Differential cytokine expression in direct and indirect co-culture of islets and mesenchymal stromal cells.

BACKGROUND: Transplantation of allogenic Langerhans islets (ISL) has been employed as an alternative to pancreas transplantation to provide endogenous supply of insulin and treat hypoglycemia unawareness in type 1 diabetes. Nevertheless, the process of islets isolation exposes the islets to hypoxia and other aggressive conditions that results in the recover of less than half of the islets present in the pancreas. Several studies demonstrated that co-culturing islets with mesenchymal stromal cells (MSC) before implantation enhances islets survival and function and this effect is mediated by cytokines. However, it remains unclear if the profile of cytokines secreted by MSC in co-culture with islets changes upon the type of co-culture: direct and indirect. MATERIALS AND METHODS: In 3 series of experiments with human islets of 3 different donors, we compared the levels of a panel of cytokines measured in the supernatant of ISL cultured alone, Wharton Jelly MSC (WJMSC) cultured alone, direct co-culture of ISL-WJMSC and indirect co-culture using a permeable transwell membrane to separate ISL and WJMSC. RESULTS: Comparing the profile of cytokines secreted by islets alone with islets in direct co- culture with WJMSC, we found higher expression of IL1b, IL17, IFγ, IL4, IL10, IL13, Granulocyte-macrophage colony-stimulating factor (GMCSF) and Leptin, in the supernatant of the co-cultures. In contrast, when comparing islets cultured alone with islets in indirect co-culture with MSC, we found no significant differences in the levels of cytokines we analyzed. CONCLUSION: Direct contact between human WJMSC and pancreatic islets is required for elevated expression of a range of immune cytokines, including both those considered inflammatory, and anti-inflammatory.

Flow cytometric analysis of bromodeoxyuridine-induced micronuclei.

The effects of DNA substitution by the thymidine analogue 5-bromodeoxyuridine (BrdU) on cell cycle progression and micronucleus induction were studied in different mammalian cell cultures. Simultaneous flow cytometric measurements of DNA content and side scatter of nuclei in Chinese hamster embryo (CHE) cells revealed a concentration-dependent temporary block in the G2/M phase of the first cell cycle. NIH 3T3 cells and human amniotic fluid fibroblast-like cells, on the contrary, did not show any cell cycle disturbances in the presence of BrdU. Micronucleus frequency increased as soon as CHE cells started to divide and reached a plateau when all cells have divided. The height of this plateau was almost equal for 60 and 100 microM BrdU. This saturation of micronucleus induction was due to a saturation of BrdU incorporation into DNA already at a doses of 60 microM as shown by the BrdU/Hoechst quenching technique. Indirect immunofluorescent staining of kinetochores with CREST antibodies revealed that nearly all BrdU-induced micronuclei were kinetochore-negative suggesting the presence of acentric chromosome fragments in these micronuclei. DNA distributions of micronuclei measured by flow cytometry showed several peaks representing micronuclei which contain DNA fragments of defined sizes induced by non-random breakage of chromosomes 1 and X as verified by flow karyotyping and C-banding.

Identification and quantification of metabolite conjugates of activated cyclophosphamide and ifosfamide with mesna in urine by ion-pair extraction and fast atom bombardment mass spectrometry.

The high bladder toxicity of the alkylating oxazaphosphorine anticancer drugs, cyclophosphamide and ifosfamide is effectively reduced by the concomitant administration of mesna (sodium 2-mercaptoethane sulphonate). The formation and rapid urinary excretion of conjugates of the activated (4-hydroxylated) oxazaphosphorine metabolites with mesna has been suggested as the pharmacological basis for the selective detoxification, but separation and identification of such metabolites in vivo have been extremely difficult due to their high polarity and chemical lability. In this study an ion-pair extraction procedure in combination with positive and negative ion fast atom bombardment mass spectrometry has been developed which enabled the identification and quantification of the conjugation products of activated oxazaphosphorine metabolites with mesna in urine. The conjugates extracted as the tetra-n-butylammonium salts are directly identified by their characteristic positive molecular ion adducts and fragment ions, and the corresponding abundant molecular anions. The pattern of molecular and fragment ion formation was established by comparison of the fast atom bombardment mass spectra of synthetic cyclophosphamide-mesna conjugates with various organic and inorganic counter ions. The ifosfamide-4-(2-thioethylsulphonate) (ifosfamide-mesna) conjugate was identified as a metabolite in the urine of rats, and in patients after administration of the combination, ifosfamide + mesna. By means of a two-step extraction and with the use of suitable analogues as internal standards, procedures for the quantification of parent oxazaphosphorine and of oxazaphosphorine-mesna conjugates by negative ion fast atom bombardment mass spectrometry have been developed, and first examples for the determination of excretion kinetics are described.

Radiation damage due to knock-on processes on carbon foils cooled to liquid helium temperature.

Radiation damage on a holey carbon foil was investigated in an electron microscope with a superconducting lens system, where the temperature of the specimen and its environment initially was 4 K. Due to an electron dose of 2 X 10(4) As/cm2 the diameter of a hole increased 5 nm. Rough calculations show that this increase can be ascribed to knock-on processes. Estimates of the rise in specimen temperature during the irradiation are given.

Improvements in electron microscopy by application of superconductivity.

Resolution tests on amorphous carbon foils were carried out in an electron microscope with a superconducting system containing 4 lenses including a shielding lens at 200 kV beam voltage. Due to the mechanical and electrical stability of the system and the absence of contamination of the specimen the highest space frequencies transferred at vertically incident beam were 6 nm-1 corresponding to a resolution of 0.17 nm, a value which approaches the theoretical resolving power of the electron optical system. It should also be feasible to apply such a lens system for microprobe analysis without strongly reducing the theoretical resolution limit, if the construction of the shielding lens is slightly changed.