Immune Competence Research Articles

Immunomodulatory effects of fucoidan in recreationally active adult males undertaking 3-weeks of intensified training

ABSTRACT The aim of the current study was to determine whether daily fucoidan supplementation (Undaria pinnatifida extract containing >85% fucoidan, 1 g/day) for three-weeks in a double blind-placebo controlled cross-over trial (ACTRN12621000872831) could modulate alterations in faecal (calprotectin, lysozyme and IgA) and salivary (lactoferrin, lysozyme and IgA) markers of mucosal immune competence typically observed in response to both acute physical activity, and a period of intensified exercise training, in healthy recreationally active men (n = 12). Participants responded positively to the intensified training with 16–19% improvement in mean power that was not different between supplement groups. Faecal biomarkers and concentrations of lactoferrin, lysozyme and IgA from resting saliva samples were largely stable over the supplementation period. Concentrations of salivary biomarkers varied significantly over time in response to acute exercise, however differences between supplementation groups were modest. For salivary lysozyme, there was a trend for a lower magnitude of increase post-exercise (p = 0.08) and limited return towards pre-exercise in response to fucoidan. For salivary IgA, a greater acute exercise response was noted for IgA in response to fucoidan (~2.7-fold higher; p = 0.02). Different dosage and supplementation protocols and inclusion of additional immune markers should be considered in subsequent assessments of any potential benefits of fucoidan supplementation in healthy active adults.

Induction of long-term tolerance to a specific antigen using anti-CD3 lipid nanoparticles following gene therapy

Development of factor VIII (FVIII) inhibitors is a serious complication in the treatment of hemophilia A (HemA) patients. In clinical trials, anti-CD3 antibody therapy effectively modulates the immune response of allograft rejection or autoimmunediseases without eliciting major adverse effects. In this study, we delivered mRNA-encapsulated lipid nanoparticles (LNPs) encoding therapeutic anti-CD3 antibody (αCD3 LNPs) to overcome the anti-FVIII immune responses in HemA mice. It was found that αCD3 LNPs encoding the single-chain antibodies (Fc-scFv) can efficiently deplete CD3+ and CD4+ effector Tcells, whereas αCD3 LNPs encoding double-chain antibodies cannot. Concomitantly, mice treated with αCD3 (Fc-scFv) LNPs showed an increase in the CD4+CD25+Foxp3+ regulatory Tcell percentages, which modulated the anti-FVIII immune responses. All Tcells returned to normal levels within 2months. HemA mice treated with αCD3 LNPs prior to hydrodynamic injection of liver-specific FVIII plasmids achieved persistent FVIII gene expression without formation of FVIII inhibitors. Furthermore, transgene expression was increased and persistent following secondary plasmid challenge, indicating induction of long-term tolerance to FVIII. Moreover, the treated mice maintained their immune competence against other antigens. In conclusion, our study establisheda potential new strategy to induce long-term antigen-specific tolerance using an αCD3 LNP formulation.

CD8 + T-cell priming is quantitatively but not qualitatively impaired in people with HIV-1 on antiretroviral therapy.

The induction of de novo CD8 + T-cell responses is essential for protective antiviral immunity, but this process is often impaired in people with HIV-1 (PWH). We investigated the extent to which the immune competence of naive CD8 + T cells, a key determinant of priming efficacy, could be preserved or restored in PWH via long-term antiretroviral therapy (ART). We used flow cytometry, molecular analyses of gene transcription and telomere length, and a fully validated priming assay to characterize naive CD8 + T cells ex vivo and evaluate the induction of antigen-specific effector/memory CD8 + T cells in vitro , comparing age-matched healthy uninfected donors (HUDs), PWH on ART, and natural HIV-1 controllers (HICs). We found that naive CD8 + T cells were numerically reduced and exhibited a trend toward shorter telomere lengths in PWH on ART compared with HUDs and HICs. These features associated with impaired priming efficacy. However, we also found that naive CD8 + T cells were fully equipped proliferatively and transcriptionally in PWH on ART, enabling the generation of antigen-specific effector/memory CD8 + T cells with functional and phenotypic attributes comparable to those primed from HUDs. Our data suggest that naive CD8 + T cells in PWH on ART are intrinsically capable of generating functionally and phenotypically intact effector/memory CD8 + T cells in response to antigen, despite evidence of senescence and an overall numerical reduction that compromises priming efficacy relative to HUDs and HICs.

