Impact of different severity hyperglycemia on erythrocyte rheological properties1.

BACKGROUND: The triad "insulin resistance, prediabetes, diabetes" is three independent neologies with characteristic features and development. In addition, each are characterized by progression and the possibility of transition from one form to other. Due to the fact that diabetes is one of the common diseases associated with high rates of disability, it is necessary to improve diagnostic methods and educational regimens for successful prevention and treatment of the disease. OBJECTIVE: We investigated Band 3 protein (B3p) level, osmotic resistance of erythrocytes, the total antioxidant activity (TAA) of blood serum, level of HbA1 in group patients with insulin resistance (IR), prediabetes, and Type 2 Diabetes Mellitus (T2DM) and comparative with health control group. METHODS: We used original, accurate research methods that measure the essence of the studied quantities. RESULTS: Disruptions of glucose and insulin homeostasis ay lead to the initiation of oxidative stress (in our study demonstrated by a decrease of TAA of blood serum) increased redox-sensitive PTP activity and aberrant band 3 phosphorylation, potentially leading to reduced erythrocyte deformability. At the same time glycation of Hb during T2DM may affect its cross-link with membrane proteins, in particular with B3p, and although appears to limit its cross-linking and decrease its clusterization ability, induces alterations in the cytoskeletal matrix, and thereby decrease erythrocytes' osmotic resistance making them more susceptible to hemolysis. CONCLUSIONS: The osmotic resistance of the erythrocytes can be used as a sensitive marker for the detection of the early stages of hyperglycemia (prediabetes). This set of clinical trials will make it possible to identify diseases that make up the triad at an early stage. Early detection of disorders and continued research in this direction will help in the development of a diagnostic scheme for the prevention of such patients. Based on our data, research into anti-oxidation drugs is very important. With the help of the array of studies described in the article and antioxidant treatment, the likelihood of successful treatment will increase.

The effect of L-theanine supplementation on the immune system of athletes exposed to strenuous physical exercise.

BACKGROUND: The aim of this study was to analyze the response of selected components of the immune system in rowers to maximal physical exercise, and to verify if this response could be modulated by supplementation with L-theanine. METHOD: The double-blind study included 20 members of the Polish Rowing Team. The subjects were randomly assigned to the supplemented group (n = 10), receiving 150 mg of L-theanine extract for 6 weeks, or to the placebo group (n = 10). The participants performed a 2000-m test on a rowing ergometer at the beginning (1st examination) and at the end of the supplementation period (2nd examination). Blood samples were obtained from the antecubital vein before each exercise test, 1 min after completing the test, and after a 24-h recovery. Subpopulations of T regulatory lymphocytes (Tregs) (CD4+/CD25+/CD127-), cytotoxic lymphocytes (CTLs) (CD8+/TCRαß+), natural killer (NK) cells (CD3-/CD16+/CD56+) and TCRδγ-positive (Tδγ) cells were determined by means of flow cytometry. The levels of interleukin 2 (IL-2), interleukin 4 (IL-4), interleukin 10 (IL-10), interferon gamma (INF-ɤ) and total antioxidant capacity (TAC) were determined with commercially available diagnostic kits. RESULTS: Supplementation with L-theanine contributed to a significant post-exercise decrease in IL-10 concentration, which was reflected by higher values of IL-2 to IL-10 and IFN-γ to IL-10 ratios. Moreover, a significant post-recovery decrease in CTL count, Treg to NK and Treg to CTL ratios was observed in the supplemented group. CONCLUSION: Despite the decrease in the number of some cytotoxic cells (CTLs) and an increase in the proportion of Tregs to CTLs, supplementation with LTE seems to exert a beneficial effect on a disrupted Th1/Th2 balance in elite athletes, as shown by the decrease in IL-10 concentration.

Potential Leukemic Cells Engraftment After Hematopoietic Stem Cell Transplantation From Unrelated Donors With Undiagnosed Chronic Leukemia.

