Lipoxin (LTX A4 5S, 6R, 15R) levels drastically decrease after 5 years of hemodialysis treatment.

The increased risk of atherosclerosis in patients with chronic kidney disease (CKD) is associated with the increased concentration of fatty acids from the omega-6 family. Products of arachidonic acid oxidation, including prostaglandins, thromboxanes, hydroxyleicosa-tetraenoic acids (HETES) and hydroxyoctadecadienoic acids (HODES) are involved in the pathogenesis of cancer and cardiovascular diseases due to increased oxidative stress. The aim of our study was to determine the relations resulting from the duration of CKD treatment. One of our main concerns is, whether and when the cascade of synthesis of inflammatory mediators may be insufficient in patients with CKD during many years of treatment. The study involved 121 patients with CKD and 87 healthy volunteers. Eicosanoid profiles 9(S)-HODE, 13(S)-HODE, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 5(S)-oxoETE, 16(RS)-HETE, and 5(S),6(R)-lipoxinA4, 5(S),6(R),15(R)-lipoxinA4 were extracted in plasma. The HPLC separations were performed by means of 1260 liquid chromatography. Patients with CKD have a significantly higher concentration of the following inflammatory mediators: 13(S)-HODE, 5(S)-HETE, 12(S)-HETE, 15(S)-HETE, 5(S)-oxoETE, 16(RS)-HETE, and 5(S),6(R), 15(R)-lipoxinA4 relative to the control group. However, the concentrations of 9(S)-HODE were lower in the CKD group. The comparison of sexes did not show significant differences in terms of CKD. A tendency for lower concentrations of HETE and HODE were observed in the group of men. 15LOX, 12LOX and 5LOX pathways in chronic kidney disease are increased, while COX are suppressed (9-HODE). The analysis of the treatment time of patients with CKD shows that incorrect levels of 5(S), 6(R) and 15(R)-lipoxinA4 are developed. We present a new evidence of possible concepts and future clinical interventions in patients suffering from chronic kidney disease for many years. These data for the first time demonstrate that lipoxin levels drastically decrease in the course of CKD. Therefore, synthetic LXA4 analogues may be used as an antioxidant therapy in CKD, which requires further research.

Diverse inhibition of forkhead box O1 activity by linoleic acid isomers - potential role in lipid metabolism in HepG2 cells and livers of C57BL/6J mice.

Conjugated dienes of linoleic acid (CLA) are constitutional and geometric isomers of linoleic acid that are commonly used as dietary supplements during body mass reduction. Their role in the reduction of lipid deposits in liver tissue is not unequivocal. CLA contain an equimolar mixture of two isomers of linoleic acid: trans-10,cis-12 CLA and cis-9,trans-11. Only one isomer - trans-10,cis-12 CLA exhibits fat-reducing properties, cis-9,trans-11 CLA does not. The main goal of this study was to determine if CLA isomers affect the activation of forkhead box O1 (FoxO1) in liver cells and tissue. FoxO1 is a protein that plays a crucial role in regulation of lipid and carbohydrates metabolism. In vitro and in vivo models of our study were HepG2 cells and C57BL/6J mice. Methods used in the study were qPCR - quantification of FoxO1 gene expression, Western blot - posttranslational phosphorylation of FoxO1, Oil Red O (ORO) - lipid staining and ELISA - quantification of apoB100. In both models trans-10,cis-12 CLA diminished FoxO1 gene expression: decrease by 44.1 ± 20.9% SD in the cells and 65.4 ± 29.8% SD in mice. The lowest accumulation of lipids (drop of 37.2 ± 1.7% SD) and the highest increase of apoB100 protein (74 ± 12.8% SD) were detected in the medium of cells cultured with trans-10,cis-12 CLA. Both isomers of linoleic acid have different effects on lipid metabolism. Isomer c9,t11 CLA accelerates lipogenesis, whereas isomer t10,c12 CLA activates secretion of lipids in HepG2 cells. In contrast to the in vitro study, unfortunately this pro-health property of t10,c12 CLA was not confirmed in the in vivo model. This casts a shadow on CLA dietary supplements that are commonly used among people with type 2 diabetes, NAFLD (non-alcoholic liver disease) or a metabolic syndrome in order to lose weight.

