L-selectin Expression Research Articles

Abstract 105: Neutrophil Cracr2a Is Crucial For Calcium Signaling And Enhances Neutrophil Adhesive Function Under Thromboinflammatory Conditions

Ca 2+ release-activated channel regulator 2A (CRACR2A) is a Ca 2+ -binding protein that facilitates store-operated Ca 2+ entry during T cell activation. However, it is unknown whether neutrophil CRACR2A regulates neutrophil functions in inflammation. Our confocal intravital microscopy (IVM) revealed that compared to wild-type (WT) mice, myeloid-specific cracr2a conditional knockout (CKO, cracr2a fl/fl;Lyz-cre ) mice exhibited a significant increase in neutrophil rolling and a decrease in neutrophil adhesion, crawling, and transmigration on ear and cremaster venules in TNF-α-induced inflammation. Deletion of neutrophil cracr2a impaired β2 integrin function without affecting the expression of L-selectin, PECAM-1, and JAM-A. Neutrophil cracr2a increased Ca 2+ mobilization after stimulation with fMLP, A23187, and thapsigargin by its rapid interaction with STIM1. Due to the lack of CRACR2A inhibitors, we generated CRACR2A-deleted HL60 cells using CRISPR/Cas9. CRACR2A deletion in differentiated HL60 cells recapitulated the defects seen in cracr2a-null neutrophils, including reduced β2 integrin function and Ca 2+ mobilization after agonist stimulation. Since excessively recruited neutrophils cause brain damage in ischemic stroke, we examined whether neutrophil cracr2a contributes to stroke pathology. Using a transient middle cerebral artery occlusion (tMCAO)-induced ischemic stroke model, we found that compared to WT mice, cracr2a CKO mice exhibited improved neurological deficits and reduced infarct volume. Longitudinal 4D IVM of mouse brain revealed that neutrophil cracr2a is crucial for neutrophil adhesion and transmigration on cerebral vessels 16-24 hours after tMCAO and upregulated αMβ2 integrin on adherent neutrophils. Further, single-cell behavior analysis of 4D IVM showed that a proinflammatory subset of neutrophils arise in WT mice, but not cracr2a CKO mice, after tMCAO. We observed that the number of circulating pro-inflammatory neutrophils was markedly decreased 24 hours after tMCAO in cracr2a CKO mice compared with WT mice. Our findings indicate that neutrophil CRACR2A is a key regulator for Ca 2+ signaling and enhances neutrophil pro-inflammatory and adhesive activities in vascular inflammation and ischemic stroke.

Resting and activated bovine neutrophils and eosinophils differ in their responses to adrenergic agonists

Polymorphonuclear cells (PMN) provide a rapid response to infection and tissue damage and stress can modify these critical innate immune defences. The study of adrenergic receptor (AR) expression and function in bovine PMNs is limited but both neutrophils and eosinophils express numerous AR genes but differ significantly in their expression of individual AR genes. A flow cytometric technique was developed to differentiate between bovine neutrophils and eosinophils so both neutrophil and eosinophil responses to adrenergic agonists could be analysed. Neutrophils and eosinophils displayed significantly different changes in CD11b, L-selectin, and CD44 expression when activated by bovine serum opsonized zymosan and recombinant bovine interferon gamma. The responses of activated and resting neutrophils and eosinophils were then compared following stimulation with endogenous adrenergic agonists, epinephrine (E) norepinephrine (NE), and synthetic agonists targeting α1-, α2-, or β-ARs. Both resting and activated neutrophils and eosinophils displayed differences in iROS, CD44, and L-selectin expression following stimulation with E and NE. Resting neutrophils displayed pro-inflammatory responses to both E and NE, while resting eosinophils displayed a pro-inflammatory response to only NE. No single synthetic adrenergic agonist fully recapitulated responses observed with either E or NE and responses to adrenergic agonists were dose-dependent. In conclusion, bovine eosinophils and neutrophils responded to multiple adrenergic agonists by altering expression of proteins involved in immune surveillance and pro-inflammatory responses. Significant differences in neutrophil and eosinophil responses to adrenergic agonists are consistent with their differences in AR gene expression. This highlights the importance of analysing separately these two PMN subpopulations when investigating the effects of either endogenous or synthetic AR agonists.

PSGL-1, ADAM8, and selectins as potential biomarkers in the diagnostic process of systemic lupus erythematosus and systemic sclerosis: an observational study.

