goal of this ...study is to document psychological dysfunction among children and adolescents who underwent confinement due to COVID-19 in Ecuador.DesignA cross-sectional, internet-based questionnaire.SettingEcuador.ParticipantsA total of 1077 caregivers of children and adolescents (4–16 years old).Outcome measuresCaregivers responded to Pediatric Symptom Checklist-35 to assess psychosocial dysfunction.ResultsThe prevalence of psychosocial dysfunction was 20.8%, with internalising symptoms being the most common (30.7%). The prevalence of psychosocial dysfunction was higher in children who had a poor family relationship during confinement (prevalence ratio (PR) 2.23; 95% CI 1.22 to 4.07), children who never helped with housework (PR 2.63; 95% CI 1.13 to 6.14) and those whose caregivers were worried about children’s need for emotional therapy (PR 2.86; 95% CI 1.97 to 4.15). Never playing video games (PR 0.34; 95% CI 0.17 to 0.69) or playing video games infrequently (PR 0.39; 95% CI 0.20 to 0.79) was a protective factor for the psychosocial problems of children and adolescents.ConclusionOur study demonstrates that children and adolescents have experienced a deterioration of mental health due to the pandemic. Family factors played an important role in the mental health of children during the lockdown. When a public crisis occurs, supportive mental health policies should be developed and implemented to promote children’s psychological welfare.
Human peripheral blood mononuclear cells (PBMCs) are extensively used for research of immune cell functions, identification of biomarkers and development of diagnostics and therapeutics for human ...diseases, among others. The assumption that "old blood samples" are not appropriate for isolation of PBMCs for functional assays has been a dogma in the scientific community. However, partial data on the impact of time after phlebotomy on the quality and stability of human PBMCs preparations impairs the design of studies in which time-controlled blood sampling is challenging such as field studies involving multiple sampling centers/sites. In this study, we evaluated the effect of time after phlebotomy over a 24 h time course, on the stability of human blood leukocytes used for immunological analyses. Blood samples from eight healthy adult volunteers were obtained and divided into four aliquots, each of which was left in gentle agitation at room temperature (24 °C) for 2 h (control), 7 h, 12 h and 24 h post phlebotomy. All samples at each time point were independently processed for quantification of mononuclear cell subpopulations, cellular viability, gene expression and cytokine secretion.
A 24 h time delay in blood sample processing did not affect the viability of PBMCs. However, a significantly lower frequency of CD3+ T cells (p < 0.05) and increased LPS-induced CXCL10 secretion were observed at 12 h post-phlebotomy. Alterations in TNFα, CCL8, CCR2 and CXCL10 gene expression were found as early as 7 h after blood sample procurement.
These data reveal previously unrecognized early time-points for sample processing control, and provide an assay-specific time reference for the design of studies that involve immunological analyses of human blood samples.
Immunological non-responders (INR) are people living with HIV (PLHIV) who fail to fully restore CD4+ T-cell counts despite complete viral suppression with antiretroviral therapy (ART). INR are at ...higher risk for non-HIV related morbidity and mortality. Previous research suggest persistent qualitative defects.
The 2000HIV study (clinical trials NTC03994835) enrolled 1895 PLHIV, divided in a discovery and validation cohort. PLHIV with CD4 T-cell count <350 cells/mm
after ≥2 years of suppressive ART were defined as INR and were compared to immunological responders (IR) with CD4 T-cell count >500 cells/mm
. Logistic and rank based regression were used to analyze clinical data, extensive innate and adaptive immunophenotyping, and
monocyte and lymphocyte cytokine production after stimulation with various stimuli.