BACKGROUND: Donor-related neoplasms are a potential complication of treatment strategies involving stem cell transplantation. Although mechanisms for detection of short-term complications after these procedures are well developed, complications with delayed onset, notably transmission of chronic diseases such as chronic myeloid leukemia (CML), have been difficult to assess. Consequently, we studied the potential of human CML cells to engraft hematopoietic tissues after intravenous implantation in mice. METHODS: Human peripheral blood cells, collected from CML patients presenting with moderately increased white blood cells count before treatment, were transplanted into sub-lethally irradiated, immunodeficient mice. Five weeks after transplantation the nuclear cells were isolated from the murine bone marrow, spleen, and peripheral blood and were used to quantitatively detect human CD45 antigen by flow cytometry; qRT-PCR was used to detect the BCR-ABL1 fusion gene, and the human or murine beta-glucuronidase housekeeping gene was used to examine human-murine chimerism. RESULTS: We found that all evaluated animals had donor chimerism at the selected interval after transplant and the presence of a specific BCR-ABL1 fusion gene transcript was also detected. CONCLUSIONS: Our results suggest that the risk of neoplasm transmission cannot be eliminated during hematopoietic stem cell transplantation from undiagnosed CML donors with borderline leukocytosis. The obtained data confirms the potential of leukemic cells to viably engraft the hematopoietic organs post-transplantation in an immunosuppressed recipient.

Correlation Between Percentage of Immuncompetent CD4+ and CD4+CD25+ Cells and Compatibility in the HLA System and Selected Parameters Assessing Transplanted Kidney Function.

BACKGROUND: Long-term kidney allograft survival is affected by many coexisting immunologic factors. Currently, only two basic immunologic parameters-HLA compatibility and panel reactive antibodies-are routinely used in kidney transplantation management. At the same time, there is a great need for immunologic biomarkers that will help inrease understanding of kidney transplant immunology and improve clinical care of kidney recipients. T regulatory cells (Tregs) represent one of the major targets of this approach. The aim of this study was to investigate possible simple associations between Tregs count in recipients' blood and other routinely assessed or easily accessible laboratory parameters. METHODS: Laboratory outcomes from medical files of transplant outpatient clinic in combination with flow cytometry analyses of particular immunocompetent cells populations were used. Flow cytometry was used to calculate Tregs recognized as TCD4+CD25high. The Spearman rank correlation test was used to verify particular associations. RESULTS: A negative correlation was found beween HLA compatibility and Tregs count as well as between platelets count and Tregs count. CONCLUSIONS: Whereas the negative correlation between Tregs and platelets counts may possibly mirror some recent findings in basic research, a negative correlation between HLA compatibility and Tregs points the direction of further research to factors triggering post-transplant immune tolerance.

The Importance of New Generation Sequencing (NGS) HLA Typing in Renal Transplantation-Preliminary Report.

INTRODUCTION: Thanks to new generation sequencing (NGS) and expansion of HLA typing with additional loci, it will be possible to increase the effectiveness of graft survival and to avoid complications related to the immune system. New pharmacogenetic factors are still being researched to develop better immunosuppressive treatment. MATERIAL AND METHODS: The incidence of polymorphic HLA loci variants was established, based on a high-resolution NGS method in kidney graft recipients. Furthermore, haplotypic analysis between examined loci was conducted to type additional loci that may influence the transplantation result. A total of 120 kidney recipients were enrolled in the study. A commercial DNA extraction kit in Tubes (QIAamp DNA Blood Mini Kit Qiagen, Germany) was used to isolate DNA from the blood. Sequencing library preparation was done with TruSight HLA set. The Conexio computer program was used to analyse the results of HLA typing. RESULTS: The patients with alleles A*02:01:01, B*44:02:01, C*03:03:01, C*01:02:01, C*05:01:01, C*07:02:01, DQB1*03:03:02, DQB1*06:04:01, or with haplotypic variation A*25:01:01-B*18:01:01- C*15:01:01 were taking the highest doses of cyclosporine (CsA), in contrast to patients with allele B*18:01:01, DQB1*06:02:01, DQB1*02:02:01, or haplotypic variation A*02:01:01- B*44:02:01-C*01:01:01, who were taking the lowest doses. The highest dose of tacrolimus (TAC) was administered to patients with alleles A*68:01:02, A*29:01:01, B*07:02:01, B*35:02:01, B*38:01:01, DRB1*12:01:01, DQB1*05:03:01, or haplotypic variations A*02:01:01-B*57:01:01-C*07:01:01, A*03:01:01-B*07:02:01-C*13:01:01, A*29:02:01-B*44:03:01- C*07:01:01, and A*01:01:01-B*08:01:01-C*03:01:01. Additionally, it was established that HLA-DRB3, HLA-DRB4, HLA-DRB5, HLA-DPA1, and HLA-DQA1 show very slight polymorphism, which suggests that there is no need for their typing for transplantation purposes. Moreover, loci HLA-C, HLA-DQB1, and HLA-DPB1, which are not routinely examined in recipient-donor matching, show genetic variability that may increase the risk of transplant rejection or shortened graft life. CONCLUSIONS: Expanding the qualification procedure to include allele genotyping could allow clinicians to establish immunosuppressive treatment schemes that would be optimally suited for recipients' phenotype.