The Neutrophil-to-Lymphocyte Ratio (NLR) Can Predict Sepsis's Presence and Severity in Malnourished Infants-A Single Center Experience.

Sepsis represents one of the leading causes of death in newborns and infants, and prompt diagnosis is essential for achieving favorable outcomes. Regarding malnourished children with concurrent infection, most studies have focused, besides blood culture, on C-reactive protein and procalcitonin. Because malnutrition has a deleterious effect on cellular immune competence, the present study characterized the acute-phase response, including hematological indices, in response to sepsis. Among the examined laboratory biomarkers, procalcitonin and neutrophil-to-lymphocyte ratio were the most accurate discriminators between sepsis patients and those with bacterial infection. Moreover, these two parameters showed a gradual increase between sepsis, severe sepsis, and septic shock patients (p < 0.001). Subgroup analysis of the sepsis group revealed positive correlations of NLR with prolonged ICU stay (<0.001), acute organ dysfunction (0.038), mechanical ventilation (<0.001), and fatality (<0.001). In summary, our results suggest that the neutrophil-to-lymphocyte ratio can be used as an auxiliary diagnostic index in discriminating the presence and severity of bacterial sepsis in malnourished infants.

Associations between immune competence phenotype and stress response in sheep

Immune competence in domestic sheep is gaining more attention in genetic selection programs that seek to enhance flock immunity and animal welfare. A goal of many programs is to identify behavioral phenotypes that indicate stress-coping strength, as well as disease resilience. The current study set out to explore the relationship between immune responsiveness, physiological stress, and behavioral response among sheep that had been selected for ‘low’ (LR) or ‘high’ (HR) response to cell-mediated and antibody-mediate reactivity to a clostridial vaccine. Multiparous ewes were placed in four experiments which exposed the animals to various threats including, dog presence, human proximity, visual isolation from flock, and physical restraint. To evaluate the consistency of behavioral phenotypes, all ewes were placed in the test circuit one year later. Basal body temperature (via iButton) and serum cortisol concentrations were collected prior to and after the circuit each year. Immune group (HR vs. LR) was not found to be related to behavioral performance during the dog, human, isolation, or restraint challenge. Immune group categorization was also unrelated to pre- and post-cortisol concentrations, and the change in cortisol concentrations during testing. There was a negative relationship identified between response to visual isolation and change in cortisol response during testing, indicating that sheep which were more active or agitated during visual isolation from the flock experienced less of an increase in serum cortisol levels and were perhaps experiencing a negative, high arousal state compared to less reactive sheep (e.g., freeze behavior) (χ2(4, N=99) = 42.72, P &amp;lt;0.0001). There was also a post hoc, positive relationship identified between weight measurements and immune group, such that individuals with greater body weight were more likely to be in the high immune responsiveness group (P= 0.01). Specifically, for every unit increase in weight, there was a ~49% chance of being categorized in the HR group. This is relevant for selection programs because producers that seek to enhance immune responsiveness and performance may be able to select sheep that carry a greater body mass, and while not found in the present study, could lead to greater flock immunity.

Skin Cancer Microenvironment: What We Can Learn from Skin Aging?

Aging is a natural intrinsic process associated with the loss of fibrous tissue, a slower cell turnover, and a reduction in immune system competence. In the skin, the continuous exposition of environmental factors superimposes extrinsic damage, mainly due to ultraviolet radiation causing photoaging. Although not usually considered a pathogenic event, photoaging affects cutaneous biology, increasing the risk of skin carcinogenesis. At the cellular level, aging is typified by the rise of senescence cells a condition characterized by reduced or absent capacity to proliferate and aberrant hyper-secretory activity. Senescence has a double-edged sword in cancer biology given that senescence prevents the uncontrolled proliferation of damaged cells and favors their clearance by paracrine secretion. Nevertheless, the cumulative insults and the poor clearance of injured cells in the elderly increase cancer incidence. However, there are not conclusive data proving that aged skin represents a permissive milieu for tumor onset. On the other hand, tumor cells are capable of activating resident fibroblasts onto a pro-tumorigenic phenotype resembling those of senescent fibroblasts suggesting that aged fibroblasts might facilitate cancer progression. This review discusses changes that occur during aging that can prime neoplasm or increase the aggressiveness of melanoma and non-melanoma skin cancer.

Comprehensive Genome and Transcriptome Analysis Identifies SLCO3A1 Associated with Aggressive Behavior in Pigs.

