Bidirectional crosstalk between the peripheral nervous system and lymphoid tissues/organs.

The central nervous system (CNS) influences the immune system generally by regulating the systemic concentration of humoral substances (e.g., cortisol and epinephrine), whereas the peripheral nervous system (PNS) communicates specifically with the immune system according to local interactions/connections. An imbalance between the components of the PNS might contribute to pathogenesis and the further development of certain diseases. In this review, we have explored the "thread" (hardwiring) of the connections between the immune system (e.g., primary/secondary/tertiary lymphoid tissues/organs) and PNS (e.g., sensory, sympathetic, parasympathetic, and enteric nervous systems (ENS)) in health and disease in vitro and in vivo. Neuroimmune cell units provide an anatomical and physiological basis for bidirectional crosstalk between the PNS and the immune system in peripheral tissues, including lymphoid tissues and organs. These neuroimmune interactions/modulation studies might greatly contribute to a better understanding of the mechanisms through which the PNS possibly affects cellular and humoral-mediated immune responses or vice versa in health and diseases. Physical, chemical, pharmacological, and other manipulations of these neuroimmune interactions should bring about the development of practical therapeutic applications for certain neurological, neuroimmunological, infectious, inflammatory, and immunological disorders/diseases.

Optimized flow cytometric detection of transient receptor potential vanilloid-1 (TRPV1) in human hematological malignancies.

The ectopic overexpression of transient receptor potential vanilloid-1 (TRPV1) has been detected in numerous solid cancers, including breast, prostate, pancreatic, and tongue epithelium cancer. However, the expression of TRPV1 in hematological malignancies remains unknown. Here we show through in silico analysis that elevated TRPV1 mRNA expression occurs in a range of hematological malignancies and presents an optimized flow cytometry method to rapidly assess TRPV1 protein expression for both cell lines and primary patient samples. Three anti-TRPV1 antibodies were evaluated for intracellular TRPV1 detection using flow cytometry resulting in an optimized protocol for the evaluation of TRPV1 in hematological malignant cell lines and patients' peripheral blood mononuclear cells (PBMC). Overexpression of TRPV1 was observed in THP-1 (acute monocytic leukemia) and U266B1 (multiple myeloma, MM), but not U937 (histiocytic lymphoma) compared to healthy PBMC. TRPV1 was also detected in all 49 patients including B-cell non-Hodgkin's lymphoma (B-NHL), MM, and others and 20 healthy controls. TRPV1 expression was increased in 8% of patients (MM = 2, B-NHL = 2). In conclusion, we provide an optimized flow cytometry method for routine expression analysis of clinical samples and show that TRPV1 is increased in a subset of patients with hematological malignancies.

Compression Socks Reduce Running-Induced Intestinal Damage.

ABSTRACT: Zadow, EK, Edwards, KH, Kitic, CM, Fell, JW, Adams, MJ, Singh, I, Kundur, A, Johnstone, ANB, Crilly, J, Bulmer, AC, Halson, SL, and, and Wu, SSX. Compression socks reduce running-induced intestinal damage. J Strength Cond Res 36(9): 2461-2464, 2022-Exercise is associated with a reduction in splanchnic blood flow that leads to the disruption of intestinal epithelium integrity, contributing to exercise-induced gastrointestinal syndrome. Strategies that promote intestinal blood flow during exercise may reduce intestinal damage, which may be advantageous for subsequent recovery and performance. This study aimed to explore if exercise-associated intestinal damage was influenced by wearing compression garments, which may improve central blood flow. Subjects were randomly allocated to wear compression socks ( n = 23) or no compression socks (control, n = 23) during a marathon race. Blood samples were collected 24 hours before and immediately after marathon and analyzed for intestinal fatty acid-binding protein (I-FABP) concentration as a marker of intestinal damage. The magnitude of increase in postmarathon plasma I-FABP concentration was significantly greater in control group (107%; 95% confidence interval [CI], 72-428%) when compared with runners wearing compression socks (38%; 95% CI, 20-120%; p = 0.046; d = 0.59). Wearing compression socks during a marathon run reduced exercise-associated intestinal damage. Compression socks may prove an effective strategy to minimize the intestinal damage component of exercise-induced gastrointestinal syndrome.