Early diagnosis and treatment of Systemic lupus erythematosus (SLE) and Systemic sclerosis (SSc) present significant challenges for clinicians. Although various studies have observed changes in serum levels of selectins between healthy donors and patients with autoimmune diseases, including SLE and SSc, their potential as biomarkers has not been thoroughly explored. We aimed to investigate serum profiles of PSGL-1 (sPSGL-1), ADAM8 (sADAM8) and P-, E- and L-selectins (sP-, sE- and sL-selectins) in defined SLE and SSc patient cohorts to identify disease-associated molecular patterns. We collected blood samples from 64 SLE patients, 58 SSc patients, and 81 healthy donors (HD). Levels of sPSGL-1, sADAM8 and selectins were analyzed by ELISA and leukocyte membrane expression of L-selectin and ADAM8 by flow cytometry. Compared to HD, SLE and SSc patients exhibited elevated sE-selectin and reduced sL-selectin levels. Additionally, SLE patients exhibited elevated sPSGL-1 and sADAM8 levels. Compared to SSc, SLE patients had decreased sL-selectin and increased sADAM8 levels. Furthermore, L-selectin membrane expression was lower in SLE and SSc leukocytes than in HD leukocytes, and ADAM8 membrane expression was lower in SLE neutrophils compared to SSc neutrophils. These alterations associated with some clinical characteristics of each disease. Using logistic regression analysis, the sL-selectin/sADAM8 ratio in SLE, and a combination of sL-selectin/sE-selectin and sE-selectin/sPSGL-1 ratios in SSc were identified and cross-validated as potential serum markers to discriminate these patients from HD. Compared to available diagnostic biomarkers for each disease, both sL-selectin/sADAM8 ratio for SLE and combined ratios for SSc provided higher sensitivity (98% SLE and and 67% SSc correctly classified patients). Importantly, the sADAM8/% ADAM8(+) neutrophils ratio discriminated between SSc and SLE patients with the same sensitivity and specificity than current disease-specific biomarkers. SLE and SSc present specific profiles of sPSGL-1, sE-, sL-selectins, sADAM8 and neutrophil membrane expression which are potentially relevant to their pathogenesis and might aid in their early diagnosis.

Prediction Modeling of CAR-T Cell Therapy for Diffuse Large B-Cell Lymphoma Using Artificial Neural Networks on Tumor Vascular Phenotype

Chimeric antigen receptor T (CAR-T) cell therapy targeting CD19 only results in complete remission in approximately 30-40% of relapsed/refractory disease patients with diffuse large B-cell lymphoma (DLBCL). Failure may result from lack of CAR-T entry into the tumor. Endogenous T cell homing is initiated by binding to post-capillary venular endothelial cells (ECs). Naïve and central memory T cells use L-selectin binding to peripheral node addressin (PNad) on high endothelial venules (HEVs) of the lymph node while circulating effector memory T cells, lacking L-selectin, use other molecules to bind cytokine-induced endothelial proteins E-selectin, ICAM-1 and VCAM-1 on flat venules of inflamed tissues. Both of these venular EC types are present in inflamed lymph nodes. We hypothesized that the expression patterns of adhesion molecules on venular ECs in pre-treatment tumor biopsies could predict CAR-T efficacy. To test this, we applied cyclic immunofluorescence to 18 archived pre-treatment DLBCL patient FFPE biopsies (8 responders and 10 non-responders, defined by relapse at 12 months). We detected venular ECs using a combination of anti-CD31 and UEA lectin on larger diameter vessels and assessed expression for PNad, E-selectin, ICAM-1, VCAM-1. We detected endogenous CD8+ T cells and evaluated their expression of L-selectin, alpha 4 integrin and PSGL-1. See table 1. DAPI nuclear staining was included in each cycle. Images of each cycle were combined in ImageJ and imported into QuPath for analysis. Using Warpy, affine transformations were applied to DAPI staining to create overlays between imaging cycles. After alignment, representative images of several samples were randomly chosen for image training using a combination of Artificial Neural Networks (ANNs) and classifiers to perform identification and mask tissue regions of vessels and T-cells within the tissue. Positive and negative cells for each marker of interest (EC PNad, ICAM-1, E-Selectin, VCAM-1, and T cell alpha 4 integrin, L-Selectin, and PSGL-1) were used to create classification algorithms to determine marker expression for ECs and T-cells within the tissue. Results were analyzed by unpaired t-test and one-way ANOVA conducted in Graph-Pad Prism 9 software. Preliminary results from quantitative analysis of pre-treatment biopsies show a significant increase in CD31+/PNAD- venular ECs compared to non-responders that were double positive for ICAM1 and VCAM-1 (p=0.0273). This analysis also revealed a significant increase in CD31+/PNAD+ HEV ECs from responders that were triple positive for E-Selectin, ICAM-1, and VCAM-1 (p=0.0242). See figure 1. Our initial semi-quantitative analysis showed a trend toward elevated numbers of tissue-infiltrating CD3 + CD8 + T cells in the pre-treatment biopsies of responders. T cells expressing PSGL-1, a glycoprotein that may carry carbohydrate moieties recognized by E-Selectin and linked to T-cell exhaustion, was increased in the non-responder cohort. These data suggest an increased extent and activation state of intra-tumoral venular ECs and increased endogenous T-cell infiltrates, which may predict the outcome of CAR-T therapy. Analysis of T-cells in the pre-treatment biopsies leveraging machine learning algorithms is in progress and both complete quantitative findings and prognostic value of this approach will be presented. While our findings require further validation thru additional biopsy analysis, they provide emerging evidence for a rationale involving vascular readiness in DLBCL responder differences to CART therapy.

