Leukocyte Profiles Research Articles

183 Circulating Biomarkers and Leukocyte Profiles in Agalactic Sows

Abstract Agalactia is characterized by reduced milk production after farrowing, resulting in economic losses due to increased piglet mortality. Although a variety of management factors have been associated with the etiology of agalactia, a specific causative mechanism has not been identified. Since clinical signs of agalactia develop in the days following farrowing, the objective of the current study was to determine if periparturient immune cell profiles and circulating biomarkers are predictive of future agalactia. Blood samples and litter weights were collected from sows (n = 374) within 24-36 hours after farrowing (timepoint 1) and sows were subsequently monitored for symptoms of agalactia and gaunt piglets. When a sow was designated as agalactic (n = 36) blood samples and litter weights were collected again (timepoint 2) and also from a parity matched healthy control sow (n = 46) of the same day of lactation. Agalactia diagnosis occurred on average 9.25 ± 2.67 d after farrowing. Average daily gain (ADG) of piglets from agalactic sows was 55% less (P < 0.01) than ADG of piglets from controls. Additionally, piglet mortality was substantially greater (P < 0.01) in litters from agalactic sows compared with controls. Circulating immune cells and metabolites were determined. Cholesterol, blood urea nitrogen, and globulin were increased 22.3, 17.8, and 34.1%, respectively, in agalactic compared with control sows at timepoint 2 (P ≤ 0.01). Aspartate aminotransferase tended to increase in agalactic sows compared with control at timepoint 1 (P = 0.08). No differences in immune cell profiles were observed between agalactic and control sows at either timepoint (P ≥ 0.14). Collectively, these data suggest metabolic changes are occurring in sows experiencing agalactia compared with healthy herd mates, but that very few of the metrics analyzed at timepoint 1 predicted future agalactia. This project was supported by Zoetis; TI-07023.

Stromal Factors as a Target for Immunotherapy in Melanoma and Non-Melanoma Skin Cancers

Immune checkpoint inhibitors (ICIs), such as anti-programmed cell death 1 (PD1) antibodies (Abs) and anti-cytotoxic T-lymphocyte associated protein 4 (CTLA4) Abs, have been widely administered for not only advanced melanoma, but also various non-melanoma skin cancers. Since profiles of tumor-infiltrating leukocytes (TILs) play important roles in immunotherapy using ICIs, it is important to evaluate cancer stromal cells such as tumor-associated macrophages (TAMs) and cancer-associated fibroblasts (CAFs), as well as stromal extracellular matrix protein, to predict the efficacy of ICIs. This review article focuses particularly on TAMs and related factors. Among TILs, TAMs and their related factors could be the optimal biomarkers for immunotherapy such as anti-PD1 Ab therapy. According to the studies presented, TAM-targeting therapies for advanced melanoma and non-melanoma skin cancer will develop in the future.

Perimesencephalic Subarachnoid Hemorrhage Has a Unique Peripheral Blood Leukocyte Profile Compared To Aneurysmal Subarachnoid Hemorrhage

The authors sought to investigate if peripheral blood leukocyte profiles on admission differed between perimesencephalic, angio-occult, and aneurysmal subarachnoid hemorrhage cohorts. We performed a retrospective analysis of 202 consecutive patients with spontaneous subarachnoid hemorrhage. We classified spontaneous subarachnoid hemorrhage as either aneurysmal or nonaneurysmal origin. Nonaneurysmal subarachnoid hemorrhage was subclassified as either perimesencephalic or angio-occult according to the distribution of hemorrhage on the initial imaging. Patient demographics, clinical parameters, radiographic metrics, and laboratory values were obtained on admission. In-hospital data including acute hydrocephalus, shunt dependence, vasospasm, and delayed cerebral ischemia were collected. Comparative analyses were conducted between cohorts. The perimesencephalic subarachnoid hemorrhage cohort exhibited significantly lower neutrophil (7.76 vs. 10.06; P=0.004), lymphocyte (1.40 vs. 1.90; P=0.024), and monocyte counts (0.52 vs. 0.73; P=0.031) than the aneurysmal subarachnoid hemorrhage cohort. There were no significant differences in peripheral blood leukocyte profiles between the angio-occult and aneurysmal subarachnoid hemorrhage cohorts. The nonaneurysmal cohort exhibited significantly lower neutrophil (8.33 vs. 10.06; P=0.005) and lymphocyte counts (1.47 vs. 1.90; P=0.011) as well as a lower lymphocyte-to-monocyte ratio (2.80 vs. 4.51; P=0.018) than the aneurysmal subarachnoid hemorrhage cohort. Perimesencephalic subarachnoid hemorrhage exhibits a unique peripheral blood leukocyte profile compared to aneurysmal subarachnoid hemorrhage. Moreover, these preliminary data demonstrate that blood leukocytes may be affected by the burden of cisternal subarachnoid hemorrhage or the presence of a ruptured aneurysm. Further large-scale prospective studies and validation are required to confirm these preliminary findings.