Pathophysiology of drug-induce peripheral neuropathy in patients with multiple myeloma.

Multiple myeloma (MM) is a disease of unknown, complex etiology that affects primarily older adults. The course of the disease and the patients' survival time are very heterogeneous, but over the last decade, clear progress in the treatment of this incurable disease has been observed. Therapeutics that have proven to be highly effective include the immunomodulatory drug thalidomide and its newer analogs, lenalidomide and pomalidomide, as well as the proteasome inhibitors bortezomib and carfilzomib. However, the administration of some of the treatments, e.g., thalidomide or bortezomib, has also been associated with the occurrence of a serious and common adverse effect, drug-induced peripheral neuropathy. The mechanism of the development of the peripheral neuropathy is poorly understood. Nevertheless, one of its potential causes could be inadequate concentrations of crucial trophic factors, including neurotrophic and/or angiogenic factors, which are responsible for the proliferation, differentiation, survival and death of neuronal and nonneuronal cells.

Cell and region specificity of Aryl hydrocarbon Receptor (AhR) system in the testis and the epididymis.

Aryl hydrocarbon receptor (AhR) plays multiple important functions in adaptive responses. Exposure to AhR ligands may produce an altered metabolic activity controlled by the AhR pathways, and consequently affect drug/toxin responses, hormonal status and cellular homeostasis. This research revealed species-, cell- and region-specific pattern of the AhR system expression in the rat and human testis and epididymis, complementing the existing knowledge, especially within the epididymal segments. The study showed that AhR level in the rat and human epididymis is higher than in the testis. The downregulation of AhR expression after TCDD treatment was revealed in the spermatogenic cells at different stages and the epididymal epithelial cells, but not in the Sertoli and Leydig cells. Hence, this basic research provides information about the AhR function in the testis and epididymis, which may provide an insight into deleterious effects of drugs, hormones and environmental pollutants on male fertility.

Autologous cord blood transfusion in preterm infants - could its humoral effect be the kez to control prematurity-related complications? A preliminary study.

Umbilical cord blood (UCB), rich in stem/progenitor cells, is partially eliminated from the bloodstream during childbirth because the cord is immediately clamped. We hypothesize that transfusion of autologous UCB to premature infants after delivery could serve as an adjuvant modality for preventing the development of prematurity-related complications. We randomly enrolled 20 preterm infants born before 32 weeks of gestational age (GA), all of whom developed anemia, necessitating transfusion of red blood cells (RBCs). Two groups, matched for GA, were selected: (1) infants (n = 5) who underwent UCB transfusion once within 5 days of birth (mean ± standard deviation, 3.2 ± 1.9 days) and (2) infants (n = 15) from whom UCB was not collected (e.g., lack of consent). The latter served as controls and received allogeneic RBC transfusions (7.8 ± 3.9 days after birth). Selected prematurity-related complications were monitored. Two weeks after UCB/RBC transfusion, peripheral blood samples were collected, and the concentrations of 41 selected growth factors and their receptors were analyzed using a multiplex protein array. UCB transfusions were found to be both feasible and tolerable. Intraventricular haemorrhage was diagnosed in two of five (40%) UCB recipients, but was found in thirteen of fifteen RBC recipients (86.7%). Twenty-two plasma proteins (e.g., insulin-like growth factors, stem cell factor, epidermal growth factors) were found with significantly different concentrations in UCB recipients compared to controls. Results demonstrate safety and feasibility of UCB transfusion in a small group of very premature neonates and should be interpreted as preliminary speculation. Transfusion of UCB could induce a specific humoral effects, and this could serve as an adjuvant modality for prevention of prematurity complications.

Morphologic Changes in the Dermis After the Single Administration of Autologous Fibroblastic Cells: A Preliminary Study.