Estradiol-Responsive miR-365a-3p Interacts with Tissue Factor 3'UTR to Modulate Tissue Factor-Initiated Thrombin Generation.

BACKGROUND: High estradiol (E2) levels are linked to an increased risk of venous thromboembolism; however, the underlying molecular mechanism(s) remain poorly understood. We previously identified an E2-responsive microRNA (miR), miR-494-3p, that downregulates protein S expression, and posited additional coagulation factors, such as tissue factor, may be regulated in a similar manner via miRs. OBJECTIVES: To evaluate the coagulation capacity of cohorts with high physiological E2, and to further characterize novel E2-responsive miR and miR regulation on tissue factor in E2-related hypercoagulability. METHODS: Ceveron Alpha thrombin generation assay (TGA) was used to assess plasma coagulation profile of three cohorts. The effect of physiological levels of E2, 10 nM, on miR expression in HuH-7 cells was compared using NanoString nCounter and validated with independent assays. The effect of tissue factor-interacting miR was confirmed by dual-luciferase reporter assays, immunoblotting, flow cytometry, biochemistry assays, and TGA. RESULTS: Plasma samples from pregnant women and women on the contraceptive pill were confirmed to be hypercoagulable (compared with sex-matched controls). At equivalent and high physiological levels of E2, miR-365a-3p displayed concordant E2 downregulation in two independent miR quantification platforms, and tissue factor protein was upregulated by E2 treatment. Direct interaction between miR-365a-3p and F3-3'UTR was confirmed and overexpression of miR-365a-3p led to a decrease of (1) tissue factor mRNA transcripts, (2) protein levels, (3) activity, and (4) tissue factor-initiated thrombin generation. CONCLUSION: miR-365a-3p is a novel tissue factor regulator. High E2 concentrations induce a hypercoagulable state via a miR network specific for coagulation factors.

Shedding of soluble glycoprotein VI is neither affected by animal-derived antibeta-2-glycoprotein 1 antibodies nor IgG fractions from patients with systemic lupus erythematosus.

: Antibeta-2-glycoprotein 1 (antiß2GP1) antibodies are associated with increased risk of thrombosis in patients with systemic lupus erythematosus (SLE). The specific effect(s) of antiß2GP1 antibodies on platelets are unclear. Platelet activation in response to antiplatelet antibodies has been shown to induce shedding of the ectodomain of the platelet collagen receptor, glycoprotein VI (GPVI), releasing soluble GPVI (sGPVI). The aim of this study was to therefore determine whether antiß2GP1 antibodies, and/or purified IgG fractions, from patients with SLE shed sGPVI from platelets. We determined sGPVI levels in platelet poor plasma from SLE patients with/without antiß2GP1 antibodies (n = 37), as well as in platelet-rich plasma from healthy donors treated with either SLE-derived IgG fractions containing antiß2GP1, animal-derived antiß2GP1, or isotype control antibodies. Levels of sGPVI were higher in three SLE-derived platelet poor plasma with antiß2GP1 antibodies (103.52 ±â€Š12.32 ng/ml) compared with those without (28.11 ±â€Š12.73 ng/ml). Neither SLE-derived IgG fractions containing antiß2GP1 antibodies, nor animal-derived antiß2GP1 antibodies induced significant shedding of sGPVI from healthy donor platelets compared with isotype controls. These results suggest that antiß2GP1 antibodies do not affect shedding of sGPVI, and therefore collagen-mediated platelet signalling pathways. The shedding activity in SLE patients may be due to factors other than antiß2GP1 antibodies, for example, metalloproteinases.

International Society on Thrombosis and Haemostasis core curriculum project: Core competencies in laboratory thrombosis and hemostasis.