Conjugated linoleic acids diminish glycogen synthase and glycogen synthase kinase-3 expression in muscle cells of C57BL/6J mice - in vitro and in vivo study.

Conjugated linoleic acids (CLA) have been extensively advertised as dietary supplements to reduce fat and increase muscle mass. However, the role of CLA in glycogen metabolism is still largely unknown. The aim of this study was to assess the effect of CLA on glycogen synthesis in vitro (CCL 136 cell line human) and CLA in vivo (C57BL/6J mice). The materials used were the CCL 136 muscle cell line and muscles of female C57BL/6J mice (n = 52), housed at animal laboratory facility and feed with "MURIGRAN", a standard feed prepared for rodents (Agropol, Poland). Chemically pure fatty acids were added to soybean oil. CLA isomers (c9,t11 CLA, t10,c12 CLA, and as a mixture (1:1)) were administered with feed. Supplementation in mice started at week 6 of age and lasted for 4 weeks. Methods used in the study were real time- PCR - quantification of gene expression, Western blot glycogen synthase kinase-3 (GSK3α 9) and glycogen synthase (GS) protein, glycogen staining by PAS. Quantitative determination of glycogen by spectrophotometry and intracellular reactive oxygen species was measured the intracellular oxidation of dichloro-dihydro-fluorescein diacetate (DCFH-DA). In vitro data showed that GS and GSK3 expression was lower in cells cultured with different CLAs and a mixture of CLAs. GS gene expression was significantly decreased in cells cultured with c9, t11 CLA (P < 0.04) and t10, c12 CLA (P < 0.05) as well as the mixture of both isomers. The GSK3α gene expression was reduced in cells cultured with a mixture of CLA (P < 0.02), whereas phosphorylation of GSK3α increased in cells cultured with c9, t11 CLA GSK3α (P < 0.05). In vivo data showed a reduction in the glycogen concentration among mice fed a diet containing t10, c 12 CLA and a mixture of CLA isomers. We conclude that both CLA isomers can affect the synthesis of glycogen in muscle cells through the regulation of GS and GSK3α gene expression.

The short chain fatty acids and lipopolysaccharides status in Sprague-Dawley rats fed with high-fat and high-cholesterol diet.

Short chain fatty acids (SCFA) are produced by the gut microbiota during the fermentation of non-digestible polysaccharides. Diet is a major factor driving the composition and metabolism of the colonic microbiota. The aim of our study was to examine how a fat-rich and cholesterol-rich diet that, which leads to many metabolic disorders, affects the SCFA profile and lipopolysaccharide (LPS) concentration. The experiment was carried out on 72 male, 8-weeks-old Sprague-Dawley rats. The study group (n = 30 rats) received high-fat and high cholesterol diet (HFHCh). The control group (n = 30) received standard food for laboratory rats. The rats from study and control groups were sacrificed after 4, 8, 12, 16 and 20 weeks after start of dietary exposure. The analysis of SFA in feces was performed using gas chromatography (Agilent Technologies 1260 A GC). The exposure to high-fat and high-cholesterol diet was associated with significant changes in SCFA levels. Relative to the control, each of HFHCh subgroup revealed a statistically significant decrease in butyrate (12.5% ± 5.7% versus 32.8% ± 9.1%) and an increase in propionate level (45.4% ± 6.2% versus 19.14% ± 7.1%). The ratio of acetate: propionate: butyrate was also changed (from 1.1: 0.6: 1 for control groups to 3 : 3,6 : 1 for HFHCh groups). The main SCFA in the HFHCh group was propionate instead of acetate. The dietary exposure resulted in significant differences in LPS concentration. After 12 weeks of HFD exposure, LPS concentration was significantly higher compared to control groups (P < 0.05). Our study showed that HFHCh diet affected butyrate and propionate production associated with an increase in LPS secretion. The hypothesis that observed changes could result in intestinal imbalance secondary to gut barrier dysfunction requires further studies.

The effect of short term treatment with probiotic VSL#3 on various clinical and biochemical parameters in patients with liver cirrhosis.