Copy number variation (CNV) represents a significant reservoir of genetic diversity within the genome and exhibits a strong association with economically valuable traits in livestock. The manifestation of aggressive behavior in pigs has detrimental effects on production efficiency, immune competency, and meat quality. Nevertheless, the impact of CNV on the aggressive behavior of pigs remains elusive. In this investigation, we employed an integrated analysis of genome and transcriptome data to investigate the interplay between CNV, gene expression changes, and indicators of aggressive behavior in weaned pigs. Specifically, a subset of pigs comprising the most aggressive pigs (MAP, n = 12) and the least aggressive pigs (LAP, n = 11) was purposefully selected from a herd of 500 weaned pigs following a mixing procedure based on their composite aggressive score (CAS). Subsequently, we thoroughly analyzed copy number variation regions (CNVRs) across the entire genome using next-generation sequencing techniques, ultimately revealing the presence of 6869 CNVRs. Using genome-wide association study (GWAS) analysis and evaluating variance-stabilizing transformation (VST) values, we successfully identified distinct CNVRs that distinguished the MAP and LAP counterparts. Among the prioritized CNVRs, CNVR-4962 (designated as the top-ranked p-value and VST value, No. 1) was located within the Solute Carrier Organic Anion Transporter Family Member 3A1 (SLCO3A1) gene. The results of our analyses indicated a significantly higher (p < 0.05) copy number of SLCO3A1 in the MAP compared to the LAP. Furthermore, this increased copy number exhibited a positive correlation with the CAS of the pigs (p < 0.05). Furthermore, we integrated genomic data with transcriptomic data from the temporal lobe to facilitate the examination of expression quantitative trait loci (eQTL). Importantly, these observations were consistent with the mRNA expression pattern of SLCO3A1 in the temporal lobe of both MAP and LAP (p < 0.05). Consequently, our findings strongly suggest that CNVs affecting SLCO3A1 may influence gene expression through a dosage effect. These results highlight the potential of SLCO3A1 as a candidate gene associated with aggressive traits in pig breeding programs.

A „faggyú-immunbiológia” betegségszintű értelmezése

Sebaceous glands are not only producing lipids to contribute to the lipid barrier but are important in maintaining the homeostasis of the skin as well, which is greatly related to their immune competence. Our aim with this review is to present key studies on their possible involvement in inflammation and their crosstalk with various cell types of the immune system that altogether led to the introduction of “sebaceous-immunobiology” as a novel field in dermatology. With a focus on diseases considered to be related to sebaceous glands, such as acne, rosacea, seborrheic dermatitis, atopic dermatitis, psoriasis, lichen planopilaris, eosinophilic pustular folliculitis, and hidradenitis suppurativa, we also aim to put forward that sebaceous glands are not only innocent bystanders but active contributors in disease development.

Abstract P200: Tnf-α Blockade During Pregnancy Does Not Impact Offspring Development In A Rat Model Of Preeclampsia

Preeclampsia (PE), new onset hypertension during pregnancy, is associated with increased tumor necrosis factor alpha (TNF-α), agonistic autoantibodies to the angiotensin II type 1 receptor (AT1-AA), and low birth weight babies. We have previously shown that Etanercept ( E ), a TNF-α inhibitor, improves PE features in the reduced uterine perfusion pressure (RUPP) rat. However, the long-term effect of E on offspring health and immune competency has not been studied. Therefore, we hypothesize that E will improve maternal health without affecting offspring outcomes in a rat model of PE. To test this hypothesis, E (0.4 mg/kg) was injected on gestation day 18 into sham and RUPP dams which were allowed to deliver. Weights were recorded at birth and 16 weeks of age when mean arterial pressure (MAP) was measured. Whole blood was collected to measure immune cells by flow cytometry. AT1-AA was measured using a cardiomyocyte bioassay. A student’s t-test was used to compare differences among male and female RUPP offspring with and without E . RUPP male and female offspring were smaller (5.21±0.19 g, n=7; 5.24±0.10 g, n=7) than sham offspring (5.94±0.14 g, n=7, p&lt;0.05; 5.86±0.16 g, n=9, p&lt;0.01) while RUPP male offspring were larger and female offspring were unchanged following perinatal E (6.01±0.29 g, n=9, p&lt;0.05; 5.28±0.14, n=9). MAP was comparable between sham (133±4 mmHg) and RUPP (132±4 mmHg) male offspring with no change seen following perinatal E (133±4 mmHg). Perinatal E did not impact adult male circulating B cells. Circulating AT1-AA was elevated in male RUPP offspring (16±2 ΔBPM, p&lt;0.01) compared to sham and was not improved with E (13±2 ΔBPM). MAP was comparable in sham (131±5 mmHg) and RUPP (130±5 mmHg) female offspring but trended downward with perinatal E (123±4 mmHg). RUPP female offspring had reduced circulating B cells (4.24±0.81 % gated, p=0.066) compared to sham females (9.56±2.07 % gated) which was normalized following perinatal E (9.04±1.42 % gated, p&lt;0.05). AT1-AA trended lower following perinatal E (7±2 ΔBPM, p=0.0888) compared to RUPP female offspring (16±3 ΔBPM). Our findings indicate that E does not worsen fetal outcomes. Moreover, E improves male birth weight and reduces adult female AT1-AA without compromising offspring immune competency.

