Immunoexpression of intermediate filaments and morphological changes in the liver and bile duct of rats infected with Fasciola hepatica.

We investigated the immunoexpression of the intermediate filament proteins, cytokeratin and desmin, and the morphological changes in the liver of rats during experimental fasciolosis at 4, 7 and 10 weeks post-infection. Rats were infected with 30 Fasciola hepatica metacercariae. Paraffin sections of the liver were stained using H & E, PAS and azan stains. Immunohistochemical reactions were performed using antibodies against cytokeratin and desmin. The experimental F. hepatica infection led to fibrosis and cirrhosis of the liver, and to inflammation of the common bile ducts. The expression of cytokeratin was increased in the epithelial cells of both the liver bile ductules at 4, 7 and 10 weeks post-infection and in the common bile ducts at 7 and 10 weeks post-infection compared to uninfected rats; expression in the common bile ducts was more intense. The myofibroblasts of the liver and smooth myocytes of the interlobular bile ducts and common bile ducts, showed a slight increase in desmin expression compared to the uninfected rats. The increased expression of cytokeratins in the hyperplastic rat common bile duct epithelium during the biliary phase of fasciolosis at 7 and 10 weeks post-infection may be explained by mechanical irritation by the parasite and an inflammatory reaction in the bile duct epithelium and in periductal fibrous tissue.

Analysis of pituitary gonadotropin concentration in blood serum and immunolocalization and immunoexpression of follicle stimulating hormone and luteinising hormone receptors in ovaries of postmenopausal women.

The participation of gonadotropins in ovarian carcinogenesis is well known and is supported by studies with inhibition of pituitary gonadotropin secretion, which results in a diminished risk of cancer. However, there are few data on localization and expression of Follicle Stimulating Hormone and Luteinising Hormone Receptors (FSHR and LHR) in ovaries of healthy postmenopausal women, and their correlation with FSH and LH concentration in blood serum is unknown. The aim of our study was to analyze gonadotropin concentration in blood serum and the expression of FSHR and LHR in ovaries of 207 postmenopausal women. Patients included in the study were divided into three groups depending on the number of years since menopause. We analyzed the concentration of FSH and LH in blood serum and the expression of FSHR and LHR in ovaries. Ovaries of postmenopausal women showed numerous morphological changes in the cortex and medulla when compared to the structure of ovaries of women at reproductive age. In all groups of patients clefts in the surface epithelium and epithelial inclusion cysts were found. The concentration of FSH and LH in the blood serum of women studied increased significantly with time from menopause. Significant differences between analyzed menopausal groups were found. The highest FSH and LH concentration in blood serum were found in women with the longest period of time from menopause. Quantitatively similar expression of FSHR and LHR was found in ovarian surface epithelial cells, in epithelial inclusion cysts and in the connective tissue cells of ovarian stroma. The intensity of the immunohistochemical reaction decreased with time from menopause and with age.

Immunohistochemical analysis of steroid receptors in ovaries of postmenopausal women--effects of aging and hormone status.

Current knowledge on immunolocalization and immunoexpression of steroid hormone receptors, especially estrogen receptor alpha (ER-alpha), progesterone receptor (PR) and androgen receptor (AR) in normal ovaries in postmenopausal women is not complete. The recognition of localization of these receptors in postmenopausal women is crucial, as many of these women receive estro-progestagene therapy, and its participation in the pathogenesis of ovarian cancer should be carefully studied. In our paper we present the results of immunohistochemical studies performed on samples from 100 post-menopausal women (aged: 48 to 60 years) who did not use hormonal therapy. The ovaries were removed during elective operation due to uterine leiomyoma, endometriosis and/or prolapsed uterine. PR, ER-alpha and AR were detected in the normal ovaries of postmenopausal women in stroma and in ovarian surface epithelium, as well as in its invagination and in epithelial inclusion cysts. The expression of PR and AR did not change, while the expression of ER-alpha decreased in time from menopause, and it was also detected in patients more than 10 years after menopause. Women older than 60 were not included in the study. The concentration of selected hormones was measured in the serum. The immunohistochemical expression of PR and AR were similar in all examined patients and did not correlate with FSH, LH, T, A, DHEAS concentrations in serum, while immunohistochemical expression of ER-alpha correlated with FSH, LH, T, A, DHEAS concentrations in serum. The significant correlation of decreasing expression of ER-alpha in normal ovarian tissue and decreasing concentrations of T, A and DHEAS in serum were found, as well as increasing serum concentrations of FSH and LH.

Induction of mixed chimerism in mice by employing different conditioning protocols and bone marrow cell transplantation.