The evidence is mounting that alterations of innate immunity and gut microbiota contribute to chronic liver disease and its complications. Modulation of intestinal microbiota is an emerging therapeutic strategy in hepatology. Probiotics through modulation of intestinal milieu have the potential to affect the course of liver disease. The data concerning the influence of probiotics on various plasma molecules and compounds involved in the pathogenesis of hyperdynamic circulatory state in liver cirrhosis is still not confluent and require further evaluation. In our study twenty patients with compensated and decompensated liver cirrhosis and ten healthy controls received probiotic VSL#3 daily for 28 days. Plasma levels of interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), plasminogen activator inhibitor (PAI), macrophage inflammatory protein 3/α (MIP-3 α/CCL20), monocyte chemotactic protein-1α (MCP-1/CCL2), human myeloperoxidase (MPO), nitric oxide (NO), prostaglandins, thromboxane (TXB2) and big-endothelin were measured at baseline, day 14 and 28 of probiotic administration. The incidence of hepatic encephalopathy was assessed with critical flicker frequency. Changes in clinical, biochemical and microbiological parameters were evaluated. The stage of liver cirrhosis correlated with an increase in plasma levels of pro-inflammatory cytokines (IL-6) and chemotactic chemokines involved in immune cell trafficking (MIP-3α/CCL20). Probiotic administration in patients with liver cirrhosis led to modulation of plasma levels of several molecules and compounds measured (MIP-3α/CCL20, NO, big-endothelin, TXB2 and MPO). The grade of encephalopathy during the course of probiotic supplementation remained unaffected in both groups of patients. VSL#3 treatment was well tolerated and safe in patients with liver disease. In patients with compensated and decompensated liver cirrhosis, VSL#3 manipulates selected plasma molecules and compounds involved in hyperdynamic circulatory dysfunction. Short term VSL#3 administration affects several clinical and biochemical parameters commonly altered in liver cirrhosis.

The bone marrow-expressed antimicrobial cationic peptide LL-37 enhances the responsiveness of hematopoietic stem progenitor cells to an SDF-1 gradient and accelerates their engraftment after transplantation.

We report that the bone marrow (BM) stroma-released LL-37, a member of the cathelicidin family of antimicrobial peptides, primes/increases the responsiveness of murine and human hematopoietic stem/progenitor cells (HSPCs) to an α-chemokine stromal-derived factor-1 (SDF-1) gradient. Accordingly, LL-37 is upregulated in irradiated BM cells and enhances the chemotactic responsiveness of hematopoietic progenitors from all lineages to a low physiological SDF-1 gradient as well as increasing their (i) adhesiveness, (ii) SDF-1-mediated actin polymerization and (iii) MAPK(p42/44) phosphorylation. Mice transplanted with BM cells ex vivo primed by LL-37 showed accelerated recovery of platelet and neutrophil counts by ∼3-5 days compared with mice transplanted with unprimed control cells. These priming effects were not mediated by LL-37 binding to its receptor and depended instead on the incorporation of the CXCR4 receptor into membrane lipid rafts. We propose that LL-37, which has primarily antimicrobial functions and is harmless to mammalian cells, could be clinically applied to accelerate engraftment as an ex vivo priming agent for transplanted human HSPCs. This novel approach would be particularly important in cord blood transplantations, where the number of HSCs available is usually limited.

Novel insight into stem cell mobilization-plasma sphingosine-1-phosphate is a major chemoattractant that directs the egress of hematopoietic stem progenitor cells from the bone marrow and its level in peripheral blood increases during mobilization due to activation of complement cascade/membrane attack complex.

The complement cascade (CC) becomes activated and its cleavage fragments play a crucial role in the mobilization of hematopoietic stem/progenitor cells (HSPCs). Here, we sought to determine which major chemoattractant present in peripheral blood (PB) is responsible for the egress of HSPCs from the bone marrow (BM). We noticed that normal and mobilized plasma strongly chemoattracts HSPCs in a stromal-derived factor-1 (SDF-1)-independent manner because (i) plasma SDF-1 level does not correlate with mobilization efficiency; (ii) the chemotactic plasma gradient is not affected in the presence of AMD3100 and (iii) it is resistant to denaturation by heat. Surprisingly, the observed loss of plasma chemotactic activity after charcoal stripping suggested the involvement of bioactive lipids and we focused on sphingosine-1-phosphate (S1P), a known chemoattracant of HSPCs. We found that S1P (i) creates in plasma a continuously present gradient for BM-residing HSPCs; (ii) is at physiologically relevant concentrations a chemoattractant several magnitudes stronger than SDF-1 and (iii) its plasma level increases during mobilization due to CC activation and interaction of the membrane attack complex (MAC) with erythrocytes that are a major reservoir of S1P. We conclude and propose a new paradigm that S1P is a crucial chemoattractant for BM-residing HSPCs and that CC through MAC induces the release of S1P from erythrocytes for optimal egress/mobilization of HSPCs.