Helicase-like transcription factor (Hltf)-deletion activates Hmgb1-Rage axis and granzyme A-mediated killing of pancreatic β cells resulting in neonatal lethality.

Epigenetic mechanisms are integral to pancreatic β cell function. Promoter hypermethylation of the helicase like-transcription factor (HLTF) gene-a component of the cellular DNA damage response that contributes to genome stability-has been implicated in age-associated changes in β cells. To study HLTF, we generated global and β cell-specific (β) Hltf knockout (KO) immune competent (IC) and immune deficient (ID) Rag2-/IL2- mice. IC global and β Hltf KO mice were neonatal lethal whereas ID global and β Hltf KO newborn mice had normal survival. This focused our investigation on the effects of Rag2 interruption with common gamma chain interruption on β cell function/survival. Three-way transcriptomic (RNAseq) analyses of whole pancreata from IC and ID newborn β Hltf KO and wild type (Hltf +/+) controls combined with spatially resolved transcriptomic analysis of formalin fixed paraffin embedded tissue, immunohistochemistry and laser scanning confocal microscopy showed DNA damage caused by β Hltf KO in IC mice upregulated the Hmgb1-Rage axis and a gene signature for innate immune cells. Perforin-delivered granzyme A (GzmA) activation of DNase, Nme1, showed damaged nuclear single-stranded DNA (γH2AX immunostaining). This caspase-independent method of cell death was supported by transcriptional downregulation of Serpinc1 gene that encodes a serine protease inhibitor of GzmA. Increased transcriptional availability of complement receptors C3ar1 and C5ar1 likely invited crosstalk with Hmgb1 to amplify inflammation. This study explores the complex dialog between β cells and immune cells during development. It has implications for the initiation of type I diabetes in utero when altered gene expression that compromises genome stability invokes a localized inflammatory response.

Cannabis smoke suppresses antiviral immune responses to influenza A in mice.

Despite its increasingly widespread use, little is known about the impact of cannabis smoking on the response to viral infections like influenza A virus (IAV). Many assume that cannabis smoking will disrupt antiviral responses in a manner similar to cigarette smoking; however, since cannabinoids exhibit anti-inflammatory effects, cannabis smoke exposure may impact viral infection in distinct ways. Male and female BALB/c mice were exposed daily to cannabis smoke and concurrently intranasally instilled with IAV. Viral burden, inflammatory mediator levels (multiplex ELISA), lung immune cells populations (flow cytometry) and gene expression patterns (RNA sequencing) were assessed in the lungs. Plasma IAV-specific antibodies were measured via ELISA. We found that cannabis smoke exposure increased pulmonary viral burden while decreasing total leukocytes, including macrophages, monocytes and dendritic cell populations in the lungs. Furthermore, infection-induced upregulation of certain inflammatory mediators (interferon-γ and C-C motif chemokine ligand 5) was blunted by cannabis smoke exposure, which in females was linked to the transcriptional downregulation of pathways involved in innate and adaptive immune responses. Finally, plasma levels of IAV-specific IgM and IgG1 were significantly decreased in cannabis smoke-exposed, infected mice compared to infected controls, only in female mice. Overall, cannabis smoke exposure disrupted host-defence processes, leading to increased viral burden and dampened inflammatory signalling. These results suggest that cannabis smoking is detrimental to the maintenance of pulmonary homeostasis during viral infection and highlight the need for data regarding the impact on immune competency in humans.

Cholesterol metabolism in T-cell aging: Accomplices or victims.