The Involvement of Neutrophil in the Immune Dysfunction Associated with BVDV Infection.

Bovine viral diarrhea virus (BVDV) induces immune dysfunction that often results in a secondary bacterial infection in the infected animals. The underlying mechanism of BVDV-induced immune dysfunction is not well understood. The role of BVDV-infected macrophage-secreted factors was investigated. BVDV-infected monocyte-derived macrophage (MDM) supernatants down-regulated the expression of neutrophil L-selectin and CD18. Regardless of the biotype, phagocytic activity and oxidative burst were downregulated by BVDV-infected MDM supernatants. However, only supernatants from cytopathic (cp) BVDV down-regulated nitric oxide production and neutrophil extracellular traps (NET) induction. Our data suggested that BVDV-induced macrophage-secreted factors caused immune dysfunction in neutrophils. Unlike lymphocyte depletion, the negative impact on neutrophils seems to be specific to cp BVDV biotype. Interestingly the majority of modified live BVDV vaccines are based on cp strain of BVDV.

Investigation of the clinical utility of adhesion molecules in the management of thyroid nodules

To better understand the relationship among cell adhesion molecules (CAM) and investigate the clinical diagnostic and prognostic application of ICAM-1 (ICAM1), LFA-1 (ITGAL), and L-selectin (SELL) proteins and mRNA corresponding expression in thyroid cancer. Gene expression was evaluated by RT–qPCR, and protein expression was evaluated by immunohistochemistry. We evaluated 275 patients (218 women, 57 men, 48.4 ± 14.5 years old), including 102 benign and 173 malignant nodules. The 143 papillary thyroid carcinoma (PTC) and 30 follicular thyroid carcinoma (FTC) patients were managed according to current guidelines and followed-up for 78.7 ± 54.2 months. Malignant and benign nodules differed concerning mRNA (p = 0.0027) and protein (p = 0.0020 for nuclear) expression of L-selectin and ICAM-1 (mRNA: p = 0.0001 and protein: p = 0.0014) and protein expression of LFA-1 (p = 0.0168), but not mRNA expression of LFA-1 (p = 0.2131). SELL expression was more intense in malignant tumors (p = 0.0027). ICAM1 (p = 0.0064) and ITGAL (p = 0.0244) mRNA expression was higher in tumors with lymphocyte infiltrate. ICAM-1 expression correlated with younger age at diagnosis (p = 0.0312) and smaller tumor size (p = 0.0443). Also, LFA-1 expression correlated with higher age at diagnosis (p = 0.0376) and was more intense at stage III and IV (p = 0.0077). In general, the protein expression of the 3 CAM decreased as the process of cellular dedifferentiation occurred. We suggest that the SELL and ICAM1 genes and L-selectin and LFA-1 protein expression may help confirm malignancy and assist in the histological characterization of follicular patterned lesions, but we were unable to correlate these CAMs with patient outcomes.

Myeloid Progenitor Inhibitory Factor-1 (CCL23) Inhibits Lung Leukocyte Recruitment in a Primate Cardiopulmonary Bypass-Induced Pulmonary Ischaemia Model.

Bone marrow (BM)-derived polymorphonuclear leukocytes (PMNs) and monocytes (MO) induced by cardiopulmonary bypass (CPB) are highly proteolytic and cause postoperative lung injury. Although CCL23/Myeloid progenitor inhibitory factor-1 is a human CC chemokine with potent suppressor effects on myeloid progenitor cells, invivo inhibitory effects on BM-derived leukocyte kinetics associated with CPB are unknown. Two-hour CPB was surgically performed in cynomolgus monkeys and BM-derived leukocytes kinetics were monitored postoperatively by flow cytometry with 5'-bromo-2'-deoxyuridine (BrdU) and cytokine ELISA. Monkeys were given CCL23 (n=5) or saline (control, n=5) intravenously daily for 3 days before BrdU labelling and peripheral blood/bronchoalveolar lavage fluid (BALF) timepoint sampling to reveal BrdU-labelled cells. Levels of cytokines, CD11b, and L-selectin were considered leukocytic activation markers. The CCL23 treatment significantly prolonged BM transit of leukocytes (PMNs, 118.4±11.7-95.5±4.1 hours [control]; MO, 91.6±5.0-62.0±3.0 hours [control]) and reduced their alveolar appearance. The BM pool size of MO was decreased by CCL23 but PMNs were unaffected. CD11b, L-selectin expression of PMNs and MO during CPB, and post-surgical increases of interleukin (IL)-6, IL-8, TNF-α, MCP-1, and PMN elastase in the BALF were not suppressed. CCL23 treatment slows turnover of PMN and MO progenitors in BM and suppresses their circulatory release and lung recruitment. CCL23 has inhibitory effects specifically on the CPB-induced BM response and could hold value for preventing CPB-induced lung injury.