Heparinized cadaveric organ donors (HCOD)--a potential source of hematopoietic cells for transplantation and gene therapy.

BACKGROUND: Hematopoietic stem cells (HSC) from unrelated HLA-matched heparinized cadaveric organ donors (HCOD) are a new potential source of cells for transplantation and gene therapy. In addition, these cells could also be used as adjuvant therapy to increase microchimerism and graft tolerance after transplantations of various solid organs. Our purpose was to develop an efficient method for harvesting hematopoietic cells from HCODs, METHODS: Bone marrow cells were harvested from pelvic bones and/or vertebral bodies from 50 adult HCODs before or up to 3 hr after disconnecting the donor from the respirator. Subsequently, we evaluated the hematological and gasometric parameters of aspirated marrow samples as well as the proliferative potential, viability, and expression of CD34 and AC133 antigens on these cells. RESULTS: We noticed that up to 2-3 hr after disconnecting the donor from the respirator bone marrow cavities do not clot and remain uninfected and that it is possible to aspirate bone marrow mononuclear cells in quantities sufficient to perform allotransplantation. Nevertheless, due to the developing hypoxia and acidosis of the hematopoietic microenvironment the number and proliferative potential of CD34+ and AC133+ cells gradually decreases. Hence, to obtain viable early hematopoietic cells, bone marrow should be aspirated without delay; optimally before HCOD is disconnected from the respirator or at the very latest 2 hr after organ harvest. CONCLUSIONS: Collectively, our results show that early hemopoietic cells may be efficiently harvested from HCOD in large quantities and used for research and/or transplantation purposes. We postulate to create an international network of banks in which hemopoietic stem cells from HCODs could be preserved for therapeutic purposes.

Evidence that human haematopoietic stem cells (HSC) do not reside within the CD34+KIT- cell population.

Contradictory reports are published concerning the c-kit receptor (KIT) expression on human haematopoietic stem cells (HHSC). Therefore, the aim of this study was to reevaluate the expression of KIT on human early haematopoietic cells, and to study the distribution of HHSC among bone marrow mononuclear KIT+ and KIT- cells. First, we found that the detection sensitivity of the KIT expression on human HEL cells as well as CD34+ depends on the type of fluorochrome employed for the immunostaining (Cy5 > PE > FITC). Based on this observation, in our strategy for isolating human HHSC we employed a Cy-5 conjugated alpha-KIT MoAbs, which stained CD34+ cells in our preliminary studies the brightest. Accordingly, we labeled human BMMNC with PE-alpha-CD34 and Cy5-alpha-KIT MoAbs and subsequently sorted various subsets of labeled cells (CD34+KIT+, CD34+KIT- and CD34-KIT-). Cells sorted by FACS were then evaluated for their ability to engraft in the immunodeficient SCID mice model. We report here that only CD34+KIT+ cells, but not CD34+KIT- or CD34-KIT- were able to establish a human-murine haematopoietic chimerism in these animals. We found that SCID mice transplanted with CD34+ KIT+ cells, possessed approximately 5-11% of mononuclear cells, which expressed human CD45 antigen 4-5 weeks after transplantation in their bone marrow and, more importantly, early human haematopoietic progenitors from the myeloid and B-lymphoid lineages. Based on these results we conclude that KIT (CD117) is a very useful marker for identifying HHSC, and that HHSC, at least in our hands, are found in the KIT+ population of CD34+ cells.

An improved serum free system for cloning human "pure" erythroid colonies. The role of different growth factors and cytokines on BFU-E formation by the bone marrow and cord blood CD34+ cells.