BACKGROUND: Laboratory analyses of blood samples are essential for diagnostics and therapy monitoring of patients with bleeding and thromboembolic diseases. Following publication of the core curriculum for clinical thrombosis and hemostasis, the International Society on Thrombosis and Haemostasis (ISTH) recognized that thrombosis and hemostasis laboratory specialists require distinct competencies that differ from medical doctors working clinically with patients. To address this gap the ISTH formed a working group of international hemostasis and thrombosis laboratory specialists to develop an evidence-based core curriculum for laboratory specialists. OBJECTIVE: This research sought consensus from the international community on core competencies required for laboratory specialists in thrombosis and hemostasis. METHODS: A draft list of 64 competencies was developed and an online stakeholder survey was circulated electronically to 15 302 ISTH members and contacts in the wider international community. The results were analyzed and used to develop the final approved core curriculum. RESULTS: Three hundred and thirty responses contained meaningful data, with broad international representation of specialists. No draft competencies were excluded, and 58 were rated as "does" or "shows how." The Leik measure of consensus for most competences was "moderate" (n = 30) or "fair" (n = 32). CONCLUSIONS: The development of an international core curriculum for laboratory specialists provides a foundation for the development and enhancement of education and quality management of the laboratory. Although there is no formal designation for laboratory specialists, international governing bodies and regulatory organizations are encouraged to consider the diagnostic core curriculum for development and accreditation of more standardized educational programs and formal assessment across jurisdictions.

Deep vein thrombosis in a well-trained masters cyclist, is popliteal vein entrapment syndrome to blame?

Whilst athletes are the epitome of health, venous thromboembolisms (VTE) including deep vein thrombosis and pulmonary embolism have been demonstrated to occur in well-trained athletes. VTE is frequently misdiagnosed and poorly treated within this population, often resulting in career or life-threatening ramifications. Furthermore, VTE risk rises with increasing age (> 40 years), potentially affecting masters athletes. A 44-year-old well-trained male cyclist volunteered to participate in a research project investigating the influence of exercise on haemostasis in well-trained athletes. The cyclist presented with elevated D-Dimer levels both pre- (2251 ng/mL) and post-exercise (2653 ng/mL). The cyclist reported constant mild-pain in the left mid-calf region, with a cold tingling sensation in their left foot. Diagnosis of DVT was confirmed via a DVT squeeze test and Doppler ultrasound, with the clot located in the left popliteal vein. During the research project, the cyclist was exposed to numerous thrombogenic risk factors including travel, dehydration, prolonged sitting and exercise. The DVT in the popliteal vein may have resulted from repetitive movements associated with cycling. Additionally, hypertrophy of the gastrocnemius muscle may have impinged the vein. When diagnosing DVT within a cycling population, PVES should not be overlooked as a contributing factor.

Crossmatching Maternal and Fetal Blood in Sheep.

We investigated the incidence of ex vivo incompatibility between ovine maternal RBC and fetal plasma. Time-mated singleton pregnant ewes (n = 8) underwent cesarean delivery of the fetus; at the time of delivery, paired maternal and fetal blood samples were collected and subsequently separated for storage as packed RBC and fresh frozen plasma. Gel column crossmatching was performed 3 to 4 wk later. All fetus-dam crossmatches were considered major crossmatches, combining fetal (recipient) plasma with dam (donor) RBC. The plasma of 8 fetuses was cross-matched with RBC from 5 dams; all autologous controls were negative, and all but one crossmatch (1 of 40, 2.5%) were considered compatible. In addition, the plasma of 3 dams was crossmatched with RBC from 5 dams; all autologous controls were negative; however, significant incompatibility was noted. In total, 4 of 13 (30.8%) dam-dam crossmatches were considered incompatible. The results of this initial study suggest that when a single animal receives multiple blood-product transfusions, the risk of an immunologic transfusion reaction can be reduced by ensuring that the blood products are obtained from a single donor, performing a crossmatch prior to transfusion, and the use of synthetic products to increase the oxygen carrying capacity of fetal blood.

Further Investigations of the Effects of Anti-ß2GP1 Antibodies on Collagen-Induced Platelet Aggregation.

Anti-beta-2-glycoprotein 1 (anti-ß2GP1) antibodies are associated with increased thrombotic risk in patients with autoimmune disease. There is conflicting evidence on the effects of anti-ß2GP1 antibodies on platelets, with both enhanced and inhibited aggregation previously reported. However, previous studies did not include isotype antibodies to ensure the observed effects were due to anti-ß2GP1 antibodies. The aims of this study were to (1) investigate the effects of anti-ß2GP1 antibodies on collagen-induced platelet aggregation in parallel with negative control (buffer normal saline) and isotype control antibodies and (2) determine the lupus anticoagulant (LA) activity of anti-ß2GP1 antibodies used. Three animal-derived anti-human-ß2GP1 antibodies (1.25, 2.5, and 5 µg/mL) incubated with healthy platelet-rich plasma were activated by collagen (2.5 µg/mL). Each anti-ß2GP1 antibody demonstrated the inhibition of aggregation compared to negative control, but not to isotype control. No anti-ß2GP1 antibody demonstrated LA activity, suggesting they were probably nonpathological. This study highlights both negative and isotype control markers are important to validate the effects of anti-ß2GP1 antibodies. Assays to measure anti-domain I-ß2GP1 antibodies are recommended to be used in conjunction with functional measures to further investigate the effects of anti-ß2GP1 antibodies.

