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.

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.

Coexistence of JAK2 or CALR mutation is a rare but clinically important event in chronic myeloid leukemia patients treated with tyrosine kinase inhibitors.

INTRODUCTION: There are 7 designated conditions under the category of myeloproliferative neoplasms (MPN), including chronic myelogenous leukemia (CML) and classical MPN, that is, polycythemia vera (PV), essential thrombocythaemia (ET), and primary myelofibrosis (PMF). Recently, reports about Philadelphia and JAK2 V617F-positive MPN cases have been described in literature. The coexistence of different molecular defects may change the clinical and laboratory manifestation of MPN and may result in an inappropriate interpretation of the response to treatment with tyrosine kinase inhibitors in CML patients. METHODS: The morphological, cytogenetic, and molecular genetic data from a retrospective analysis of 592 adult patients aged 18-86 years diagnosed with CML were analyzed. RESULTS: In 5 CML patients, the presence of JAK2 V617F or CALR mutation was confirmed. Three of 4 TKI-treated patients did not reach complete hematologic response due to the persistence of thrombocytosis and/or splenomegaly. In all of them (in 3 with JAK2 V617F mutation and 1 with CALR mutation), thrombocytosis was present at the time when complete cytogenetic response was documented. In 3 out of 4 reported CML patients, thrombocytosis and/or splenomegaly were still present even at the time when deep molecular response was reached. CONCLUSION: In our opinion, a detailed evaluation and appropriate interpretation of clinical and laboratory data in such a category of patients seem to be extremely important, especially when a decision about the TKI change due to therapy failure is considered.

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.

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.

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.

Stem cells for neural regeneration--a potential application of very small embryonic-like stem cells.

The goal of regenerative medicine is to ameliorate irreversible destruction of brain tissue by harnessing the power of stem cells in the process of neurogenesis. Several types of stem cells, including mesenchymal stem cells, hematopoietic stem cells, as well as neural cells differentiated from embryonic stem cell lines, have been proposed as potential therapeutic vehicles. In this review paper we will discuss a perspective of stem cell therapies for neurological disorders with special emphasis on potential application of cells isolated from adult tissues. In support of this our group found that murine bone marrow contains a mobile population of Oct-4+CXCR4+SSEA-1+Sca-1+lin⁻CD45⁻ very small embryonic-like stem cells (VSELs) that are mobilized into peripheral blood in a murine stroke model. The number of these cells in circulation increases also after pharmacological mobilization by administration of granulocyte colony stimulating factor (G-CSF). Recently we found that VSELs are present in various non-hematopoietic adult organs and, interestingly, our data indicate that the brain contains a high number of cells that display the VSEL phenotype. Based on our published data both in human and mice we postulate that VSELs are a mobile population of epiblast/germ line-derived stem cells and play an important role as an organ-residing reserve population of pluripotent stem cells that give rise to stem cells committed to particular organs and tissues--including neural tissue. In conclusion human VSELs could be potentially harnessed in regenerative medicine as a source of stem cells for neurogenesis.

The effect of stem cell mobilisation with granulocyte colony-stimulating factor on the morphology of the haematopoietic organs in mice.

The cellular mobilisation of mice with granulocyte colony-stimulating factor (G-CSF) results in an egress of haematopoietic stem/progenitor cells from the bone marrow and an increase in their level in the peripheral blood. While the mobilisation process with different agents is widely studied, little is known about the morphology of the murine haematopoietic organs during the mobilisation. The purpose of this study was to examine the morphology of the bone marrow, spleen and liver in mice mobilised with G-CSF. To address this issue mice were injected subcutaneously with G-CSF for 6 consecutive days. Morphological analysis revealed an increase in the number of mature neutrophils close to the wall of sinusoids in the bone marrow as well as hypertrophy of the red pulp in the spleen. At the same time no morphological changes were noticed in the livers of G-CSF-mobilised mice. In conclusion, G-CSF induces discrete ultrastructural changes in the bone marrow, which intensify the transendothelial traverse of haematopoietic stem and progenitor cells from it. The changes in the spleen are related to repopulation of this organ by mobilised early haematopoietic cells circulating in the peripheral blood. We also noticed that the process of migration of haematopoietic cells from the bone marrow into the peripheral blood began on day 2 and was most pronounced on day 4 after stimulation with G-CSF.