Serum Cortisol and Its Correlation with Leucocyte Profile and Circulating Lipids in Donkeys (Equus asinus).

Simple SummaryThere are relatively few studies investigating serum cortisol levels in donkeys. The aim of this study was to evaluate basal cortisol values in donkeys, and its physiological role on the immune system and lipid levels in relation to age and pregnancy. The results showed that age did not influence cortisol levels while pregnancy increased its concentrations. Different correlations were found between cortisol, leucocyte types, and serum triglycerides depending upon age and pregnancy state. Although correlation is, of course, not equivalent to causation, we can hypothesize that the multiple functions of cortisol on the immune system and lipid metabolism vary across donkey lifespan and are related to physiological state.The values for basal serum cortisol concentrations of horses are available in many studies. However, there are limited data about serum cortisol in donkeys. The present study aimed to determine the baseline values for serum cortisol, to evaluate the influence of age and pregnancy on its levels, and to correlate its values with leucocyte profile, serum cholesterol, and triglycerides. Serum samples were collected from 97 healthy donkeys. Cortisol was analyzed by chemo-luminescent assay. The median and the 2.5th and 97.5th percentiles of serum cortisol measured and calculated in all donkeys were 5.64, 3.40, and 10.54 µg/dL, respectively. Females (n.91) were divided into three groups: Group A (young), Group B (adult), and Group C (pregnant at the 9th–11th months). The effect of age and physiological status was investigated by the Mann–Whitney test. Group C showed significantly higher levels than Group B (p < 0.05). Significant correlations were found in Group B with monocytes (r = 0.37, p < 0.01) and triglycerides (r = 0.30, p < 0.05), and in Group C with monocytes (r = 0.79, p < 0.01), basophils (r = 0.6, p < 0.05), and neutrophil/lymphocyte ratio (r = −0.63, p < 0.05). Higher cortisol values related to late pregnancy are also found in this species. These preliminary results provide evidence for a relationship between cortisol and the immune system as well as cortisol and lipid metabolism modulated by age and pregnancy when parameters are within normal values.

Physiological stress responses to nonmimetic model brood parasite eggs: Leukocyte profiles and heat-shock protein Hsp70 levels.

Obligate avian brood parasites lay their eggs in the nest of other bird species, known as hosts. Brood parasitism often imposes severe fitness costs on hosts, selecting for the evolution of effective antiparasitic defences, such as recognition and rejection of brood parasite eggs. Glucocorticoids have been recently found to mediate host physiological and behavioral adjustments in response to brood parasite eggs; however, it remains unclear whether brood parasitism triggers a general response involving multiple physiological elements. In this study, we experimentally investigated whether a salient brood parasitic stimulus (the presence of a nonmimetic model egg in the nest) causes physiological adjustments in adult Eurasian blackbirds (Turdus merula) at immune (leukocyte profiles) and cellular (heat-shock protein Hsp70 synthesis) level. Also, we explored whether these physiological changes are mediated by variations in corticosterone (CORT) levels. We found that experimental brood parasitism caused an increase in heterophils and a decrease in lymphocytes, leading to higher heterophils and lymphocytes ratios in parasitized birds. Nevertheless, we did not find tradeoffs between immune function and CORT levels. Hsp70 synthesis was not affected by our experimental manipulation. Our findings provide evidence that brood parasite eggs trigger a general stress response in egg-rejecter hosts, including changes in cellular immune profiles.

Patients with hypothermic sepsis have a unique gene expression profile compared to patients with fever and sepsis.

