Annual variations in animal\'s physiological functions are an essential strategy to deal with seasonal challenges which also vary according to the time of year. Information regarding annual adaptations in the immune-competence to cope with seasonal stressors in reptiles is scarce. The present research plan was designed to analyze the presence of circannual immune rhythms in defense responses of the leucocytes in an ophidian, Natrix piscator. Peripheral blood leucocytes were obtained, counted, and superoxide anion production, neutrophil phagocytosis, and nitrite release were tested to assess the innate immune functions. Peripheral blood lymphocytes were separated by centrifugation (utilizing density gradient) and the cell proliferation was measured. The Cosinor rhythmometry disclosed the presence of significant annual rhythms in the number of leucocytes, superoxide anion production, nitric oxide production, and proliferation of stimulated lymphocytes. The authors found that respiratory burst activity and proliferative responses of lymphocytes were crucial immune responses that showed the annual rhythm. It was summarized that the immune function of the N. piscator is a labile attribute that makes the animal competent to cope with the seasonal stressor by adjustment in the potency of response.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder identified by hematological abnormalities including anemia, leukopenia, and thrombocytopenia. Complement system disturbance is implicated in the pathogenesis of SLE. In this work, we aim to study how a full assessment of the complement system, which includes the evaluation of its three pathways, relates to blood cell counts in a population of patients with SLE. New-generation functional assays of the classical, alternative, and lectin pathways of the complement system were conducted in 284 patients with SLE. Additionally, serum levels of inactive molecules (C1q, C2, C3, C4, factor D) and activated molecules (C3a), as well as regulators (C1-inhibitor and factor H), were evaluated. Complete blood cell counts were analyzed. Multivariable linear regression analysis was performed to study the relationship of hematological profiles with this full characterization of the complement system. After multivariable adjustments that included age, sex, SLICC-DI (damage), and SLEDAI (activity) scores, as well as the use of aspirin, prednisone, methotrexate, azathioprine, and mycophenolate mofetil, several relationships were observed between the C pathways and the individual products and blood cells profile. Lower values of C1q and C2 were associated with lower hemoglobin levels. Lower leukocyte counts showed significantly lower values of C4, C1 inhibitor, C3, factor D, and alternative pathway functional levels. Neutrophil counts showed significant negative relationships only with the alternative pathway and C1-inh. In the case of lymphocytes, associations were found, especially with functional tests of the classical and alternative pathways, as well as with C2, C4, C3, and C3a. On the contrary, for platelets, significance was only observed, after multivariable adjustment, with lower C2 concentrations. In conclusion, the serum complement system and hematological profile in SLE are independently linked, after adjustment for disease activity and damage. These relationships are basically negative and are predominantly found in lymphocytes.

UNASSIGNED: Cardiac surgery often necessitates considerable post-operative vasoactive-inotropic support. Given an encouraging literature on the prognostic potential of leucoglycemic index (LGI) [serum glucose (mg/dl) × total leucocytes count (cells/mm3)/1000], we aimed to evaluate whether intensive care unit (ICU)-admission LGI can predict post-operative vasopressor-inotropic requirements following cardiac surgery on cardio-pulmonary bypass (CPB).
UNASSIGNED: The data of patients undergoing cardiac surgery at our tertiary care center between January 2015 and December 2020 was retrospectively reviewed. The vasopressor-inotropic requirement was estimated using the VIS (vasoactive-inotropic score) values over the first post-operative 72 hrs. Subsequently, VISi (indexed VIS) was computed as maxVIS[0-24hrs] + maxVIS[24-48hrs] +2 × maxVIS[48-72hrs]/10), and the study participants were divided into h-VISi (VISi ≥3) and l-VISi (VISi <3).
UNASSIGNED: Out of 2138 patients, 479 (22.40%) patients categorized as h-VISi. On univariate analysis: LGI, age, European System for Cardiac Operative Risk Evaluation score (EuroSCORE II), left-ventricle ejection fraction, prior congestive heart failure (CHF), chronic renal failure, angiotensin-converting enzyme inhibitors, combined surgeries, CPB and aortic cross-clamp (ACC) duration, blood transfusion, and immediate post-operative glucose were significant h-VISi predictors. Subsequent to multi-variate analysis, the predictive performance of LGI (OR: 1.09; 95% CI: 1.03-1.14; P = 0.002) prior CHF (OR: 2.35; 95% CI: 1.44-3.82; P = 0.001), CPB time (OR: 1.08; 95% CI: 1.02-1.14; P = 0.019), ACC time (OR: 1.03; 95% CI: 1.02-1.04; P = 0.008), and EuroSCORE II (OR: 1.14; 95% CI: 1.06-1.21; P < 0.001) remained significant. With 1484.75 emerging as the h-VISi predictive cut-off, patients with LGI ≥ 1484.75 also had a higher incidence of vasoplegia, low-cardiac output syndrome, new-onset atrial fibrillation, acute kidney injury, and mortality. LGI additionally exhibited a significant positive correlation with duration of mechanical ventilation and ICU stay (R = 0.495 and 0.564, P value < 0.001).
UNASSIGNED: An elevated LGI of greater than 1484.75 independently predicted a VISindex ≥3 following adult cardiac surgery on CPB.

