Macrophages Provide Essential Support for Erythropoiesis, and Extracellular ATP Contributes to a Erythropoiesis-Supportive Microenvironment during Repeated Psychological Stress.

Psychological stress is a significant contributor to various chronic diseases and affects multiple physiological processes including erythropoiesis. This study aimed to examine the tissue-specific contributions of macrophages and extracellular ATP, as a signal of disturbed tissue homeostasis, to erythropoiesis under conditions of repeated psychological stress. Adult male BALB/c mice were subjected to 2 h daily restraint stress for seven consecutive days. Clodronate-liposomes were used to deplete resident macrophages from the bone marrow and spleen two days prior to the first restraint procedure, as well as newly recruited macrophages, every third day for the duration of the experiment. Repeated stress induced a considerable increase in the number of erythroid progenitor cells as well as in the percentage of CD71+/Ter119+ and CD71-/Ter119+ cells in the bone marrow and spleen. Macrophage depletion completely abolished the stimulative effect of repeated stress on immature erythroid cells, and prevented stress-induced increases in ATP levels, P2X7 receptor (P2X7R) expression, and ectonucleotidase CD39 activity and expression in the bone marrow and spleen. The obtained results demonstrate the stimulative effects of repeated stress on erythroid cells, extracellular ATP levels, P2X7R expression, CD39 activity and expression within the bone marrow and spleen, as well as the essential role of macrophages in stress-induced changes.

Structural and functional properties of a plant NRAMP-related aluminum transporter.

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe2+ and Mn2+ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg2+ uptake in prokaryotes and another the transport of Al3+ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg2+ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al3+ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al3+ and Ga3+, which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

Interplay between stress and cancer-A focus on inflammation.

Stress is an integral part of life. While acute responses to stress are generally regarded as beneficial in dealing with immediate threats, chronic exposure to threatening stimuli exerts deleterious effects and can be either a contributing or an aggravating factor for many chronic diseases including cancer. Chronic psychological stress has been identified as a significant factor contributing to the development and progression of cancer, but the mechanisms that link chronic stress to cancer remain incompletely understood. Psychological stressors initiate multiple physiological responses that result in the activation of the hypothalamic-pituitary-adrenal (HPA) axis, sympathetic nervous system, and the subsequent changes in immune function. Chronic stress exposure disrupts the homeostatic communication between the neuroendocrine and immune systems, shifting immune signaling toward a proinflammatory state. Stress-induced chronic low-grade inflammation and a decline in immune surveillance are both implicated in cancer development and progression. Conversely, tumor-induced inflammatory cytokines, apart from driving a tumor-supportive inflammatory microenvironment, can also exert their biological actions distantly via circulation and therefore adversely affect the stress response. In this minireview, we summarize the current findings on the relationship between stress and cancer, focusing on the role of inflammation in stress-induced neuroendocrine-immune crosstalk. We also discuss the underlying mechanisms and their potential for cancer treatment and prevention.

Putative Role of Neutrophil Extracellular Trap Formation in Chronic Myeloproliferative Neoplasms.

Myeloproliferative neoplasms (MPNs) are hematologic malignancies characterized by gene mutations that promote myeloproliferation and resistance to apoptosis via constitutively active signaling pathways, with Janus kinase 2-signal transducers and the activators of transcription (JAK-STAT) axis as a core part. Chronic inflammation has been described as a pivot for the development and advancement of MPNs from early stage cancer to pronounced bone marrow fibrosis, but there are still unresolved questions regarding this issue. The MPN neutrophils are characterized by upregulation of JAK target genes, they are in a state of activation and with deregulated apoptotic machinery. Deregulated neutrophil apoptotic cell death supports inflammation and steers them towards secondary necrosis or neutrophil extracellular trap (NET) formation, a trigger of inflammation both ways. NETs in proinflammatory bone marrow microenvironment induce hematopoietic precursor proliferation, which has an impact on hematopoietic disorders. In MPNs, neutrophils are primed for NET formation, and even though it seems obvious for NETs to intervene in the disease progression by supporting inflammation, no reliable data are available. We discuss in this review the potential pathophysiological relevance of NET formation in MPNs, with the intention of contributing to a better understanding of how neutrophils and neutrophil clonality can orchestrate the evolution of a pathological microenvironment in MPNs.

Gender-specific differences in hemodialysis patients: a multicenter longitudinal study from Serbia.

