Subpopulation Level Research Articles

Prevalence of lymphopenia in the American population: Insights from demographic, BMI, and lifestyle factors.

To determine the difference in the prevalence of lymphopenia in the American population according to demographic characteristics, body mass index (BMI) and living habits. A total of 33,365 participants aged over 1 were included in the 2009-2018 National Health and Nutrition Survey (NHANES). All analyses used weighted samples and considered the layering and clustering of the design. Using white participants as a reference, the prevalence of lymphopenia in Mexican-American participants was significantly lower than that of white participants (P = 0.018). There was no significant difference in the prevalence of lymphopenia between black participants (P = 0.376) and white participants. The prevalence of lymphopenia was 1.81% (95%CI, 1.53%-2.10%) for white participants, 1.08% (95%CI, 0.78%-1.39%) for black participants, and 0.42% (95%CI, 0.17%-0.68%) for Mexican-American participants. The prevalence of lymphopenia increases with age, reaching a peak of 6.84% among elderly participants aged 75 and above. In terms of the gender difference, the prevalence of lymphopenia in men is significantly higher than that in women (P<0.001). Individuals who smoke (P<0.001), consume alcohol (P = 0.032), engage in regular exercise (P = 0.031), have sleep disorders (P<0.001) and those classified as having an unhealthy weight (P<0.001) had a higher average lymphocyte count. The prevalence of lymphopenia in participants with sleep disorders is significantly higher than those without sleep disorders (P = 0.014). However, no significant differences were observed among the classification variables of smoking, drinking, exercise, and BMI. In the diagnosis and treatment of lymphopenia, clinicians should consider the influence of factors such as race, gender, age, sleep disorders, and other unhealthy lifestyle habits to improve the accuracy of diagnosis and treatment, thereby reducing the high mortality risk associated with lymphopenia. Consequently, we propose a novel perspective that the diagnosis and treatment of lymphopenia should be tailored to the lymphocyte levels of specific subpopulations, rather than applying a generalized approach.

Characterizing local pig breeds as reservoirs for the domestic pig genetic variability worldwide via contributions to gene diversity and allelic richness.

Contributions to gene diversity and allelic richness were computed in a total of 2,260 domestic pig genotypes, sharing 53,626 autosomal SNPs, belonging to 98 pig subpopulations worldwide (41 Asian, 696 genotypes; 20 American, 262 genotypes; and 37 European, 686 genotypes), using 616 pig samples belonging to six different Cosmopolitan pig breeds as an outgroup, to ascertain if local pig subpopulation can be considered reservoirs of genetic diversity for the whole domestic pig species worldwide. Assessments were carried out for the whole dataset and separately for the American-European and Asian subsets. Effective population size was computed at the subpopulation level using molecular coancestry and linkage disequilibrium information to ensure that estimates of contributions to diversity were not affected by demographic issues. Most American and European pig subpopulations tended to have favorable contributions to both gene diversity and allelic richness. However, contributions to allelic richness were more consistent than those obtained for gene diversity, whether the computations are performed using either the whole dataset or the American-Asian subset, suggesting that allelic richness can be a key parameter to identify putative reservoirs for the species. The Asian pig subpopulations never contributed favorably to the allelic richness of the domestic pig metapopulation. Although these results can partially be explained by the highly divergent origins of the American-European and the Asian pig subpopulations, it cannot be discarded that the results obtained for the Asian subpopulations are biased due to a worse calling performance of the pig SNP arrays used for genotyping. The use of other potentially less biased sources of genotypic information is advisable to compare the Asian and American-European pig subpopulations genetic diversity.

High prevalence of polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae and its within-host evolution to resistance among critically ill scenarios.

