Erythropoietin-stimulated endothelial cells support erythroid cell differentiation of CD34(+) haematopoietic progenitors.

BACKGROUND AND OBJECTIVES: Endothelial cells provide a unique medium for the proliferation and white lineage differentiation of haematopoietic progenitor cells (HPC). Whether this quality can be exploited to facilitate the differentiation of erythroid precursors is not yet known. MATERIALS AND METHODS: Haematopoietic progenitor cells derived from cord blood were cultured for 3 weeks in erythropoietin-stimulated supernatants with (n = 6) or without cyclosporine A (CSA, n = 6). Cell count, phenotype and morphology were assessed on a weekly basis, and the haemoglobin content was analysed. These cultures were compared with erythroid differentiation induced by cytokines only (n = 6). RESULTS: Endothelial supernatants combined with CSA led to equivalent numbers of CD71(+) erythroblasts after 1 week as cytokines only. The purity of glycophorin-positive, CD45-negative cells was higher in cells generated in endothelial supernatants than in cytokine-based media. Additional prostaglandin E2 induced a change from fetal to adult haemoglobin. CONCLUSION: For the generation of erythroblasts from HPC, endothelial supernatants are a simple and cost-effective alternative to culture conditions based on cytokines.

Established and emerging pathogens in Ixodes ricinus ticks collected from birds on a conservation island in the Baltic Sea.

Tick-borne pathogens such as Lyme borreliosis spirochaetes, Anaplasma phagocytophilum, Rickettsia spp. and Babesia spp. cause a great variety of diseases in animals and humans. Although their importance with respect to emerging human diseases is increasing, many issues about their ecology are still unclear. In spring 2007, 191 Ixodes ricinus (Acari: Ixodidae) ticks were collected from 99 birds of 11 species on a bird conservation island in the Baltic Sea in order to test them for Borrelia spp., A. phagocytophilum, Rickettsia spp. and Babesia spp. infections. Identification of the pathogens was performed by polymerase chain reaction (PCR), restriction fragment length polymorphism and sequence analysis. The majority of birds with ticks testing positive were European robins and thrushes. Borrelia DNA was detected in 14.1%, A. phagocytophilum in 2.6%, rickettsiae in 7.3% and Babesia spp. in 4.7% of the ticks. Co-infections with different pathogens occurred in six ticks (3.1%). The fact that 11 ticks (five larvae, six nymphs) were infected with Borrelia afzelii suggests that birds may, contrary to current opinion, serve as reservoir hosts for this species. Among rickettsial infections, we identified Rickettsia monacensis and Rickettsia helvetica. As we detected five Rickettsia spp. positive larvae and two birds carried more than one infected tick, transmission of those pathogens from birds to ticks appears possible. Further characterization of Babesia infections revealed Babesia divergens and Babesia microti. The occurrence of Babesia spp. in a total of five larvae suggests that birds may be able to infect ticks, at least with Ba. microti, a species considered not to be transmitted transovarially in ticks.

Optimum storage conditions for cord blood-derived hematopoietic progenitor cells prior to isolation.

Optimum storage conditions of cord blood-derived hematopoietic progenitor cells before isolation remain unknown. We therefore evaluated CD34+ cells isolated from cord blood units (n=57) within 1 h after collection and following storage for 24, 48 and 72 h at either room temperature (RT) or 4 degrees C. Isolated CD34+ cells were analyzed for their cell count, immunophenotype, apoptosis rate, clonogenicity and transmigration capacity in response to stroma-derived factor 1alpha using direct-paired comparisons (n=27). CD34+, CD133+ and CD45+ positivity after isolation remained the same under all conditions. After 24 h, CD34+ cell counts and numbers of CFU-GM colonies dropped regardless of the storage temperature. After 48 h, the number of CD34+ cells increased compared to 24 h, if the cord blood had been stored at RT resulting in almost three times more CD34+ cells than at 4 degrees C. These cells had a lower early apoptosis rate and formed four times more BFU-E than those stored at 4 degrees C with equivalent plating efficiencies. CD34+ cells kept at RT for 48 h had the highest transmigration capacities, which paralleled an increased CXCR-4 expression. Cord blood should be stored at RT before CD34+ isolation and a storage time for 48 h should be preferred to 24 h.