The pathophysiology of hypothermia during sepsis is unclear. Using genomic profiling of blood leukocytes, we aimed to determine if hypothermia is associated with a different gene expression profile compared to fever during sepsis. Patients with sepsis and either hypothermia or fever within 24 hours after ICU admission were included in the study (n = 168). Hypothermia was defined as body temperature below 36 °C. Fever was defined as body temperature equal to or above 38.3°C. We compared blood gene expression (whole‐genome transcriptome in leukocytes) in hypothermic septic compared to febrile septic patients in an unmatched analysis and matched for APACHE IV score and the presence of shock. In total, 67 septic patients were hypothermic and 101 patients were febrile. Hypothermia was associated with a distinct gene expression profile in both unmatched and matched analyses. There were significant differences related to the up‐ and downregulation of canonical signalling pathways. In the matched analysis, the top upregulated gene was cold‐inducible mRNA binding protein (CIRBP) which plays a role in cold‐induced suppression of cell proliferation. In addition, we found three signalling pathways significantly upregulated in hypothermic patients compared to febrile patients; tryptophan degradation X, phenylalanine degradation IV and putrescine degradation III. In conclusion, there are distinct signalling pathways and genes associated with hypothermia, including tryptophan degradation and CIRBP expression, providing a possible link to the modulation of body temperature and early immunosuppression. Future studies may focus on the canonical signalling pathways presented in this paper to further investigate spontaneous hypothermia in sepsis.

Splenic and PB immune recovery in neoadjuvant treated gastrointestinal cancer patients

In recent years, immune therapy, notably immune checkpoint inhibitors (ICI), in conjunction with chemotherapy and surgery has demonstrated therapeutic activity for some tumor types. However, little is known about the optimal combination of immune therapy with standard of care therapies and approaches. In patients with gastrointestinal (GI) cancers, especially pancreatic ductal adenocarcinoma (PDAC), preoperative (neoadjuvant) chemotherapy has increased the number of patients who can undergo surgery and improved their responses. However, most chemotherapy is immunosuppressive, and few studies have examined the impact of neoadjuvant chemotherapy (NCT) on patient immunity and/or the optimal combination of chemotherapy with immune therapy. Furthermore, the majority of chemo/immunotherapy studies focused on immune regulation in cancer patients have focused on postoperative (adjuvant) chemotherapy and are limited to peripheral blood (PB) and occasionally tumor infiltrating lymphocytes (TILs); representing a minority of immune cells in the host. Our previous studies examined the phenotype and frequencies of myeloid and lymphoid cells in the PB and spleens of GI cancer patients, independent of chemotherapy regimen. These results led us to question the impact of NCT on host immunity. We report herein, unique studies examining the splenic and PB phenotypes, frequencies, and numbers of myeloid and lymphoid cell populations in NCT treated GI cancer patients, as compared to treatment naïve cancer patients and patients with benign GI tumors at surgery. Overall, we noted limited immunological differences in patients 6 weeks following NCT (at surgery), as compared to treatment naive patients, supporting rapid immune normalization. We observed that NCT patients had a lower myeloid derived suppressor cells (MDSCs) frequency in the spleen, but not the PB, as compared to treatment naive cancer patients and patients with benign GI tumors. Further, NCT patients had a higher splenic and PB frequency of CD4+ T-cells, and checkpoint protein expression, as compared to untreated, cancer patients and patients with benign GI tumors. Interestingly, in NCT treated cancer patients the frequency of mature (CD45RO+) CD4+ and CD8+ T-cells in the PB and spleens was higher than in treatment naive patients. These differences may also be associated, in part with patient stage, tumor grade, and/or NCT treatment regimen. In summary, the phenotypic profile of leukocytes at the time of surgery, approximately 6 weeks following NCT treatment in GI cancer patients, are similar to treatment naive GI cancer patients (i.e., patients who receive adjuvant therapy); suggesting that NCT may not limit the response to immune intervention and may improve tumor responses due to the lower splenic frequency of MDSCs and higher frequency of mature T-cells.

Tissue eosinophilia correlates with mice susceptibility, granuloma formation, and damage during Toxocara canis infection.