BACKGROUND: Aging is a multifactorial process defined by an accumulation of damage in all tissues and organs, including the skin, throughout the lifespan of an individual. The reduction of both cellular and extracellular matrix components of the dermis during the aging process is followed by the alteration of the morphology of the skin tissue. This study was conducted to assess skin morphology in men before and 3 months after the intradermal injection of autologous fibroblastic cells. METHODS: Tissue biopsies were surgically obtained before and 3 months after the treatment with autogenously harvested fibroblasts expanded in vitro, as well as after injection of phosphate-buffered saline. The thickness of collagen fiber bundles and number of fibroblasts in the dermis were analyzed in morphometric studies. The morphologic evaluation, using different methods of staining has been performed to analyze of extracellular matrix proteins, including collagen and reticular fibers, fibrillin-1-rich microfibrils, elastic fibers, and hyaluronic acid. RESULTS: After administration of the cells, we found a noticeable increase in the number of fibroblasts within the dermis, a significant enlargement in diameter of the collagen fiber bundles, and an improvement in the density of reticular fibers, fibrillin-1-rich microfibrils, and elastic fibers compared with the initial, steady-state condition. CONCLUSIONS: The administration of autogenous fibroblasts could be an effective and safe adjunctive therapy to conventional health care treatment to prevent and reduce the age-related accumulation of dermal tissue damage.

Changes in the Immune System of Female Wistar Rats After Exposure to Immunosuppressive Treatment During Pregnancy.

This experimental study assessed the impact of medications frequently used after kidney transplantation on the immune system of pregnant female Wistar rats. The study evaluates medications, both approved and contraindicated during pregnancy in common therapeutic combinations. The study was conducted on 32 female Wistar rats, subjected to immunosuppressive regimens most commonly used in therapy of human kidney transplant recipients (cyclosporine A, mycophenolate mofetil and prednisone; tacrolimus, mycophenolate mofetil and prednisone; and cyclosporine A, everolimus and prednisone). The animals received drugs by oral gavage 2 weeks before pregnancy and at 3 weeks of pregnancy. We found drug regimen-dependent differences in cytometry from spleen. Many subpopulations of lymphocytes were suppressed in rats treated with cyclosporine A, mycophenolate mofetil and prednisone and tacrolimus, mycophenolate mofetil and prednisone; the number of NK cells was increased in group of rats treated with cyclosporine A, everolimus and prednisone. We also found changes in histological examination of thymus and spleen of all treated dams. In cytokine assay, we noticed increasing levels of IL-17 with increasing doses of concanavalin A in control group and in group of dams treated with cyclosporine A, mycophenolate mofetil and prednisone. This increase was blocked in rats treated with tacrolimus, mycophenolate mofetil and prednisone and cyclosporine A, everolimus and prednisone. Qualitative, quantitative and morphological changes of immune system in pharmacologically immunosuppressed females have been observed. Thymus structure, spleen composition and splenocytes IL-17 production were mostly affected in drug regimen-dependent manner.

Expression of neurotrophins and their receptors in human CD34+ bone marrow cells.

Bone marrow (BM) CD34+ cells have the ability to secrete growth factors, cytokines, and chemotactic factors. We sought to better characterize this population and to investigate whether human BM CD34+ cells express neurotrophins (NTs) and their relevant receptors. We also compared their expression levels with BM nucleated cells (NCs). BM CD34+ cells were evaluated with respect to the expression levels of neurotrophins using qRT-PCR, immunofluorescent staining, and Western blotting. Next, the expression of specific (TrkA, TrkB, TrkC) and non-specific (p75NTR) neurotrophin receptors was detected by qRT-PCR and immunofluorescent staining in BM CD34+ cells. Using qRT- PCR, we show that even in the absence of inducing factors, CD34+ cells spontaneously express neurotrophins such as NGF, BDNF, NT-3, and NT-4. In addition, the NT expression levels in BM CD34+ cells are considerably higher than in NCs. Furthermore, we confirmed intracellular NT expression in BM CD34+ cells at the protein level using immunofluorescent staining and Western blotting. Using qRT-PCR, we found that immunomagnetically separated BM CD34+ cells spontaneously express high-affinity neurotrophin receptors (TrkA, TrkB, and TrkC) and the low-affinity receptor p75NTR at higher levels than NCs. Immunomagnetic CD34+ cell separation enables for the rapid and gentle sorting of stem/progenitor cells (SPCs) to prepare specific cell types for use in research and clinical applications. Our study suggests that BM CD34+ cells have the potential to support trophic factors for neural tissue and could contribute towards the protection and regeneration of neural cells.

