Comparison of mouse, rat and rabbit models for adipose - Derived stem cells (ASC) research.

PURPOSE OF THE STUDY: Cellular therapies are becoming more popular and there is a big demand for suitable animal model for research in field of tissue engineering. Both the small (rodents) and large animals have their advantages for cellular therapy experiments. Appropriate animal research model would be a bridge between basic research and clinical medicine. The aim of this study was to compare mouse, rat and rabbit as animal models useful for adipose - derived stem cell research. MATERIALS AND METHODS: Quantity, phenotype, clonogenic and differentiation potential of cells isolated from different localizations of adipose tissue from WAG and LEW/W rat strains, rabbit and mouse were analysed. RESULTS: The highest number of cells from 1 g of tissue were isolated from cervical white fat of LEW/W rat. ASCs isolated from rat had also the highest clonogenic potential. Phenotype and capability to differentiate into osteogenic, adipogenic and chondrogenic lineages are at the same level for rat and rabbit. CONCLUSIONS: Rat as a research model can be a rational solution between large animal models and typical laboratory mice because of their size, genetic homogenity, availability of genetically modified stains and possibility to perform research mimicking clinical applications.

Biocompatibility of electrospun human albumin: a pilot study.

Albumin is rarely used for electrospinning because it does not form fibres in its native globular form. This paper presents a novel method for electrospinning human albumin from a solution containing pharmaceutical grade protein and 25% polyethylene oxide (PEO) used as the fibre-forming agent. After spontaneous cross-linking at body temperature, with no further chemicals added, the fibres become insoluble and the excess PEO can be washed out. Albumin deposited along the fibres retains its native characteristics, such as its non-adhesiveness to cells and its susceptibility for degradation by macrophages. To demonstrate this we evaluated the mechanical properties, biocompatibility and biodegradability of this novel product. After subcutaneous implantation in mice, albumin mats were completely resorbable within six days and elicited only a limited local inflammatory response. In vitro, the mats suppressed cell attachment and migration. As this product is inexpensive, produced from human pharmaceutical grade albumin without chemical modifications, retains its native protein properties and fulfils the specific requirements for anti-adhesive dressings, its clinical use can be expedited. We believe that it could specifically be used when treating paediatric patients with epidermolysis bullosa, in whom non-healing wounds occur after minor hand injuries which lead to rapid adhesions and devastating contractures.

Transcriptional activity of telomerase complex in CD34- stem cells of cord blood in dependence of preparation time.

The aim of the study was to determine whether the expression of telomerase subunits encoding genes changes during the process of cord blood preparation. It should establish if the commonly accepted 24 hours time interval in stem cells kriopreservation procedure significantly influences their immortalization and so decreases the "quality" of cord blood stem cells. Investigation includes 69 women. Spontaneous labour was the inclusion condition. The material was collected at birth after clamping of umbilical cord by direct vasopuncture. CD34- cells were extracted from cord blood (MACS, Miltenyi Biotec; Bisley, Surrey, UK). The expression profile of telomerase activators and inhibitors encoding genes was determined using HG_U133A oligonucleotide microarray (Affymetrix). We used a real-time quantitative RT-PCR assay to quantify the telomerase TERT, hTR and TP1 subunits mRNA copy numbers in CD34- cells in 0, 6, 12 and 24 hours after cord blood collection. We observed significant decrease of numbers of copies of TERTA+B mRNA within the successive hours of observation. Significant decrease of numbers of TERTA mRNA copies was confirmed after 24 hours. However, we observed significant increase of numbers of copies of TERTB mRNA after 6 hours of observation. Similar level was maintained during another 6h. The significantly lower number of copies of TERTB mRNA was observed after 24h. We also observed significant increase of number of copies of TERT mRNA after 6 hours. Number of copies of TERT mRNA significantly decreased after another 6h, remaining, however, on a higher then initial one. The significant lower number of copies of TERT mRNA was observed 24h after delivery. The possible explanation of those results is discussed in the paper.

Expression of SDF-1-CXCR4 axis and an anti-remodelling effectiveness of foetal-liver stem cell transplantation in the infarcted rat heart.