We aimed to explore the prevalence and within-host evolution of resistance in polymyxin-heteroresistant carbapenem-resistant Klebsiella pneumoniae (PHR-CRKP) in critically ill patients. We performed an epidemiological analysis of consecutive patients with PHR-CRKP from clinical cases. Our study investigated the within-host resistance evolution and its clinical significance during polymyxin exposure. Furthermore, we explored the mechanisms underlying the dynamic evolution of polymyxin resistance at both subpopulation and genetic levels, involved population analysis profile test, time-killing assays, competition experiments, and sanger sequencing. Additionally, comparative genomic analysis was performed on 713 carbapenemase-producing K. pneumoniae strains. We enrolled 109 consecutive patients, and PHR-CRKP was found in 69.7% of patients without previous polymyxin exposure. 38.1% of PHR-CRKP isolates exhibited polymyxin resistance and led to therapeutic failure in critically ill scenarios. An increased frequency of resistant subpopulations was detected during PHR-CRKP evolution, with rapid regrowth of resistant subpopulations under high polymyxin concentrations, and a fitness cost in an antibiotic-free environment. Mechanistic analysis revealed that diverse mgrB insertions and pmrB hypermutations contributed to the dynamic changes in polymyxin susceptibility in dominant resistant subpopulations during PHR evolution, which were validated by comparative genomic analysis. Several deleterious mutations (e.g. pmrBLeu82Arg, pmrBSer85Arg) were firstly detected during PHR-CRKP evolution. Indeed, specific sequence types of K. pneumoniae demonstrated unique deletions and deleterious mutations. Our study emphasizes the high prevalence of pre-existing heteroresistance in CRKP, which can lead to polymyxin resistance and fatal outcomes. Hence, it is essential to continuously monitor and observe the treatment response to polymyxins in appropriate critically ill scenarios.

A predictive model for progression to clinical arthritis in at-risk individuals with arthralgia based on lymphocyte subsets and ACPA.

The presence of autoantibodies against citrullinated proteins (ACPA) significantly increases the risk of developing rheumatoid arthritis (RA). Dysregulation of lymphocyte subpopulations was previously described in RA. To propose the predictive model for progression to clinical arthritis based on peripheral lymphocyte subsets and ACPA in individuals who are at risk of RA. Our study included 207 at-risk individuals defined by the presence of arthralgias and either additional ACPA positivity or meeting the EULAR definition for clinically suspect arthralgia. For the construction of predictive models, 153 individuals with symptom duration ≥12 months who have not yet progressed to arthritis were included. The lymphocyte subsets were evaluated using flow cytometry and anti-CCP using ELISA. Out of all individuals with arthralgia, 41 progressed to arthritis. A logistic regression model with baseline peripheral blood lymphocyte subpopulations and ACPA as predictors was constructed. The resulting predictive model showed that high anti-CCP IgG, higher percentage of CD4+ T cells, and lower percentage of T and NK cells increased the probability of arthritis development. Moreover, the proposed classification decision tree showed, that individuals having both high anti-CCP IgG and low NK cells have the highest risk of developing arthritis. We propose a predictive model based on baseline levels of lymphocyte subpopulations and ACPA to identify individuals with arthralgia with the highest risk of progression to clinical arthritis. The final model includes T cells and NK cells, which are involved in the pathogenesis of RA. This preliminary model requires further validation in larger at-risk cohorts.

Spatio-temporal metapopulation trends: The coconut crabs of Zanzibar.

Species experience a variety of environmental and anthropogenic conditions across their ranges leading to spatial variation in population dynamics. Understanding population dynamics under different conditions is important but it is challenging to allocate limited effort to spatial and temporal subpopulation monitoring. Using GLMMs, we analyze survey data of a metapopulation of coconut crabs spanning 7 years and 15 sites in and near the Pemba archipelago, Zanzibar, to estimate trends in population size (based on catch per unit effort), weight and sex ratio at the meta- and subpopulation level and investigate anthropogenic drivers of these trends. We found that the overall metapopulation has remained stable in terms of size and composition over the survey period, but observed diverging trends in population size and sex ratio at some subpopulations. Formal protection of sites was associated with positive population trends. Of nine sites for which we could estimate site-specific trends, three showed increasing and two decreasing trends, whereas four sites had stable subpopulations. Although anthropogenic factors affected the average weight, and the incidence of small and large individuals, we found no temporal trends in any weight-related measures. Furthermore, there were no apparent patterns between weight-related measures and subpopulation trends. The metapopulation was biased toward males, and exploitation appeared to be associated with declining trends in the proportion of females, likely an artifact of a strong decline in the proportion of females in one of only two exploited sites in the dataset. Educational campaigns implemented in 2020 at six sites were not related to higher population sizes in later surveys. The variable trends in subpopulation sizes and composition highlight the need for spatially replicated monitoring in metapopulations. The analyses further provide a detailed baseline for future subpopulation studies of this vulnerable species in one of its last remaining metapopulations in the Western Indian Ocean.