PURPOSE: The study was undertaken with the aim to determine gender-specific differences in incident hemodialysis (HD) patient and their changes over time. METHODS: The retrospective longitudinal closed cohort study involved 441 incident patients starting HD in 2014 and followed for 1-59 (median 43, IQR 40) months. Demographic, clinical data, treatment characteristics, laboratory findings and outcome were abstracted from the patients' medical records. RESULTS: The relative number of males on HD was about twice that of females throughout the five years investigated. At the beginning of the study, no significant differences were found in the main demographic and clinical characteristics except that diabetes was more often the underlying disease in men than in women. Systolic blood pressure decreased over time significantly more in females than in males. Throughout the study spKt/V was significantly higher in females than in males, but it increased in patients of both genders. There were no gender differences for comorbidities, vascular access and the majority of laboratory findings except for higher serum levels of creatinine and CRP in men than in women. Relatively more females were treated with erythropoiesis stimulating agents and phosphate binders than males. Age and malignancy were selected as significant predictors of mortality for both genders, and, in addition, polycystic kidney disease, serum level of albumin and CRP for men, but spKt/V for women. CONCLUSION: Some significant gender differences were observed throughout, while others appeared during the study but none of them were due to gender inequalities in the applied treatment.

Inflammation Promotes Oxidative and Nitrosative Stress in Chronic Myelogenous Leukemia.

Chronic inflammation is characterized by the production of reactive oxygen species (ROS), reactive nitrogen species, and inflammatory cytokines in myeloproliferative neoplasms (MPNs). In addition to these parameters, the aim of this study was to analyze the influence of ROS on the proliferation-related AKT/mTOR signaling pathway and the relationship with inflammatory factors in chronic myelogenous leukemia (CML). The activity of the antioxidant enzymes superoxide dismutase, glutathione peroxidase, and catalase is reduced in erythrocytes while levels of the oxidative stress markers malondialdehyde and protein carbonyl are elevated in the plasma of patients with CML. In addition, nitrogen species (nitrotyrosine, iNOS, eNOS) and inflammation markers (IL-6, NFkB, and S100 protein) were increased in granulocytes of CML while anti-inflammatory levels of IL-10 were decreased in plasma. CML granulocytes exhibited greater resistance to cytotoxic H2O2 activity compared to healthy subjects. Moreover, phosphorylation of the apoptotic p53 protein was reduced while the activity of the AKT/mTOR signaling pathway was increased, which was further enhanced by oxidative stress (H2O2) in granulocytes and erythroleukemic K562 cells. IL-6 caused oxidative stress and DNA damage that was mitigated using antioxidant or inhibition of inflammatory NFkB transcription factor in K562 cells. We demonstrated the presence of oxidative and nitrosative stress in CML, with the former mediated by AKT/mTOR signaling and stimulated by inflammation.

Neutrophil Death in Myeloproliferative Neoplasms: Shedding More Light on Neutrophils as a Pathogenic Link to Chronic Inflammation.

Neutrophils are an essential component of the innate immune response, but their prolonged activation can lead to chronic inflammation. Consequently, neutrophil homeostasis is tightly regulated through balance between granulopoiesis and clearance of dying cells. The bone marrow is both a site of neutrophil production and the place they return to and die. Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders characterized by the mutations in three types of molecular markers, with emphasis on Janus kinase 2 gene mutation (JAK2V617F). The MPN bone marrow stem cell niche is a site of chronic inflammation, with commonly increased cells of myeloid lineage, including neutrophils. The MPN neutrophils are characterized by the upregulation of JAK target genes. Additionally, MPN neutrophils display malignant nature, they are in a state of activation, and with deregulated apoptotic machinery. In other words, neutrophils deserve to be placed in the midst of major events in MPN. Our crucial interest in this review is better understanding of how neutrophils die in MPN mirrored by defects in apoptosis and to what possible extent they can contribute to MPN pathophysiology. We tend to expect that reduced neutrophil apoptosis will establish a pathogenic link to chronic inflammation in MPN.

Correlation between leukocyte-platelet aggregates and thrombosis in myeloproliferative neoplasms.