Immunoadsorption patients with multiple sclerosis: an open-label pilot study.

BACKGROUND: Immunoadsorption (IA) is occasionally applied in patients with acute relapses of multiple sclerosis (MS). This pilot study was undertaken to determine whether IA might help in secondary progressive and relapsing-remitting multiple sclerosis. DESIGN: IA was performed at 1-week intervals in 12 patients with secondary progressive or relapsing-remitting MS. These patients had an extended disability status scale (EDSS) score of 4.5-7 and an EDSS increase of 0.5 within 6 months before inclusion in the study despite conventional drug therapy. The change in the EDSS and that in the MS functional composite (MSFC) score, which consisted of quantitative tests of arm function, ambulation, visual acuity and cognition, served as the primary outcome variables, which were measured at baseline and at 3, 6 and 12 months. Changes in quality of life and cerebral lesions by magnetic resonance imaging (MRI) were also assessed at baseline and after the last immunoadsorption (month 3). RESULTS: A significant reduction of the median EDSS change was observed after the treatment period, which reversed 3 months after the immunoadsorptions had been stopped. Ten of 12 patients remained stable during the first year of follow-up with no significant changes of the MSFC scores. No significant changes in magnetic resonance imaging T2-hyperintense brain lesions or in the number of gadolinium-positive lesions and in the patients' quality of life were observed. Western blot analyses demonstrated a reduction of serum myelin-specific antibodies, which were collected in the adsorber eluates. CONCLUSIONS: Removal of immunoglobulins, including myelin-specific antibodies by immunoadsorption, seems to delay disease progression as defined by EDSS, MSFC and MRI, while the patients' quality of life did not deteriorate.

Tumor necrosis factor alpha enhances the adenoviral transduction of CD34+ hematopoietic progenitor cells.

The purpose of this study was to improve the transduction efficiency of adenoviral vectors (Ad) in human CD34+ hematopoietic progenitor cells. CD34+ cells from cord blood or mobilized peripheral blood were incubated with tumor necrosis factor-alpha (TNF-alpha). After removal of free TNF-alpha, the cells were infected with an Ad encoding green fluorescent protein (GFP). One day later, viable cells were counted and analyzed for GFP and CD34 by flow cytometry. To visualize vectoral trafficking, CD34+ cells were incubated with fluorophore-conjugated Ad. Plating efficiencies of hematopoietic progenitors before and after transduction were evaluated by methylcellulose assays. Pretreatment with TNF-alpha increased the transduction efficiency more than twofold (39.2% versus 15.5%) in a dose-dependent manner and strongly improved the survival of GFP-positive CD34+ cells. Time course experiments showed that TNF-alpha incubation times as short as 10 minutes were still effective. Neutralizing antibodies to TNF receptor II and RGD peptides diminished the TNF-alpha-dependent increase in transduction efficiency. No TNF-alpha-dependent increase in adenoviral receptors (coxsackie-adenovirus receptor, alphavbeta3-integrin) occurred. Analysis of viral binding demonstrated a significantly higher incidence of local concentrations of Ad along the cell surface (caps) in virus-positive cells of the TNF-alpha-treated group. Plating efficiency, especially the formation of granulocyte-macrophage colony forming units, was enhanced by TNF-alpha pretreatment. We conclude that brief incubation with TNF-alpha before addition of the Ad significantly increased the Ad transduction efficiency in CD34+ cells, and improved post-transduction survival of progenitors of the granulocyte-macrophage lineage. This finding correlates with increased Ad capping at the cell surface and suggests an alteration of Ad trafficking.

Optimized culture conditions for the generation of dendritic cells from peripheral blood monocytes.