A systematic review of validity of US survey measures for assessing substance use and substance use disorders

BackgroundThe steep rise in substance use and substance use disorder (SUD) shows an urgency to assess its prevalence using valid measures. This systematic review summarizes the validity of measures to assess the prevalence of substance use and SUD in the US estimated in population and sub-population-based surveys.MethodsA literature search was performed using nine online databases. Studies were included in the review if they were published in English and tested the validity of substance use and SUD measures among US adults at the general or sub-population level. Independent reviews were conducted by the authors to complete data synthesis and assess the risk of bias.ResultsOverall, 46 studies validating substance use/SUD (n = 46) measures were included in this review, in which 63% were conducted in clinical settings and 89% assessed the validity of SUD measures. Among the studies that assessed SUD screening measures, 78% examined a generic SUD measure, and the rest screened for specific disorders. Almost every study used a different survey measure. Overall, sensitivity and specificity tests were conducted in over a third of the studies for validation, and 10 studies used receiver operating characteristics curve.ConclusionFindings suggest a lack of standardized methods in surveys measuring and reporting prevalence of substance use/SUD among US adults. It highlights a critical need to develop short measures for assessing SUD that do not require lengthy, time-consuming data collection that would be difficult to incorporate into population-based surveys assessing a multitude of health dimensions.Systematic review registrationPROSPERO CRD42022298280.

High concentrations of Maraviroc do not alter immunological and metabolic parameters of CD4 T cells

Maraviroc (MVC) is an antiretroviral drug capable of binding to CCR5 receptors and block HIV entry into target cells. Moreover, MVC can activate NF-kB pathway and induce viral transcription in HIV-infected cells, being proposed as a latency reversal agent (LRA) in HIV cure strategies. However, the evaluation of immunological and metabolic parameters induced by MVC concentrations capable of inducing HIV transcription have not been explored in depth. We cultured isolated CD4 T cells in the absence or presence of MVC, and evaluated the frequency of CD4 T cell subpopulations and activation markers levels by flow cytometry, and the oxidative and glycolytic metabolic rates of CD4 T cells using a Seahorse Analyzer. Our results indicate that a high concentration of MVC did not increase the levels of activation markers, as well as glycolytic or oxidative metabolic rates in CD4 T cells. Furthermore, MVC did not induce significant changes in the frequency and activation levels of memory cell subpopulations. Our data support a safety profile of MVC as a promising LRA candidate since it does not induce alterations of the immunological and metabolic parameters that could affect the functionality of these immune cells.

Multi-index comprehensive evaluation of the efficacy and response mechanism of immunotherapy in non-small cell lung cancer.

This research conducted multi-index comprehensive evaluations of the immunotherapeutic efficacy and response in non-small cell lung cancer (NSCLC). Forty-five patients with epidermal growth factor receptor (EGFR)/anaplastic lymphoma kinase (ALK) wild-type advanced NSCLC who received immunotherapy were included. Immunohistochemistry was adopted to detect the expression levels of programmed death ligand 1 (PD-L1) with X-ray cross-complementing protein 1 (XRCC1) and excision repair cross-complementing group 1 (ERCC1) proteins in tumor tissues. Flow cytometry was utilized to measure the levels of T-cell subsets in peripheral blood before and after treatment. PCR-RELP method was employed to evaluate XRCC1 and ERCC1 gene polymorphisms in peripheral blood. According to the treatment effect, patients evaluated as complete response (CR), partial response (PR), and stable disease (SD) were categorized into the immune response group, and patients evaluated as progressive disease (PD) were categorized into the immune unresponsive group. The correlation between PD-L1 protein expression, XRCC1 and ERCC1 protein expression, gene polymorphisms, T-cell subpopulation levels, and treatment efficacy was analyzed. The therapeutic efficacy of patients with positive PD-L1 expression was better than that of patients with negative PD-L1 expression (P < 0.05). After treatment, peripheral blood CD3+ and CD4+ cell levels and Thl/Th2 cell levels were higher and CD8+ T cells were lower in the immune response group than in the immune unresponsive group (P < 0.05). Among the patients in the immune response group, peripheral blood CD3+ and CD4+ cell levels were higher and CD8+ T cells were lower in patients with positive PD-L1 expression than in patients with negative PD-L1 expression (P < 0.05). In the XRCC1 gene, the proportion of patients in the immune response group carrying the Arg/Trp + Trp/Trp genotype was higher than that of patients in the immune unresponsive group (P < 0.05). In the ERCC1 gene, the proportion of patients in the immune response group carrying the C/T + T/T genotype was higher than that of patients in the immune unresponsive group (P < 0.05). The positive expression rates of XRCC1 and ERCC1 in patients in the immune unresponsive group were higher than those in the immune response group (P < 0.05). PD-L1 protein expression, XRCC1 and ERCC1 protein expression, and gene polymorphisms are associated with immunotherapy outcome in EGFR/ALK wild-type advanced NSCLC patients, and may be biological indicators for predicting immunotherapy outcome in EGFR/ALK wild-type advanced NSCLC patients.