We have developed an efficient serum free culture model for cloning human erythroid progenitors. Accordingly, human bone marrow or cord blood CD34+ cells if plated in our serum free medium and stimulated with a mixture of EpO + KL, grow erythroid colonies exclusively. Cells isolated from these cultures express glycophorin-A (GPA-A), are CD33-, IIb/IIIa-, and finally all become hemoglobinized. By employing this system we also found out that cord blood CD34+ mononuclear cells (MNC) contain more BFU-E than adult marrow CD34+ MNC, moreover, the erythroid colonies formed by cord blood progenitors are significantly larger then the ones formed by the marrow cells. We have also compared the influence of different cytokines and growth factors, which were reported in the literature to costimulate BFU-E growth on cloning efficiency of human BFU-E cultured in our serum free medium. We found that from 20 different growth factors and cytokines tested, EpO dependent bone marrow BFU-E growth is costimulated only by KL, and to lesser degree also by IL-3, GM-CSF, TpO and IL-9. In contrast to marrow cells we observed that cord blood BFU-E in addition to KL, IL-3, GM-CSF, TpO, LIF and IL-9 were also costimulated by NGF-beta, FGF-1, FGF-2 and STK-IL. We found simultaneously that TPO which possess only negligible costimulatory effect on erythroid colony formation by bone marrow CD34+ cells, significantly costimulated the formation of erythroid colonies grown by cord blood CD34+ cells. Therefore, the cord blood CD34+ cells are largely committed to erythroid differentiation, and, moreover, they respond to a wider spectrum of the growth factors than their bone marrow counterparts.

CD34+, kit+, rhodamine123(low) phenotype identifies a marrow cell population highly enriched for human hematopoietic stem cells.

We hypothesized that human hematopoietic cells displaying a CD34+, kit-, rhodamine123(low) phenotype would be highly enriched for cells with stem-like properties. To test this hypothesis, we employed fluorescence activated cell sorting (FACS) to isolate cells with this phenotype from normal light density marrow mononuclear cells (MNC). CD34+, kit+, rhodamine123(low) cells comprised from 0.05-0.01% of the total MNC population. They were small, had scant cytoplasm, and contained nuclei with dense, hyperchromatic chromatin and inconspicuous nucleoli. Additional immunophenotyping revealed that these cells were CD33-, CD38-, CD20-, and glycophorin A-. When plated in semisolid cultures containing optimal concentrations of IL-3, GM-CSF, KL, EPO, IL-6, and IL-1 these cells did not form colonies. However, when cultured over irradiated stromal cells, cobblestone areas were observed to form after 3 weeks, and harvested cells were able to initiate long-term cultures. To further demonstrate that these cells were indeed stem like, we also tested their ability to engraft and mature in immunocompromised (SCID) mice. Irradiated (400 cGy) SCID mice were transplanted with 2 x 10(3) candidate stem cells which were then injected with recombinant human growth factors every other day. Two months post-transplant the animals were sacrificed. PCR and FACS analysis of marrow and spleen cell samples revealed the presence of cells expressing human CD45 consistent with engraftment of human stem cells and the establishment of murine-human chimerism. Moreover, MNC isolated from transplanted mice formed unambiguously human BFU-E, CFU-GM and B cell colonies when stimulated with the appropriate growth factors. Accordingly, we have identified a relatively rapid and simple mechanism for isolating primitive human hematopoietic cells with stem cell-like properties. We anticipate that this strategy will be useful for experimental and therapeutic applications that require human stem cells in quantity.

Role of vascular endothelial growth factor (VEGF) and placenta-derived growth factor (PlGF) in regulating human haemopoietic cell growth.

Vascular endothelial growth factor (VEGF) and placental derived growth factor (PlGF) stimulate cell proliferation and differentiation by binding to their specific receptors, Flk-1/KDR and Flt-1 respectively. Flk-1/KDR-deficient murine embryos manifest failure of blood-island formation and vasculogenesis. The aim of this study was to directly evaluate the importance of VEGF, PlGF/Flt-1 and Flk-1/KDR receptor ligand interactions in regulating normal and malignant human haemopoiesis. Addition of VEGF and PlGF failed to enhance survival or cloning efficiency of human haemopoietic progenitors isolated from adult bone marrows, fetal livers or cord blood samples. This finding may be explained by the apparent absence of mRNA encoding Flt-1 and Flk-1/KDR receptors on stem cell rich CD34+ c-kit-R+ Rh123(low) cells. Further studies revealed that Fit-1 R mRNA, but not Flk-1/KDR mRNA was first detectable in the more mature cells isolated from haemopoietic colonies. Accordingly, VEGF receptors are either absent, or expressed at very low level, on human haemopoietic stem/progenitor cells. Of interest, normal and malignant human haemopoietic cells appeared to secrete VEGF protein. However, in contrast to normal haemopoietic progenitors, VEGF co-stimulated HEL cell proliferation as well as CFU-GM colony formation from approximately 15% of the chronic myeloid leukaemia (CML) and acute myeloid leukaemia (AML) patients studied. Therefore, although VEGF appeared to have minimal effects on normal haemopoietic cell growth it would appear to drive malignant haemopoietic cell proliferation to some degree. Of more importance, however, we speculate that VEGF may play an very important role in leukaemogenesis by stimulating growth of vascular endothelium, thereby providing a sufficient blood supply to feed the growing haematological tumour.