Prospective flow cytometry analysis of leucocyte subsets in critically ill patients who develop sepsis: a pilot study.

Sepsis in critically ill patients with injury bears a high morbidity and mortality. Extensive phenotypic monitoring of leucocyte subsets in critically ill patients at ICU admission and during sepsis development is still scarce. The main objective of this study was to identify early changes in leukocyte phenotype which would correlate with later development of sepsis. Patients who were admitted in a tertiary ICU for organ support after severe injury (elective cardiac surgery, trauma, necessity of prolonged ventilation or stroke) were sampled on admission (T1) and 48-72h later (T2) for phenotyping of leukocyte subsets by flow cytometry and cytokines measurements. Those who developed secondary sepsis or septic shock were sampled again on the day of sepsis diagnosis (Tx). Ninety-nine patients were included in the final analysis. Nineteen (19.2%) patients developed secondary sepsis or septic shock. They presented significantly higher absolute monocyte counts and CRP at T1 compared to non-septic patients (1030/µl versus 550/µl, p = 0.013 and 5.1mg/ml versus 2.5mg/ml, p = 0.046, respectively). They also presented elevated levels of monocytes with low expression of L-selectin (CD62Lneg monocytes) (OR[95%CI] 4.5 (1.4-14.5), p = 0.01) and higher SOFA score (p < 0.0001) at T1 and low mHLA-DR at T2 (OR[95%CI] 0.003 (0.00-0.17), p = 0.049). Stepwise logistic regression analysis showed that both monocyte markers and high SOFA score (> 8) were independently associated with nosocomial sepsis occurrence. No other leucocyte count or surface marker nor any cytokine measurement correlated with sepsis occurrence. Monocyte counts and change of phenotype are associated with secondary sepsis occurrence in critically ill patients with injury.

Association between uterine toxicity induced by chlorpyrifos and downregulation of heparin-binding epidermal growth factor and L-selectin genes.

Various factors are effective in reducing the fertility rate. This experiment aimed to investigate chlorpyrifos (CPF), an organophosphate, that could alter the structure of the uterus and the molecules involved in parental and fetal. CPF was injected intraperitoneally in thirty mice for five days in a week (six weeks). The animals were euthanized on the 5th day of gestation, then their blood and uterus were collected for biochemical and histopathological assays. Exposure to CPF resulted in a significant reduction in maternal weight gain and the number of litters. Alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) were significantly increased in blood serum of the CPF group compared with the control. The number of uterus glands, endometrium thickness, and the uterine cavity were changed following CPF injection. Additional investigation indicated that the expressions of L-selectin, L-selectin ligand, and heparin-binding epidermal growth factor (HB-EGF) as initial adhesion of mice blastocysts and maternal endometrium biomarkers were downregulated in the CPF group. Nevertheless, any mortality and abnormal clinical symptoms were not observed in the treated mice. This study revealed a potential molecular mechanism of continuous CPF-induced toxicity in fetal-maternal attachment without clinical symptoms.

Evaluation of cell adhesion molecules (LFA-1 and L-selectin) in ankylosing spondylitis patients after treatment with β-D-mannuronic acid (M2000).

To examine β-D-mannuronic acid (M2000) effects on L-selectin shedding and leucocyte function-associated antigen-1 (LFA-1) expression as mechanisms of action of this drug in patients with ankylosing spondylitis (AS). To investigate the molecular consequences of β-D-mannuronic acid on L-selectin shedding, flow cytometry method was used. Furthermore, the effect of it on LFA-1 gene expression was analyzed by using quantitative real time (qRT)-PCR technique. The LFA-1 expression in patients with AS was higher than controls (P=0.046). The LFA-1 expression after 12 wk therapy with β-D-mannuronic acid was meaningfully decreased (P=0.01). After 12 wk treatment with β-D-mannuronic acid, the frequency of CD62L-expressing CD4+ T cells in patients with AS, was not considerably altered, compared to the patients before therapy (P=0.5). Furthermore, after 12 wk therapy with β-D-mannuronic acid, L-selectin expression levels on CD4+ T-cells in patients with AS, were not remarkably changed, compared to the expression levels of these in patients before treatment (P=0.2). The results of this study for the first time showed that β-D-mannuronic acid can affect events of adhesion cascade in patients with AS. Moreover, β-D-mannuronic acid presented as an acceptable benefit to AS patients and could aid in the process of disease management.