Temporal and Spatial Influences on Fawn Summer Survival in Pronghorn Populations: Management Implications from Noninvasive Monitoring.

Monitoring vital rates allows managers to estimate trends in growth rates of ungulate populations. However, connecting the influence of nutrition on ungulate demography is challenging. Noninvasive sampling offers a low-cost, low-effort alternative for measuring nutritional indices, allowing for an increased understanding of the mechanistic relationships between environmental factors, nutrition, and specific population vital rates. We examined the temporal influence of intrinsic and extrinsic factors on pronghorn (Antilocapra americana) fawn recruitment. We collected fresh fecal samples from adult female pronghorn in five subpopulations spanning three sampling periods associated with critical maternal life-history stages (late gestation, early lactation, breeding season) for 2 years to investigate both intra- and interannual influences. Intrinsic factors were fecal glucocorticoid metabolites (FGMs), nutritional indices (fecal nitrogen (FN) and 2,6-diaminopimelic acid (DAPA)), and dietary composition (protein intake of forbs, graminoids, legumes, other, shrubs), while the extrinsic factor was vegetative greenness (normalized difference vegetation index (NDVI)). We found variations in DAPA, protein intake of forbs, variation in forb protein intake, and protein intake of legumes during late gestation positively influenced fawn recruitment. Fecal nitrogen during early lactation showed the strongest positive influence on the recruitment of any measured parameter. Finally, breeding season NDVI and the variation in DAPA values positively influenced the subsequent year's fawn recruitment. Our longitudinal study enabled us to investigate which parameter was most important to specific periods of fawn development and recruitment. We combined the results across five subpopulations, but interpretation and subsequent management decisions should be made at the subpopulation level such that pronghorn subpopulations with low recruitment can be positively influenced by increasing nitrogen on the landscape available to adult females during the early lactation period. As the use of noninvasive monitoring methods continues to expand, we believe our methodologies and results can be broadly applied to other ungulate monitoring programs.

Abstract PO4-03-09: Profile of circulating natural killer cells in stage III triple negative breast cancer before and after neoadjuvant chemotherapy

Abstract Background: Triple negative breast cancer (TNBC) is the most aggressive subtype of breast cancer. The treatment for locally advanced disease is based on neoadjuvant chemotherapy and immunotherapy, with complete pathological response (pCR) serving as a predictor of disease-free survival. Although the presence of immune cell infiltration in the tumor has a favorable prognostic impact, there is a lack of predictive biomarkers for complete pathological response to neoadjuvant chemotherapy (NAC). Objective: to analyze the profile of circulating Natural Killer (NK) cells and NK subpopulations (NK FAS+ and NK PD1+) in association with the occurrence of complete pathological response after neoadjuvant chemotherapy in women with clinical stage III TNBC. Methods: This was a longitudinal prospective and translational cohort study, including women aged 18 to 60 years with stage III TNBC diagnosed between March 2015 and July 2017, with a follow-up of 24 months. A comparison control group consisted of 30 healthy women aged 18 to 60 years without prior or current cancer diagnosis and no family history of breast cancer. Peripheral blood samples were collected before NAC and before surgery. Flow cytometry was used to analyze the percentage levels of NK cells and NK subpopulations (NK FAS+ and NK PD1+). The Shapiro-Wilk test was initially applied to assess the normality of quantitative variables. The non-parametric Mann-Whitney test was used for comparison between two groups. The statistical significance level was set at p&amp;lt; 0.05. Overall survival (OS) was estimated using the Kaplan-Meier method. Statistical analysis was performed using GraphPad Prism v9.5.1. Results: The median age was 45 years. Stage IIIA was identified in 28% of the patients, while IIIB was observed in 72%. Regarding pathological response, 14 patients (48%) achieved pCR, while 15 patients (52%) had a non-complete pathological response (non-pCR). The 2-year OS was 86%, with no median reached. Higher percentage levels of NK cells, NK FAS+, and NK PD1+ were observed in the patient group compared to the control group, with significant differences. There was a significant increase in the percentage levels of NK cells after NAC compared to pre-NAC levels, and a reduction in NK PD1+ levels. NK cell levels were significantly higher in patients with stage IIIA compared to stage IIIB, while NK FAS+ levels were lower. NK cell and NK FAS+ subpopulation levels were significantly higher in patients who achieved pCR compared to non-pCR patients, with no difference in NK PD1+ cell levels. Conclusions: Elevated levels of circulating NK cells and NK FAS+ subpopulation collected from peripheral blood before NAC may serve as potential biomarkers for pCR in patients with TNBC. Citation Format: Luiz Jose de Barros Batista, Jurema Telles, Candice Santos, Ariani Souza, Marcelo Salgado, Leuridan Torres. Profile of circulating natural killer cells in stage III triple negative breast cancer before and after neoadjuvant chemotherapy [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-03-09.