The influence of the tumor necrosis factor-alpa-308G>A polymorphism on the efficacy of immunosuppressive therapy in patients after kidney transplantation.

Cytokines play an important role in the immune response. The calcineurin inhibitors (cyclosporine CsA, tacrolimus TAC) widely used after renal transplantation to prevent allograft rejection are immunosuppressive drugs suppressing the production of cytokines. These drugs are characterized by interindividual variability and require monitoring their blood concentrations to predict their optimal dosage. Therefore, the aim of the study was to determine the correlation between therapeutic effects of immunosuppressants and the tumor necrosis factor-α (TNF-α)-308G>A polymorphism in renal transplant patients. A total of 412 patients receiving TAC and CsA were included in the study. Genotype frequencies were determined using the real-time PCR method. Patients with the GG genotype received higher doses of TAC as compared to carriers of the GA genotype (5.24 mg versus 3.35 mg) and had lower mean drug concentration in blood (5.86 ng/ml versus 6.92 ng/ml). Similar results were also obtained for CsA (GG: 185.33 mg versus GA: 153.30 mg, P < 0.05). The comparison of the TNF-α-308G>A polymorphism with the biochemical parameters did not reveal a potential risk for transplant rejection. These results indicate that the TNF-α-308G>A polymorphism may influence the dosage of immunosuppressive drugs in patients after transplantation as far as individualization of drug therapy is concerned.

Study on size effect of the silica nanospheres with solid core and mesoporous shell on cellular uptake.

The properties of mesoporous silica nanoparticles including large surface area, large pore volume, easy surface functionalization and control of structure and pore size has made them promising drug carriers. In this study, the effect of different diameters (50 nm, 70 nm, 90 nm, 110 nm and 140 nm) of silica nanospheres with a solid core and mesoporous shell (mSiO2/SiO2) on cellular internalization in mouse fibroblast cells (L929) was evaluated. The physical properties of the nanostructures were characterized with various methods, such as transmission electron microscopy with x-ray dispersion spectroscopy, thermogravimetric analysis, Fourier transform infrared spectroscopy and zeta potential. In order to define the cellular uptake, the nanostructures were labelled with fluorescent dye Alexa647, and imaging and quantitative methods were applied: laser scanning confocal microscopy, flow cytometry and thermogravimetry. Our results indicate that cellular uptake of the studied nanospheres is size-dependent, and nanospheres of 90 nm in diameter showed the most efficient cell internalization. Thus, particle size is an important parameter that determines cellular uptake of nanoparticles and should be considered in designing drug delivery carriers.

Measurement of Internal and External Pressure of Transplanted Kidney: An Underestimated Method of Diagnosis for Renal Grafts.

Hardness, or tensity (tonus), of transplanted kidney can change in the course of various pathologic conditions. Manual examination (with palpation), which is most frequently used to evaluate this transplanted organ, is not objective. First attempts of objective evaluation were described in the medical literature in the 1980s. They consisted of evaluation of intrarenal pressure by puncturing the kidney, connecting an intravenous drip line, and measuring the pressure in centimeters of water column. Examination of a group of subjects revealed significant differences in mean measurements, especially in patients with acute rejection process compared with the control group. However, use of this method was not continued, owing to its invasiveness. Our own diagnostic method, described here, is measurement of external kidney pressure (tonus). Two types of devices (tonometers) are described, as well as a project of a future tonometer functioning on the basis of electronically measured differences in values of forces used above the graft and above the symmetric part of the abdomen causing identical deflection of abdominal wall. Thirty-two patients (including control group) were examined with the use of this method. Statistically significant differences were revealed between patients with acute graft rejection and chronic graft nephropathy compared with the control group. The method described here can be a valuable supplement to other currently used noninvasive means of renal graft evaluation, including ultrasonography, Doppler, and elastographic examinations.

Effects of growth hormone therapeutic supplementation on hematopoietic stem/progenitor cells in children with growth hormone deficiency: focus on proliferation and differentiation capabilities.