The influence of 3,3',5-triiodo-L-thyronine on human haematopoiesis.

OBJECTIVES: Thyroid hormones mediate many physiological and developmental functions in humans. The role of the 3,3',5-triiodo-L-thyronine (T3) in normal human haematopoiesis at the cellular and molecular levels has not been determined. In this study, it was revealed that the human haematopoietic system might be directly depended on T3 influence. MATERIALS AND METHODS: We detected the TRalpha1 and TRbeta1 gene expression at the mRNA level in human cord blood, peripheral blood and bone marrow CD34(+)-enriched progenitor cells, using the RT-PCR method. Furthermore, we performed Western blotting to prove TRalpha1 and TRbeta1 expression occurs at the protein level in human cord blood, peripheral blood and bone marrow CD34(+) cells. In addition, the examined populations of cells were exposed in serum-free conditions to increasing doses of T3 and were subsequently investigated for clonogenic growth of granulocyte-macrophage colony-forming unit and erythrocyte burst-forming unit in methylcellulose cultures, and for the level of apoptosis, by employing annexin V staining and the terminal deoxynucleotidyltransferase-mediated dUTP nick-end labelling method. We investigated expression levels of apoptosis-related Bax and antiapoptotic Bcl-2 and Bcl-x(L) genes in the examined cells. RESULTS: We found that exposure to higher and lower than normal concentration of thyroid hormone significantly influenced clonogenecity and induced apoptosis in human haematopoietic progenitor cells. CONCLUSIONS: This study expands the understanding of the role of thyroid disorders in normal human haematopoiesis and indicates a direct influence of T3 on this process.

Mobilization of human hematopoietic stem/progenitor-enriched CD34+ cells into peripheral blood during stress related to ischemic stroke.

The bone marrow-derived stem/progenitor cells were demonstrated to play an important role in a regeneration of damaged tissue. Based on these observations we asked whether the stroke-related stress triggers mobilization of stem/progenitor cells from the bone marrow into the peripheral blood, which subsequently could contribute to regeneration of damaged organs. To address this issue, the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 hrs as well as after the 48 (2nd day) and 144 hrs (6th day) since the manifestation of symptoms. In these patients we evaluated the percentage of hematopoietic stem/progenitor-enriched CD34+ cells by employing flow cytometry and the number of hematopoietic progenitor cells for the granulocyto-monocytic (CFU-GM) and erythroid (BFU-E)-lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke triggers the mobilization of hematopoietic stem/progenitor cells from the bone marrow into peripheral blood. These circulating stem/progenitor cells may play an important role in the process of regeneration of the ischemic tissue.

Expression of stem cell markers on mononuclear cells derived from heparinized cadaveric organ donors before and after disconnection from the respirator.

We have attempted to evaluate the level of the earliest human hematopoietic cell marker expression (CD34, CD117, CD133, CD184) on cells obtained from heparinized cadaveric organ donors before and after disconnection from the respirator. Moreover, we compared various cell populations: (1) coexpressing CD34/CD117; (2) CD34/CD133; (3) highly enriched hematopoietic stem cells (CD34+CXCR4+CD45+); and (4) highly enriched tissue-committed stem cells (CD34+CXCR4+CD45-). Finally, we analyzed whether the level of hematopoietic stem cell marker expression depended on the age of the donor. The expression of the membrane receptors (CD34, CD45, CD117, CD133, CD184) was studied by flow cytometry. We observed that the proportion of mononuclear cells expressing these markers slightly decreased in bone marrow harvested after disconnection from the respirator compared with the samples obtained before disconnection. Moreover, the proportion of cells expressing CD117 antigen depended on age of the donor.

Human hematopoietic stem/progenitor-enriched CD34(+) cells are mobilized into peripheral blood during stress related to ischemic stroke or acute myocardial infarction.