Aging has a significant impact on the function and metabolism of T cells. Cholesterol, the most important sterol in mammals, is known as the "gold of the body" because it maintains membrane fluidity, rigidity, and signal transduction while also serving as a precursor of oxysterols, bile acids, and steroid hormones. Cholesterol homeostasis is primarily controlled by uptake, biosynthesis, efflux, and regulatory mechanisms. Previous studies have suggested that there are reciprocal interactions between cholesterol metabolism and T lymphocytes. Here, we will summarize the most recent advances in the effects of cholesterol and its derivatives on T-cell aging. We will furthermore discuss interventions that might be used to help older individuals with immune deficiencies or diminishing immune competence.

Evolutionary safety of lethal mutagenesis driven by antiviral treatment.

Nucleoside analogs are a major class of antiviral drugs. Some act by increasing the viral mutation rate causing lethal mutagenesis of the virus. Their mutagenic capacity, however, may lead to an evolutionary safety concern. We define evolutionary safety as a probabilistic assurance that the treatment will not generate an increased number of mutants. We develop a mathematical framework to estimate the total mutant load produced with and without mutagenic treatment. We predict rates of appearance of such virus mutants as a function of the timing of treatment and the immune competence of patients, employing realistic assumptions about the vulnerability of the viral genome and its potential to generate viable mutants. We focus on the case study of Molnupiravir, which is an FDA-approved treatment against Coronavirus Disease-2019 (COVID-19). We estimate that Molnupiravir is narrowly evolutionarily safe, subject to the current estimate of parameters. Evolutionary safety can be improved by restricting treatment with this drug to individuals with a low immunological clearance rate and, in future, by designing treatments that lead to a greater increase in mutation rate. We report a simple mathematical rule to determine the fold increase in mutation rate required to obtain evolutionary safety that is also applicable to other pathogen-treatment combinations.

Health Consequences of Thymus Removal in Adults

BackgroundThe function of the thymus in human adults is unclear, and routine removal of the thymus is performed in a variety of surgical procedures. We hypothesized that the adult thymus is needed to sustain immune competence and overall health.MethodsWe evaluated the risk of death, cancer, and autoimmune disease among adult patients who had undergone thymectomy as compared with demographically matched controls who had undergone similar cardiothoracic surgery without thymectomy. T-cell production and plasma cytokine levels were also compared in a subgroup of patients.ResultsAfter exclusions, 1420 patients who had undergone thymectomy and 6021 controls were included in the study; 1146 of the patients who had undergone thymectomy had a matched control and were included in the primary cohort. At 5 years after surgery, all-cause mortality was higher in the thymectomy group than in the control group (8.1% vs. 2.8%; relative risk, 2.9; 95% confidence interval [CI], 1.7 to 4.8), as was the risk of cancer (7.4% vs. 3.7%; relative risk, 2.0; 95% CI, 1.3 to 3.2). Although the risk of autoimmune disease did not differ substantially between the groups in the overall primary cohort (relative risk, 1.1; 95% CI, 0.8 to 1.4), a difference was found when patients with preoperative infection, cancer, or autoimmune disease were excluded from the analysis (12.3% vs. 7.9%; relative risk, 1.5; 95% CI, 1.02 to 2.2). In an analysis involving all patients with more than 5 years of follow-up (with or without a matched control), all-cause mortality was higher in the thymectomy group than in the general U.S. population (9.0% vs. 5.2%), as was mortality due to cancer (2.3% vs. 1.5%). In the subgroup of patients in whom T-cell production and plasma cytokine levels were measured (22 in the thymectomy group and 19 in the control group; mean follow-up, 14.2 postoperative years), those who had undergone thymectomy had less new production of CD4+ and CD8+ lymphocytes than controls (mean CD4+ signal joint T-cell receptor excision circle [sjTREC] count, 1451 vs. 526 per microgram of DNA [P=0.009]; mean CD8+ sjTREC count, 1466 vs. 447 per microgram of DNA [P<0.001]) and higher levels of proinflammatory cytokines in the blood.ConclusionsIn this study, all-cause mortality and the risk of cancer were higher among patients who had undergone thymectomy than among controls. Thymectomy also appeared be associated with an increased risk of autoimmune disease when patients with preoperative infection, cancer, or autoimmune disease were excluded from the analysis. (Funded by the Tracey and Craig A. Huff Harvard Stem Cell Institute Research Support Fund and others.)