AbstractAn increase in peripheral blood eosinophils in helminth infections is expected, and these cells are known to promote immunity against these parasites. However, studies have suggested that in some specific helminths, eosinophils may promote the needs and longevity of these parasites, and their role in these infections remains undefined, including in Toxocara canis infection. Thus, this study aimed to investigate the role of eosinophils in the context of larval migration of T. canis and the immunopathological aspects of infection. For this, we used wild-type mice and mice genetically deficient for the transcription factor GATA-binding factor 1 (GATA1−/−), infected with 1000 eggs of T. canis. At 0, 3, 14 and 63 days post-infection, parasite load, tissue cytokine production, leucocyte profile, bronchoalveolar lavage cells and histopathological analyses were carried out. Collectively, our results demonstrate that the presence of eosinophils mediates susceptibility to T. canis, inducing leucocytosis and the formation of granulomas, increasing the pulmonary and cerebral parasite load, and reducing the number of neutrophils, which may be necessary to control the infection.

Neutrophil extracellular traps (NETs) modulate inflammatory profile in obese humans and mice: adipose tissue role on NETs levels.

Neutrophilextracellular traps (NETs) are a recently discovered neutrophil defense mechanism whichmodulates several inflammatory conditions contributing to metabolic profile alterations. Therefore, the present study aimed to evaluate the production of NETs in obese patients and mice, verifying the possible mechanisms associated with the release of NETs by the adipose tissue. The present study investigated NETs production in human adipose tissue and also showing the neutrophils using intravital microscopy in mouse epididymal adipose tissue. Blood and white adipose tissues were obtained from eutrophic and obese individuals and from mice. Lipid, glycemic and leukocyte profiles were evaluated, as well as the levels of NETs and its markers. Bioinformatics and proteomics analyses were performed and the identified key proteins were measured. The main findings showed that the inflammatory markers interleukin-8 (IL-8), heat shock protein 90 (HSP90) and the E1 heat shock protein family (HSPE1) can be modulated by the NETs levels in obesity. Obesity has also been associated with increased cholesterol, glucose intolerance, ionic calcium and NETs. We also observed an increase in catalase and a decreased superoxide dismutase activity. Bioinformatics and proteomics analyses revealed that IL-8, HSP90 and HSPE1 were associated with obesity, inflammation and NETs release. In conclusion, the present study shows an increase in NETs production during obesity associated with important inflammatory markers in adipose.

DETERMINATION OF LEUKOCYTE PROFILE IN CHRONIC ALCOHOL CONSUMPTION

Chronic alcohol consumption can induce Alcoholic Liver Disease (ALD) promoting biological alterations and liver damage; however, the immunological changes usually are underestimated. It has been reported, the increase of leucocytes in alcoholic hepatitis patients 1 , but the regulation of other cell lineages has not fully evaluated. To evaluate the leukocyte profile in patients with liver damage induces by chronic and acute alcohol consumption. A Cross-sectional study. Patients were classified as follow: (1) Controls with alcohol consumption <10g/day, AUDIT <8 (CT); (2) Chronic alcohol consumption AUDIT> 8, without clinical or biochemical data of liver damage (OH, alcoholism); (3) Cirrhotic patients due to alcohol (CiOH) and (4) Patients with alcoholic hepatitis (AH). Leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, and basophils were determined by hematic biometry. U-Mann Whitney was used for statistical analysis, p <0.05 was considered significant. 570 patients were included. The mean in age was: 29.5±10.8 for CT; 31±12.6 for OH; 47.6±7.7 for CiOH and 41.2±9.2 years old (p<0.001). Alcohol consumption was higher in CiOH 240 (320, 120; p<0.001) and AH 320 (480, 160; p<0.05). Albumin decreases in CiOH 2.9 (3.5, 2.2; p<0.001) and AH 1.9 (2.3, 1.6; p<0.001). On the other hand, AST, ALT and GGT increase in CiOH and AH, 49.5 (75.3, 38; p<0.001), 155 (177, 121; p<0.001) for AST, 32.5 (47.3, 24; p<0.001), 49 (75, 35.3; p<0.001) for ALT and 91.5 (191.8, 48; p<0.001), 224 (525.5, 104; p<0.001) for GGT. There was no a significant difference in eosinophils and basophils. The statistical number of leukocyte profile is described in Table 1. Data is expressed as the median with interquartile values (Q3-Q1). a) Differences between Alcoholism and Controls; b) Cirrhosis and Controls; c) Alcoholic Hepatitis and Controls; d) Alcoholism and Cirrhosis; e) Alcoholism and Alcoholic Hepatitis; f) Cirrhosis and Alcoholic Hepatitis. €p<0.05; +p<0.01; *p<0.001. During alcoholism, lymphocytes decrease, whereas neutrophils increase; this could be related to a susceptibility to recurrent respiratory and gastrointestinal infections. Lymphocytes and neutrophils decrease in CiOH; the reduction in neutrophils could be explained because the stimuli in CiOH decrease. In patients with AH, monocytes and neutrophils increase, that in a consequence increases the inflammatory state, promoting liver fibrosis and mortality. Chronic alcohol consumption, liver cirrhosis and alcoholic hepatitis promote cellular alterations, this phenomenon is more evident in AH. Our findings can be used to design novel detection strategies for the treatments of chronic and acute alcohol consumption. Conflict of interest: The authors declare that there is no conflict of interest. This work was partially financed by CONACyT SALUD-2016-272579 y PAPIIT-UNAM TA200515