Silk fibroin (SF) microparticles were administered in the diet of gilthead seabream with or without experimental skin wounds to study the effects on cellular immunity and liver. A commercially available diet was enriched with varying amount of SF: 0, 50 and 100 mg kg-1 (representing the control, SF50 and SF100 diets respectively). The animals were fed for 30 days and half of them were sampled. Similar experimental wounds were then performed on the rest of fish, and they continued to be fed the same diet. At 7 days post-wounding, samples were taken from the wounded fish. Cellular immunity was studied on head kidney leucocytes (phagocytosis, respiratory and peroxidase content) and liver status (histological study and gene expression) were studied. Our results showed that experimental wounds affect both cellular immunity (by decreasing leucocyte respiratory burst and peroxidase activity) and altered liver histology (by inducing vascularisation and congestion of blood vessels). In addition, it influences the expression of genes that serve as markers of oxidative stress, endoplasmic reticulum stress and apoptosis. The highest dose of SF (SF100) increased the phagocytic capacity of leucocytes the most, as well as the expression of genes related to blood vessel formation in the liver. Furthermore, increased expression of antioxidant genes (cat and gsr) and decreased expression of genes related to reticulum endoplasmic stress (grp94 and grp170) and apoptosis (nos and jnk) were detected in these fish fed with SF100 and wounded. In conclusion, fed fish with SF100 had many beneficial effects as cellular immunostimulant and hepatoprotection in wounded fish. Its use could be of great interest for stress management in farmed fish conditions.

OBJECTIVE: To assess the association of postoperative C-reactive protein (CRP), leucocytes and vital signs in the first three postoperative days (PODs) with major complications after oncological colorectal resections in a tertiary referral centre for colorectal cancer in The Netherlands.
METHODS: A retrospective cohort study, including 594 consecutive patients who underwent an oncological colorectal resection at Maastricht University Medical Centre between January 2016 and December 2020. Descriptive analyses of patient characteristics were performed. Logistic regression models were used to assess associations of leucocytes, CRP and Modified Early Warning Score (MEWS) at PODs 1-3 with major complications. Receiver operating characteristic curve analyses were used to establish cut-off values for CRP.
RESULTS: A total of 364 (61.3%) patients have recovered without any postoperative complications, 134 (22.6%) patients have encountered minor complications and 96 (16.2%) developed major complications. CRP levels reached their peak on POD 2, with a mean value of 155 mg/L. This peak was significantly higher in patients with more advanced stages of disease and patients undergoing open procedures, regardless of complications. A cut-off value of 170 mg/L was established for CRP on POD 2 and 152 mg/L on POD 3. Leucocytes and MEWS also demonstrated a peak on POD 2 for patients with major complications.
CONCLUSIONS: Statistically significant associations were found for CRP, Δ CRP, Δ leucocytes and MEWS with major complications on POD 2. Patients with CRP levels ≥ 170 mg/L on POD 2 should be carefully evaluated, as this may indicate an increased risk of developing major complications.