INTRODUCTION: The impact of activated blood and endothelial cells on the thrombosis in myeloproliferative neoplasms (MPN) has not yet been clarified. We prospectively analyzed correlation between circulating leukocyte-platelet aggregates and soluble selectins to thrombosis occurrence in MPN, in the context of standard and cardiovascular risk factors, and different clinical and biological characteristics. METHODS: Flow cytometric analysis of neutrophil-platelet (Neu-Plt) and monocyte-platelet (Mo-Plt) aggregates in peripheral blood, as well as quantification of soluble E-/L-/P-selectins by enzyme immunoassay, was performed on 95 newly diagnosed MPN patients. RESULTS: During the follow-up, thrombosis occurred in 12.6% MPN patients (arterial 9.4%, venous 3.2%), with a mean time of 39 months. The overall incidence rate of main thrombotic events was 4.36 per 100 patient-years. The incidence of arterial hypertension (HTA) was significantly higher in patients with thrombosis, compared to those without thrombosis (P < .05). The level of soluble P-selectin was significantly higher in patients with thrombosis compared to those without thrombosis (346.89 ng/mL vs 286.39 ng/mL, P = .034). The mean level of Neu-Plt (26.7% vs 22.4%) and Mo-Plt (17.8% vs 12.3%) aggregates did not differ significantly between the groups with and without thrombosis. A multivariate COX proportional hazard regression model confirmed an independent predictive significance of Mo-Plt aggregates (HR = 1.561, 95% CI: 1.007-2.420, P = .046), as well as the cumulative effect of Mo-Plt aggregates and HTA (HR = 1.975, 95%CI: 1.215-3.212, P = .006) for thrombosis occurrence. CONCLUSION: Monocyte-platelet aggregates represent an independent risk factor for thrombosis occurrence, further on supported by HTA.

Exploring immunogenicity of tick salivary AV422 protein in persons exposed to ticks: prospects for utilization.

In order to determine whether conserved tick salivary protein AV422 is immunogenic, the goal of our study was to detect specific IgG response within at-risk populations. Study groups included 76 individuals, differing in occurrence of recently recorded tick bites and health status. Western blotting with recombinant (r) protein derived from Ixodes ricinus (Ir) was performed. IgG response to Borrelia/Rickettsia, as indicators of previous tick infestations, was also assessed. Additionally, a detailed in silico AV422 protein sequence analysis was performed, followed by modelling of the interactions between peptides and corresponding MHC II molecules by molecular docking. Anti-rIrAV422 seroprevalences among individuals exposed to ticks were high (62.5, 57.9 and 66.7%) and anti-Borrelia/Rickettsia seroprevalences were 54.2, 15.8 and 44.4% among individuals with/without recent tick bite and patients suspected of tick-borne disease, respectively. In silico analysis of AV422 protein sequence showed a high level of conservation across tick genera, including also the predicted antigenic determinants specific for T and B cells. Docking to the restricted MHC II molecules was performed for all predicted AV422 T cell epitopes, and the most potent (highly immunogenic) epitope determinants were suggested. The epitope prediction reveals that tick salivary protein AV422 may elicit humoral immune response in humans, which is consistent with the high anti-rIrAV422 seroprevalence in tested at-risk subjects. Tick-borne diseases are a growing public health concern worldwide, and AV422 is potentially useful in clinical practice and epidemiological studies.

The effects of incubation media on the assessment of the shape of human erythrocytes by flow cytometry: a contribution to mathematical data interpretation to enable wider application of the method.

Flow cytometry (FC) analysis of erythrocyte shape and related biomechanical properties, such as osmotic fragility, have not moved from a research tool to regular clinical testing. The main reason is existing evidence that various pre-analytical factors influence the mathematical interpretation of the data obtained. With an aim to contribute to the standardization and broaden the use of FC for human erythrocyte shape assessment, freshly prepared peripheral blood erythrocytes isolated from healthy donors were incubated in iso and hypo-osmotic solutions (pure saline, saline with potassium and calcium, and phosphate buffered saline) and examined by FC using values of forward scatter (FSC) and side scatter (SSC). Kurtosis, skewness, Pearson's second skewness coefficient of dissymmetry (PCD), and spherical index, calculated from FSC distributions, were used for the erythrocyte shape evaluation. In all isotonic media FSC distribution and FSC-based morphology parameters showed huge inter-individual and inter-medium variation. With decreasing osmolality, in all media and samples, the size of the erythrocytes increased, and swelling index and kurtosis decreased. However, changes in skewness and PCD were influenced by the medium used and the sample tested. Compared to FSC, SSC signal in isotonic and its change in hypotonic media showed lower inter-individual variation and was not influenced by the type of medium. We propose a spherical index and kurtosis as FSC-based indicators of erythrocyte shape. As more resistant to the influence of the preanalytical treatment, SSC data appeared to be unfairly neglected for the assessment of erythrocyte shape, in comparison to the usually employed FSC data.