Mixed chimerism has been suggested to produce allograft tolerance. Since this phenomenon is not fully understood, the aim of our study was to evaluate various protocols for chimerism induction in a mouse model. B6.SJL-Ptprc(a)Pep3(b) mice were injected with 20 to 30 x 10(6) bone marrow cells from Balb C mice. Conditioning consisted of total body gamma irradiation with 9.5, 5, and 3 Gy on "-1 day" of the experiment, with 200 mg/kg cyclophosphamide (CP) ("+2 day"). Additionally, one group of mice received blocking antibody against CD40L on days 0, 1, 4, and 7. The presence of mixed chimerism in peripheral blood was assessed at 1, 2, 3, 4, 6, and 8 weeks using flow cytometry to detect CD45.1 or CD45.2 antigen expression. Moreover, the chimerism was examined in CD4, CD8, CD45/B220, Mac-1alpha subpopulations in peripheral blood and bone marrow cells at 8 weeks. We also compared chimerism in peripheral blood, bone marrow, and spleen leukocyte populations. We observed that the most effective conditioning method with relatively low toxicity was based on concomitant use of 5 Gy total body irradiation and CP. The percentage of donor cells differed among peripheral blood subpopulations and bone marrow cells, but was similar in leukocyte populations derived from various sources. Our experiments sought to optimize the induction of stable mixed chimerism.

An optimization of protocol for mixed chimerism induction in mice model.

Studies on mixed chimerism are currently focused primarily on obtaining less toxic conditioning protocols. With these issues in mind, we have undertaken the attempt to optimize the procedure of mixed chimerism induction in mice. In order to reduce toxicity, we used decreasing doses of total body irradiation (TBI) together with combination of blocking antibodies. We also tried to eliminate immunosuppression (cyclophosphamide - CP) treatment after bone marrow transplantation. B6.SJL-PtprcaPep3b mice were injected with 20-30 x 106 bone marrow cells from Balb C mice. Mice were treated with TBI (3 - 1.5 - 0 Gy) on "-1" day of the experiment and blocking antibodies against CD40L ("0", and "4" days) and additionally anti-CD8 ("-2" day) and/or anti-NK1.1 ("-3" day). Mice in certain groups also received CP (175 mg/kg) on "2" day. Presence of mixed chimerism was assessed in peripheral blood cells by flow cytometry on the 1st, 2nd, 3rd, 4th, 6th and 8th weeks of the experiment by detecting of CD45.1 (characteristic for B6.SJL-PtprcaPep3b strain) and CD45.2 (characteristic for Balb C strain) antigens expression. We also analyzed the percentage of peripheral blood CD8 T-cells (CD3e/CD8a) and NK cells (Ly-49D/NK1.1). We found that reduction of TBI dose and elimination of CP decrease the rate of mixed chimerism formation. The highest percentage of donor cells was obtained in the group of animals treated with 3 Gy of TBI, CP and combination of anti-CD40L, anti-CD8, and anti-NK1.1 antibodies. The 3 Gy TBI was necessary to induce stable mixed chimerism, but it could be obtained without the CP use. The percentage of CD3e/CD8a and Ly-49D/NK1.1 cells was significantly lower in the groups of mice treated by corresponding antibodies. Moreover, we observed the lowest number of peripheral blood Ly-49D/NK1.1 cells in the group of animals with highest mixed chimerism. Our experiments in mice model can help in better understanding of mixed chimerism phenomenon and in selecting the method of mixed chimerism induction with lowest possible toxicity. This also might improve the protocols of stable mixed chimerism induction in humans, and in the future, the effectiveness of vascularized organ transplantation.

Human postmenopausal ovary--hormonally inactive fibrous connective tissue or more?

The ovary undergoes several changes after the menopause. In this period, the main structural changes in both the cortex and medulla were observed. In the cortex, they included: 1) reduction of its thickness; 2) epithelial inclusions forming cysts; 3) blurring the line between medulla and cortex; 4) reduction of follicles number; 5) tendency to fragmentation of corpora albicantia; 6) surface epithelium invaginations. Whereas the changes in the medulla included: 1) fibrosis and scars in stroma; 2) architectonical changes in blood vessels with hyalinization of walls and constriction of lumen. The loss of follicles and several changes in the ovary are due to apoptotic processes. Despite age related atrophic changes, the postmenopausal ovary is not devoid of hormonal activity. Our results are coherent with the reports of other researchers, and reveal that postmenopausal ovary produces trace quantities of steroid hormones, mainly androgens, and confirm the presence of steroid receptors and activity of main enzymes involved in steroidogenesis process.

