Low-dose irradiation prior to bone marrow transplantation results in ATM activation and increased lethality in Atm-deficient mice.

Ataxia telangiectasia is a genetic instability syndrome characterized by neurodegeneration, immunodeficiency, severe bronchial complications, hypersensitivity to radiotherapy and an elevated risk of malignancies. Repopulation with ATM-competent bone marrow-derived cells (BMDCs) significantly prolonged the lifespan and improved the phenotype of Atm-deficient mice. The aim of the present study was to promote BMDC engraftment after bone marrow transplantation using low-dose irradiation (IR) as a co-conditioning strategy. Atm-deficient mice were transplanted with green fluorescent protein-expressing, ATM-positive BMDCs using a clinically relevant non-myeloablative host-conditioning regimen together with TBI (0.2-2.0 Gy). IR significantly improved the engraftment of BMDCs into the bone marrow, blood, spleen and lung in a dose-dependent manner, but not into the cerebellum. However, with increasing doses, IR lethality increased even after low-dose IR. Analysis of the bronchoalveolar lavage fluid and lung histochemistry revealed a significant enhancement in the number of inflammatory cells and oxidative damage. A delay in the resolution of γ-H2AX-expression points to an insufficient double-strand break repair capacity following IR with 0.5 Gy in Atm-deficient splenocytes. Our results demonstrate that even low-dose IR results in ATM activation. In the absence of ATM, low-dose IR leads to increased inflammation, oxidative stress and lethality in the Atm-deficient mouse model.

Bone marrow transplantation improves the outcome of Atm-deficient mice through the migration of ATM-competent cells.

Ataxia telangiectasia (A-T) is a highly pleiotropic disorder. Patients suffer from progressive neurodegeneration, severe bronchial complications, immunodeficiency, hypersensitivity to radiotherapy and elevated risk of malignancies. Leukemia and lymphoma, along with lung failure, are the main causes of morbidity and mortality in A-T patients. At present, no effective therapy for A-T exists. One promising therapeutic approach is bone marrow transplantation (BMT) that is already used as a curative therapy for other genomic instability syndromes. We used an established clinically relevant non-myeloablative host-conditioning regimen and transplanted green fluorescent protein (GFP)-expressing ataxia telangiectasia mutated (ATM)-competent bone marrow-derived cells (BMDCs) into Atm-deficient mice. GFP expression allowed tracking of the potential migration of the cells into the tissues of recipient animals. Donor BMDCs migrated into the bone marrow, blood, thymus, spleen and lung tissue of Atm-deficient mice showing an ATM-competent phenotype. BMT inhibited thymic lymphomas, normalized T-lymphocyte populations, improved weight gain and rearing activity of Atm-deficient mice. In contrast, no GFP(+) cells were found in the cerebellum or cerebrum, and we detected decreased size index in MRI imaging of the cerebellum in 8-month-old transplanted Atm-deficient mice in comparison to wild-type mice. The repopulation with ATM-competent BMDCs is associated with a prolonged lifespan and significantly improved the phenotype of Atm-deficient mice.

Conditioned medium from renal tubular epithelial cells initiates differentiation of human mesenchymal stem cells.

OBJECTIVES: Mesenchymal-epithelial interactions play a pivotal role in tubular morphogenesis and in maintaining the integrity of the kidney. During renal repair, similar mechanisms may regulate cellular reorganization and differentiation. We have hypothesized that soluble factors from proximal tubular epithelial cells (PTC) induce differentiation of adipose-derived adult mesenchymal stem cells (ASC). This hypothesis has been tested using cultured ASC and PTC. MATERIAL AND METHODS: Conditioned medium was prepared from injured PTC and transferred to ASC cultures. ASC proliferation was analysed by a fluorometric and photometric assay. Signal transduction was analysed by phosphorylation of extracellular signal-regulated kinase 1 and 2 (ERK1/ERK2). Grade of ASC differentiation was assessed by morphological analysis and cell expression of characteristic markers. RESULTS: Conditioned medium significantly induced proliferation and phosphorylation of ERK1/ERK2 of ASC. After 12 days of incubation, cell morphology changed to an epithelial-like monolayer. Expression of cytokeratin 18 was induced by conditioned medium, while alpha-smooth muscle actin, CD49a and CD90 expression decreased. These alterations strongly indicate onset of the differentiation process to the epithelial lineage. In summary, soluble factors from PTC induce signal transduction and differentiation of ASC. CONCLUSIONS: Our study shows that conditioned medium from renal tubular epithelial cells provides a convenient source of inductive signals to initiate differentiation of ASC towards epithelial lineage. We deduce that these interactions may play an important role during renal repair mechanisms.

Safety and immunogenicity of a cluster specific immunotherapy in children with bronchial asthma and mite allergy.