Antenatal Care and Weight Gain in Adolescent Compared to Adult Pregnancy.

BACKGROUND: The aim of this study is to compare the antenatal care, body weight, and weight gain in pregnancy between the adolescent and adult pregnancies and, thus, examine the impact of adolescence on the studied parameters. METHODS: This prospective study includes 300 pregnant women who were the patients of University Clinical Center Tuzla, Clinic for Gynecology and Obstetrics from January 2011 to December 2014. The women were divided into two groups: an experimental group consisted of 150 adolescent pregnant women aged 13-19 years and a control group consisted of 150 adult pregnant women aged 20-35 years. The following parameters were analyzed: age of pregnant women, number of antenatal controls in pregnancy, prepregnancy body weight, weight gain in pregnancy, parity, and obstetric history data. RESULTS: A significantly higher number of adolescent pregnant women belongs to a subgroup from one to two examinations during pregnancy (P < 0.000013) and to subgroups from three to five examinations (P < 0.000001). A significantly smaller number of adolescent pregnant women performed their first antenatal control in the first 2 lunar months (P < 0.01). A subgroup with optimal body weight (from 51 to 69 kg) are the most prevalent among adolescent pregnant women (P < 0.000001). A significantly larger number of adolescent pregnant women had an optimal weight gain of 7.8 to 12.99 kg (P < 0.001). CONCLUSIONS: The adolescent pregnant women have suboptimal antenatal care, which could lead to adverse maternal and birth outcomes, but have optimal body weight and weight gain during pregnancy.

Structural characteristics of circulating immune complexes in calves with bronchopneumonia: Impact on the quiescent leukocytes.

Calf bronchopneumonia is accompanied by increased level of circulating immune complexes (CIC), and we analysed size, and protein and lipid constituents of these CIC with an attempt to elucidate the connection between the CIC structural properties and their capacity to modulate leukocyte function. CIC of heathy calves (CICH) and calves with naturally occurring bronchopneumonia (CICD) were isolated by PEG precipitation and analysed by electrophoresis and chromatography. The predominant CIC proteins were IgG, albumin, and transferrin. Affinity isolated serum and CIC IgG coprecipitated several proteins, but only 75 and 80 kDa proteins bound CIC IgG, exclusively. 60 and 65 kDa proteins co-precipitated with CICD IgG, unlike CICH IgG. In both CICH and CICD, oleic acid-containing phospholipids predominated. In CICD, the content of oleic and vaccenic acid was higher than in CICH, while myristic, palmitic, stearic, linoleic and arachidonic acid showed lower content. Dynamic light scattering displayed difference in particle size distribution between CICH and CICD; 1280 nm large particles were present only in CICD. The effect of CICH and CICD on mononuclear cells (MNC) and granulocytes was analysed in vitro. CICH and CICD, with slight difference in intensity, stimulate MNC apoptosis, promote cell cycle arrest of unstimulated MNC, and cell cycle progression of PHA stimulated MNC. Both CIC reduced granulocyte apoptosis after 24 h while after 48 h this effect was detected for CICD only. These results indicate that structural differences of CICH and CICD might interfere with the CIC functional capacity, which we consider important for evaluation of CIC immunoregulatory function.

Vitamin E-Bonded Membranes Do Not Influence Markers of Oxidative Stress in Hemodialysis Patients with Homozygous Glutathione Transferase M1 Gene Deletion.