Altered DNA Methylome Profiles of Blood Leukocytes in Chinese Patients with Mild Cognitive Impairment and Alzheimer's Disease

Altered DNA Methylome Profiles of Blood Leukocytes in Chinese Patients with Mild Cognitive Impairment and Alzheimer's Disease

Leukocyte profile variation in Dupont\u2019s Lark (Chersophilus duponti) in Spain and Morocco

Stress in birds has been widely studied through the measurement of heterophil-to-lymphocyte ratio (H/L ratio). In this study we aimed to assess for the first time the potential variation of stress, measured as H/L ratio, associated to geography (between-country variation) and seasonality (between seasons and within the breeding season), as well as the leukocyte profiles, in the threatened Dupont’s Lark (Chersophilus duponti), using samples from Spain and Morocco. Furthermore, we tested whether variation in H/L ratio was associated with variables such as population density, presence of blood parasites and individual body condition. We found that H/L ratio did not vary between countries, but individuals captured during the breeding season showed higher values of H/L compared to non-breeding ones. Neither male density, nor date within the breeding season had an effect on the H/L ratio. In Spain, individuals with higher body condition showed lower H/L ratio regardless of whether they were malaria-infected. In Morocco, malaria-infected individuals showed higher values of H/L ratio than the non-infected birds. Moreover, we found that our average values of H/L ratio in Morocco were within the ranges of other passerines, but not in Spain. Individuals with higher H/L ratios may be more stressed or present higher capability to face stressful situations. Although H/L ratio is a useful and relatively easy way to obtain measure of stress, the impact that the environment might have on stress and the way it is explained by H/L ratio must be addressed carefully. This study provides new insight for this species’ biology and provides useful reference information to test the status and survival of other populations.

EFFECT OF DIETARY SUPPLEMENTATION WITH ASTAXANTHIN ON THE HEMATOLOGICAL AND BIOCHEMICAL RESPONSE OF NILE TILAPIA (Oreochromis niloticus)

This study aimed to evaluate the effect of astaxanthin on the hematological, biochemical and somatic response of Nile tilapia ( Oreochromis niloticus ) orally administered in the feed for a period of 60 days. For the study, 105 tilapia (n=35) from the same spawn were used, constituting the following treatments: Control = animals (not treated with astaxanthin); T100 and T200 = fish treated with 100 and 200 mg of astaxanthin/kg of feed, respectively. There were no hematological alterations in red blood cells counts, hematocrit, hemoglobin, MCV and MCHC. In addition, astaxanthin-fed tilapia presented a better thrombocyte and leukocyte responses with a marked decrease in the number of thrombocytes, lymphocytes and neutrophils. Among treatments, there were no changes in serum levels of total protein, albumin, globulins, aspartate aminotransferase and alkaline phosphate. Treatment with astaxanthin resulted in a decrease in triglyceride and glucose levels, as well as increase in hepatosomatic indices. The results of hematological and biochemical analyzes of tilapias demonstrated the clinical safety of this carotenoid, not causing harmful effects to the health of fish. Therefore, the antioxidant activity of this compound in tilapias resulted in an improvement in the leukocyte profile and contributed to hypolipidemic effects at the dose of 200mg of astaxanthin/kg of feed.