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.

Morphological and molecular characterization of novel population of CXCR4+ SSEA-4+ Oct-4+ very small embryonic-like cells purified from human cord blood: preliminary report.

Recently, we purified from adult murine bone marrow (BM) a population of CXCR4(+), Oct-4(+) SSEA-1(+), Sca-1(+) lin(-) CD45(-) very small embryonic-like (VSEL) stem cells and hypothesized that similar cells could be also present in human cord blood (CB). Here, we report that by employing a novel two-step isolation procedure -- removal of erythrocytes by hypotonic lysis combined with multiparameter sorting -- we could isolate from CB a population of human cells that are similar to murine BM-derived VSELs, described previously by us. These CB-isolated VSELs (CB-VSEL) are very small (3-5 micro m) and highly enriched in a population of CXCR4(+)AC133(+)CD34(+)lin(-) CD45(-) CB mononuclear cells, possess large nuclei containing unorganized euchromatin and express nuclear embryonic transcription factors Oct-4 and Nanog and surface embryonic antigen SSEA-4. Further studies are needed to see if human CB-isolated VSELs similar to their murine BM-derived counterparts are endowed with pluripotent stem cell properties.

Soluble HLA class I molecules exert differentiated influence on renal graft condition.

The function of soluble HLA (sHLA) antigens in the process of immunoregulation and especially in graft tolerance versus rejection has not yet been established. It has been suggested that donor-derived sHLA may exert an immunotolerant influence on the graft. We sought to determine the role of sHLA class I in kidney graft survival by evaluating the influence of these molecules on allotypic lymphocytotoxic antibodies and the concentration of gamma interferon (INF-gamma). Analysis of sHLA was performed indirectly utilizing their ability to inhibit lymphocytotoxic reaction dependent on complement activation. To demonstrate the inhibitory properties of sHLA, we modified the NIH microcytotoxic test. Furthermore, we determined the concentration of INF-gamma in all sera samples for comparison with the intensity of the cytotoxic test. The comparison of the intensity of cytotoxic test inhibition with the concentration of INF-gamma revealed that high concentrations of this cytokine were associated with stronger inhibition of the cytotoxic test, thus with higher concentrations of sHLA class I molecules in recipient sera. We observed that high concentrations of sHLA class I molecules in recipient sera significantly inhibited cytotoxic reactions, which could contribute to a protective influence of sHLA on renal grafts. On the other hand, the observed increase of INF-gamma concentration might be caused by sHLA themselves, which would produce a detrimental influence on a transplanted organ. Therefore we concluded that the role of sHLA class I molecules in renal graft condition remains ambiguous.

Association of cytokine gene polymorphisms and the release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone.

Immunosuppressive drugs are widely used in the therapy of autoimmune disorders to suppress autoreactive T cells. The immune system is regulated by the release of cytokines. Cytokine are potent immunomodulatory molecules that act as mediators of inflammation and the immune response. Primarily secreted by T cell and macrophages, they influence cellular activation, differentiation, and function. Cytokine production is under genetic control. This is evidenced by the identification of polymorphism in cytokine gene regulatory regions that correlate with intra-individual variations in actual cytokine production. The aim of the study was to examine whether the individual differences in the polymorphic cytokine genes can lead to individual variation in release of cytokines after treatment with methotrexate and glucocorticosteroids. The study was carried out on mononuclear cells isolated from peripheral blood of 72 healthy subjects. The cells were activated with PHA and incubated with increasing concentrations of methotrexate (0.1-10 microM) and dexamethasone (0.01-1 microM). Levels of IL-2, IL-4, IL-6, IL-10, and TNFalpha in the culture supernatants were quantified by flow-cytometry using Th1/Th2 kit and correlated with cytokine gene polymorphisms. The increased concentrations of DEX resulted in comparable cytokine concentrations in cultures from subjects with low and high cytokine genotypes. Despite MTX treatment, the cytokine levels were significantly increased in individuals homozygous for the high producer allele. These results suggest that the cytokine gene variants may influence the efficacy of therapy with some immunosuppressive and anti-inflammatory drugs.

Involvement of P-glycoprotein in the release of cytokines from peripheral blood mononuclear cells treated with methotrexate and dexamethasone.