BACKGROUND: Forty percent of patients with aneurysmatic subarachnoid hemorrhage (aSAH) develop acute hydrocephalus requiring treatment with cerebrospinal fluid (CSF) drainage. CSF cell parameters are used in the diagnosis of nosocomial infections but also reflect sterile inflammation after aSAH. We aimed to study the temporal changes in CSF parameters and compare external ventricular drain (EVD)-derived and lumbar spinal drain-derived samples.
METHODS: We retrospectively identified consecutive patients with aSAH treated at our neurointensive care unit between January 2014 and May 2019. We mapped the temporal changes in CSF leucocyte count, erythrocyte count, cell ratio, and cell index during the first 19 days after aSAH separately for EVD-derived and spinal drain-derived samples. We compared the sample sources using a linear mixed model, controlling for repeated sampling.
RESULTS: We included 1360 CSF samples from 197 patients in the analyses. In EVD-derived samples, the CSF leucocyte count peaked at days 4-5 after aSAH, reaching a median of 225 × 106 (interquartile range [IQR] 64-618 × 106). The cell ratio and index peaked at 8-9 days (0.90% [IQR 0.35-1.98%] and 2.71 [IQR 1.25-6.73], respectively). In spinal drain-derived samples, the leucocyte count peaked at days 6-7, reaching a median of 238 × 106 (IQR 60-396 × 106). The cell ratio and index peaked at 14-15 days (4.12% [IQR 0.63-10.61%]) and 12-13 days after aSAH (8.84 [IQR 3.73-18.84]), respectively. Compared to EVD-derived samples, the leucocyte count was significantly higher in spinal drain-derived samples at days 6-17, and the cell ratio as well as the cell index was significantly higher in spinal drain-derived samples compared to EVD samples at days 10-15.
CONCLUSIONS: CSF cell parameters undergo dynamic temporal changes after aSAH. CSF samples from different CSF compartments are not comparable.

OBJECTIVE: Cyclophilin A (CyPA) induces leucocyte recruitment and platelet activation upon release into the extracellular space. Extracellular CyPA therefore plays a critical role in immuno-inflammatory responses in tissue injury and thrombosis upon platelet activation. To date, CD147 (EMMPRIN) has been described as the primary receptor mediating extracellular effects of CyPA in platelets and leucocytes. The receptor for advanced glycation end products (RAGE) shares inflammatory and prothrombotic properties and has also been found to have similar ligands as CD147. In this study, we investigated the role of RAGE as a previously unknown interaction partner for CyPA.
RESULTS: Confocal imaging, proximity ligation, co-immunoprecipitation, and atomic force microscopy were performed and demonstrated an interaction of CyPA with RAGE on the cell surface. Static and dynamic cell adhesion and chemotaxis assays towards extracellular CyPA using human leucocytes and leucocytes from RAGE-deficient Ager-/- mice were conducted. Inhibition of RAGE abrogated CyPA-induced effects on leucocyte adhesion and chemotaxis in vitro. Accordingly, Ager-/- mice showed reduced leucocyte recruitment and endothelial adhesion towards CyPA in vivo. In wild-type mice, we observed a downregulation of RAGE on leucocytes when endogenous extracellular CyPA was reduced. We furthermore evaluated the role of RAGE for platelet activation and thrombus formation upon CyPA stimulation. CyPA-induced activation of platelets was found to be dependent on RAGE, as inhibition of RAGE, as well as platelets from Ager-/- mice showed a diminished activation and thrombus formation upon CyPA stimulation. CyPA-induced signalling through RAGE was found to involve central signalling pathways including the adaptor protein MyD88, intracellular Ca2+ signalling, and NF-κB activation.
CONCLUSIONS: We propose RAGE as a hitherto unknown receptor for CyPA mediating leucocyte as well as platelet activation. The CyPA-RAGE interaction thus represents a novel mechanism in thrombo-inflammation.