BACKGROUND: Increased oxidative stress is a hallmark of end-stage renal disease. Hemodialysis (HD) patients lacking glutathione transferase M1 (GSTM1) enzyme activity exhibit enhanced oxidative DNA damage and higher mortality rate than those with active GSTM1 enzyme. To our knowledge, this is the first study to use the vitamin E-bonded membranes (VEM) in patients with homozygous GSTM1 gene deletion, and we aimed to determine the effect of VEM on oxidative and inflammatory status in HD patients with homozygous GSTM1 gene deletion. METHODS: GSTM1 genotypes were determined by polymerase chain reaction (PCR) in 170 chronic HD patients. Those with GSTM1-null genotype were randomized and 80 were included in the study. Forty of them were dialyzed for three months with VEM, while the other forty were dialyzed with high-flux same-surface polysulfone dialyzers. Markers of protein and lipid oxidative damage and inflammation (thiol groups, malondialdehyde (MDA), Interleukin-6 (IL-6)), together with plasma antioxidant activity (glutathione peroxidase (GPX), superoxide dismutase (SOD)) were determined. RESULTS: Seventy-five patients finished the study. There were no differences at baseline in markers of protein and lipid oxidative damage, inflammation and plasma antioxidant activity. After three months of therapy, GPX, MDA, and thiol groups increased significantly in both groups, but without statistical significance between groups. SOD and C reactive protein (CRP) did not change significantly during the three-month period. IL-6 increased in the control group, and at the same time, decreased in the VEM group, but without statistical significance. Hemoglobin (Hb) value, red blood cells, erythropoiesis resistance index (ERI), serum ferritin and iron did not change significantly within or between groups. Regarding other laboratory parameters, proteins, albumins, triglycerides, serum phosphorus, serum bicarbonate and Kt/V showed significant improvements within groups but with no significant difference between groups. CONCLUSIONS: Our data shows that therapy with VEM over three months had no benefit over standard polysulfone membrane in decreasing by-products of oxidative stress and inflammation in dialysis patients lacking GSTM1 enzyme activity.

Nitric oxide-dependent expansion of erythroid progenitors in a murine model of chronic psychological stress.

Anaemia occurs frequently in patients with heart failure and its current treatment lacks clear targets. Emerging evidence suggests that erythroid progenitor cell expansion is an integral part of physiological response to anaemia associated with chronic stress. Understanding the underlying mechanism may provide a novel approach to anaemia management. In this study, we aimed to examine a role for nitric oxide (NO) in the regulation of bone marrow erythroid progenitor response to chronic stress. For this purpose, adult male mice were subjected to 2 h daily restraint stress for 7 or 14 consecutive days. The role of NO was assessed by subcutaneous injection with NG-nitro-L-arginine methyl ester, 30 min prior to each restraint. Chronic exposure to stress resulted in significantly increased number of bone marrow erythroid progenitors, and blockade of NO biosynthesis prior to daily stress completely prevented stress-induced erythroid progenitor cell expansion. Furthermore, chronic stress exposure led to altered expression of neural, endothelial and inducible nitric oxide synthases (NOS) in the bone marrow, both on mRNA and protein level. Decreased expression of neural and endothelial NOS, as well as reduced expression of NF-kappaB/p65 in bone marrow nuclear cell fraction, was accompanied by elevated bone marrow expression of inducible NOS in chronically stressed animals. This is the first study to demonstrate a role for NO in adaptive response of erythroid progenitors to chronic stress. Targeting NO production may be beneficial to improve bone marrow dysfunction and reduced erythroid progenitor cell expansion in chronic heart failure patients.

Impact of Angiotensin-converting Enzyme and Matrix Metalloproteinase-3 Gene Polymorphisms on Risk for Developing Vascular Access Failure in Hemodialysis Patients - A Pilot Study.

For adequate hemodialysis, functional vascular access is obligatory. Neointimal hyperplasia (NIH) has a central role in stenosis and thrombosis development, which represent the most frequent causes of vascular access failure. Polymorphism of different genes that have a significant role in endothelial function may have an impact on NIH development. Therefore, the aim of our study is to determine the effect of angiotensin-converting enzyme (ACE) I/D and matrix metalloproteinase-3 (MMP3) 5A/6A polymorphism on risk for developing vascular access failure in hemodialysis patients. The study included 200 patients on regular hemodialysis at Nephrology Department, University Medical Center Zvezdara. Retrospective analysis included a collection of general and vascular access data from medical records. Genetic analysis was performed by using polymerase chain reaction-restriction fragment length polymorphism method (PCR-RFLP). Patients were divided into two groups: Group 1-patients who have never experienced vascular access failure and Group 2-patients who have at least one spontaneous vascular access failure. There was no difference in age, gender, hemodialysis vintage, main diagnosis, presence of hypertension, and diabetes mellitus between the two groups. There were no statistically significant differences in the frequencies of ACE and MMP3 genotypes between the two groups. Without statistical significance, it was found that homozygotes for I allele had two times higher risk for developing vascular access failure than homozygotes for D allele (OR 2.00; 95%CI: 0.727-5.503; P = 0.180). In addition, patients with 5A allele have 1.7 times higher risk for developing vascular access failure compared with patients without this allele (OR 1.745; 95% CI: 0.868-3.507; P = 0.118). Patients with vascular access failure do not have different genotype distribution regarding ACE gene and MMP3 gene polymorphism as compared with patients without vascular access failure. Still, homozygotes for I allele and homozygotes for 5A allele have higher risk for developing vascular access failure compared with other patients.