TRPV1 Channels in Immune Cells and Hematological Malignancies.

Transient receptor potential vanilloid-1 (TRPV1) is a member of the TRP family of channels that are responsible for nociceptive, thermal, and mechanical sensations. Originally associated exclusively with sensory neurons, TRPV1 is now known to be present in almost all organs, including cells of the immune system, where TRPV1 has been shown to play a pivotal role in inflammation and immunity. Monocytes, macrophages, and dendritic cells express TRPV1, with both mouse and human studies suggesting that TRPV1 activation protects against endotoxin-induced inflammation. In contrast, TRPV1 (and other TRP channels) appears to be required for T-cell receptor activation by mitogens. Additionally, studies in cell lines derived from hematological and other malignancies suggest altered expression/function of TRPV1 might serve as a target for novel cytotoxic therapies.

Expression of CCR6 on B cells in systemic lupus erythematosus patients.

B cells are known to play a dominant pathogenic role in autoimmune conditions such as systemic lupus erythematosus (SLE) and rheumatoid arthritis. In recent times, the chemokine receptor CCR6 and its cognate ligand CCL20 have been shown to play a role in the fundamental kinetics of germinal centres and B cell responses. As CCR6 is found on B cells and is upregulated after activation, we investigated the expression of CCR6 on naive, pre-germinal centre (GC), GC/plasma cell and memory B cells in peripheral B cells of SLE patients and healthy controls using flow cytometry. Pre-germinal centre B cells are found in lower proportions and the expression of CCR6 is increased on B cells of SLE patients, suggesting a role for the chemokine pair in the pathogenesis of the disease. Further studies are needed to explore these preliminary results.

Capsaicin-Induced Death of Human Haematological Malignant Cell Lines Is Independent of TRPV1 Activation.

The effect of the plant-derived vanilloid, capsaicin (CAP), on the metabolic activity of THP-1, U266B1 and U937 hematological malignancy cells was determined. CAP reduced metabolic activity in a concentration-dependent manner in the three cell lines. A biphasic effect was observed on THP-1 cells (EC50: IC50 (95% CI) 32.9 (19.9-54.3)/219 (144-246) µmol/l). U266B1 cells were more resistant to CAP than THP-1 and U937. Metabolic activity was significantly inhibited by CAP in U937 compared to U266B1 cells (IC50: 197 versus 431 µmol/l, respectively, p < 0.008). Transient receptor potential vanilloid-1 (TRPV1) and CB1 antagonists (SB452533 and AM251, respectively) suppressed the CAP-induced increase in THP-1 cell metabolic activity (p < 0.001). AM251 and SB452533 appeared to act as partial agonists and displayed a synergistic effect with CAP in U937 cells. CAP inhibits the metabolic activity of malignant hematological cells through non-TRPV1-dependent mechanisms.

Changes to fibrinolysis in patients with systemic lupus erythematosus are associated with endothelial cell damage and inflammation, but not antiphospholipid antibodies.