BACKGROUND AND OBJECTIVES: Dendritic cells (DCs) are promising adjuvants for clinical immunotherapy, but they are scantily distributed. Therefore, numerous in vitro methods have been developed to expand these cells while maintaining their normal functions. Current culture systems generally require the use of fetal bovine serum (FBS)-supplemented media in order to attain DCs with high immunostimulatory activity. However, the presence of exogenous animal proteins sets limits for their use in clinical trials. The purpose of this study was to establish a simple, efficient and FBS-free method for the generation of human DCs for clinical application. MATERIALS AND METHODS: We compared monocyte-derived DCs generated in a standard FBS-supplemented medium vs. DCs generated in an autologous plasma (AutoPl)-supplemented medium, with regard to their yield, function and longevity. Peripheral blood monocytes were isolated from buffy coats by two consecutive 2-h adherence steps in tissue culture flasks. The adherent cells were differentiated into DCs within 2 weeks by adding granulocyte-macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), c-kit ligand and tumour necrosis factor-alpha (TNF-alpha). Every 2-3 days, the cells in suspension were analysed for their immunophenotype and apoptosis rate by flow cytometry. Their function was demonstrated by their allostimulatory and migratory capacity, as well as by their proteolytic activity. RESULTS: We show that more than 30 x 10(6) DCs can be achieved per unit of buffy coat using either AutoPl- or FBS-supplemented media. The purity of the DCs was 53.4% and 65% (P > 0.05) in AutoPl- and FBS-based medium, respectively. DCs grown in AutoPl media showed a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1aneg phenotype, while FBS-generated DCs exhibited a CD80high CD83+ CD86high CD14neg HLA-DR+ CD1ahigh phenotype. The apoptosis rate in both culture conditions increased from 10% to 25% over 1 week. AutoPl-generated DCs were shown to be equally strong stimulators for proliferation of allogeneic T lymphocytes as FBS-generated DCs. In addition, the capacity to migrate in response to macrophage inflammatory protein-1alpha (MIP-1alpha) and stromal-cell-derived factor 1alpha (SDF-1alpha) was similar in both groups, whereas the response to MIP-3beta was reduced in AutoPl-derived cells. Zymography analysis of supernatants from 5-day-old cultures demonstrated that AutoPl-generated DCs produced higher amounts of matrix metalloproteinases, suggesting that they have an enhanced capability to traffic through peripheral tissues. CONCLUSIONS: Our findings indicate that plastic-adherent peripheral blood cells, when cultured with GM-CSF, IL-4, c-kit-ligand and TNF-alpha in autologous human plasma-supplemented media, are a potent source of functional DCS that may be of value for human therapy.

Significance of a T-lymphocytotoxic crossmatch in liver and combined liver-kidney transplantation.

BACKGROUND: In contrast to kidney transplants a positive crossmatch is no contraindication for liver transplantation (OLT). In liver transplantation, antibody mediated rejections are rarely reported and a liver graft is suspected to have protective effects for kidney grafts when transplanted simultaneously. The aim of this study was to evaluate the effect of a positive crossmatch on outcome after OLT and combined liver and kidney transplantation (CLKTx). METHODS: We analyzed retrospectively the impact of a positive crossmatch on graft survival and rejection episodes after OLT (793pats) and CLKTx (18pats, 2.2%). Immunosuppression consisted of either Cyclosporine- or Tacrolimus-based regimens. RESULTS: A total of 50/811 (6%) of patients had a positive crossmatch, 45/793 (5.6%) with liver transplantation alone and 5/18 (28%) of patients with CLKTx. Follow-up ranged from 1 to 122.5 months (median 45.8 months). One- and 5-year graft survival rates of liver transplants alone with a positive crossmatch were 89.6% and 75.3%, respectively and were 88% and 77.5% in crossmatch negative recipients. Additionally, the incidence of acute and steroid-resistant rejection (44% and 15.5%) was not significantly increased in patients with a positive crossmatch when compared with patients with a negative crossmatch (38% and 19%). None of the patients with a positive crossmatch and CLKTx underwent a hyperacute-rejection episode after transplantation, and kidney graft survival 100%. CONCLUSIONS: In conclusion, a positive crossmatch is no contraindication for OLT and CLKTx. Furthermore, not having to wait for results of donor/recipient crossmatching can shorten cold ischemia time and may improve the clinical outcome.