Cell-Mediated Immunity in Multiple Sclerosis Patients Who Discontinued Therapy with an Integrin Inhibitor

Introduction. Natalizumab (NTZ) is a humanized monoclonal antibody (mAb) that selectively inhibits α4-integrin adhesion molecule located on the surface of lymphocytes and prevents their trafficking into the central nervous system (CNS).&#x0D; The aim of this study was to identify characteristics of lymphocyte population and subpopulation pattern in the peripheral blood (PB) of multiple sclerosis (MS) patients who discontinued NTZ due to an increased risk of developing developing progressive multifocal leukoencephalopathy.&#x0D; Materials and methods. We conducted an open-label prospective observational study in 26 MS patients. Of those, 6 patients had rapidly progressive MS, 10 patients discontinued NTZ and had confirmed relapses afterwards, and 10 patients received NTZ and had no relapses during the washout period. Ten apparently healthy individuals were used as controls. Cell-mediated immunity parameters were evaluated by flow cytometry using a panel of mAbs to differentiation antigens of PB lymphocytes.&#x0D; Results. Patients who discontinued NTZ had significantly decreased absolute lymphocyte counts in PB, decreased T-cytotoxic, NKT and B1 lymphocyte subpopulation levels, and decreased activated T-cell (CD3+HLA–DR+) levels, which may be related to their redistribution, passing through the blood-brain barrier, and trafficking into the central nervous system. CD20+ В-cell levels did not differ from normal. Additional immune predictors of MS relapses after NTZ discontinuation can include decreased absolute count of PB lymphocytes and decreased percentage of CD3+CD8+ T-cell, NKT-cell, and B1-cell (CD19+CD5+) subpopulations. Significantly increased levels of CD25+- and CD38+-activated B-cells compared with the normal levels in naïve patients and subjects without relapses after NTZ discontinuation may suggest a high activation potential of the circulating B-cell pool and, therefore, a high risk of MS relapses.&#x0D; Conclusions. The changes in the lymphocyte subpopulation pattern in the PB of MS patients after NTZ discontinuation may have a prognostic value for assessing the risk of relapses; they justified switching patients to anti-B-cell therapy.

Novel typing scheme reveals emergence and genetic diversity of Chlamydia pecorum at the local management scale across two koala populations

To overcome shortcomings in discriminating Chlamydia pecorum strains infecting the koala (Phascolarctos cinereus) at the local level, we developed a novel genotyping scheme for this pathogen to inform koala management at a fine-scale subpopulation level. We applied this scheme to two geographically distinct koala populations in New South Wales, Australia: the Liverpool Plains and the Southern Highlands to South-west Sydney (SHSWS). Our method provides greater resolution than traditional multi-locus sequence typing, and can be used to monitor strain emergence, movement, and divergence across a range of fragmented habitats. Within the Liverpool Plains population, suspected recent introduction of a novel strain was confirmed by an absence of genetic diversity at the earliest sampling events and limited diversity at recent sampling events. Across the partially fragmented agricultural landscape of the Liverpool Plains, diversity within a widespread sequence type suggests that this degree of fragmentation may hinder but not prevent spread. In the SHSWS population, our results suggest movement of a strain from the south, where diverse strains exist, into a previously Chlamydia-free area in the north, indicating the risk of expansion towards an adjacent Chlamydia-negative koala population in South-west Sydney. In the south of the SHSWS where koala subpopulations appear segregated, we found evidence of divergent strain evolution. Our tool can be used to infer the risks of strain introduction across fragmented habitats in population management, particularly through practices such as wildlife corridor constructions and translocations.