BACKGROUND: Cluster specific immunotherapy (SIT) is a modern form of allergen immunotherapy allowing safe administration of high allergen doses in a short time interval compared to classic SIT. In the current study, we investigated the safety profile and immunological effect of cluster SIT in children with allergic asthma due to house dust mite allergy. METHODS: A total of 34 children (6-18 years) with allergic asthma were assigned to cluster (n = 22) or classic SIT (n = 12). To achieve a maintenance dose of allergen extract, cluster patients received 14 injections of house dust mite allergen within 6 weeks, whereas the classic SIT group received 14 injections within 14 weeks. Safety was monitored by recording adverse events. Immunogenicity was measured by specific IgG(Mite) and IgG4(Mite), by antibody-blocking properties on basophil activation, and by the T cell subset transcription factors Foxp3, T-bet, and GATA-3. RESULTS: There were no significant differences in local and systemic side effects between the two groups. In the cluster group, serum levels of specific IgG(Mite) (p < 0.001) and specific IgG4(Mite) (p < 0.001) significantly increased after 8 weeks, while it took 12 weeks in the classic SIT group. These data were confirmed by blocking CD63 expression as well as release of cysteinyl leukotrienes after in vitro basophil stimulation. No differences in transcription factor expression were found in the two groups. CONCLUSION: Cluster SIT is safe in children. Additionally, our data demonstrated an even more rapid induction of specific immune tolerance. Cluster SIT is an attractive alternative to conventional up-dosing schedules with fewer consultations for the patients.

C-reactive protein induced activation of MAP-K and RANTES in human renal distal tubular epithelial cells in vitro.

AIMS: C-reactive protein (CRP) is a component of the acute-phase reaction to inflammation, severe tissue injury, and infection. Investigations have shown that CRP concentration is highly increased in the urine during acute renal graft dysfunction and, therefore, may affect tubular cell metabolism. Nevertheless, no data about the effects of CRP on human renal tubular epithelial cells are available. METHODS: Human renal distal tubular cells (DTC) were isolated immunomagnetically and cultured. Cells were stimulated with affinity chromatography pure native CRP from human ascites (10 - 0.001 microg/ml). Phosphorylation of MAP-K was assessed by Westernblot analysis. Release of RANTES and interleukin-6 was evaluated with an enzyme immunoassay. Cytotoxic effects of CRP were determined by a commercially available Live/Dead assay and MTT assay. Effects on cell proliferation were analyzed by a fluorimetric assay. RESULTS: Westernblot analysis clearly showed that CRP activates the MAP-K pathway of DTC. CRP upregulated RANTES expression of DTC in a significant and dose-dependent manner. CRP (10 microg/ml) induced a 12.3-fold upregulation, CRP 1 or 0.1 microg/ml induced a 6.3-/2.8-fold RANTES upregulation, respectively. Interleukin-6 synthesis was not influenced. Cytotoxic, proliferative or apoptotic effects were not observed at the concentrations used. CONCLUSIONS: We demonstrated an activating effect of CRP on DTC in vitro. In vivo, this effect of CRP might be part of the immune activation cascade during episodes of renal graft rejection or bacterial infections.

Induction of RANTES, HLA-DR, and intercellular adhesion molecule-1 on highly purified distal tubular cells from human kidney.

BACKGROUND: Expression of proinflammatory molecules by tubular epithelial cells plays an important role in renal allograft rejection and inflammatory kidney diseases. Different studies from patients with acute rejection point to the involvement of distal tubular segments. At present no in vitro system for the human distal tubule is established. METHODS: Human distal tubular cells were isolated immunomagnetically. Cultured cells were stimulated with cytokines (interferon-gamma, tumor necrosis factor-alpha, interleukin-1beta, or a cytokine mix). Secretion of RANTES (regulated upon activation, normal T-cell expressed and secreted) was evaluated with an enzyme-linked immunoassay. Expression of HLA-DR and intercellular adhesion molecule (ICAM)-1 was assessed by flow cytometric analysis and immunofluorescence studies. RESULTS: Our data clearly indicate that distal tubular cells express RANTES, HLA-DR, and ICAM-1 in response to a mixture of specific cytokines. Dexamethasone inhibited the induced expression of RANTES and HLA-DR significantly, but not that of ICAM-1. CONCLUSIONS: We demonstrate an appropriate in vitro system for the human distal tubule. The present study proves the involvement of the distal tubular segment during inflammatory kidney diseases.

Effects of mycophenolic acid on human renal proximal and distal tubular cells in vitro.