SDF-1, a chemokine secreted by injured tissues, may be instrumental in chemoattracting CXCR4(+) stem cells (SCs) for repair of infarcted myocardium. We hypothesize that the myocardial SDF-1 expression determines also the engraftment and beneficial effects of SCs transplanted into the infarcted heart. Myocardial infarction (MI) was induced in rats by coronary artery ligation. The animals were either sacrificed at 2, 7, 16, 21 or 28 days after MI or were re-operated at 2, 7 or 14 days after MI to receive SCs transplantation, and were sacrificed 14 days later. SCs transplantation consisted of 3 x 15 microl injections of SCs isolated from foetal rat liver (FLSCs) into the myocardium bordering the infarction zone (5 x 10(6) cells/heart, labelled with PKH2 Green Fluorescent Cell Linker, approximately 20% CXCR4(+)). In the MI border zone, SDF-1 and CXCR4 immunostaining was transiently increased after MI, picking at 2 days and down regulating to the sham level by 21 days after MI. Simultaneously, an increased incorporation of CXCR4(+) and CD133(+) cells into capillaries was evident. AMD1300, a blocker of CXCR4, prevented the post-MI expression of CXCR4. In the MI border zone, the cardiomyocyte cross-sectional diameter increased and capillary/cardiomyocyte ratio decreased systematically during the 28 post-MI days, while an interstitial collagen accumulation demonstrated transient increase. FLSCs did not survive in the non-infarcted hearts. In infarcted hearts, FLSCs survived best when they were injected at 2 days after MI. The survival was negligible again when the injection was performed at 14 days after MI. FLSCs transplanted at 2 days after MI caused a further rise in SDF-1, CXCR4, and CD133 expression, compared with the untreated infarcted hearts. Only FLSCs transplanted at 2 days, but not later, attenuated cardiomyocyte hypertrophy and increased capillary/cardiomyocyte ratio in the MI border zone. These results suggest that myocardial signalling for homing of the endogenous and the exogenous SCs is transiently activated early after MI, that SDF-1 is instrumental in this process, and that there is only a narrow time-window after MI when SCs transplantation results in their efficient myocardial engraftment and beneficial anti-remodelling effect.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells in leukapheresis product and bone marrow for clinical transplantation: a comparison of three methods.

Flow cytometric enumeration of CD34+ hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts. In the present study, we review and compare the major counting techniques of stem and progenitor cells. The methods are: the Milan/Mullhouse protocol, two-platform ISHAGE (International Society of Hematotherapy and Graft Engineering) and single-platform ISHAGE analysis system. According to the Milan/Mulhouse protocol, HSCs are identified by CD34 antibody staining and easy gating strategy. The ISHAGE guidelines for detection of CD34+ cells are based on a four-parameter flow cytometry method (CD34PE/CD45PerCP staining, side and forward angle light scatter) thus employing multiparameter gating strategy. With two-platform ISHAGE protocol, an absolute CD34+ count is generated by incorporating the leukocyte count from an automated hematology analyser. The single-platform ISHAGE method to determine the absolute CD34+ count directly from a flow cytometer includes the use of Trucount tubes (Becton Dickinson) with a known number of fluorescent beads. CD34+ cells were quantified in mobilized peripheral blood, collected by leukapheresis, and bone marrow from 42 samples from patients with hematological malignancies. The differences against the means display low disagreement between the Milan/Mulhouse and ISHAGE protocols, with discrepancies of up to 2.5% (two-platform ISHAGE)--2.6% (single-platform ISHAGE) in enumeration of CD34+ cells in leukapheresis product and 4.8% (two-platform ISHAGE)--4.9% (single-platform ISHAGE) in bone marrow. Our results show high correlation among all three methods. Since the three protocols are compatible, choosing the most convenient in terms of costs, simplicity and compliance with clinical results appears to be a logical consequence.

Human cord blood-derived cells attain neuronal and glial features in vitro.

Neural stem cells are clonogenic, self-renewing cells with the potential to differentiate into brain-specific cell lines. Our study demonstrates that a neural-stem-cell-like subpopulation can be selected and expanded in vitro by the use of human umbilical cord blood cells, which are a relatively easily available starting material. Through a combination of antigen-driven magnetic cell sorting and subfractionation according to cell surface adhesive properties, we have isolated a clonogenic fraction devoid of hematopoietic or angiogenetic properties but with relatively high self-renewal potency. The resulting clones express nestin, a neurofilament protein that is one of the most specific markers of multipotent neural stem cells. In the presence of selected growth factors or in the rat brain co-culture system, the progeny of these cells can be oriented towards the three main neural phenotypes: neurons, astroglia and oligodendroglia. The cells show high commitment (about 30% and 40% of the population) to neuronal and astrocytic fate, respectively. Interestingly, upon differentiation, the neural-type precursor cells of cord blood origin also give rise to a relatively high proportion of oligodendrocytes - 11% of the total population of differentiating cells.