Analysis of the Effects of the Addition and Subtraction Therapy of Jianpi Jiedu Decoction on T-lymphocyte Subpopulations’ Changes in Colorectal Cancer Patients’ Clinical Symptoms

Objective: To investigate the clinical efficacy of applying the addition and subtraction of Jianpi Jiedu decoction (JPJD) in patients suffering from colorectal cancer and changes in their clinical symptoms. Methods: Seventy-two colorectal cancer cases were selected and randomly divided into the control group and the observation group, where both groups received the same comprehensive treatment and chemotherapy intervention after surgery. The observation group received the addition and subtraction therapy of JPJD based on the control group. The Chinese medicine (TCM) symptom scores, the intestinal bacterial flora, and the changes in the T-lymphocyte subsets of the two groups were compared. Results: The observation group had lower TCM evidence points, more Escherichia coli, less Bifidobacterium and Lactobacillus, and higher T-lymphocyte subpopulation levels than that of the control group after treatment (P &lt; 0.05). Conclusion: The application of addition and subtraction therapy of JPJD in conjunction with postoperative radiotherapy for colorectal cancer patients further improved the patient's clinical symptoms and intestinal flora environment, and effectively enhanced their immunity. which is worthy of promotion.

Bioinformatics-based screening and analysis of the key genes involved in the influence of antiangiogenesis on myeloid-derived suppressor cells and their effects on the immune microenvironment.

This study aimed to screen differentially expressed genes (DEGs) involved in the influence of antiangiogenic therapy on myeloid-derived suppressor cell (MDSC) infiltration and investigate their mechanisms of action. Data on DEGs after the action of antiangiogenic drugs in a pan-cancer context were obtained from the Gene Expression Omnibus (GEO) database. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses were performed using the clusterProfiler package in R software. Single-sample gene set enrichment analysis was performed using the gene set variation analysis package to evaluate the levels of immune cells and the activity of immune-related pathways. The relationships of DEGs with the infiltration levels of MDSCs and specific immune cell subpopulations were investigated via gene module analysis. The top 10 key genes were subsequently obtained from PPI network analysis using the cytoHubba plugin of the Cytoscape platform. When the DEGs of the four datasets were intersected, a DEG in the intersection of three datasets and 12 DEGs in the intersection of two datasets were upregulated, and 28 DEGs in the intersection of two datasets were downregulated. GO and KEGG pathway enrichment analyses revealed that the DEGs were associated with multiple important signaling pathways closely related to tumor onset and development, including cell differentiation, cell proliferation, the cell cycle, and immune responses. Most downregulated genes in lung adenocarcinoma (LUAD) were positively correlated with MDSC expression. Only MGP was negatively correlated; the correlation between CACNG6 and MDSC expression was statistically insignificant. In lung squamous cell carcinoma (LUSC), the relationships of PMEPA1, PCDH7, NEURL1B, and CACNG6 with MDSC expression were statistically insignificant; MGP was negatively correlated with MDSC expression. The top 10 key genes with the highest degree scores obtained using the cytoHubba plugin of Cytoscape were AURKB, RRM2, BUB1, NUSAP1, PRC1, TOP2A, NCAPH, CENPA, KIF2C, and CCNA2. Most of these genes were upregulated in LUAD and associated with immune cell infiltration and prognosis in tumors. An analysis of the relationships between DEGs and infiltration by other specific immune cells revealed the presence of consistent patterns in the downregulated genes, which exhibited positive correlations with the levels of Th2 cells, γδ T cells, and CD56dim NK cells, and negative correlations with other infiltrating immune cells. Antiangiogenic therapy may regulate MDSC infiltration through multiple important signaling pathways closely associated with tumor onset and development, such as cell differentiation, cell proliferation, the cell cycle, and immune responses. Antiangiogenic drugs may exert effects by affecting various types of infiltrating cells associated with immune suppression.

Leukocytes and lactate responses to cycling and running at the same target heart rate

AbstractHeart Rate (HR) is widely used for erobic exercise intensity prescriptions and/or studies of exercise training. It is often assumed that exercising at a given HR results in similar physiological response, regardless of exercise modality. This study aimed to gauge cellular immune mobilization to submaximal exercise at a given target HR on a cycle ergometer (CE) and treadmill (TM). Thirteen healthy male adults (23.2 ± 3.5 y.o) completed 4 laboratory visits. Participants performed two graded exercise tests to exhaustion on CE and TM and two 30‐min constant exercise challenges at 70% HR reserve on CE or TM in random order. Rating of Perceived Exertion (RPE) was recorded every 5 min, and blood was drawn before and after exercise to measure leukocytes subpopulation levels, lactate, and IL‐6. HR was successfully “clamped” during the exercise in CE and TM (CE 156.7 ± 1.1; TM 159.3 ± 1.6 bpm). Cycling was perceived as more strenuous than running and was accompanied by a greater increase in lactate post‐exercise (p &lt; 0.0001; 6.2 ± 0.3 vs. 2.9 ± 0.3 mmol/L). IL‐6 and leukocytes subpopulations were significantly elevated post‐exercise (p &lt; 0.003) with no difference between exercise modalities (monocytes; CE 57.6% TM 61.2%, granulocytes; CE 41.37%, TM 50.1%, lymphocytes; CE 91.03%, TM 78.8%). The findings revealed that HR is not sufficient in and of itself to fully assess the metabolic stress associated with a given exercise modality. However, despite different metabolic and subjective stress, the IL‐6 and leukocyte counts relative changes were similar in the two modalities.