Intraoperative cell salvage: The impact on immune cell numbers.

Patient outcomes are influenced by many confounding factors peri-operatively, including the type of surgery, anaesthesia, transfusion, and immune competence. We have previously demonstrated (in-vitro) that compared to allogeneic blood transfusion (ABT), intraoperative cell salvage (ICS) improves immune competence. The peri-operative immune response is complex. Altered or impaired immune responses may predispose patients to develop adverse outcomes (i.e., post-operative wound infection, pneumonia, urinary tract infection etc.) Surgical patients may develop infection, even without the confirmed presence of a definite microbiological pathogen. With all these factors in mind it is important to consider changes in immune cell numbers (and sub-populations) and functional capacity during peri-operative transfusion. In this TRIMICS-Cell (Transfusion Related Immune Modulation and Intraoperative Cell Salvage-Cell numbers) study (n = 17, October 2018-November 2019) we prioritized and analysed peri-operative changes in the number and proportions of immune cell populations and sub-populations (B cells (CD20+), NK (natural killer) cells (CD56+), monocytes (CD14+), T cells (total CD3+ and sub-populations: T helper cells (CD4+), cytotoxic T cells (CD8+), effector T cells (CD4+ CD127+), activated effector T cells (CD4+ CD25+ CD127+) and regulatory T cells (CD4+ CD25+ CD127-)), plasmacytoid dendritic cells (pDC; Lineage-, HLA-DR+, CD11c-, CD123+), classical dendritic cell (cDC) (Lineage-, HLA-DR+, CD11c+), and cDC activation (Lineage-, HLA-DR+, CD11c+), co-stimulatory/adhesion molecules and pDC (CD9+, CD38+, CD80+, CD83+, CD86+, CD123+). Firstly we analysed the whole cohort of study patients and secondly according to the relevant transfusion modality (i.e., three study groups: those who received no transfusion, received ICS only (ICS), or both ICS and allogeneic packed red blood cells (pRBC) (ICS&RBC)), during major orthopaedic surgery. For the whole study cohort (all patients), changes in immune cell populations were significant: leucocytes and specifically neutrophils increased post-operatively, returning towards pre-operative numbers by 48h post-operatively (48h), and lymphocytes reduced post-operatively returning to pre-operative numbers by 48h. When considering transfusion modalities, there were no significant peri-operative changes in the no transfusion group for all immune cell populations studied (cell numbers and proportions (%)). Significant changes in cell population numbers (i.e., leucocytes, neutrophils and lymphocytes) were identified in both transfused groups (ICS and ICS&RBC). Considering all patients, changes in immune cell sub-populations (NK cells, monocytes, B cells, T cells and DCs) and functional characteristics (e.g., co-stimulation markers, adhesion, activation, and regulation) were significant peri-operatively and when considering transfusion modalities. Interestingly DC numbers and functional capacity were specifically altered following ICS compared to ICS&RBC and pDCs were relatively preserved post-operatively following ICS. A transient peri-operative alteration with recovery towards pre-operative numbers by 48h post-surgery was demonstrated for many immune cell populations and sub-populations throughout. Immune cell sub-populations and functional characteristics were similar peri-operatively in those who received no transfusion but changed significantly following ICS and ICS&RBC. Interesting changes that require future study are a post-operative monocyte increase in the ICS&RBC group, changes in cDC considering transfusion modalities, and possibly preserved pDC numbers post-operatively following ICS. Future studies to assess changes in immune cell sub-populations, especially during peri-operative transfusion, while considering post-operative adverse outcomes, is recommended.

An in vitro model of human hematopoiesis identifies a regulatory role for the aryl hydrocarbon receptor

AbstractIn vitro models to study simultaneous development of different human immune cells and hematopoietic lineages are lacking. We identified and characterized, using single-cell methods, an in vitro stromal cell–free culture system of human hematopoietic stem and progenitor cell (HSPC) differentiation that allows concurrent development of multiple immune cell lineages. The aryl hydrocarbon receptor (AHR) is a ligand–activated transcription factor influencing many biological processes in diverse cell types. Using this in vitro model, we found that AHR activation by the highly specific AHR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin, drives differentiation of human umbilical cord blood–derived CD34+ HSPCs toward monocytes and granulocytes with a significant decrease in lymphoid and megakaryocyte lineage specification that may lead to reduced immune competence. To our knowledge, we also discovered for the first time, using single-cell modalities, that AHR activation decreased the expression of BCL11A and IRF8 in progenitor cells, which are critical genes involved in hematopoietic lineage specification processes at both transcriptomic and protein levels. Our in vitro model of hematopoiesis, coupled with single-cell tools, therefore allows for a better understanding of the role played by AHR in modulating hematopoietic differentiation.