We investigated the direct effects of growth hormone (GH) replacement therapy (GH-RT) on hematopoiesis in children with GH deficiency (GHD) with the special emphasis on proliferation and cell cycle regulation. Peripheral blood (PB) was collected from sixty control individuals and forty GHD children before GH-RT and in 3rd and 6th month of GH-RT to measure hematological parameters and isolate CD34(+)-enriched hematopoietic progenitor cells (HPCs). Selected parameters of PB were analyzed by hematological analyzer. Moreover, collected HPCs were used to analyze GH receptor (GHR) and IGF1 expression, clonogenicity, and cell cycle activity. Finally, global gene expression profile of collected HPCs was analyzed using genome-wide RNA microarrays. GHD resulted in a decrease in several hematological parameters related to RBCs and significantly diminished clonogenicity of erythroid progenies. In contrast, GH-RT stimulated increases in clonogenic growth of erythroid lineage and RBC counts as well as significant up-regulation of cell cycle-propagating genes, including MAP2K1, cyclins D1/E1, PCNA, and IGF1. Likewise, GH-RT significantly modified GHR expression in isolated HPCs and augmented systemic IGF1 levels. Global gene expression analysis revealed significantly higher expression of genes associated with cell cycle, proliferation, and differentiation in HPCs from GH-treated subjects. (i) GH-RT significantly augments cell cycle progression in HPCs and increases clonogenicity of erythroid progenitors; (ii) GHR expression in HPCs is modulated by GH status; (iii) molecular mechanisms by which GH influences hematopoiesis might provide a basis for designing therapeutic interventions for hematological complications related to GHD.

Mixed chimerism induction influences cytokine release from chimeric mice cells.

Interest in mixed chimerism has evolved from its role in the induction of alloantigen tolerance. However, its precise impact on the host organism remains to be elucidated. In the present work, we analyzed cytokine secretion from chimeric mice cells to assess the influence of different mixed chimerism induction protocols on immune system function in recipient mice. To our knowledge, there have been no reports on using this parameter for the optimization of the mixed chimerism induction method. B6.SJL-PtprcaPep3b or C57BL/6J mice were used as recipients and Balb/c as donors. We utilized four protocols which consisted of: 3Gy total body irradiation (day -1), the injection of 20-30×10(6) bone marrow cells (day 0), and a combination of CD40L (days 0 and 4), CD8 (day -2), and NK1.1 (day -3) blocking antibodies and cyclophosphamide (175mg/kg - day 2). The concentrations of cytokines (IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF) were evaluated in the supernatants of unstimulated or phytohemagglutinin-stimulated chimeric spleen, bone marrow and peripheral blood cells in the 8th week of experiment. The induction of tolerance to Balb/c mouse antigens was initially tested in chimeric mice by assessing the presence of Vß5 and Vß11 TCR-expressing lymphocytes. The cytokine production was considerably increased, especially in chimeric mice treated by cyclophosphamide. Also the mixed chimerism itself seems to affect IFN-γ, IL-2, IL-4, IL-6, IL-17A, and TNF secretion. Using the optimized induction protocol, we established that chimeric mice cells secreted lower IFN-γ, IL-2, IL-4 and higher IL-6, IL-17A, and TNF levels as compared to control animals. We found that both donor and recipient cells markedly participated in the cytokine production. In conclusion, our optimization study based on cytokine assessment contributes to establishing an effective protocol of mixed chimerism induction with no cyclophosphamide use and better understanding of the influence of this phenomenon on the recipient organism.

Increased circulating endothelial progenitor cells in patients with haemorrhagic and ischaemic stroke: the role of endothelin-1.

Ischaemic stroke induces endothelial progenitor cell (EPC) mobilisation from bone marrow into peripheral blood. Circulating EPCs play an important role in post-injury regeneration of vasculature, whereas endothelial cells (ECs) have been shown to reflect endothelial damage and may be responsible for increased Endothelin-1 (ET-1) expression. We investigated herein the association between numbers of circulating ECs and EPCs, the levels of soluble factors regulating their migration and function, and the clinical outcome in patients with haemorrhagic (HS) or ischaemic stroke (IS). Sixteen patients with HS and eighteen with IS were assessed during the first 24h, day 3, and day 7 after stroke and compared them with twenty-three control subjects. We found elevated EPC and EC concentrations using flow cytometry and increase in VEGF, SDF-1, HGF, and ET-1 plasma levels by ELISA in the HS patients, while ET-1 mRNA expression in peripheral blood cells was elevated in the IS patients. Significant correlations were observed between EPCs or ECs and Big ET-1 protein or mRNA levels in HS but not in the IS patients. We suggest that ET-1 may play a role in pathophysiology of stroke and subsequent EPC mobilisation; however, further studies aimed at the precise elucidation of this issue are required.