UNASSIGNED: To assess the impact of varying centrifugation speeds on platelet and leucocyte-rich plasma (L-PRP) in liquid and gel form cellularity and growth factor concentrations for potential use against ocular surface disorders.
UNASSIGNED: L-PRP was collected from 16 healthy subjects using three different centrifugation speeds: 580, 1000, and 2000 g, each for 8 min at 25°C. Platelet and leukocyte counts were automatically evaluated. The concentrations of vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor beta 1 (TGF-B1) were measured using enzyme-linked immunosorbent assays. L-PRP gel cellularity was assessed through hematoxylin-eosin and Masson\'s trichrome staining, categorized as moderate or abundant, and statistically analyzed. L-PRP gel membrane\'s chemical composition was analyzed using Fourier-transform infrared spectroscopy (FTIR), crystallization was investigated with X-ray diffraction (XRD), and ultrastructure was assessed using surface electron microscopy (SEM). Additionally, membrane degradation was evaluated over a 7-day period.
UNASSIGNED: No significant differences in cellularity and growth factor concentrations among centrifugation speeds (p > 0.05) were found. Moderate cellularity predominated at 580 g and 2000 g, while abundant cellularity was observed at 1000 g. No significant differences were found techniques (p = 0.16). Masson\'s trichrome staining suggested the existence of abundant fibrin at 1000 g but without significant differences (p = 0.07). FTIR analysis exhibited the characteristic fibrin bands at all speeds, and XRD indicated a keratin-like pattern. SEM revealed greater porosity at 580 g and fibrin membrane degradation was lower at this speed (p = 0.0001).
UNASSIGNED: Centrifugation speed did not significantly affect growth factor concentration or cellularity in both liquid and gel L-PRP. Further studies should explore the impact of different separation techniques for L-PRP used in ophthalmic applications.

We present a simple and quantitative assay system that accurately models human endothelium by use of primary human umbilical vein endothelial cells (HUVECs) in cell culture plates coated with gelatin, a matrix that mimics basal lamina, the matrix that is tightly associated with the vascular endothelium and is critical for its proper function. We describe using this system to quantitatively measure adhesion of the inflammatory cells - monocytic THP-1 cell line to the HUVEC monolayer. The THP-1 cells are fluorescently labeled which allows to quantify the number of the fluorescent THP-1 cells adhering to the endothelium under a microscope and the level of florescence - a quantitative measure of the number of adhering fluorescent THP-1 cells using a fluorescent plate reader. After optimization, we were able to detect increased adhesion of the THP-1 cells to the endothelium in response to the inflammatory cytokine TNFα in a dose-dependent manner like what has been observed in vivo.

Inflammation is a complex pathophysiological process underlying many clinical conditions. Platelets contribute to the thrombo-inflammatory response. Platelet P2Y12 receptors amplify platelet activation, potentiating platelet aggregation, degranulation and shape change. The contents of platelet alpha granules, in particular, act directly on leucocytes, including mediating platelet-leucocyte aggregation and activation via platelet P-selectin. Much evidence for the role of platelet P2Y12 receptors in inflammation comes from studies using antagonists of these receptors, such as the thienopyridines clopidogrel and prasugrel, and the cyclopentyltriazolopyrimidine ticagrelor, in animal and human experimental models. These suggest that antagonism of P2Y12 receptors decreases markers of inflammation with some evidence that this reduces incidence of adverse clinical sequelae during inflammatory conditions. Interpretation is complicated by pleiotropic effects such as those of the thienopyridines on circulating leucocyte numbers and of ticagrelor on adenosine reuptake. The available evidence suggests that P2Y12 receptors are prominent mediators of inflammation and P2Y12 receptor antagonism as a potentially powerful strategy in a broad range of inflammatory conditions. LINKED ARTICLES: This article is part of a themed issue on Platelet purinergic receptor and non-thrombotic disease. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v181.4/issuetoc.