P-glycoprotein (P-gp), a product of the MDR1 gene, is an important factor in the turnover of many drugs and xenobiotics. Recent reports have suggested that P-gp can also be involved in the transport of cytokines. The aim of this study was to examine the role of P-gp in cytokine release from phytohaemagglutinin (PHA)-stimulated peripheral blood mononuclear cells (MNCs) as well as in the release of cytokines from MNCs treated with methotrexate (MTX) and dexamethasone (DEX). The study was carried out on PHA-stimulated MNC from 10 healthy subjects. Flow cytometry was applied to measure interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha levels in the culture supernatants. In the experiments verapamil (VER) and P-gp specific monoclonal antibodies (mAb) (clone 17F9) were used to inhibit P-gp function. P-gp inhibitors suppressed the release of IL-2, IL-4, IFN-gamma and TNF-alpha from PHA-stimulated MNC, whereas release of IL-6 and IL-10 remained unaffected. VER and mAb significantly decreased the release of IL-2, IL-4, TNF-alpha and INF-gamma in MNC cultures treated with MTX or DEX. The results of this study suggest that P-gp may be involved in the transmembrane transport of some cytokines. Moreover, it seems that blocking of P-gp function may influence the release of some cytokines from MNCs, displaying an additive inhibitory effect to DEX and MTX.

The evaluation of apoptosis and reactive oxygen species in hematopoietic cells derived from heparinized cadaveric organ donors stored short-term at 4 degrees C.

Heparinized cadaveric organ donors are an important source of human organs and potentially of hematopoietic cells for transplantation purposes. The aim of this study was to evaluate the kinetics of programmed cell death in the hematopoietic cells harvested from these individuals and stored short-term at 4 degrees C. We also attempted to assess hematopoietic cell oxidation by measuring reactive oxygen species (ROS) generated by the mitochondria of stored cells. We found that these bone marrow cells harvested and stored at 4 degrees C for 7 days did not display a significant increase in programmed cell death. However, prolonged storage resulted in lower ROS production, indirectly giving evidence of activation of intracellular signaling proteins.

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.

Involvement of P-gp in the process of apoptosis in peripheral blood mononuclear cells.

Multidrug resistance mediated by the drug-efflux protein P (P-gp) is one of mechanisms that cells use to escape death induced by drugs and other agents. The aim of the study was to evaluate the effect of P-gp inhibition on apoptosis of PHA-activated peripheral blood mononuclear cells (MNC) as well as apoptosis induced by methotrexate (MTX), dexamethasone (DEX), methylprednisolone (MP) and cortisone (COR). Apoptosis was quantified by flow cytometry using Annexin V/PI and terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL). P-gp expression was inhibited using verapamil (VER) and P-gp specific monoclonal antibodies (mAb). VER and mAb enhanced the apoptosis of PHA-activated MNC. Moreover these agents significantly increased the apoptosis induced by MTX, DEX, MP and COR. The results of this study suggest that P-gp is involved in the process of apoptosis in peripheral blood mononuclear cells.

Effect of stem cell mobilization with cyclophosphamide plus granulocyte colony-stimulating factor on morphology of haematopoietic organs in mice.

Both granulocyte colony-stimulating factor (G-CSF) and cyclophosphamide (CY) are employed in the clinic as mobilizing agents to stimulate the egress of haematopoietic stem/progenitor cells (HSPC) from bone marrow (BM) into peripheral blood (PB). However, although both compounds are effective, the simultaneous administration of G-CSF + CY allows for optimal mobilization. The aim of this study was to compare morphological changes in major haematopoietic organs in mice mobilized by G-CSF + CY. We employed the standard G-CSF + CY mobilization protocol, in which mice were injected at day 0 with a single dose of CY followed by daily injection of G-CSF for 6 consecutive days. We noticed that the cytoreductive effect of CY on BM and spleen tissue was compensated at day 2 by the pro-proliferative effect of G-CSF. Furthermore, as evidenced by histological examination of BM sections at day 4, egress of haematopoietic cells from BM was accelerated by 2 days as compared to mobilization by G-CSF or CY alone; also, by day 6 there was accumulation of early haematopoietic (Thy-l(low) c-kit+) cells in the spleens and livers of mobilized animals. This implies that HSPC that are mobilized from BM and circulate in PB may 'home' to peripheral organs. We envision that such an accumulation of these cells in the spleen (which is a major haematopoietic organ in mouse) allows them to participate in haematopoietic reconstitution. Their homing to other sites (for example the liver) is evidence that BM-derived stem cells are playing a pivotal role in organ/tissue regeneration. The potential involvement of major chemoattractants for stem cells, like stromal-derived factor-1 which is induced by CY in various regenerating organs such as the liver, requires further study. We conclude that inclusion of CY into mobilization protocols on the one hand efficiently increases the egress of HSPC from the BM, but on the other hand may lead to the relocation of BM stem cell pools to peripheral tissues.