Knockdown of NF-κB activating protein promotes pancreatic cancer growth and metastasis through mTOR signaling pathway.

NF-κB activating protein (NKAP) acts as a transcriptional suppressor in the Notch signaling pathway, It plays a role in hematopoiesis maintenance, immune cell development, maturation, and functional competency acquisition. NKAP has been found to act as an oncogene in many tumors, but it has not been reported in PAAD.The purpose of this study was to investigate the effect of NKAP on the growth and metastasis of pancreatic adenocarcinoma(PAAD). In this study, western blot and qRT-PCR showed that highly expressed NKAP was found in PAAD cell lines, and small interfering RNA (siRNA) was employed to reduce the expression of NKAP in PAAD cell lines. The results of CCK-8, clony formation, Transwell and flow cytometry showed that knockdown of NKAP significantly inhibited biological function of PAAD cells, and increased cell apoptosis. Study also observed that knockdown of NKAP inhibited the expression levels of apoptosis proteins and cyclin in PAAD cells. In addition, mTOR's degree of phosphorylation and the expression of its downstream target p70S6K can both be activated by NKAP. This effect was also confirmed in salvage experiments performed with Rapamycin(RaPa), an inhibitor of mTOR. At the end of the experiment, It was investigated how NKAP affected the drug sensitivity of gemcitabine used to treat PAAD. The results showed that knocking down NKAP could increase the drug sensitivity of gemcitabine. NKAP as an oncogene regulates the development of PAAD cells. The research found that the mTOR signaling pathway is engaged in the oncogenic role of NKAP in PAAD for the first time.

Melatonin Administration to Pregnant Ewes for Coccidiosis Control in Their Offspring.

In livestock research, there has been a growing interest in the impact of melatonin on both health and disease conditions. The hypothesis of the present study was that melatonin treatment prenatally could support the immune competence and growth of experimentally infected lambs. This is the first study that aimed to investigate the impact of melatonin administration throughout pregnancy on immunity and oocyst excretion of pre-partum ewes and their offspring after experimental infection with Eimeria species. Thirty pregnant ewes were allocated into five equal groups, ΚΜ, ΚC, CM, CC, and NC, and gave birth to 47 lambs. Ewes of the KM and KC groups were orally challenged with a cocktail of Eimeria-sporulated oocysts (mainly consisting of Eimeria ovinoidalis), on day 120 of pregnancy, as well as all the lambs at the age of 5-9 days apart from those born from the NC group (environmental control). Fecal samples were collected from all ewes before infection and at parturition and from all lambs 14 times (S0-S13), before infection and during the following 8 weeks, for counting oocysts per gram of feces (OPG). Immunoglobulin (IgG) and cytokine (IL-1β, IL-6, IL-10, IFN-γ) levels were determined in ewes' plasma collected before infection and at parturition, in lambs' plasma at 24 and 72 h after their birth, and in colostrum samples at parturition and 72 h later. Body weight of lambs was recorded five times from birth until the age of 60 days. Accordingly, the leucogram was evaluated in blood samples collected six times within the same period. On average, IgG concentration was higher (p < 0.05) in the blood of KM-ewes compared to KC and CC groups and in colostrum of KM-ewes compared to other groups (p < 0.001). KM-lambs had greater IgG titer and IFN-γ level than the other groups (p < 0.05). The IL-10/ IFN-γ ratio in KM-ewes was lower than the CC group (p = 0.06). Overall, the growth rate of lambs did not differ among groups (p > 0.05). Total oocysts' excretion in KM- and CM-lambs was reduced by 94.9% (p = 0.05) and 92.6% (p = 0.025), respectively, compared to KC-lambs, following the 3-week period after challenge, when E. ovinoidalis predominated in all groups. The dominant type of leucocytes was monocytes in all experimentally infected lambs, but not in NC-lambs, while overall lymphocytes were lower in KC-lambs than in NC-lambs (p < 0.05). Considering that almost all young indoor-reared lambs are exposed to coccidia species during their early life, melatonin treatment prenatally could suggest an alternative management tool in alleviating infection pressure.