[Results of the activity of Jose Carreras Memorial Cord Blood Bank in Warsaw]. / Podsumowanie dzialalnosci Banku Krwi Pepowinowej im. Jose Carrerasa w Warszawie.

Following the first successful cord blood transplantation in 1988, several hundreds of patients were treated using same protocol. The main limitation of the wide use of cord blood as a source of haematopoietic cells is the number of available units of this tissue. To make possible selection of HLA-matched cells for individual patient, several thousands of cord blood samples must be collected and stored in liquid nitrogen. The network of cooperating cord blood banks with join accessible database is necessary. In this paper the activity of Jose Carreras memorial Cord Blood Bank established in Warsaw was described. Since the middle of January the collection of cord blood units for clinical purposes was started. During first three months 80 samples of cord blood was collected. Collections were obtained from normal full-term deliveries after the third stage of labour. For the banking the collection over 60 ml or contain over 4 x 10(8) of mononuclear cells were qualified. Whole blood samples and plasma samples obtained following volume reduction were used for HLA and bacteriology tests. After volume reduction the number of nucleated cells (WBC), mononuclear cells (MNC) and hematopoietic cells (CD34+) were evaluated. After processing the cord blood samples were frozen using control freezer and were stored in liquid nitrogen storage tanks. According to results of cord blood transplantation hundred percent of banked samples are suitable for recipients weighing 10 kg and only 7 percent for these weighing 50 kg.

[Comparison of the selected methods of cord processing for transplantation purposes]. / Porównanie wybranych metod preparatyki krwi pepowinowej przeznaczonej dla celów transplantacyjnych.

Human umbilical cord blood (UCB) has been successfully used as a source of allogeneic hematopoietic cells for transplantation. Banking of the UCB requires its volume reduction to decrease storage space, costs and volume of infused DMSO. In order to select an optimal method for volume reduction we compared several methods of cord blood processing, namely buffy coat centrifugation, red cell lysis, hydroxyethyl starch (HES)-, methylcellulose- and gelatin-sedimentations. The viability of cells and the recoveries of total white blood cells, mononuclear cells and CD34+ cells was evaluated. We also compared the efficacy of red cells depletion from the original UCB sample. Buffy coat centrifugation, red cell lysis, HES, gelatin or methylcellulose resulted in high mononuclear cell recoveries, whereas high hematopoietic cell recovery was observed only after HES sedimentation and buffy coat processing. The HES sedimentation procedure compared to buffy coat processing is more time and labor consuming and resulted in higher red blood cell and platelets depletion. Both methods can be recommended as a method at choice for the umbilical cord blood processing before banking.

New cytokine dressings. I. Kinetics of the in vitro rhG-CSF, rhGM-CSF, and rhEGF release from the dressings.

Wound repair-stimulatory activities of various cytokines and growth factors depend on successful delivery of these factors to the injured sites. Here were present the design and preparation of the new collagen- and polyurethane-based dressings containing the recombinant human cytokines-rhG-CSF, rhGM-CSF or rhEGF. To test the efficacy of the retrieval of the incorporated cytokines, their controlled release from the dressings was carried out over three consecutive days using polyurethane sponge as a collector of the extracts. The maximum quantities of the released rhG-CSF, rhGM-CSF and rhEGF reached approximately 25, 50, and 10%, respectively, of the total cytokine contents of the dressings, as assessed by the specific ELISA tests. These data indicate that collagen- and polyurethane dressings containing rhGM-CSF and rhG/CSF may serve as effective tools for the topical delivery of cytokines to wounded tissues.

New cytokine dressings. II. Stimulation of oxidative burst in leucocytes in vitro and reduction of viable bacteria within an infected wound.

Recently, we have developed the new dressings containing rhG-CSF or rhGM-CSF. In the present study we investigated either in vitro or in vivo biological activity of the dressings. Human whole blood samples were incubated with extracts from the collagen- or polyurethane-based dressings containing rhG-CSF or/and rhGM-CSF and phagocytic and oxidative metabolic activities were quantitated using Phagotest or Bursttest kits. The results indicate that both the number of phagocyting cells and the intensity of phagocytosis per cell, as well as the level of the oxidative burst in particular, were stimulated by one or both of the cytokines extracted. Next, the experimental skin wounds in mice were infected with 107 CFU of Pseudomonas aeruginosa strain ATCC 27853 and treated locally with the rhG-CSF-containing dressing. The analysis of the biopsies taken from the wounds indicated that in mice treated with the cytokine-containing dressing on the third day the log of CFU per biopsy was 5.0 vs. 6.2 in the control (P<0.001), and on the 8th day was lower than 4 vs. 5.4 in control (P<0.0001). Our findings clearly suggest that the newly designed dressings containing the incorporated CSFs can be used as effective topical cytokine-delivery system in the treatment of bacterial infections in wounds.