Association of lymphocyte subsets with the efficacy and prognosis of PD‑1 inhibitor therapy in advanced gastric cancer: results from a monocentric retrospective study

PurposeThis retrospective study aimed to investigate the changes in peripheral blood lymphocyte subsets before and after immunotherapy in patients with advanced gastric cancer and their relationship n with the therapeutic efficacy and clinical prognosis.MethodsPeripheral blood lymphocyte subsets, including CD4 + T cells, CD8 + T cells, CD4+/CD8 + ratio, NK cells, Treg cells, and B cells, were collected from 195 patients with advanced gastric cancer who were admitted to the First Hospital of Shanxi Medical University with immunotherapy from January 2020 to October 2021, at the time of diagnosis of advanced gastric cancer, before immunotherapy and after 3 cycles of immunotherapy. T-tests were used to examine the factors influencing the patients’ peripheral blood lymphocyte subsets and the changes after immunotherapy. To examine the relationship between lymphocyte subsets and treatment outcomes, ROC curves were plotted using a logistic regression. Kaplan–Meier curve was drawn, and the Log Rank test was carried out to compare the differences in PFS between the different groups. Cox proportional hazards regression model was used to analyze the factors affecting PFS after calibration of other variables.ResultsThe proportion of peripheral blood lymphocyte subsets in patients with advanced gastric cancer was affected by age and PD-L1 level. Compared to the baseline, the treatment effective group had higher proportions of CD4 + T cells, a higher CD4+/CD8 + ratio, NK cells and Treg cells, and lower proportions of CD8 + T cells and B cells in the peripheral blood after three cycles of immunotherapy. In the treatment-naive group, there were no significant differences in the lymphocyte subsets. With cut-off values of 30.60% and 18.00%, baseline CD4 + T cell and NK cell ratios were independent predictors of immunotherapy efficacy and PFS. Treg cell ratio, gender, PD-L1 levels, and MMR status all predicted PFS independently.ConclusionThe proportion of peripheral blood lymphocyte subsets was modified in patients who responded to PD-1 inhibitors. Different lymphocyte subpopulation levels can be used as biomarkers to predict immunotherapy efficacy and clinical prognosis in patients with advanced gastric cancer.

Time-stratified case-crossover studies for aggregated data in environmental epidemiology: a tutorial.

The case-crossover design is widely used in environmental epidemiology as an effective alternative to the conventional time-series regression design to estimate short-term associations of environmental exposures with a range of acute events. This tutorial illustrates the implementation of the time-stratified case-crossover design to study aggregated health outcomes and environmental exposures, such as particulate matter air pollution, focusing on adjusting covariates and investigating effect modification using conditional Poisson regression. Time-varying confounders can be adjusted directly in the conditional regression model accounting for the adequate lagged exposure-response function. Time-invariant covariates at the subpopulation level require reshaping the typical time-series data set into a long format and conditioning out the covariate in the expanded stratum set. When environmental exposure data are available at geographical units, the stratum set should combine time and spatial dimensions. Moreover, it is possible to examine effect modification using interaction models. The time-stratified case-crossover design offers a flexible framework to properly account for a wide range of covariates in environmental epidemiology studies.

Flow cytometry of DNMT1 as a biomarker of hypomethylating therapies.

The 5-azacytidine (AZA) and decitabine (DEC) are noncytotoxic, differentiation-inducing therapies approved for treatment of myelodysplastic syndrome, acute myeloid leukemias (AML), and under evaluation as maintenance therapy for AML postallogeneic hematopoietic stem cell transplant and to treat hemoglobinapathies. Malignant cell cytoreduction is thought to occur by S-phase specific depletion of the key epigenetic regulator, DNA methyltransferase 1 (DNMT1) that, in the case of cancers, thereby releases terminal-differentiation programs. DNMT1-targeting can also elevate expression of immune function genes (HLA-DR, MICA, MICB) to stimulate graft versus leukemia effects. In vivo, there is a large inter-individual variability in DEC and 5-AZA activity because of pharmacogenetic factors, and an assay to quantify the molecular pharmacodynamic effect of DNMT1-depletion is a logical step toward individualized or personalized therapy. We developed and analytically validated a flow cytometric assay for DNMT1 epitope levels in blood and bone marrow cell subpopulations defined by immunophenotype and cell cycle state. Wild type (WT) and DNMT1 knock out (DKO) HC116 cells were used to select and optimize a highly specific DNMT1 monoclonal antibody. Methodologic validation of the assay consisted of cytometry and matching immunoblots of HC116-WT and -DKO cells and peripheral blood mononuclear cells; flow cytometry of H116-WT treated with DEC, and patient samples before and after treatment with 5-AZA. Analysis of patient samples demonstrated assay reproducibility, variation in patient DNMT1 levels prior to treatment, and DNMT1 depletion posttherapy. A flow-cytometry assay has been developed that in the research setting of clinical trials can inform studies of DEC or 5-AZA treatment to achieve targeted molecular pharmacodynamic effects and better understand treatment-resistance/failure.

