Type of monocyte immunomagnetic separation affects the morphology of monocyte-derived dendritic cells, as investigated by scanning electron microscopy.

Dendritic cells (DCs) are increasingly being used for multiple applications and are useful tools for many immunotherapeutic strategies. The understanding of the possible impact of the DCs-generation methods on the biological capacities of these cells is therefore essential. Although the immunomagnetic separation is regarded as a fast and accurate method yielding cells with the high purity and efficiency, still little is known about its impact on the properties of the generated DCs. The aim of this study was to compare the morphology of the monocyte derived dendritic cells (MoDCs), generated from monocytes selected with anti-CD14 mAbs (positive separation) and treated with anti-CD3, -CD7, -CD16, -CD19, -CD56, -CD123, glycophorin A (negative separation), using laser scanning microscopy. We found that the type of the immunomagnetic separation method used strongly influences the shape and cell dimension of the MoDCs. We observed that the height of both immature and LPS-matured DCs generated from monocytes isolated by negative separation was significantly higher compared to the cells obtained by positive separation.

Virulence of clinical Mycobacterium tuberculosis strains in Lodz, Poland.

SETTING: The transmission of tuberculosis (TB) in the population and the development of disease are determined not only by the patient's immunological status, but also by the virulence of Mycobacterium tuberculosis strains. OBJECTIVE: To examine the virulence of M. tuberculosis clinical isolates with recognised transmission collected from 2006 to 2007 in a population in Lodz, Poland. METHODS: A total of 36 isolates were studied to determine their sensitivity to human neutrophil peptide 1 (HNP-1) and intracellular growth within THP-1 cells. Bacterial strains were cultured using HNP-1 at different concentrations. After incubation, the number of colony-forming units (cfu) was determined by bacteria plating. The intracellular survival was examined on days 3, 6 and 8 post-THP-1 infection by cfu enumeration. RESULTS: Overall, 69% of the isolates showed greater resistance to the highest HNP-1 concentration (15 g/ml) than the virulent H37Rv strain, and the growth of 10 strains was totally inhibited. On day 8, 56% of the strains displayed higher cfu numbers than the virulent H37Rv strain. CONCLUSION: The results suggest that isolates from our urban population represent highly virulent phenotypes. We could not find any significant difference in virulence between strains with unique genotypes and those in clusters.

Interactions between an M. tuberculosis strain overexpressing mtrA and mononuclear phagocytes.

PURPOSE: It was previously shown that the bacterial two-component regulatory signal transduction (2CR) system MtrAB may be associated with the ability of M. tuberculosis (Mtb) to survive in macrophages. In the present work Mtb mutants: Rv-78 with overexpression of mtrA and Rv-129 with elevated level of phosphorylation-defective MtrA were used for further investigation of the potential influence of the MtrAB system on Mtb interaction with human monocytes. MATERIAL/METHODS: Flow cytometry was used to determine the expression of MHC class II molecules. The expression of genes for inducible nitric oxide synthase (iNOS) and cathepsin G was quantified by RT-PCR. The association of Mtb strains with Rab5 and Rab7 positive vacuoles was investigated applying confocal microscopy. IL-10 and IL-12 secretion by monocytes as well as the Mtb susceptibility to cathepsin G were investigated. RESULTS: Mutation-carried and wild type Mtb strains inhibited MHC class II expression on monocytes to a similar extent. Monocyte stimulation with mycobacteria led to the increased production of IL-10 but no detectable amounts of IL-12 or NO were observed. Expression of the gene for iNOS was not detected while that for cathepsin G was shown, however its intensity was not associated with MtrA mutation. Mtb mutant strains were more effectively enclosed in phagosomes containing the late endosome marker Rab7 as compared to the control. CONCLUSIONS: The results may confirm the importance of the MtrAB system in mycobacterial capacity for successful survival in phagocytes, especially in the context of high degree of colocalization of Mtb Rv-78 to mature phagosomes.

Resorcylic acid lactone L-783,277 inhibits the growth of the human adrenal cancer cell line H295R in vitro.

The resorcylic acid lactone L-783,277, isolated from a Phoma sp. (ATCC 74403), is a potent and specific inhibitor of MEK (Map kinase kinase) that exerts very interesting pharmacological activities including anti-neoplastic properties. However, the role of this compound in the regulation of endocrine-related cancer cell growth and tumor progression remains unknown. In the present study we have evaluated the effect of L-783,277 on the viability, proliferation and cell cycle of the human adrenocortical carcinoma cell line H295R. L-783,277 inhibited viability (IC50 of 22 microM) and cell proliferation (IC50 of 21 microM) of H295R. At concentrations of 10(-6)-10(-8)M this effect was associated with the accumulation of H295R cells in S-phase, whereas at concentrations of 10(-9)-10(-10)M a prolonged G1-phase and reduced transition into S-phase were observed. Our findings demonstrate for the first time the anti-proliferative action of L-783,277 on the human adrenocortical H295R cell line.