[Levels of interleukin-6 in cerebrospinal fluid of patients with meningitis]. / Stezenie interleukiny-6 w plynie mózgowo-rdzeniowym u chorych na zapalenie opon mózgowo-rdzeniowych.

UNLABELLED: Interleukin-6 (IL-6) is a multifunctional cytokine that regulates the immune response, differentiation of B cells, activation of T lymphocytes and acute phase reactions. AIM OF THE STUDY: Quantitation of interleukin-6 in cerebrospinal fluid from patients with purulent bacterial meningitis (PBM) and viral meningitis (VM) on the first day of hospitalization. PATIENTS AND METHODS: IL-6 activity was measured in cerebrospinal fluid (CSF) of 15 children with VM (predominantly 13/15 caused by mumps virus) in age ranging from 2.5 to 15 years and 13 patients with PBM (age ranging from 18 to 52 years). The control group consisted of 10 patients with meningeal syndrome and normal laboratory estimation of CSF (white cells count, concentration of glucose and protein). The IL-6 assays were performed using immunoenzymatic method, ELISA (Amersham) technique. RESULTS: Increased levels of IL-6 were detected in the CSF of patients with bacterial meningitis and with viral meningitis. CSF IL-6 levels were 6 times greater in patients with purulent bacterial versus viral meningitis. The highest level of IL-6 was observed in a 18 years old patient with pneumococcal meningitis who died. CONCLUSION: We suggest that cytokine levels may be valuable in distinguishing patients with purulent bacterial meningitis from viral meningitis and may help in prognosis of patients with purulent meningitis.

Organization of the cord blood bank in Warsaw, Poland: current status and future prospects.

The preliminary results of the currently established cord blood bank in Warsaw, Poland have been reported. 163 cord blood samples (mean volume 102 ml) were collected during/or after delivery of the placenta. Average cell numbers per sample were as follows: WBC 13.4 x 10(8); BFU-E 5.7 x 10(4); GM-CFC 4.1 x 10(5); CD34+ 4.5 x 10(6). The quantities and quality of collected cells are suitable for their use for clinical transplantation.

G-CSF prevents the suppression of bone marrow hematopoiesis induced by IL-12 and augments its antitumor activity in a melanoma model in mice.

BACKGROUND: IL-12 has been successfully used in experimental tumor therapy. However, administration of this cytokine induces dose-dependent suppression of hematopoiesis that could potentially limit its use in clinical trials. We decided to examine whether the myelosuppressive activity of IL-12 could be corrected by the administration of G-CSF. MATERIALS AND METHODS: In the initial experiments the influence of IL-12 and/or G-CSF on bone marrow and spleen GM-CFC was evaluated. To examine whether C-CSF could influence the antitumor activity of IL-12 the combination therapy with these agents was carried out starting on day seven following inoculation of melanoma MmB16 cells into the footpads of B6D2F1 mice. To obtain insight into the mechanism of the observed augmented antitumor activity of the combination therapy with IL-12 and G-CSF, the influence of these cytokines on macrophage activity (cytotoxicity and nitric oxide release) was analyzed. RESULTS: In accord with our expectations, the application of G-CSF partially prevented the suppression of bone marrow myelopoiesis in IL-12 treated mice. Unexpectedly, G-CSF also showed potentiation of antitumor effects of IL-12 in this melanoma model. The augmented antitumor activity of combined IL-12/G-CSF immunotherapy could result from the enhanced stimulation of macrophage NO production and cytotoxicity. CONCLUSION: The simultaneous administration of IL-12 and G-CSF partially prevented suppression of bone marrow myelopoiesis in IL-12-treated mice. Moreover, treatment with these cytokines also results in potentiated antitumor effects in a murine melanoma model.

[Chemotactic monocyte and activation factor (MCAF/MCP1)in material from bronchoalveolar lavage fluid of patients with chronic bronchitis]. / Czynnik chemotaktyczny i aktywujacy monocyty (MCAF/MCP1) w materiale z plukania oskrzelowo-pecherzykowego u chorych na przewlekle zapalenie oskrzeli.

