Widefield deconvolution epifluorescence microscopy combined with fluorescence in situ hybridization reveals the spatial arrangement of bacteria in sponge tissue.

Widefield deconvolution epifluorescence microscopy (WDEM) combined with fluorescence in situ hybridization (FISH) was performed to identify and characterize single bacterial cells within sections of the mediterranean sponge Chondrosia reniformis. Sponges were embedded in paraffin wax or plastic prior to the preparation of thin sections, in situ hybridization and microscopy. Serial digital images generated by widefield epifluorescence microscopy were visualized using an exhaustive photon reassignment deconvolution algorithm and three-dimensional rendering software. Computer processing of series of images taken at different focal planes with the deconvolution technique provided deblurred three-dimensional images with high optical resolution on a submicron scale. Results from the deconvolution enhanced widefield microscopy were compared with conventional epifluorescent microscopical images. By the application of the deconvolution algorithm on digital image data obtained with widefield epifluorescence microscopy after FISH, the occurrence and spatial arrangement of Desulfovibrionaceae closely associated with micropores of Chondrosia reniformis could be visualized.

Induction of proto-oncogene and cytokine expression in human peripheral blood monocytes and the monocytic cell line THP-1 after stimulation with mycoplasma-derived material MDHM.

Mycoplasma fermentans-derived high-molecular-weight material (MDHM) was originally described to induce differentiation of murine thymocytes to cytolytic effector T-cells by stimulating IL-6 release from adherent cells. This study shows that human peripheral blood monocytes (PBMo) also respond to MDHM with increases in IL-1 beta, IL-6 and TNF alpha expression, both at the mRNA and protein level. The induced expression of IL-1 beta and TNF alpha mRNA in the monocytic THP-1 cell line increased as quickly as in primary cells. In contrast to PBMo, THP-1 and 14 other monocytic/myeloid leukemia-derived cell lines did not secrete measurable amounts of the cytokines upon treatment with MDHM. IL-1 beta and IL-6 genes contain AP-1 binding sites as regulatory elements, the AP-1 protein being composed of c-jun and c-fos gene products. In THP-1 cells c-jun mRNA expression increased after incubation with MDHM while positive c-fos expression remained unaffected. Although these data suggest AP-1 regulated cytokine mRNA expression, results from PBMo are not in accordance with this notion. In the primary cells MDHM-induced elevation of cytokine mRNA levels was preceded by a downregulation of c-fos expression while positive c-jun expression was not modulated. c-myc mRNA expression, constitutively high in THP-1 cells, was induced in MDHM-stimulated PBMo. In conclusion, MDHM-stimulated induction of cytokine mRNA expression was accompanied by different proto-oncogene responses in PBMo and THP-1 cells. These differences may represent different regulatory pathways of the two cell systems. Alternatively, these data support the notion that neither AP-1 nor the c-myc protein are involved in the MDHM-induced increase in IL-1 beta, IL-6 or TNF alpha mRNA levels. Furthermore, the present results demonstrate clearly that mycoplasma products can have a profound impact on the activation status of eukaryotic cells.