Pleiotropic Effects of Hydroxyurea on Red Blood Cell and Neutrophil Phenotype in Patients with Sickle Cell Disease; Data from the Notice Study

Introduction The pathophysiology of sickle cell disease (SCD) is complex and goes beyond red blood cell (RBC) sickling alone. Neutrophils have been proposed to play an important role in SCD pathophysiology, but their exact contribution remains to be elucidated. Factors such as chronic hemolysis and oxidative stress can influence neutrophil adhesion, activity and aging. We designed a prospective study (Notice) to investigate a broad set of neutrophil characteristics in SCD patients. To correlate treatment effects of hydroxyurea (HU) on neutrophils with those on RBCs in SCD, we also studied RBC sickling and adhesion. Here, we will focus on the effects of hydroxyurea on RBC deformability and neutrophil adhesion, activity and aging. Aim: To assess the effects of HU on RBC deformability and neutrophil phenotypes with respect to adhesion, activation and aging. Methods Blood (EDTA) was collected from SCD patients with HbSS or HbSβ0 in steady-state. A subgroup of patients was included before and during treatment with HU. For measurement of antigen expression by flow cytometry, total leukocyte fraction after lysis of red blood cells (without Percoll gradient) was used and neutrophils were gated based on their forward and side scatter. For in vitro neutrophil adhesion, neutrophils were isolated using a Percoll gradient and lysis buffer, as previously described. (Kuijpers et al. Blood 2007) Neutrophils were labeled with calcein and placed in an uncoated 96-well MaxiSorp plate. After washing, adherent cells were lysed and fluorescence was measured. (Kuijpers et al. J Allergy Clin Immunol 2017). Adhesion of RBCs was measured by flowing whole blood (20x106/ml)over laminin-coated Ibidi slides for 30 minutes with visualization by microscope. Percentage of mean RBC adhesion was normalized to a healthy control (HC). Data were analyzed using SPSS and GraphPad Prism and are presented as mean ±SE or ±SD as appropriate. Conventional statistics were used. Results A total of 49 patients (median age 25.0 (IQR 20.0 - 32.5) years, 39% female) were included. Twenty patients (41%) used a stable dose of HU at baseline. In 10 patients, blood was drawn before and after starting HU treatment. HU treatment improved hemoglobin F and markers of hemolysis and was associated with a decrease in the point of sickling (PoS) of RBCs (from median 24.8 mmHg [IQR 16.0-28.2] to 16.8 [15.0-20.8], P=0.13). Adhesion of RBC was more than 3-fold higher in SCD patients than in HC. At baseline, there was no difference in RBC adhesion between patients with and without HU (P=0.86). After starting HU treatment, RBC adhesion decreased significantly from a median of 318% (normalized to control) to 149% (P=0.016, Figure 1a-b). Adhesion of neutrophils was significantly higher in patients compared to HC (median 18.3% versus 12.6% P <0.0001). No difference was observed between patients with and without HU at baseline. Neutrophil adhesion did not decrease after starting HU treatment (Figure 1c-d). Neutrophil surface expression of CD11b/CD18 was measured as markers of adhesion. While CD11b expression was comparable between SCD patients and HC, CD18 expression was significantly higher in SCD patients (mean MFI 12193 vs 10586, P=0.016; Figure 2b. After treatment with HU both CD11b and CD18 expression decreased, though the differences did not reach statistical significance (P=0.22 and 0.56, respectively). Neutrophils of SCD patients displayed a more aged phenotype compared to neutrophils of HC with lower L-selectin (median MFI 3108 vs 4656, P=0.021) and higher CXCR4 (median MFI 1150 vs 886, P=0.003) expression. Activation marker CD64 was also higher in SCD patients compared to HC (median MFI 1841 vs 1065, P=0.0006). HU treatment seemed to modify all markers more towards a HC phenotype although no statistically significant changes were observed. Conclusion Adhesion of both RBCs and neutrophils is increased in SCD patients compared to HC. HU treatment was associated with significantly improved RBC rheology as shown by decreased PoS and decreased adhesion to laminin. Despite a trend to improvement in expression of adhesion markers (CD11b/CD18), in vitro adhesion of neutrophils did not decrease. These findings could be due to the small sample size of patients starting with HU or by a difference in vitro behaviour of changed composition of neutrophil populations after treatment. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Recent advances in "sickle and niche" research - Tribute to Dr. Paul S Frenette.