Serum-derived extracellular vesicles from breast cancer patients contribute to differential regulation of T-cell-mediated immune-escape mechanisms in breast cancer subtypes.

Intracellular communication within the tumour is complex and extracellular vesicles (EVs) have been identified as major contributing factors for the cell-to-cell communication in the local and distant tumour environments. Here, we examine the differential effects of breast cancer (BC) subtype-specific patient serum and cell-line derived EVs in the regulation of T cell mediated immune responses. Ultracentrifugation was used to isolate EVs from sera of 63 BC patients, 15 healthy volunteers and 4 human breast cancer cell lines. Longitudinal blood draws for EV isolation for patients on neoadjuvant chemotherapy was also performed. Characterization of EVs was performed by Nanoparticle Tracking Analysis (NTA), transmission electron microscopy (TEM) and immunoblotting. CD63 staining was performed on a tissue microarray of 218 BC patients. In-house bioinformatics algorithms were utilized for the computation of EV associated expression scores within The Cancer Genome Atlas (TCGA) and correlated with tumour infiltrating lymphocyte (TIL) scores. In vitro stimulation of PBMCs with EVs from serum and cell-line derived EVs was performed and changes in the immune phenotypes characterized by flow cytometry. Cytokine profiles were assessed using a 105-plex immunoassay or IL10 ELISA. Patients with triple negative breast cancers (TNBCs) exhibited the lowest number of EVs in the sera; whilst the highest was detected in ER+HER2+ cancers; reflected also in the higher level of CD63+ vesicles found within the ER+HER2+ local tumour microenvironment. Transcriptomic analysis of the TCGA data identified that samples assigned with lower EV scores had significantly higher abundance of CD4+ memory activated T cells, T follicular cells and CD8 T cells, plasma, and memory B cells; whilst samples with high EV scores were more enriched for anti-inflammatory M2 macrophages and mast cells. A negative correlation between EV expression scores and stromal TIL counts was also observed. In vitro experiments confirmed that circulating EVs within breast cancer subtypes have functionally differing immunomodulatory capabilities, with EVs from patients with the most aggressive breast cancer subtype (TNBCs) demonstrating the most immune-suppressive phenotype (decreased CD3+HLA-DR+ but increased CD3+PD-L1 T cells, increased CD4+CD127-CD25hi T regulatory cells with associated increase in IL10 cytokine production). In depth assessment of the cytokine modulation triggered by the serum/cell line derived exosomes confirmed differential inflammatory cytokine profiles across differing breast cancer subtypes. Studies using the MDA-231 TNBC breast cancer cell-line derived EVs provided further support that TNBC EVs induced the most immunosuppressive response within PBMCs. Our study supports further investigations into how tumour derived EVs are a mechanism that cancers can exploit to promote immune suppression; and breast cancer subtypes produce EVs with differing immunomodulatory capabilities. Understanding the intracellular/extracellular pathways implicated in alteration from active to suppressed immune state may provide a promising way forward for restoring immune competence in specific breast cancer patient populations.

Lymphocyte immunophenotyping in dogs with lymphopenia of common causes

Lymphocyte immunophenotyping can be useful for evaluating immune competence and predicting the disease prognosis. It is essential to gain knowledge about canine lymphocyte immunophenotypes in various conditions. The study deals with the characteristics of lymphopenia in dogs, with an emphasis on lymphocyte immunophenotyping by flow cytometry. Blood samples from 44 dogs with lymphopenia were included in the study. All lymphopenias sent from veterinary clinics to the diagnostic laboratory were analyzed. The hematological and biochemical abnormalities were investigated, as well as the effect of the age. Lymphopenias were classified according to the level of C-reactive protein (CRP). The percentage of T cells, B cells, Th cells and Tc cells, and T/B and Th/Tc ratios were determined by flow cytometry. Lymphopenias often occurred in dogs over 7 years of age (79.5 %). The most common were postoperative lymphopenia (31.8 %) and inflammatory diseases (29.5 %), most commonly affecting the gastrointestinal tract. Frequent abnormalities were monocytosis (56.8 %), increased CRP (72.7 %) and decreased albumin/globulin ratio (50.0 %). The percentage of Th lymphocytes was significantly lower in the group with elevated CRP than in the group with basal CRP (P = 0.0329). A negative correlation was found between the level of CRP and the percentage of Th lymphocytes (r = −0.3278, P = 0.0390). This study provided new insights into the appearance, incidence and classification of canine lymphopenia.