The discovery cohort consisted of 62 INR and 1224 IR, the validation cohort of 26 INR and 243 IR. INR were older, had more advanced HIV disease before starting ART and had more frequently a history of non-AIDS related malignancy. INR had lower absolute CD4+ T-cell numbers in all subsets. Activated (HLA-DR+, CD38+) and exhausted (PD1+) subpopulations were proportionally increased in CD4 T-cells. Monocyte and granulocyte immunophenotypes were comparable. INR lymphocytes produced less IL-22, IFN-γ, IL-10 and IL-17 to stimuli. In contrast, monocyte cytokine production did not differ. The proportions of CD4+CD38+HLA-DR+ and CD4+PD1+ subpopulations showed an inversed correlation to lymphocyte cytokine production.
INR compared to IR have hyperactivated and exhausted CD4+ T-cells in combination with lymphocyte functional impairment, while innate immune responses were comparable. Our data provide a rationale to consider the use of anti-PD1 therapy in INR.
Even during long-term combination antiretroviral therapy (cART), people living with HIV (PLHIV) have a dysregulated immune system, characterized by persistent immune activation, accelerated immune ...ageing and increased risk of non-AIDS comorbidities. A multi-omics approach is applied to a large cohort of PLHIV to understand pathways underlying these dysregulations in order to identify new biomarkers and novel genetically validated therapeutic drugs targets.
The 2000HIV study is a prospective longitudinal cohort study of PLHIV on cART. In addition, untreated HIV spontaneous controllers were recruited. In-depth multi-omics characterization will be performed, including genomics, epigenomics, transcriptomics, proteomics, metabolomics and metagenomics, functional immunological assays and extensive immunophenotyping. Furthermore, the latent viral reservoir will be assessed through cell associated HIV-1 RNA and DNA, and full-length individual proviral sequencing on a subset. Clinical measurements include an ECG, carotid intima-media thickness and plaque measurement, hepatic steatosis and fibrosis measurement as well as psychological symptoms and recreational drug questionnaires. Additionally, considering the developing pandemic, COVID-19 history and vaccination was recorded. Participants return for a two-year follow-up visit. The 2000HIV study consists of a discovery and validation cohort collected at separate sites to immediately validate any finding in an independent cohort.
Overall, 1895 PLHIV from four sites were included for analysis, 1559 in the discovery and 336 in the validation cohort. The study population was representative of a Western European HIV population, including 288 (15.2%)
-women, 463 (24.4%) non-whites, and 1360 (71.8%) MSM (Men who have Sex with Men). Extreme phenotypes included 114 spontaneous controllers, 81 rapid progressors and 162 immunological non-responders. According to the Framingham score 321 (16.9%) had a cardiovascular risk of >20% in the next 10 years. COVID-19 infection was documented in 234 (12.3%) participants and 474 (25.0%) individuals had received a COVID-19 vaccine.
The 2000HIV study established a cohort of 1895 PLHIV that employs multi-omics to discover new biological pathways and biomarkers to unravel non-AIDS comorbidities, extreme phenotypes and the latent viral reservoir that impact the health of PLHIV. The ultimate goal is to contribute to a more personalized approach to the best standard of care and a potential cure for PLHIV.
Background:
Obtaining high quality RNA from skin biopsies is complex due the physical composition and high content of nucleases of this tissue. This becomes particularly challenging when using ...compromised skin samples with necrotic, inflammed or damaged areas, such as those from patients suffering skin conditions, which affect more than 900 million people annually. We evaluated the impact of the biopsy size and tissue preservation method on the quality and quantity of RNA extracts.
Methods:
Skin lesion biopsies were obtained from patients with cutaneous leishmaniasis (CL). Biopsy specimens of 2 mm (n = 10) and 3 mm (n = 59) were preserved in Allprotect® reagent, and 4 mm biopsies in OCT (n = 54). Quality parameters were evaluated using Nanodrop and Bioanalyzer. The informativeness of the extracted samples for downstream analyses was evaluated using RT-qPCR and RNA-Seq.