We investigated whether changes to fibrinolysis were associated with other manifestations of systemic lupus erythematosus (SLE), including antiphospholipid (APL) antibody status, endothelial damage, and inflammation. Ninety-four patients (36 SLE patients, 58 healthy controls) were recruited from Tasmania, Australia. Circulating levels of plasminogen, α2-antiplasmin, tissue-type plasminogen activator, plasminogen activator inhibitor-1, and thrombin-activatable fibrinolysis inhibitor (TAFI) were measured, as well as APL antibodies (including lupus anticoagulant, anticardiolipin, and antibeta-2 glycoprotein-1 antibodies), soluble E-selectin, and interleukin-6. Whereas there was a significant decrease in plasminogen (patient vs. control; median) (210 vs. 444 ng/ml; P < 0.0001) and increase in α2-antiplasmin (0.53 vs. 0.09 µg/ml; P = 0.0007), there was increased t-PA (0.65 vs. 0.40 ng/ml; P = 0.0001) and decreased TAFI (8.8 vs. 10.0 ng/ml; P = 0.002) in SLE patients compared to healthy controls. Plasminogen was significantly associated with α2-antiplasmin (rho = -0.563, P < 0.001); TAFI (rho = 0.410, P = 0.011); soluble E-selectin (rho = 0.531, P = 0.001); and interleukin-6 (rho = 0.489, P = 0.002) in SLE patients; however, APL antibody status was not associated with any of the markers measured. This study has demonstrated that fibrinolysis is significantly altered in patients with SLE compared to controls, and associated with endothelial cell damage and inflammation, but not APL antibody status.

ß2GP1, Anti-ß2GP1 Antibodies and Platelets: Key Players in the Antiphospholipid Syndrome.

Anti-beta 2 glycoprotein 1 (anti-ß2GP1) antibodies are commonly found in patients with autoimmune diseases such as the antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Their presence is highly associated with increased risk of vascular thrombosis and/or recurrent pregnancy-related complications. Although they are a subtype of anti-phospholipid (APL) antibody, anti-ß2GP1 antibodies form complexes with ß2GP1 before binding to different receptors associated with anionic phospholipids on structures such as platelets and endothelial cells. ß2GP1 consists of five short consensus repeat termed "sushi" domains. It has three interchangeable conformations with a cryptic epitope at domain 1 within the molecule. Anti-ß2GP1 antibodies against this cryptic epitope are referred to as 'type A' antibodies, and have been suggested to be more strongly associated with both vascular and obstetric complications. In contrast, 'type B' antibodies, directed against other domains of ß2GP1, are more likely to be benign antibodies found in asymptomatic patients and healthy individuals. Although the interactions between anti-ß2GP1 antibodies, ß2GP1, and platelets have been investigated, the actual targeted metabolic pathway(s) and/or receptor(s) involved remain to be clearly elucidated. This review will discuss the current understanding of the interaction between anti-ß2GP1 antibodies and ß2GP1, with platelet receptors and associated signalling pathways.

Serum metabolic profile predicts adverse central haemodynamics in patients with type 2 diabetes mellitus.

AIMS: People with type 2 diabetes mellitus (T2DM) have abnormal peripheral and central haemodynamics at rest and during exercise, probably due to metabolic perturbations, but mechanisms are unknown. We used untargeted metabolomics to determine the relationships between metabolic perturbations and haemodynamics (peripheral and central) measured at rest and during exercise. METHODS: Serum samples from 39 participants with T2DM (62 ± 9 years; 46 % male) and 39 controls (52 ± 10 years; 51 % male) were analysed by liquid chromatography-mass spectrometry, nuclear magnetic resonance spectroscopy and principal component analysis. Scores on principal components (PC) were used to assess relationships with haemodynamics including peripheral and central BP, central augmentation index (AIx) and central augmentation pressure (AP). RESULTS: Participants with T2DM had higher resting and exercise haemodynamics (peripheral and central BP, central AIx and central AP) compared to controls (p < 0.05). PC that comprised of a signature metabolic pattern of T2DM was independently associated with resting and exercise central AIx and central AP (p < 0.05). CONCLUSIONS: Serum metabolic profile was associated with central, but not peripheral, haemodynamics in T2DM participants, suggesting that metabolic irregularities may explain abnormal central haemodynamics in T2DM patients.

Association of von Willebrand factor blood levels with exercise hypertension.