CCRG-04. AUTOMATED REAL TIME TRACKING OF GLIOBLASTOMA CELL CYCLE TRANSITIONS IN ASSEMBLOIDS LINKS G2 CELLS TO INFILTRATION

Abstract Glioblastoma (GB) is the most aggressive primary brain tumour in adults associated with survival rates below 5% despite treatment. Tumour repopulating residual cells in the surgical cavity resist treatment due to their molecular heterogeneity. Single-cell transcriptomic studies have linked GB heterogeneity to the cell cycle; however, it remains unclear to what extent cell cycle states affect the infiltrative capacity of GB. The “go or grow” paradigm predicts that migration and proliferation are separate spatiotemporal events. We used a brain tumour-cerebral organoid (assembloid) approach to test the migratory potential of GB cell cycle phases during assembloid infiltration. Human induced pluripotent cells were differentiated into cerebral organoids that self-assemble with spheroids of patient-derived GB cells, expressing a fluorescent cell cycle reporter (FUCCI). Utilising live-cell confocal imaging, the process of GB assembloid infiltration was captured. Resultant videos were analysed with automated image analysis capable of tracking cell cycle transitions and cellular migration in confocal microscopy planes, enabling the longitudinal quantification of GB migration and invasion. Our analysis revealed a subset of highly infiltrative GB cells, containing a significant number of cycling G2 phase cells that demonstrated a ~1.4-fold increase in directional infiltrative capacity compared to their non cycling G1 phase counterparts. To isolate and characterize the G2 migratory GB cell state, we developed a phenotypic separation assay using microenvironmental cues. Specifically, we found that these cues markedly affect GB migration routes enabling the isolation of the highly migratory GB cells for further investigation. Our results question the “go or grow” hypothesis at the level of GB cellular subpopulations. We hypothesise that the observed G2 migratory capacity of GB cells could be critical to tumour recurrence following surgery. Further research is underway to define the underlying mechanisms of G2 GB cell migration.

The Role of Dorsal Raphe Nucleus Serotonergic Systems in Emotional Learning and Memory in Male BALB/c Mice

Selective serotonin reuptake inhibitors (SSRIs) are the first-line pharmacological treatment for a variety of anxiety-, trauma- and stressor-related disorders. Although they are efficacious, therapeutic improvements require several weeks of treatment and are often associated with an initial exacerbation of symptoms. The dorsal raphe nucleus (DR) has been proposed as an important target for the modulation of emotional responses and the therapeutic effects of SSRIs. Using a fear-conditioning paradigm we aimed to understand how SSRIs affect emotional learning and memory, and their effects on serotonergic circuitry. Adult male BALB/c mice were treated with vehicle (n = 16) or the SSRI fluoxetine (18 mg/kg/d) acutely (n = 16), or chronically (21d, n = 16), prior to fear conditioning. Treatment was stopped, and half of the mice (n = 8/treatment group) were exposed to cued fear memory recall 72 h later. Activation of DR serotonergic neurons during fear conditioning (Experiment 1) or fear memory recall (Experiment 2), was measured using dual-label immunohistochemistry for Tph2 and c-Fos. Acute and chronic fluoxetine treatment reduced associative fear learning without affecting memory recall and had opposite effects on anxiety-like behaviour. Acute fluoxetine decreased serotonergic activity in the DR, while chronic treatment led to serotonergic activity that was indistinguishable from that of control levels in DRD and DRV subpopulations. Chronic fluoxetine facilitated fear extinction, which was associated with rostral DRD inhibition. These findings provide further evidence that SSRIs can alter aspects of learning and memory processes and are consistent with a role for discrete populations of DR serotonergic neurons in regulating fear- and anxiety-related behaviours.