UNLABELLED: The aim of this study was to evaluate concentrations of monocyte chemotactic and activating factor (MCAF/MCP1) in bronchoalveolar lavage fluid (BAL) from patients with chronic bronchitis. 32 patients (aged 46 +/- 2 years) with chronic bronchitis and 14 patients (aged 30.5 +/- 3.4 years) of control group took part in this study. ELISA test (kits from R&D, USA) was used to measure concentrations of this chemokine in BALF. The results indicate for significant increase of MCAF/MCP1 in BALF from patients with chronic bronchitis (mean +/- SEM = 393 +/- 105.9 pg/ml) as compared from those of control group (middle +/- SEM = 36.4 +/- 10.9 pg/ml). CONCLUSION: MCAF/MCP1 may participate in creation of inflammatory changes of airways in patients with chronic bronchitis.

[The effect of bronchial inhalation provocation tests on levels of interleukin-8 in material from broncho-alveolar fluid of patients with atopic bronchial asthma]. / Wplyw wziewnych prób prowokacji oskrzeli na stezenie interleukiny-8 w materiale z plukania oskrzelowo-pecherzykowego u chorych na atopowa astme oskrzelowa.

Airway inflammation is a very important feature of asthma and occurs simultaneously with increased hyperreactivity. We have examined whether the local inflammation provoked by histamine and allergen challenge of patients with atopic bronchial asthma is associated with the appearance, in vivo interleukin-B (IL-8) in bronchoalveolar lavage fluid (BAL). Bronchoscopy and BAL for further IL-8 investigation were performed before and 24 h after challenge test with histamine (n = 11), grass pollen antigen (n = 8) and PBS (n = 5). ELISA test was used to measure IL-8 concentration (pg/ml) (kits from R&D, USA). There was observed increased level of IL-8 (p < 0.05) after histamine and allergen challenge test. This increased level of IL-8 was correlated with neutrophils in BAL (Kendall's correlation coefficient = +0.5). We conclude that IL-8 may participate in creation of bronchial hyperreactivity in atopic bronchial asthma.

[Interleukin-6 and interleukin-8 in bronchoalveolar lavage fluid material from patients with chronic bronchitis]. / Interleukina-6 i interleukina-8 w materiale z plukania oskrzelowo-pecherzykowego u chorych na przewlekle zapalenie oskrzeli.

The aim of this study was evaluation of interleukin-6 (IL-6) and interleukin-8 (IL-8) in creation of inflammation of lower airways in patients with chronic bronchitis. 32 patients with chronic bronchitis and 14 subjects of control group took part in this study. Spirometry (Jaeger eq.), bronchofibroscopy and bronchoalveolar lavage (Olympus eq.) were performed in every patient. Cytology and concentration of IL-6 and IL-8 (kits from R&D) were measured in 1 ml of lavage fluid recovered. The increased levels of IL-6 and IL-8 in BAL were correlated with clinical parameters. We conclude that these two cytokines participate in creation of inflammatory changes of lower respiratory tract in chronic bronchitis.

[The effect of specific immunotherapy on the concentration of some chemokines in BALF in patients with atopic bronchial asthma]. / Wplyw immunoterapii swoistej na stezenia niektórych chemokin w materiale z BAL u chorych na atopowa astme oskrzelow.

The cellular basis the mechanism of specific hyposensitization is still unclear. We studied the effect of immunotherapy on activity of interleukin-8 (IL-8) and monocyte activating and chemoattractant protein (MCAF/MCP1) in bronchoalveolar lavage fluid (BALF). 20 patients with atopic asthma took part in this study. 14 were treated with grass pollen extracts (Allergovit, Allergopharma, Germany) for 7 weeks and 6 of this group were not treated and served as control group. ELISA test (R&D, USA) was used to assess IL-8 and MCAF/MCP1 activity in one ml of BALF recovered before and after immunotherapy. The results indicate that immunotherapy induces the suppression of MCAF/MCP1 production and the increase of IL-8 production in BALF. These chemokines may have reciprocal control on inflammatory cells, which participate in creating of inflammatory changes in asthma.

Populations of thymocytes and peripheral blood leucocytes in leukaemia-bearing mice treated with G-CSF.

The influence of granulocyle-colony stimulating factor (G-CSF) on thymocyte subsets and peripheral blood leucocytes in leukaemia L1210-bearing mice was evaluated. Leukaemia-bearing mice have a markedly reduced L3T4+Lyt2+ thymocyte subpopulation. We observed "correction" of relative values of thymocyte subpopulations induced by treatment of mice with a G-CSF preparation. The suggestion is presented that this could result from liberation of lymphocyte T precursor cells from the bone marrow and increased homing of them into the thymus. The direct or indirect influence of G-CSF on the L3T4+Lyt2+ subpopulation of thymocytes is discussed.