SummaryIn this retrospective, we review the two research topics that formed the basis of the outstanding career of Dr. Paul S. Frenette. In the first part, we focus on sickle cell disease (SCD). The defining feature of SCD is polymerization of the deoxygenated mutant hemoglobin, which leads to a vicious cycle of hemolysis and vaso-occlusion. We survey important discoveries in SCD pathophysiology that have led to recent advances in treatment of SCD. The second part focuses on the hematopoietic stem cell (HSC) niche, the complex microenvironment within the bone marrow that controls HSC function and homeostasis. We detail the cells that constitute this niche, and the factors that these cells use to exert control over hematopoiesis. Here, we trace the scientific paths of Dr. Frenette, highlight key aspects of his research, and identify his most important scientific contributions in both fields.

Application of Ultrasonic Intelligent Imaging in L-Selectin Regulating Embryo Implantation in Mongolian Sheep Endometrium.

In order to explore the practical application of ultrasonic imaging in the pregnancy stage of Mongolian sheep and the role of L-selectin in the embryo implantation process of Mongolian sheep, this paper systematically observed the early embryonic development by B-mode ultrasonic imaging wave diagnostic instrument with 5 MHz rectal probe and detected the expression of sLex and L-selectin in embryonic cells (jar cells) and endometrial cells (RL95-2 cells) by immunoassay to show the role of L-selectin in embryonic adhesion. The results were as follows: the correct rate of fetal sex determination by ultrasound imaging increased with the increase of pregnancy days and reached 93% at 84 days; sLex/L-selectin on the surface of Jar/RL95-2 cells is involved in the adhesion between embryo and endometrium; and when the concentration of L-selectin was 30 μg/ml, the implantation success rate of fertilized eggs and embryos was the highest, reaching 95%. It is proved that ultrasonic intelligent imaging exploration can summarize the imaging characteristics of the early development law of sheep fetus, which provides a basis for B-ultrasound to monitor fetal growth and predict fetal age. While discussing the molecular mechanism of implantation, it provides a new idea and means for the clinical intervention of contraception and pregnancy assistance with oligosaccharide as the target.

Impacts of Various Nutraceutical Milk Replacer Supplements on the Health and Performance of High-Risk Holstein Calves

The objectives of this study were to determine the impacts of supplementing various nutraceuticals in milk replacers, including a blend of probiotics, β-glucan extract, mannanoligosaccharide extract, or a non-immunoglobulin rich extract, from colostrum on the performance and health of high-risk calves and to determine carry-over effects into the immediate post-weaned period. One hundred bull calves were acquired from a local calf ranch within 24 h of birth and randomly assigned to one of five dietary treatments added to milk replacer only: (1) Control (CON), no additive; (2) Immu-PRIME (ImmPr), 1.5 g/d ImmPr first 3 d only (Sterling Technology, Brookings, SD, USA); (3) Beta glucan (BG), 1 g/d BG extract (ImmuOligo, Irvine, CA, USA); (4) Mannanoligosaccharide + Bacillus subtilis (MOS+Bs), 3 g/d CEREVIDA EXCELL-M + 4 x 109 CFU/d Bacillus subtilis (MB Nutritional Sciences, Lubbock, TX, USA); and (5) PROVIDA CALF (PRO), proprietary blend of 2 x 109 CFU/d of a Lactobacillus casei and an Enterococcus faecium + 2 x 109 CFU/d Saccharomyces cerevisiae (MB Nutritional Sciences, Lubbock, TX, USA). Calves were weaned at d 56, comingled, and treatment carry-over effects evaluated through d 84. Starter intake was measured daily and BW weekly. Peripheral blood samples were collected and analyzed for hematology and serum was analyzed for haptoglobin concentrations. Polymorphonuclear leukocyte (PMNL) function was assessed through surface L-selectin expression, phagocytic, and oxidative burst activities against Escherichia coli. Data were analyzed using Proc Mixed in SAS (SAS 9.4, Cary, NC). The BG calves consumed the most starter from d 1 to 28 and pre-weaned average daily gain was greater for both the PRO and BG when compared to the CON group. There was a tendency for BG, MOS+Bs, and PRO to have reduced serum haptoglobin throughout the study. Total leukocyte, neutrophil, and lymphocyte counts were reduced among MOS+Bs calves, whereas BG calves tended to have the greatest neutrophil:lymphocyte ratio. Oxidative burst function for PMNL was reduced among BG and PRO treatments, suggesting a decreased need for neutrophil function. Supplementing BG, MOS+Bs, and PRO all influenced the performance and health of high-risk calves, but mechanistically appear to be different.

Human Coronary Plaque T Cells Are Clonal and Cross-React to Virus and Self.