Results:
The success rate, based on quality parameters of RNA extraction from tissue biopsies stored in OCT and 2 mm biopsies stored in Allprotect®, was 56% (30/54) and 30% (3/10), respectively. For 3 mm skin biopsies stored in Allprotect® was 93% (55/59). RNA preparations from 3 mm-Allprotect® biopsies had an average RIN of 7.2 ± 0.7, and their integrity was not impacted by sample storage time (up to 200 days at -20°C). RNA products were appropriate for qRT-PCR and RNA-seq. Based on these results, we propose a standardized method for RNA extraction from disrupted skin samples. This protocol was validated with lesion biopsies from CL patients (n = 30), having a success rate of 100%.
Conclusions:
Our results indicate that a biopsy size of 3 mm in diameter and preservation in Allprotect® for up to 200 days at -20°C, are best to obtain high quality RNA preparations from ulcerated skin lesion biopsy samples.
Early host-pathogen interactions drive the host response and shape the outcome of natural infections caused by intracellular microorganisms. These interactions involve a number of immune and ...non-immune cells and tissues, along with an assortment of host and pathogen-derived molecules. Our current knowledge has been predominantly derived from research on the relationships between the pathogens and the invaded host cell(s), limiting our understanding of how microbes elicit and modulate immunological responses at the organismal level. In this study, we explored the early host determinants of healing and non-healing responses in human cutaneous leishmaniasis (CL) caused by
. We performed a comparative transcriptomic profiling of peripheral blood mononuclear cells from healthy donors (PBMCs, n=3) exposed to promastigotes isolated from patients with chronic (CHR, n=3) or self-healing (SH, n=3) CL, and compared these to human macrophage responses. Transcriptomes of
infected PBMCs showed enrichment of functional gene categories derived from innate as well as adaptive immune cells signatures, demonstrating that
modulates adaptive immune cell functions as early as after 24h post interaction with PBMCs from previously unexposed healthy individuals. Among differentially expressed PBMC genes, four broad categories were commonly modulated by SH and CHR strains: cell cycle/proliferation/differentiation, metabolism of macromolecules, immune signaling and vesicle trafficking/transport; the first two were predominantly downregulated, and the latter upregulated in SH and CHR as compared to uninfected samples. Type I IFN signaling genes were uniquely up-regulated in PBMCs infected with CHR strains, while genes involved in the immunological synapse were uniquely downregulated in SH infections. Similarly, pro-inflammatory response genes were upregulated in isolated macrophages infected with CHR strains. Our data demonstrate that early responses during
infection extend beyond innate cell and/or phagocytic host cell functions, opening new frontiers in our understanding of the triggers and drivers of human CL.
The inflammatory response is prominent in the pathogenesis of dermal leishmaniasis. We hypothesized that regulatory T cells (Tregs) may be diminished in chronic dermal leishmaniasis (CDL) and ...contribute to healing during treatment.
The frequency and functional capacity of Tregs were evaluated at diagnosis and following treatment of CDL patients having lesions of ≥6 months duration and asymptomatically infected residents of endemic foci. The frequency of CD4(+)CD25(hi) cells expressing Foxp3 or GITR or lacking expression of CD127 in peripheral blood was determined by flow cytometry. The capacity of CD4(+)CD25(+) cells to inhibit Leishmania-specific responses was determined by co-culture with effector CD4(+)CD25(-) cells. The expression of FOXP3, IFNG, IL10 and IDO was determined in lesion and leishmanin skin test site biopsies by qRT-PCR. Although CDL patients presented higher frequency of CD4(+)CD25(hi)Foxp3(+) cells in peripheral blood and higher expression of FOXP3 at leishmanin skin test sites, their CD4(+)CD25(+) cells were significantly less capable of suppressing antigen specific-IFN-γ secretion by effector cells compared with asymptomatically infected individuals. At the end of treatment, both the frequency of CD4(+)CD25(hi)CD127(-) cells and their capacity to inhibit proliferation and IFN-γ secretion increased and coincided with healing of cutaneous lesions. IDO was downregulated during healing of lesions and its expression was positively correlated with IFNG but not FOXP3.