Double-Spin Leukocyte-Rich Platelet-Rich Plasma Is Predominantly Lymphocyte Rich With Notable Concentrations of Other White Blood Cell Subtypes

PurposeTo comprehensively characterize a double-spin leukocyte-rich platelet-rich plasma (LR-PRP) formulation and to compare it with whole blood (WB) by quantitatively assessing platelet and WB cell subtype concentrations in each.MethodsProspective human ex vivo analysis with 12 healthy adult men with ages ranging from 25 to 31 was performed in a controlled laboratory setting. The main outcome measure was the leukocyte profile of human LR-PRP.ResultsIn LR-PRP, lymphocytes were the predominant WB cell type (11.94 ± 2.97 × 103 cells/μL) followed by neutrophils (3.72 ± 1.28 × 103 cells/μL). The mean cumulative percentage of granulocytes was 23% ± 8% and agranulocytes was 77% ± 18%. There was a significant difference observed between granulocyte and agranulocyte percentage within both WB (P = .004, [95% CI: (7%,31%)]) and LR-PRP (P < .0001, [95% CI: (42%,66%)]) groups. In addition, there was a significant difference observed between the WB and LR-PRP granulocyte percentages (P < .0001, [95% CI: (29%,43%)]) and between the WB and LR-PRP agranulocyte percentages (P < .0001, [95% CI: (30%,42%)]).ConclusionsOur study found that LR-PRP is predominantly lymphocyte rich with notable concentrations of other WB cell subtypes, including neutrophils, monocytes, eosinophils, basophils, and large unstained cells. While these subtypes are not routinely reported, they may play a role in modulating the local inflammatory environment. We also found significant differences in WB cell subtype concentrations between WB and LR-PRP.Clinical RelevancePRP has been routinely used in many clinical practices without clear indications for its use and lacks standardization in its formulation. This study provides a comprehensive characterization of a broadly used PRP, LR-PRP, and further characterizes subtypes of WBC cells present in LR-PRP that have not been previously reported. Comprehensively reporting these subtypes in clinical trials of PRP is crucial to understanding how these cells participate in PRP’s therapeutic potential. This type of data can help standardize future PRP formulations and improve patient outcomes.

The Effect of Insect Repellent Exposure on Leukocyte Profile and Histopathologic Findings in Lungs

Organophosphate compounds in insect repellent have a role in contributing to mosquito mortality but have toxic effects for humans when exposed for a long time. The research is aimed to analyze the effect of insect repellent exposure in blood leukocyte profile and histopathologic findings in lungs. The study used thirty males Rattus novergicus, which were divided into three groups, such as electric liquid insect repellent (P1) with contain 0.031% dimefluthrin, anti-mosquito coils (P2) with 0.014% dimefluthrin, and electric mat mosquito repellent with 0.566% dimefluthrin for 8 hours in 20 days respectively. Leukocyte profiles were determined by using the blood smear method, and the lung’s health was identified by histopathologic findings. Based on the results study showed mosquito coils exposure increase the lymphocytes count. Meanwhile, the electric liquid-repellent increased the basophil’s numbers. The electric mat exposure had more eosinophils, neutrophils stab, neutrophils segment, and monocytes in the blood. The leukocyte profile of each group showed there were no statistically significant differences (P-value &gt; 0.05). Based on histopathology, lung findings showed that the electric mat exposure contributed to cells degeneration 7.5% and pleural thickening 30%. The higher dimefluthrin concentrations in insect repellents could affect leukocyte profile and lungs health.

Secretome Profiling of Atlantic Salmon Head Kidney Leukocytes Highlights the Role of Phagocytes in the Immune Response to Soluble β-Glucan

β‐Glucans (BG) are glucose polymers which are produced in bacteria and fungi but not in vertebrate organisms. Being recognized by phagocytic leukocytes including macrophages and neutrophils through receptors such as dectin-1 and Complement receptor 3 (CR3), the BG are perceived by the innate immune system of vertebrates as foreign substances known as Pathogen Associated Molecular Patterns (PAMPs). The yeast-derived BG has been recognized for its potent biological activity and it is used as an immunomodulator in human and veterinary medicine. The goal of the current study was to characterize the immunostimulatory activity of soluble yeast BG in primary cultures of Atlantic salmon (Salmo salar) head kidney leukocytes (HKLs) in which phagocytic cell types including neutrophils and mononuclear phagocytes predominate. The effect of BG on the secretome of HKL cultures, including secretion of extracellular vesicles (EVs) and soluble protein55s was characterized through western blotting and mass spectrometry. The results demonstrate that, along with upregulation of proinflammatory genes, BG induces secretion of ubiquitinated proteins (UbP), MHCII-containing EVs from professional antigen presenting cells as well as proteins derived from granules of polymorphonuclear granulocytes (PMN). Among the most abundant proteins identified in BG-induced EVs were beta-2 integrin subunits, including CD18 and CD11 homologs, which highlights the role of salmon granulocytes and mononuclear phagocytes in the response to soluble BG. Overall, the current work advances the knowledge about the immunostimulatory activity of yeast BG on the salmon immune system by shedding light on the effect of this PAMP on the secretome of salmon leukocytes.