Oxidative and nitrosative stress in myeloproliferative neoplasms: the impact on the AKT / mTOR signaling pathway.

PURPOSE: A common feature of malignancies is increased reactive oxygen species (ROS) and reactive nitrogen species (RNS). We analyzed the influence of oxidative and nitrosative stress on the activation of AKT/mTOR signaling pathway in myeloproliferative neoplasms (MPN). METHODS: Oxidative stress-induced gene expression in circulatory CD34+ cells of MPN patients was studied by microarray analysis. Biomarkers of oxidative and nitrosative stress were determined using spectrophotometry in plasma and erythrocyte lysate. The levels of nitrotyrosine, inducible NO synthase (iNOS) and AKT/mTOR/p70S6K phosphorylation were determined by immunocytochemistry and immunoblotting in granulocytes of MPN patients. RESULTS: Antioxidants superoxide dismutase 2 (SOD2) and glutathione peroxidase 1 (GPx1) gene expression were increased in circulatory CD34+ cells, while SOD1 and GPx enzymes were reduced in the erythrocytes of MPN. Plasma malonyl-dialdehyde and protein carbonyl levels were elevated in MPN. The total antioxidant capacity in plasma and erythrocyte catalase (CT) activities was the most prominent in primary myelofibrosis (PMF) with JAK2V617F heterozygosity. The total nitrite/nitrate (NOx) level was augmented in the plasma of PMF patients (p<0.001), while nitrotyrosine and iNOS were generally increased in the granulocytes of MPN patients. Activation of AKT/mTOR signaling was the most significant in PMF (p<0.01), but demonstrated JAK2V617F dependence and consequent p70S6K phosphorylation in the granulocytes of essential thrombocytemia (ET) and polycythemia vera (PV) patients. Hydrogen peroxide stimulated mTOR pathway, iNOS and nitrotyrosine quantities, the last one prevented by the antioxidant n-acetyl-cysteine (NAC) in the granulocytes of MPN. CONCLUSION: Our study showed increased levels of oxidative and nitrosative stress parameters in MPN with JAK2V617F dependence. The ROS enhanced the constitutive activation of AKT/mTOR signaling and nitrosative parameters in MPN.

Assessment of using recombinant Ixodes ricinus AV422 saliva protein for confirmation of tick bites in hunting dogs as naturally infested hosts.

Due to the recorded spreading of ticks in past years, a higher incidence of tick-borne diseases (TBDs) can be expected in the future in endemic areas, but can also pose an emerging public health concern in areas where they have not yet been recognized. Assessment of the exposure of vulnerable hosts to ticks would be a very helpful tool for TBD epidemiological studies, as well as for their proper managing. To confirm previous tick bites, the method of choice is detection of antibodies in host serum as markers developed against injected tick saliva proteins during feeding. We recently showed that the recombinant form of Ixodes ricinus AV422 saliva protein (rIrAV422) can serve for detection of markers in experimentally infested rats. Here we examine whether it can be used in the same manner in naturally exposed hosts. We chose hunting dogs as good sentinel animals. The study group consisted of 15 dogs that varied in breed, age, sex, previous tick infestation history and repellent treatment. Western blot analysis with rIrAV422 as an antigen confirmed the presence of tick bite markers in all analysed dogs. For some of the dogs, their previous tick infestation history was unclear, which emphasizes the usefulness of rIrAV422 for revealing it. Since hunting dogs are naturally infested with different ticks, the potential of rIrAV422 in assessment of general exposure to ticks is highlighted. Use of rIrAV422 can also be helpful in veterinary practice and research as a tool for validation of the efficiency of tick repellent products.

Ixodes ricinus immunogenic saliva protein, homologue to Amblyomma americanum AV422: Determining its potential for use in tick bite confirmation.