BACKGROUND: Mycophenolic acid has been shown to be effective for the prevention and treatment of renal allograft rejection. Rejection episodes were found to be associated with an infiltration of lymphocytes and macrophages/monocytes into the diseased kidney. Expression of RANTES, HLA-DR and ICAM-1 may be important for the pathogenesis of this leukocyte infiltration. Therefore the aim of this study was to evaluate the effect of the antiproliferative and immunosuppressive agent mycophenolic acid (MPA) on cell growth and cytokine-induced expression of RANTES, HLA-DR and ICAM-1 of highly purified proximal (PTC) and distal tubular cells (DTC) from human kidney. METHODS: Human PTC and DTC were cultured in the presence of different concentrations of MPA (0.25-50 microM) or MPA plus guanosine (100 microM). Total cell number (DNA content) was determined after 4 days of cell culture by a non-radioactive fluorescence assay. Cells were stimulated by a combination of cytokines (IL1beta+gammaIFN+TNFalpha=cytomix) or cytomix plus MPA. Secretion of RANTES protein was evaluated with an enzyme-linked-immunosorbent assay. Cell surface expression of HLA-DR and ICAM-1 was assessed by flow cytometric analysis. RESULTS: MPA inhibited cell growth of PTC and DTC in a dose-dependent manner. This effect was totally abolished by the addition of guanosine. Cytokine-induced RANTES expression was synergistically increased in the presence of MPA, an effect that was partially prevented by the addition of guanosine. Cytokine stimulation resulted in de novo expression of HLA-DR and a marked increase of ICAM-1 expression, which was partially inhibited by dexamethasone. Addition of MPA did not influence this stimulated expression. CONCLUSIONS: We demonstrate that MPA has an effect on cell growth and chemokine release of tubular epithelial cells, and that these effects are dependent on the inhibition of cellular guanosine production. The clinical consequences of this possible pro-inflammatory effect of MPA on RANTES release may be abolished by a concomitant treatment with steroids.

Transdifferentiation of distal but not proximal tubular epithelial cells from human kidney in culture.

Human renal proximal and distal (thick ascending limb and early distal convoluted tubule) epithelial cells have been isolated according to their specific antigen expression. The cells were well characterized by flow cytometry, enzyme cytochemistry and electron microscopy and cultured for up to 3 months. Cultured tubular cells coexpressed cytokeratin and vimentin as intermediate filament proteins. While primary isolated cells, proximal as well as distal, revealed the phenotypic characteristics of their nephron origin, cultured distal cells showed the tendency to dedifferentiate/transdifferentiate. Distal cells lost their characteristic expression of Tamm-Horsfall glycoprotein and started de novo expression of the proximal marker proteins aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV. The expression of these antigens by distal cells could be shown by flow-cytometric analysis and fluorescence microscopy. Enzyme activity assays revealed the activity of aminopeptidase M, gamma-glutamyl transferase and dipeptidyl peptidase IV, but not of the proximal marker enzyme alkaline phosphatase. This antigenic shift could not be prevented in different culture media, and the original phenotype could not be restored. Cultured cells displayed characteristic hormonal stimulation patterns indicative of their proximal and distal origins, as shown by activation of adenylate cyclase by different peptide hormones. These results indicate that distal tubular cells possibly transdifferentiate to a more proximal phenotype in view of loss of the distal marker enzyme Tamm-Horsfall protein and de novo expression of proximal marker enzymes like dipeptidyl peptidase IV and aminopeptidase M.

Isolation of proximal and distal tubule cells from human kidney by immunomagnetic separation. Technical note.

After collagenase digestion and Percoll density gradient centrifugation of human renal tissue, tubular epithelial cells of the proximal and the distal segments were isolated with an immunomagnetic method using MACS microbeads. To enrich proximal tubular (PT) cells we used a monoclonal antibody (mAb) against aminopeptidase M (APM, CD 13), specific of the proximal tubule. Distal tubular (DT) cells were isolated through a mAb recognizing Tamm-Horsfall glycoprotein (THG), a specific antigen for the thick ascending limb and the early distal convoluted tubule. Cells of the proximal primary isolate were histochemically strongly positive for aminopeptidase M (98.6%), however, cells of the distal portion were negative (98.7%). Ultrastructural analysis of PTC primary isolates revealed highly preserved brush border microvilli, well-developed endocytosis apparati and numerous mitochondria, whereas DTC primary isolates showed smaller cells with basolateral invaginations and less apical microvilli. Characterization by immunofluorescence indicated the coexpression of cytokeratin and vimentin, whereas staining for desmin, smooth muscle actin, a fibroblast-specific marker and von Willebrand factor was negative. Cultured PT and DT cells displayed different adenylate cyclase responsiveness to hormonal stimulation. PTH (10(-6) M) increased cAMP production in distal cells up to 32.8-fold of the basal level and in proximal only up to 3.5-fold (10(-8) M, DT 14.4x and PT 2.25x). Calcitonin stimulated adenylate cyclase in DT in a dose dependent fashion (10(-6) M, 4.3x; 10(-8) M, 2.25x), whereas only a low calcitonin response was found in PT cells (10(-6) M, 1.6x; 10(-8) M, 1.4x). AVP (10(-6) M) activated the distal cAMP-production only up to 1.9x of the basal level, but the proximal cAMP-production was negligible (only 1.3x the basal level). The data of this study indicate the proximal and distal tubule origin of the cultured cells that were isolated according to their segment-specific antigens.