Melanocortin 1 Receptor Deficiency in Hematopoietic Cells Promotes the Expansion of Inflammatory Leukocytes in Atherosclerotic Mice.

Melanocortin receptor 1 (MC1-R) is expressed in leukocytes, where it mediates anti-inflammatory actions. We have previously observed that global deficiency of MC1-R signaling perturbs cholesterol homeostasis, increases arterial leukocyte accumulation and accelerates atherosclerosis in apolipoprotein E knockout (Apoe-/-) mice. Since various cell types besides leukocytes express MC1-R, we aimed at investigating the specific contribution of leukocyte MC1-R to the development of atherosclerosis. For this purpose, male Apoe-/- mice were irradiated, received bone marrow from either female Apoe-/- mice or MC1-R deficient Apoe-/- mice (Apoe-/- Mc1re/e) and were analyzed for tissue leukocyte profiles and atherosclerotic plaque phenotype. Hematopoietic MC1-R deficiency significantly elevated total leukocyte counts in the blood, bone marrow and spleen, an effect that was amplified by feeding mice a cholesterol-rich diet. The increased leukocyte counts were largely attributable to expanded lymphocyte populations, particularly CD4+ T cells. Furthermore, the number of monocytes was elevated in Apoe-/- Mc1re/e chimeric mice and it paralleled an increase in hematopoietic stem cell count in the bone marrow. Despite robust leukocytosis, atherosclerotic plaque size and composition as well as arterial leukocyte counts were unaffected by MC1-R deficiency. To address this discrepancy, we performed an in vivo homing assay and found that MC1-R deficient CD4+ T cells and monocytes were preferentially entering the spleen rather than homing in peri-aortic lymph nodes. This was mechanistically associated with compromised chemokine receptor 5 (CCR5)-dependent migration of CD4+ T cells and a defect in the recycling capacity of CCR5. Finally, our data demonstrate for the first time that CD4+ T cells also express MC1-R. In conclusion, MC1-R regulates hematopoietic stem cell proliferation and tissue leukocyte counts but its deficiency in leukocytes impairs cell migration via a CCR5-dependent mechanism.

Foraging strategies and physiological status of a marine top predator differ during breeding stages

Habitat characteristics determine the presence and distribution of trophic resources shaping seabirds' behavioural responses which may result in physiological consequences. Such physiological consequences in relation to foraging strategies of different life-history stages have been little studied in the wild. Thus, we aim to assess differences in oxidative status, condition (fat stores, i.e. triglyceride levels, TRI), stress (Heterophil/Lymphocyte (H/L) ratio), and leukocyte profiles between incubation and chick rearing highlighting the role of foraging strategies in a seabird (Calonectris diomedea). Chick rearing was more energetically demanding and stressful than incubation as demonstrated by high stress levels (H/L ratio and leukocytes) and lower body stores (assessed by TRI and the increment of weight) due to the high energy requirements of rearing chicks. Also, our results make reconsider the simplistic trade-off model where reproduction increases metabolism and consequently the rate of oxidative stress. In fact, high energy expenditure (VeDBA) during chick rearing was correlated with low levels of oxidative damage likely due to mechanisms at the level of mitochondrial inner membranes (uncoupling proteins or low levels of oxygen partial pressure). Further (more distant) and longer (more days) foraging trips were performed during incubation, when antioxidants showed low levels compared to chick rearing due to incubation fasting, a change in diet, or a combination of these factors; but unlikely because of oxidative shielding since no relation was found between oxidative damage and antioxidant capacity. Males showed higher numbers of monocytes which were positively correlated with antioxidant capacity compared to females, suggesting sexual differences in immune profiles. Species-specific costs and energetic demands of different breeding phases trigger behavioural and physiological adjustments.