An extended lymphocytotoxicity test for patients treated with lymphocytotoxic antibodies.

BACKGROUND AND OBJECTIVES: After organ transplantation, HLA antibodies (HLA-Ab) stimulated by transplant or transfusion are usually indistinguishable from passive therapeutic lymphocytotoxic antibodies (LyAb), e.g. antilymphocyte globulin (ALG), antithymocyte globulin (ATG) and OKT3, by standard lymphocytotoxicity tests (LCTs). Herein, an extended technique capable of distinguishing between these two types of antibodies is described. MATERIALS AND METHODS: Serum samples from 11 patients who received therapeutic antibodies were screened for HLA-Ab by LCTs. The administered LyAb were murine OKT3 (n = 2), equine ALG (n = 3), rabbit ATG (n = 2), and combinations of ALG and OKT3 (n = 1), ATG and ALG (n = 1), and ATG and OKT3 (n = 2). To discriminate between therapeutically applied antibodies and active HLA alloimmunization, the sera were preincubated with species-specific anti-Fc IgG, thus blocking the therapeutic antibodies. RESULTS: The sera of all patients treated showed strong positive lymphocytotoxic reactions [panel reactive antibodies (PRA) 50-100%]. After incubation with the corresponding Fc antibodies, in 9 of the 11 samples the amount of PRA decreased to the level present before transplantation (0-18%). In the remaining cases, new-onset alloimmunization was detected. CONCLUSION: This extended LCT allows the screening of serum samples for active HLA-Ab even in the presence of therapeutic LyAb.

CA 125 production and release by ovarian cancer cells in vitro.

The antigenic determinant CA 125 is a high molecular weight glycoprotein which is elevated in more than 80% of patients with epithelial ovarian cancer. Despite its good performance as a human tumor marker, only little is known about its physiological function. According to recent publications, CA 125 production and release appear to be related to cellular growth. In order to investigate this putative relationship more closely, we analyzed the pattern of CA 125 production and release by ovarian cancer cells during exponential cell growth, during cell cycle arrest by colchicine and during inhibition of cellular protein synthesis by cycloheximide. The results were correlated with the cell cycle distribution. According to our results, the main determinant of CA 125 release into the culture supernatant is the total cell count. Although cell cycle arrest in the G2 + M phase by means of colchicine treatment resulted in the death of most cells, which was reflected by an increased release of CA 125, no differences in the intracellular production rate between colchicine treated and untreated cells were seen. In contrast, treatment of cells with cycloheximide not only resulted in decreasing cell numbers but also in a complete inhibition of CA 125 production by surviving cells.

Changes in the apical surface of chloride cells following acclimation of lampreys to seawater.

Scanning electron microscopy (SEM) was used to study the changes that occur in the morphological relationships between chloride and pavement cells in the gills during acclimation of young adult lampreys to seawater. Because chloride cells are located predominantly between lamellae and are thus obscured from view, the lamellae were removed with the use of a micromanipulator installed in a SEM. In gills of animals maintained in river water, chloride cells could then be seen to be dislike and typically to form single rows between successive lamellae. After acclimation to seawater, the apical surfaces of chloride cells lose their microvilli and change in shape from small circles to rectangles that extend the full width between successive lamellae. These changes result in an increase in the length of the paracellular pathway between chloride cells. Previous work has shown that the number of strands of the zonulae occludentes sealing this pathway declines under these conditions. This presumably leads to an increase in paracellular permeability of the gill epithelium, thereby providing the low-resistance paracellular shunt required for the passive movement of sodium into the environment during osmoregulation in seawater. The above changes are reversed by transfer of lampreys downward to 10% seawater.