PURPOSE: A hypertensive response to moderate intensity exercise (HRE) is associated with increased cardiovascular risk. The mechanisms of an HRE are unclear, although previous studies suggest this may be due to haemostatic and/or haemodynamic factors. We investigated the relationships between an HRE with haemostatic and hemodynamic indices. METHODS: Sixty-four participants (57 ± 10 years, 71 % male) with indication for exercise stress testing underwent cardiovascular assessment at rest and during moderate intensity exercise, from which 20 participants developed an HRE (defined as moderate exercise systolic BP ≥ 170 mmHg/men and ≥ 160 mmHg/women). Rest, exercise and post-exercise blood samples were analysed for haemostatic markers, including von Willebrand factor (vWf), and haemodynamic measures of brachial and central blood pressure (BP), aortic stiffness and systemic vascular resistance index (SVRi). RESULTS: HRE participants had higher rest vWf compared with normotensive response to exercise (NRE) participants (1,927 mU/mL, 95 % CI 1,240-2,615, vs. 1,129 mU/mL, 95 % CI 871-1,386; p = 0.016). vWf levels significantly decreased from rest to post-exercise in HRE participants (p = 0.005), whereas vWf levels significantly increased from rest to exercise in NRE participants (p = 0.030). HRE participants also had increased triglycerides, rest BP, aortic stiffness and exercise SVRi (p < 0.05 for all). Rest vWf predicted exercise brachial systolic BP (ß = 0.220, p = 0.043; adjusted R (2) = 0.451, p < 0.001) independent of age, sex, body mass index, triglycerides, rest brachial systolic BP and aortic stiffness. CONCLUSIONS: Increased rest blood levels of vWf are independently associated with moderate intensity exercise systolic BP. These findings implicate abnormalities in haemostasis as a possible factor contributing to HRE at moderate intensity.

Tissue factor pathway inhibitor: then and now.

Tissue factor pathway inhibitor (TFPI) is the major physiological regulator of tissue factor (TF)-induced blood coagulation. TFPI inhibits the TF-activated factor VII (FVIIa) complex in an activated factor X (FXa)-dependent manner, helping to control thrombin generation and ultimately fibrin formation. The importance of TFPI is demonstrated in models of hemophilia where lower levels of FVIII or FIX are insufficient to overcome its inhibitory effect, resulting in a bleeding phenotype. There are two major isoforms in vivo; TFPIα contains three Kunitz-type inhibitory domains (designated K1, K2, and K3), is secreted by endothelial cells and requires protein S to enhance its anticoagulant activity. In contrast, TFPIß contains only the K1 and K2 domains, but it is attached to the endothelial surface via a glycosylphosphatidylinositol anchor. This review will initially provide a brief history of the major discoveries related to TFPI, and then discuss new insights into the physiology of TFPI, including updates on its association with protein S and FV, as well as the current understanding of its association with disease.

Vanilloid-like agents inhibit aggregation of human platelets.

INTRODUCTION: Plant-derived and endogenous vanilloid-like agents exert their effects on cells through transient receptor potential vanilloid-1 (TRPV1). Little is known about the effects of these agents on platelet aggregation. We investigated the effect of various vanilloid-like agents on in-vitro platelet aggregation and tested whether this action is mediated through TRPV1. Understanding the mechanism of action of these compounds in platelets is important in that these compounds may be developed as novel anti-platelet agents. MATERIALS AND METHODS: The effects of plant-derived (capsaicin; dihydrocapsaicin, DHC) and endogenous vanilloid-like agents (N-oleoyldopamine, OLDA; N-arachidonoyl-dopamine, NADA) on platelet aggregation were investigated using ADP (5, 10µM), collagen (4, 8µg/mL) and arachidonic acid (AA, 300, 400µg/mL) as agonists. The direct effects of these agents on platelet viability were also determined using an LDH release assay. RESULTS: Capsaicin, OLDA and NADA inhibited ADP-induced platelet aggregation in a concentration-dependent manner. OLDA and NADA, but not capsaicin and DHC, inhibited collagen-induced aggregation, whereas AA-induced aggregation was inhibited by capsaicin, DHC and NADA, but not OLDA. Inhibition of aggregation was not due to direct toxicity of these agents towards platelets. The TRPV1 antagonist, SB-452533, did not affect inhibition of ADP-induced platelet aggregation by capsaicin and OLDA. CONCLUSIONS: These results demonstrate that the endovanilloids, OLDA and NADA, and plant-derived vanilloid, capsaicin, inhibit ADP-induced platelet aggregation. Collagen-induced aggregation was inhibited only by endovanilloids, whereas AA-induced aggregation was inhibited by capsaicin, DHC and NADA. This inhibition was not due to direct toxic effects of these agents, nor was inhibition of ADP-induced aggregation TRPV1 mediated.