The influence of antisense oligonucleotides against STAT5 on the regulation of normal haematopoiesis in a bone marrow model.

Cytokines and growth factors that take part in the regulation of haematopoietic cell development activate many signalling pathways in target cells. The STAT5 (signal transducers and activators of transcription) proteins are members of a family of signal transducers and activators of transcription that can be activated after cytokine stimulation. Their binding to promoters of different genes influences cell proliferation, differentiation and survival. It is suggested that they play an important role in haematopoiesis, however, the question of the real function of STAT5 proteins requires further examination. The aim of our study was to investigate the role of STAT5 in the proliferation and apoptosis of normal haematopoietic bone marrow cells derived from heparinized cadaveric organ donors (HCOD). We applied antisense oligodeoxynucleotides (ODNs) to block STAT5A and STAT5B at the mRNA level and the reverse transcription polymerase chain reaction method to study STAT5 mRNA expression in the cells after incubation with ODNs. Moreover, we performed Western blot analysis of the STAT5A protein after exposure to antisense STAT5A. We analysed the clonogenicity of the colony-forming unit of granulocytes-macrophages and the burst-forming unit of erythrocytes in methylcellulose cultures according to the type and the dose of ODNs. We also examined apoptosis induced in bone marrow mononuclear and CD34(+) cells by employing annexin V staining and the TUNEL method using flow cytometry (FACScan). We found that the perturbation of STAT5 expression decreased the clonogenicity of bone marrow haematopoietic cells. However, we did not observe any significant increase in the percentage of apoptotic cells after incubation with antisense ODNs. It was concluded that the STAT5 proteins play a significant role in the proliferation of human bone marrow cells harvested from HCOD. These proteins might be critical in the regulation of haematopoiesis, especially under stress conditions.

The expansion of CD4+CD28- T cells in patients with chronic kidney graft rejection.

CD4+CD28- T cells are oligoclonal lymphocytes rarely found in healthy subjects, but are present in high frequencies in patients with inflammatory diseases. Contrary to paradigm, they are functionally active and produce interferon gamma and cytolytic proteins, are cytotoxic in vessels and may contribute to tissue damage. The size of the peripheral blood CD4+CD28- T cell compartments was determined in 20 healthy individuals, 20 patients after renal transplantation with stable graft function, and 20 with chronic graft rejection by two-color FACS analysis. In patients with stable graft function, the median frequency of CD4+CD28- T cells was 3.1% and was significantly higher in comparison to the control group (1.4%) (P <.01). The highest subset CD4+CD28- cells was detected in patients with chronic graft rejection (10.65%). The amount of CD4+CD28- cells was significantly higher in this group in comparison to patients with stable graft function (P <.01). The evaluated number of CD4+CD28- cells in patients after renal transplantation, especially in graft recipients with chronic graft rejection, suggests a role of these cells in chronic graft destruction.

The influence of STAT5 antisense oligonucleotides on the proliferation and apoptosis of selected human leukaemic cell lines.

The signal transducers and activators of transcription--STAT5A and STAT5B--take part in the regulation of many essential physiopathological processes. They influence the cell cycle, apoptosis and the proliferation of different types of cell lines. The STAT5 proteins are induced in response to multiple haematopoietic cytokines. Because they are constitutively active in certain haemato-oncologic diseases, it is also suggested that they play an important role in leukaemogenesis. However, function of these proteins in haematopoietic cell transformation and proliferation is not clear. The aim of this study was to evaluate the impact of perturbation of STAT5 expression [using oligodeoxynucleotide (ODN) against STAT5 mRNA], on the clonogenicity and survival of selected human leukaemic cell lines, HEL, HL-60, K562, TF-1. We analysed the effect of ODN pre-treatment on the cell clonogenicity in methylcellulose cultures according to the time and the temperature of exposure. Moreover, we attempted to estimate apoptosis induced in examined cells, by flow cytometry using combined Annexin V-PI staining and the TUNEL method. We also applied the RT-PCR method to analyse Bax and Bcl-xL gene expression. We found that the perturbation of STAT5 expression with antisense oligonucleotides caused a decrease in the proliferative potential of human K562 and TF-1 cell lines. Also, we observed higher induction of apoptotic cell death in the K562 and TF-1 cells incubated with the antisense STAT5A ODNs. We did not notice any impact of ODNs on the HL-60 and HEL cells. Our studies using STAT5 antisense oligonucleotides showed that these proteins may be critical in the regulation of growth and apoptosis of some types of leukaemic blasts.