Effect of hepatocyte growth factor on early human haemopoietic cell development.

Hepatocyte growth factor (HGF) stimulates cell proliferation, differentiation and migration by binding to its receptor, MET R. Whether the HGF/MET R axis plays an important regulatory role in human haemopoietic cell growth is an unresolved issue. To investigate this situation, we employed several complementary strategies including RT-PCR, FACS analysis, and mRNA perturbation with oligodeoxynucleotides (ODN). We found that very primitive, FACS sorted, CD34+ Kit+ marrow mononuclear cells (MNC) failed to express RT-PCR detectable MET R mRNA. In contrast, MET R expression was easily detectable by RT-PCR in marrow stroma fibroblasts, in cells isolated from BFU-E and CFU-GM colonies, and in unselected normal MNC. Subsequent FACS analysis revealed that MET R protein was detectable on approximately 5% of the latter cells. HGF, at concentrations of 1-50 ng/ml, had no demonstrable effect on survival or cloning efficiency of normal CD34+ MNC in serum-free cultures. Antisense ODN mediated perturbation of MET R mRNA expression in normal CD34+ MNC, with FACS documented decline in protein expression, had no effect on the ability of these cells to give rise to haemopoietic colonies of any lineage. We also examined the biology of HGF/MET R expression in malignant haemopoietic cells. Using the strategies described above, we found that MET R mRNA was expressed in many human haemopoietic cell lines, and that the protein was expressed at high levels on HTLV transformed T lymphocytes. Wild-type CML and AML blast cells also expressed MET mRNA, and HGF was able to co-stimulate CFU-GM colony formation in approximately 20% of cases studied. Therefore, although the HGF/MET R axis appears to be dispensable for normal haemopoietic cell growth, it may play a role in the growth of malignant haemopoietic progenitor cells.

Recombinant human thrombopoietin (TPO) stimulates erythropoiesis by inhibiting erythroid progenitor cell apoptosis.

Thrombopoietin (TPO) has been reported to stimulate erythropoiesis, but the stimulatory mechanism has not been defined. To address this issue, we performed serum-free cell-culture experiments with recombinant human TPO and purified human progenitor cells. We found that TPO alone was able to stimulate the megakaryocyte colony formation in serum-free cultures, but erythroid colonies were never observed. Only in the presence of EPO (erythropoietin) +IL-3 was TPO able to stimulate a small increase (approximately 25%) in erythroid colony formation. Accordingly, we hypothesized that TPO might have an effect on erythroid progenitor cell viability, rather than a direct stimulatory effect. To test this idea, CD34+ cells were cultured for 7d in serum-free methylcellulose in the presence or absence of TPO, after which time KL+ EPO was added to the cultures. Cells which were pre-cultured for 7 d in the presence of TPO gave rise to approximately 6 times as many burst forming unit-erythroid (BFU-E) colonies as cells which were pre-cultured in the absence of TPO. Further, when primitive CD34+, Kit+ MNC were cultured for 3-7 d under serum-free conditions in the presence or absence of TPO, significantly fewer cells cultured in the presence of TPO displayed apoptotic changes when compared to cells cultured in the absence of TPO. Taken together, these results suggest that TPO has little direct stimulatory effect on erythroid progenitor cells, but might indirectly enhance erythropoiesis by preventing very early erythroid progenitor cells from undergoing apoptotic cell death.

[The influence of thrombopoietin (TpO) on human erythropoiesis. In vitro studies--clinical implications]. / Wplyw trombopoetyny (TpO) na ludzka erytropoeze. Badania in vitro--implikacje kliniczne.