The disparity between CD25(hi)Foxp3(+) CD4 T cell frequency in peripheral blood, Foxp3 expression at the site of cutaneous responses to leishmanin, and suppressive capacity provides evidence of impaired Treg function in the pathogenesis of CDL. Moreover, the concurrence of increased Leishmania-specific suppressive capacity with induction of a CD25(hi)CD127(-) subset of CD4 T cells during healing supports the participation of Tregs in the resolution of chronic dermal lesions. Treg subsets may therefore be relevant in designing immunotherapeutic strategies for recalcitrant dermal leishmaniasis caused by Leishmania (Viannia) species.
Addition of the immunomodulator pentoxifylline (PTX) to antimonial treatment of mucosal leishmaniasis has shown increased efficacy. This randomized, double-blind, placebo-controlled trial evaluated ...whether addition of pentoxifylline to meglumine antimoniate (MA) treatment improves therapeutic response in cutaneous leishmaniasis (CL) patients. Seventy-three patients aged 18−65 years, having multiple lesions or a single lesion ≥ 3 cm were randomized to receive: intramuscular MA (20 mg/kg/day × 20 days) plus oral PTX 400 mg thrice daily (intervention arm, n = 36) or MA plus placebo (control arm, n = 37), between 2012 and 2015. Inflammatory gene expression was evaluated by RT-qPCR in peripheral blood mononuclear cells from trial patients, before and after treatment. Intention-to-treat failure rate was 35% for intervention vs. 25% for control (OR: 0.61, 95% CI: 0.21−1.71). Per-protocol failure rate was 32% for PTX, and 24% for placebo (OR: 0.50, 95% CI: 0.13−1.97). No differences in frequency or severity of adverse events were found (PTX = 142 vs. placebo = 140). Expression of inflammatory mediators was unaltered by addition of PTX to MA. However, therapeutic failure was associated with significant overexpression of il1β and ptgs2 (p < 0.05), irrespective of study group. No clinical benefit of addition of PTX to standard treatment was detected in early mild to moderate CL caused by Leishmania (V.) panamensis.
Antiretroviral treatment (ART) has contributed to increased life expectancy of HIV-1 infected children. In developed countries, an increasing number of children reaching adulthood are transferred to ...adult units. The objectives were to describe the demographic and clinical features, ART history, antiviral drug resistance and drug susceptibility in HIV-1 perinatally infected adolescents transferred to adult care units in Spain from the Madrid Cohort of HIV-1 infected children.
Clinical, virological and immunological features of HIV-1 vertically infected patients in the Madrid Cohort of HIV-infected children were analyzed at the time of transfer. Pol sequences from each patient were recovered before transfer. Resistance mutations according to the InternationaI AIDS Society 2011 list were identified and interpreted using the Stanford algorithm. Results were compared to the non-transferred HIV-1 infected pediatric cohort from Madrid.
One hundred twelve infected patients were transferred to adult units between 1997 and 2011. They were mainly perinatally infected (93.7%), with a mean nadir CD4+-T-cells count of 10% and presented moderate or severe clinical symptoms (75%). By the time of transfer, the mean age was 18.9 years, the mean CD4+T-cells count was 627.5 cells/ml, 64.2% presented more than 350 CD4+T-cells/ml and 47.3% had ≤ 200 RNA-copies/ml. Most (97.3%) were ART experienced receiving Highly Active ART (HAART) (84.8%). Resistance prevalence among pretreated was 50.9%, 76.9% and 36.5% for Protease Inhibitors (PI), Nucleoside Reverse Transcriptase Inhibitors (NRTI) and Non-NRTI (NNRTI), respectively. Resistance mutations were significantly higher among transferred patients compared to non-transferred for the PI+NRTI combination (19% vs. 8.4%). Triple resistance was similar to non-transferred pediatric patients (17.3% vs. 17.6%).
Despite a good immunological and virological control before transfer, we found high levels of resistance to PI, NRTI and triple drug resistance in HIV-1 infected adolescents transferred to adult units.
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