Coronary artery disease is an incurable, life-threatening disease that was once considered primarily a disorder of lipid deposition. Coronary artery disease is now also characterized by chronic inflammation' notable for the buildup of atherosclerotic plaques containing immune cells in various states of activation and differentiation. Understanding how these immune cells contribute to disease progression may lead to the development of novel therapeutic strategies. We used single-cell technology and in vitro assays to interrogate the immune microenvironment of human coronary atherosclerotic plaque at different stages of maturity. In addition to macrophages, we found a high proportion of αβ T cells in the coronary plaques. Most of these T cells lack high expression of CCR7 and L-selectin, indicating that they are primarily antigen-experienced memory cells. Notably, nearly one-third of these cells express the HLA-DRA surface marker, signifying activation through their TCRs (T-cell receptors). Consistent with this, TCR repertoire analysis confirmed the presence of activated αβ T cells (CD4<CD8), exhibiting clonal expansion of specific TCRs. Interestingly, we found that these plaque T cells had TCRs specific for influenza, coronavirus, and other viral epitopes, which share sequence homologies to proteins found on smooth muscle cells and endothelial cells, suggesting potential autoimmune-mediated T-cell activation in the absence of active infection. To better understand the potential function of these activated plaque T cells, we then interrogated their transcriptome at the single-cell level. Of the 3 T-cell phenotypic clusters with the highest expression of the activation marker HLA-DRA, 2 clusters expressed a proinflammatory and cytolytic signature characteristic of CD8 cells, while the other expressed AREG (amphiregulin), which promotes smooth muscle cell proliferation and fibrosis, and, thus, contributes to plaque progression. Taken together, these findings demonstrate that plaque T cells are clonally expanded potentially by antigen engagement, are potentially reactive to self-epitopes, and may interact with smooth muscle cells and macrophages in the plaque microenvironment.

Blocking of inflammatory heparan sulfate domains by specific antibodies is not protective in experimental glomerulonephritis.

Glomerulonephritis is an acquired serious glomerular disease, which involves the interplay of many factors such as cytokines, chemokines, inflammatory cells, and heparan sulfate (HS). We previously showed that blocking of inflammatory heparan sulfate domains on cultured glomerular endothelium by specific anti-HS single chain antibodies reduced polymorphonuclear cell (PMN) adhesion and chemokine binding. We hypothesized that injection of anti-HS antibodies in PMN-driven experimental glomerulonephritis should reduce glomerular influx of PMNs and thereby lead to a better renal outcome. In contrast to our hypothesis, co-injection of anti-HS antibodies did not alter the final outcome of anti-glomerular basement membrane (anti-GBM)-induced glomerulonephritis. Glomerular PMN influx, normally peaking 2 hours after induction of glomerulonephritis with anti-GBM IgG was not reduced by co-injection of anti-HS antibodies. Four days after induction of glomerulonephritis, albuminuria, renal function, glomerular hyalinosis and fibrin deposition were similar in mice treated and not treated with anti-HS antibodies. Interestingly, we observed transient effects in mice co-injected with anti-HS antibodies compared to mice that did not receive anti-HS antibodies: (i) a decreased renal function 2 hours and 1 day after induction of glomerulonephritis; (ii) an increased albuminuria after 2 hours and 1 day; (iii) an increased glomerular fibrin deposition after 1 day; (iv) a reduced glomerular macrophage influx after 1 day; (v) a sustained glomerular presence of PMNs at day 1 and 4, accompanied by an increased renal expression of IL-6, CXCL1, ICAM-1, L-selectin, CD11b and NF-κB. The mechanism underlying these observations induced by anti-HS antibodies remains unclear, but may be explained by a temporarily altered glycocalyx and/or altered function of PMNs due to the binding of anti-HS antibodies. Nevertheless, the evaluated anti-HS antibodies do not show therapeutic potential in anti-GBM-induced glomerulonephritis. Future research should evaluate other strategies to target HS domains involved in inflammatory processes during glomerulonephritis.

Expression of L-selectin (CD62L) and CD44 on bovine lym-phocytes and WC1.1+ γδ T cells under stimulation with Staph-ylococcus aureus

Expression of L-selectin (CD62L) and CD44 on bovine lym-phocytes and WC1.1+ γδ T cells under stimulation with Staph-ylococcus aureus

Effect of injectable trace mineral supplementation on peripheral polymorphonuclear leukocyte function, antioxidant enzymes, health, and performance in dairy cows in semi-arid conditions