The role of thrombopoietin (TpO) in regulations erythropoiesis is still controversial. This fact prompt us to present our own data. We found, that TpO is not able to replace erythropoietin (EpO) in stimulation of the growth of human erythroid colonies. TpO was found to be only a very weak costimulator of EpO dependent erythroid progenitors growth. This effect is however much weaker than those observed after costimulation with kit ligand or interleukin-3. The rationale for TpO application for treatment of anemia in humans seems therefore doubtful. This conclusion seems now to be supported by the results of the first trials performed in anemic patients.

An optimization study on unprogrammed cryopreservation of human CD34+ bone marrow cells and their subsequent storage in an -80 degrees C mechanical freezer.

Among the factors which enable successful bone marrow transplantation, the ability to store and subsequently recover sufficient viable marrow cells to reestablish hematopoiesis is crucial. In most centers, marrow is typically prepared for long term storage by programmed, controlled rate freezing with subsequent placement into the vapor phase of liquid nitrogen. Though clearly effective, this procedure is time consuming and equipment intensive. Since cost containment is an important issue for all medical procedures, we evaluated the utility of a faster and less costly method to accomplish the same purpose. We found that when CD34+ bone marrow cells are immediately frozen and stored at -80 degrees C, the number and quality of stem/progenitor cells recovered after thawing was comparable to those reported recovered after conventional processing. Herein we report optimized conditions for cryopreserving and storing CD34+ bone marrow cells at -80 degrees C and for subsequent thawing of the stored product. With these methods post-thaw recovery of CFU-Mix, for example, equaled or exceeded 80% of predicted numbers, even after six months of storage. Further, though progenitor cell recovery does not necessarily correlate with speed of engraftment, these results nonetheless suggest that storage of human CD34+ cells at -80 degrees C in a mechanical freezer is a convenient, inexpensive, and reliable method for storing marrow for transplantation.

Influence of leukemia inhibitory factor (LIF) on the survival, proliferation and differentiation of human erythroid progenitor cells. In vitro studies under serum free conditions.

Different inflammatory cytokines are involved in the pathogenesis of the anemia of chronic disease (ACD), by inhibiting the proliferation of erythroid progenitors. Leukemia inhibitory factor (LIF), is an important inflammatory cytokine, and the purpose of this study was to evaluate its effect on the proliferation and viability of human erythroid progenitors. We have found that LIF slightly increased the survival of human early progenitor cells cultured under serum free conditions. LIF also slightly costimulated in vitro growth of human erythroid colonies. This last effect seems to be a direct one, because we found that LIF receptor (LIF-R) is expressed in cells isolated from growing in vitro human erythroid BFU-E colonies. These data, and the data reported by others in in vivo models, where LIF administration to experimental animals did not change the values of erythropoietic parameters, demonstrate that this inflammatory cytokine itself is not involved in the pathogenesis of ACD.

Effect of Interleukin-1 α and Interleukin-1 ß on Erythroid Progenitor Cell Growth in Serum Free Cultures: An In Vitro Study Relevant to the Pathogenesis of the Anemia of Chronic Disease.

It has been postulated that interleukin-lα (IL-lα) and interleukin-1ß (IL-1ß) play a role in the pathogenesis of the anemia of chronic disease by inhibiting the proliferation of human erythroid progenitor cells. In the course of investigating this hypothesis we found that IL-1 type I receptor (IL-1R) mRNA is expressed on erythroid progenitor enriched, primitive human hematopoietic cells (CD34+, c-kit-R(bright)) and on cells isolated from human erythroid burst forming colonies (BFU-E). Nevertheless, when CD34+, c-kit-R(bright) cells were exposed to IL-1α and IL-1ß in vitro, cloning efficacy of BFU-E and CFU-E in a serum free culture system was not inhibited. Moreover, in apparent contradiction to the hypothesis being tested, we found that both IL-1 isoforms actually increased the survival of human BFU-E in serum free, growth factor free medium. Accordingly, these results suggest that if IL-1 plays a role in the pathogenesis of the anemia of chronic disease, it is not due to a direct suppressive effect on erythroid cell growth. Rather, our data support the hypothesis that IL-1 may cause the elaboration of another inhibitory cytokine (s) by cells of the marrow microenvironment.