Total white blood cell counts but not HL ratio changes in the transition from post-juvenile molt to autumn migration in the first-year Eurasian blackcap (Sylvia atricapilla).

Theoretically, seasonal changes in immune functioning in animals are shaped by the trade-off between a probability of encountering pathogens and availability of resources. We used leukocyte profile (absolute and relative leukocyte counts) as a simple measure of immune system condition to study how it changes during the transition from postjuvenile molt to autumn migration in a free-living migratory songbird, the Eurasian blackcap (Sylvia atricapilla). We observed the higher white blood cells (WBC) and lymphocyte counts in molting birds compared to migrating individuals, but we did not find differences in heterophils and ratio of heterophils to lymphocytes (HL ratio). We suppose that the high number of WBC in molting blackcaps could reflect the heightened ability of their immune system to resists infections. The lower WBC counts in migrants compared to molting birds were mostly due to reduced lymphocyte numbers, thus representing in a downregulation of specific immunity. An absence of heterophil differences between molt and migration might indicate that various components of immunity can change relatively independently (or at different pace). Fat scores had no effect on WBC counts and HL ratio. Therefore, we found no strong evidence for a resource-immune functionality trade-off during transition from postjuvenile molt to autumn migration in immature Eurasian blackcap. This study is an important step in understanding how immune system in general and leukocyte profile in particular changes in transition between life-history stages in migratory songbirds.

EVALUATION OF THE LEUKOCYTE INDEX IN SICKLE CELL ANEMIA WITH THE USE OF HYDROXYUREA

The term sickle cell disease (SCD) is used to describe a group of genetic disorders in which there is a predominance of HbS, a variant hemoglobin originated from a single nucleotide polymorphism (SNP) responsible for the exchange of a glutamic acid by valine in position 6 of ß-globin chain. The most severe form of the disease is homozygous (HbSS). Thus, sickle cell disease patients may present different clinical manifestations and inflammatory processes. A pharmacotherapeutic used for the treatment is Hydroxyurea. The action of this drug includes increasing fetal hemoglobin levels and improving clinical severity and hematological parameters. This study aimed to investigate hematological stress in patients with sickle cell anemia treated (or not) with hydroxyurea, and classified regarding the risk of severity by the Calculator of severity of sickle cell disease. DNA samples from 200 patients with sickle cell anemia, in steady state of the disease, using and not using hydroxyurea, were submitted to molecular analysis to confirm HbSS. Then, chronic stress was characterized from the neutrophil-lymphocyte ratio (NLR) through the analysis of the blood count and clinical history. NLR is an important inflammatory marker, its calculation is indicated for patients with sickle cell disease and values above one may indicate hematological stress. Statistical analyzes were performed using IBM SPSS Statistics 20 software. Data normality was verified using the Shapiro-Wilk test. Assessed for non-normality, the non-parametric Wilcoxon test was performed to compare the means between the leukocyte profile and the use of hydroxyurea. Then, a comparison was made between the risk of death of patients who were (or not) treated with hydroxyurea. The results were shown as mean and standard deviation, with a significance level of 0.05. The means and standard deviation were NLR/With HU (1.76 ±1.53) and NLR/Without HU (1.65 ±1.07). In order to verify the difference between the means, we used the Wilcoxon test whose value was p = 0.975. Thus, we observed that there was no difference between leukocyte stress and the use of Hydroxyurea. When comparing the risk of death of patients which use the drug (1.96 ±0.73) and those who do not use it (2.22 ±0.75), we noticed that there were significant differences between the groups (p = 0.01). Therefore, the drug can contribute to reduce the risk of death. The non-relation between leukocyte stress and the use of hydroxyurea reported in this study is not well documented in the literature. However, we observed an increase in NLR in critically ill patients with sickle cell disease classified by the Calculator of severity of sickle cell disease. The role of hydroxyurea in the risk of life for patients with sickle cell anemia is well documented. It is known that it can help reducing the number of hospitalizations, length of stay, less occurrence of acute chest syndrome, lower morbidity and mortality rate, contributing to an increase of up to 40% in patient survival. Our results corroborate literature data. There was no relationship between stress through the NLR and the use of hydroxyurea, but there is a relationship between the severity of the disease and the use of the medication. Relating leukocyte stress with disease severity as well as the impact of hydroxyurea on these indices is important to understand the clinical manifestations of sickle cell anemia patients.