The objective of this study was to evaluate the effect of subcutaneous injections of 15 mg/mL Cu, 5 mg/mL Se, 60 mg/mL Zn, and 10 mg/mL Mn on health, performance, polymorphonuclear leukocyte (PMNL) function, circulating glutathione peroxidase (GPx) and superoxide dismutase (SOD) concentrations, and inflammation of dairy cows undergoing the transition period in high temperature-humidity index. A total of 923 multiparous cows from 2 commercial dairy farms were randomly allocated into 1 of 2 treatment groups as follows: control and injectable trace mineral supplementation (ITMS). Cows in the ITMS group received 7 mL of subcutaneous injections at dry-off (208 ± 3 d of gestation), 260 ± 3 d of gestation, and at 35 ± 3 d in milk (DIM). Data regarding health traits, reproductive performance, milk yield, and survivability were extracted from farm database software, and animals were followed-up until 300 DIM. For a subset of 142 cows from one herd, blood samples were collected at enrollment, and at 3 ± 1, 7 ± 1, 10 ± 1, and 35 ± 3 DIM to evaluate hematology, PMNL function, GPx and SOD concentrations, and circulating haptoglobin. Logistic regression was used to assess health and pregnancy per artificial insemination at first service. Cox proportional hazards models were used to evaluate hazard of pregnancy and culling. Mixed linear regression models accounting for repeated measures were used to assess all continuous variables collected over time. Parity, twinning, and previous gestation length were considered as potential confounders. Farm was included as a random effect. The ITMS cows tended to have lower incidence of metritis and stillbirth compared with control group. However, ITMS treatment did not influence the incidence of other diseases (e.g., mastitis, retained placenta), milk yield, reproductive performance, culling, and leukocyte count. Neutrophil-to-lymphocyte ratio, PMNL phagocytosis, and oxidative burst as well as intensity of the oxidative burst were greater for ITMS-treated cows in comparison to control cows. The ITMS cows had decreased expression of the adhesion molecule L-selectin on PMNL surface. The serum concentration of GPx and SOD were not affected by ITMS treatment. In conclusion, ITMS tended to reduce the incidence of metritis and stillbirth parturition, improved PMNL function, and improved the inflammatory status of dairy cows undergoing the transition period in high temperature-humidity index conditions. However, these findings did not translate into improved milk yield, reproductive performance, and survivability.

Kaposi's sarcoma-associated herpesvirus ubiquitin ligases downregulate cell surface expression of l-selectin.

Kaposi's sarcoma-associated herpesvirus (KSHV) is an oncogenic etiological factor for Kaposi's sarcoma and primary effusion lymphoma in immunocompromised patients. KSHV utilizes two immune evasion E3 ubiquitin ligases, namely K3 and K5, to downregulate the expression of antigen-presenting molecules and ligands of natural killer (NK) cells in the host cells through an ubiquitin-dependent endocytic mechanism. This allows the infected cells to evade surveillance and elimination by cytotoxic lymphocytes and NK cells. The number of host cell molecular substrates reported for these ubiquitin ligases is limited. The identification of novel substrates for these ligases will aid in elucidating the mechanism underlying immune evasion of KSHV. This study demonstrated that K5 downregulated the cell surface expression of l-selectin, a C-type lectin-like adhesion receptor expressed in the lymphocytes. Tryptophan residue located at the centre of the E2-binding site in the K5 RINGv domain was essential to downregulate l-selectin expression. Additionally, the lysine residues located at the cytoplasmic tail of l-selectin were required for the K5-mediated downregulation of l-selectin. K5 promoted the degradation of l-selectin through polyubiquitination. These results suggest that K5 downregulates l-selectin expression on the cell surface by promoting polyubiquitination and ubiquitin-dependent endocytosis, which indicated that l-selectin is a novel substrate for K5. Additionally, K3 downregulated l-selectin expression. The findings of this study will aid in the elucidation of a novel immune evasion mechanism in KSHV.

Biomarkers to Guide the Timing of Surgery: Neutrophil and Monocyte L-Selectin Predict Postoperative Sepsis in Orthopaedic Trauma Patients.

Deciding whether to delay non-lifesaving orthopaedic trauma surgery to prevent multiple organ failure (MOF) or sepsis is frequently disputed and largely based on expert opinion. We hypothesise that neutrophils and monocytes differentially express activation markers prior to patients developing these complications. Peripheral blood from 20 healthy controls and 162 patients requiring major orthopaedic intervention was collected perioperatively. Neutrophil and monocyte L-selectin, CD64, CD11, CD18, and CXCR1 expression were measured using flow cytometry. The predictive ability for MOF and sepsis was assessed using the Receiver Operating Characteristic (ROC) comparing to C-reactive protein (CRP). Neutrophil and monocyte L-selectin were significantly higher in patients who developed sepsis. Neutrophil L-selectin (AUC 0.692 [95%CI 0.574–0.810]) and monocyte L-selectin (AUC 0.761 [95%CI 0.632–0.891]) were significant predictors of sepsis and were not significantly different to CRP (AUC 0.772 [95%CI 0.650–0.853]). Monocyte L-selectin was predictive of MOF preoperatively and postoperatively (preop AUC 0.790 [95%CI 0.622–0.958]). CD64 and CRP were predictive of MOF at one-day postop (AUC 0.808 [95%CI 0.643–0.974] and AUC 0.809 [95%CI 0.662–0.956], respectively). In the perioperative period, elevated neutrophil and monocyte L-selectin are predictors of postoperative sepsis. Larger validation studies should focus on these biomarkers for deciding the timing of long bone/pelvic fracture fixation.