Tick bites often go unnoticed, so specific reliable tests are needed to confirm them for prompt diagnosis and treatment of tick-borne diseases. One of the promising candidates for developing such a test is AV422, a tick saliva protein that has been conserved across tick genera. In this study, we demonstrate the potential of the AV422 homologue from Ixodes ricinus to be used for tick bite detection for both Prostriata and Metastriata. We expressed recombinant (r) I. ricinus (Ir) AV422 in E. coli and subjected it to Western blot analysis using rat antibodies to saliva proteins of both I. ricinus (Prostriata) and Dermacentor reticulatus (Metastriata) larvae. Our data demonstrate that rIrAV422 specifically bound to antibodies from sera of rats used for both I. ricinus and D. reticulatus larvae feeding, but not to antibodies from control serum, emphasizing its specificity since tick bites were the sole cause of sera reactivity.

Angiogenic factors are increased in circulating granulocytes and CD34+ cells of myeloproliferative neoplasms.

It has been shown that angiogenesis and inflammation play an important role in development of most hematological malignancies including the myeloproliferative neoplasm (MPN). The aim of this study was to investigate and correlate the levels of key angiogenic molecules such as hypoxia-inducible factor-1α (HIF-1α), vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) in peripheral blood and bone marrow cells of MPN patients, along with JAK2V617F mutation allele burden and effects of therapy. HIF-1α and VEGF gene expression were decreased, while eNOS mRNA levels were increased in granulocytes of MPN patients. Furthermore, positively correlated and increased VEGF and eNOS protein levels were in negative correlation with HIF-1α levels in granulocytes of MPN patients. According to immunoblotting, the generally augmented angiogenic factors demonstrated JAK2V617F allele burden dependence only in granulocytes of PMF. The angiogenic factors were largely reduced after hydroxyurea therapy in granulocytes of MPN patients. Levels of eNOS protein expression were stimulated by Calreticulin mutations in granulocytes of essential thrombocythemia. Immunocytochemical analyses of CD34+ cells showed a more pronounced enhancement of angiogenic factors than in granulocytes. Increased gene expression linked to the proinflammatory TGFß and MAPK signaling pathways were detected in CD34+ cells of MPN patients. In conclusion, the angiogenesis is increased in several cell types of MPN patients supported by the transcriptional activation of inflammation-related target genes, and is not limited to bone marrow stroma cells. It also appears that some of the benefit of hydroxyurea therapy of the MPN is mediated by effects on angiogenic factors. © 2016 Wiley Periodicals, Inc.

Bone marrow microvessel density and plasma angiogenic factors in myeloproliferative neoplasms: clinicopathological and molecular correlations.

Increased angiogenesis in BCR-ABL1 negative myeloproliferative neoplasms (MPNs) has been recognized, but its connection with clinical and molecular markers needs to be defined. The aims of study were to (1) assess bone marrow (BM) angiogenesis measured by microvessel density (MVD) using CD34 and CD105 antibodies; (2) analyze correlation of MVD with plasma angiogenic factors including vascular endothelial growth factor, basic fibroblast growth factor, and interleukin-8; (3) examine the association of MVD with clinicopathological and molecular markers. We examined 90 de novo MPN patients (30 polycythemia vera (PV), primary myelofibrosis (PMF), essential thrombocythemia (ET)) and 10 age-matched controls. MVD was analyzed by immunohistochemistry "hot spot" method, angiogenic factors by immunoassay and JAK2V617F, and CALR mutations by DNA sequencing and allelic PCR. MVD was significantly increased in MPNs compared to controls (PMF > PV > ET). Correlation between MVD and plasma angiogenic factors was found in MPNs. MVD was significantly increased in patients with JAK2V617F mutation and correlated with JAK2 mutant allele burden (CD34-MVD: ρ = 0.491, p < 0.001; CD105-MVD: ρ = 0.276, p = 0.02) but not with CALR mutation. MVD correlated with leukocyte count, serum lactate dehydrogenase, hepatomegaly, and splenomegaly. BM fibrosis was significantly associated with CD34-MVD, CD105-MVD, interleukin-8, and JAK2 mutant allele burden. JAK2 homozygote status had positive predictive value (100%) for BM fibrosis. Patients with prefibrotic PMF had significantly higher MVD than patients with ET, and we could recommend MVD to be additional histopathological marker to distinguish these two entities. This study also highlights the strong correlation of MVD with plasma angiogenic factors, JAK2 mutant allele burden, and BM fibrosis in MPNs.