The hematopoietic and non-hematopoietic stem/progenitor cells harvested directly from the bone marrow (BM) or G-CSF mobilized peripheral blood were demonstrated to play an important role in regeneration of damaged organs (1, 2). Here, we asked if the stroke- or acute heart infarct-related stress triggers mobilization of stem/progenitor-enriched CD34(+)cells from the BM into the peripheral blood, which subsequently could contribute to regeneration of damaged tissues. To address this question the peripheral blood samples were harvested from patients with ischemic stroke during the first 24 h of manifestation of symptoms and on the second and sixth day afterwards or during the first 24 h of acute cardiac pain as well as on the second and sixth day of infarct. We measured in these patients (i) percentage of circulating hematopoietic stem/progenitor-enriched CD34(+) cells in peripheral blood by employing fluorescence activated cell sorter (FACS) and (ii) number of hematopoietic progenitor cells for the granulocyte-monocytic colony-forming unit (CFU-GM) and erythoid burst-forming unit (BFU-E) lineages circulating in peripheral blood. We concluded that stress related to ischemic stroke or acute myocardial infarction triggers the mobilization of hematopoietic stem/progenitor-enriched CD34(+) cells from the BM into peripheral blood. These circulating stem/progenitor-enriched CD34(+) cells may contribute to the regeneration of ischemic tissues, however, this possibility requires further studies.

The influence of angiotensin-converting enzyme gene of donor and recipient on the function of transplanted kidney.

One of the genes that is supposed to influence renal graft function is the one encoding angiotensin I-converting enzyme (ACE). It shows polymorphism in the presence (I allele) or absence (D allele) of a 287-base pair fragment. The question arises whether ACE gene polymorphism of the recipient and donor influences renal graft survival. This prospective study included 94 recipients who underwent ACE genotyping (DD, DI, II) and measured their creatinine clearance after cimetidine administration. These factors were correlated with the occurrence of acute or chronic rejection and of pharmacologic treatment of hypertension. In 27 recipients it was possible to obtain the ACE genotype of the donor. Among the recipients, 36 proved to be DD genotype, 38 ID, and 20 II. Among the donors, 10 proved to be DD genotype, 10 ID, and 7 II. The changes in creatinine clearance after cimetidine administration were not significantly different among any of the genotype subgroups. Significantly higher creatinine concentrations were found among recipients with II genotype compared to the combined group of ID and DD among patients not treated with ACE inhibitors, but not among those receiving ACE I after kidney transplantation. No differences were found in the frequency of rejection episodes among the subgroups with different ACE genotypes. No significant influence of donor ACE genotype on renal graft function was observed. In summary, the I/D genotype was not an independent prognostic factor for renal graft survival in the first 4 years after transplantation. Possibly the use of ACE I alters the influence of genotype on some parameters.

An optimization of isolation of early hematopoietic cells from heparinized cadaveric organ donors.

Heparinized cadaveric organ donors are a potential source of hematopoietic cells for transplantation purposes. The aim of this study was to optimize the harvest of early hematopoietic cells from cadaver donors. Accordingly, we noticed that bone marrow cells harvested from cadaver donors should be resuspended in RPMI or Iscove's medium supplemented with heparin or ACD. Bags with harvested marrow should contain 20% atmosphere air during short-term storage/transportation. Finally, we noticed that BMMNC survive short-term storage better if the collected marrow is not depleted of erythrocytes.

Effect of genomic and subgenomic leader sequences of potato leafroll virus on gene expression.

The effect of the genomic and subgenomic leader sequence of potato leafroll polerovirus on the efficiency of translation of the downstream located genes has been studied. The results obtained in vitro and in vivo indicate that neither leader sequence functions as translational enhancer, a generally important feature of leader sequences. Deletion analyses demonstrated that both leader sequences not only decrease translation of the downstream located genes but also alter the ratio of the synthesized proteins. A correlation between the in vitro and in vivo results can be established in the case of the subgenomic leader sequence.