Perinatal exposure to lead induces morphological, ultrastructural and molecular alterations in the hippocampus.

The aim of this paper is to examine if pre- and neonatal exposure to lead (Pb) may intensify or inhibit apoptosis or necroptosis in the developing rat brain. Pregnant experimental females received 0.1% lead acetate (PbAc) in drinking water from the first day of gestation until weaning of the offspring; the control group received distilled water. During the feeding of pups, mothers from the experimental group were still receiving PbAc. Pups were weaned at postnatal day 21 and the young rats of both groups then received only distilled water until postnatal day 28. This treatment protocol resulted in a concentration of Pb in rat offspring whole blood (Pb-B) below the threshold of 10 µg/dL, considered safe for humans.We studied Casp-3 activity and expression, AIF nuclear translocation, DNA fragmentation, as well as Bax, Bcl-2 mRNA and protein expression as well as BDNF concentration in selected structures of the rat brain: forebrain cortex (FC), cerebellum (C) and hippocampus (H). The microscopic examinations showed alterations in hippocampal neurons.Our data shows that pre- and neonatal exposure of rats to Pb, leading to Pb-B below 10 µg/dL, can decrease the number of hippocampus neurons, occurring concomitantly with ultrastructural alterations in this region. We observed no morphological or molecular features of severe apoptosis or necrosis (no active Casp-3 and AIF translocation to nucleus) in young brains, despite the reduced levels of BDNF. The potential protective factor against apoptosis was probably the decreased Bax/Bcl-2 ratio, which requires further investigation. Our findings contribute to further understanding of the mechanisms underlying Pb neurotoxicity and cognition impairment in a Pb-exposed developing brain.

Activation of phospholipase A(2) by low levels of fluoride in THP1 macrophages via altered Ca(2+) and cAMP concentration.

Phospholipases (PLA's) participate in the regulation of physiological and pathological processes in the cell, including the release of pro-inflammatory mediators and stimulation of inflammatory processes. It is also well known that fluoride can increase the inflammatory reactions. Therefore we decided to examine the effect of fluorides in concentrations determined in human serum on cPLA(2) and sPLA(2) activity. The incubation of macrophages in fluoride solutions significantly increased the amount of synthesized cellular cAMP, intracellular calcium and sPLA(2) activity in a dose-dependent pattern. The cPLA(2) activity, estimated by the amount of released arachidonic acid, increased significantly when 10 µM NaF was used. The results of our study suggest that fluoride may change the activity of phospholipases in macrophage cells. Probably, increased cAMP concentration activates protein kinase C (PKC) and thus stimulates PLA(2). cAMP also regulates the passage of Ca(2+) through ion channels, which additionally influence PLA(2) throughout Ca(2+)-calmodulin dependent protein kinase.

Blood arachidonic acid and HDL cholesterol influence the phagocytic abilities of human monocytes/macrophages.

AIMS: Many immunomodulators may intensify the process of phagocytosis in monocytes. Among them are the fatty acids contained in cellular membrane phospholipids. But in the available literature there are no reports on how blood fatty acids and lipoproteins can modulate the phagocytic abilities of cells. Therefore, the aim of this study was the evaluation of the phagocytic activity of monocytes isolated from the blood of healthy human subjects with defined fatty acids and lipoprotein content. METHODS: Cells obtained from 24 donors were used for measuring phagocytic activity and for the quantification of serum fatty acids, total cholesterol, TG, HDL, and LDL, respectively. Phagocytosis was determined using a PHAGOTEST kit, fatty acids using a GC chromatograph, and lipids using test kits. RESULTS/CONCLUSIONS: The analysis shows an influence of serum HDL concentrations on the process of phagocytosis in the isolated cells and suggests that the concentration of arachidonic acid in blood is a factor that determines the phagocytic ability of monocytes. Moreover, the concentration of conjugated linoleic acid trans-10 cis-12 has considerable influence on phagocytosis.