Metabolism of methyl-branched iodo palmitic acids in cultured hepatocytes

The metabolic fate of methyl-branched iodo fatty acids was studied in primary culture of rat hepatocytes. We compared 16-iodo-2-R,S-methyl palmitic acid (2-Me), which can be oxidized, with 16-iodo-3-R,S-methyl palmitic acid (3-Me) which can be oxidized only after an initial oxydation and with 16-iodo-2,2-dimethyl palmitic acid (2,2-Me₂) and 16-iodo-3,3-dimethyl palmitic acid (3,3-Me₂) which cannot be oxidized at all. The normal fate of natural fatty acids was given by comparative experiments with [1-¹⁴C] palmitic acid. Monomethyl-branched iodo fatty acids were taken up in the same range as palmitic acid but more than dimethyl-branched iodo fatty acids. After a 15-h incubation, acido-soluble products (ASP) accounted for 75% of the radioactivity taken up as 16-iodo-2-methyl palmitic acid, 50% as other methyl-branched iodo fatty acids and only 30% as palmitic acid, which indicated that all the methyl-branched iodo fatty acids underwent a strong deiodination process. Fatty acids were esterified in the following order: palmitic acid >16-iodo-3-R,S-methyl palmitic acid>16-iodo-2-R,S-methyl palmitic acid>16-iodo-2,2-dimethyl palmitic acid>16-iodo-3,3-dimethyl palmitic acid. Cultured hepatocytes, labelled for 3 h with the various fatty acids and reincubated for 12 h without fatty acid, secreted large amounts of free dimethylbranched iodo fatty acids as compared to the monomethyl ones and palmitic acid. Only hepatocytes prelabelled with 16-[¹²⁵I]iodo-2,2-dimethyl palmitic acid exhibited an appreciable secretion of labeled triglycerides, but at a lower rate than with [1-¹⁴C] palmitic acid. Conversely, the 16-iodo-monomethyl palmitic acids remained chiefly in hepatocyte triglycerides. Minute amounts of 16-iodo-methyl-branched-palmitic acids were found in hepatocyte or secred phospholipids as compared with palmitic acid. This metabolic fate of methyl-branched iodo palmitic acids argues against their utilization as imaging probes to monitor in vivo the synthesis and the secretion of triglycerides by the liver.     

Epidermal growth factor receptor regulates normal urothelial regeneration

Members of the epidermal growth factor (EGF) family and their receptors are involved in many cellular processes, including proliferation, migration, and differentiation. We have previously reported that these growth factors are expressed and have specific regulatory functions in an organ-like culture model of normal human urothelial cells. Here, we used this model to investigate the involvement of EGF receptor (EGFR) in human urothelial regeneration. Three 4-mm-diameter damaged areas were made in confluent normal human urothelial cell cultures with a biopsy punch. Regeneration was measured, on fixed stained cultures, with an image analyzer, at 4, 24, and 48 hours after injury. Cell proliferation was assessed by 5-bromo-2-deoxyuridine incorporation. To identify EGF family factors potentially involved in the healing process, we studied the effect of these factors on damaged confluent cultures and the level of expression of mRNAs extracted from these cultures. EGFR inhibition of the proliferation and migration of urothelial cells was tested with (1). a specific tyrosine kinase inhibitor (AG1478) and (2). a blocking anti-EGFR antibody (LA22). Exogenously added amphiregulin, EGF, transforming growth factor-alpha and heparin-binding EGF (HB-EGF) stimulated urothelial regeneration. The damaged areas were repaired by regrowth within 48 hours. Both AG1478 and LA22 inhibited the repair (by 50% and 30%, respectively), as well as proliferation and migration. This regeneration was accompanied by increased HB-EGF mRNA expression in cultures of cells from four of six subjects, but no corresponding change in EGFR protein level was observed. These results indicate that the EGFR signaling pathway is involved in urothelial regeneration. Our data support an autocrine role of HB-EGF in this process and suggest that the EGFR pathway is a potential therapeutic target for modulating urothelial cell proliferation.     

Antifungal coating by biofunctionalized polyelectrolyte multilayered films

The surface of medical devices is a common site of bacterial and fungal adhesion, first step to the constitution of a resistant biofilm leading frequently to chronic infections. In order to prevent such complications, several physical and chemical modifications of the device surface have been proposed. Here, we experiment a new type of topical antifungal coating using the layer-by-layer technique. The nanometric multilayer film obtained by this technique is functionalized by the insertion of a chromogranin A-derived antifungal peptide (CGA 47-66, chromofungin). We show that the embedded peptide keeps its antifungal activity by interacting with the fungal membrane and penetrating into the cell. In vitro studies demonstrate that such an antifungal coating is able to inhibit the growth of yeast Candida albicans by 65% and completely stop the proliferation of filamentous fungus Neurospora crassa. The cytotoxicity of such a coating was also assessed by growing human gingival fibroblasts at its surface. Finally, the antifungal coating of poly(methylmethacrylate), a widely used material for biomedical devices, is successfully tested in an in vivo oral candidiasis rat model. Taken together, these results assessed the functionalized multilayer films containing a new potent antifungal non-toxic peptide, as a novel and promising technique for local antifungal protection.     

Prevalence and characterization of a binary toxin (actin-specific ADP-ribosyltransferase) from Clostridium difficile

In addition to the two large clostridial cytotoxins (TcdA and TcdB), some strains of Clostridium difficile also produce an actin-specific ADP-ribosyltransferase, called binary toxin CDT. We used a PCR method and Southern blotting for the detection of genes encoding the enzymatic (CDTa) and binding (CDTb) components of the binary toxin in 369 strains isolated from patients with suspected C. difficile-associated diarrhea or colitis. Twenty-two strains (a prevalence of 6%) harbored both genes. When binary toxin production was assessed by Western blotting, 19 of the 22 strains reacted with antisera against the iota toxin of C. perfringens (anti-Ia and anti-Ib). Additionally, binary toxin activity, detected by the ADP-ribosyltransferase assay, was present in only 17 of the 22 strains. Subsequently, all 22 binary toxin-positive strains were tested for the production of toxins TcdA and TcdB, toxinotyped, and characterized by serogrouping, PCR ribotyping, arbitrarily primed PCR, and pulsed-field gel electrophoresis. All binary toxin-positive strains also produced TcdB and/or TcdA. However, they had significant changes in the tcdA and tcdB genes and belonged to variant toxinotypes III, IV, V, VII, IX, and XIII. We could differentiate 16 profiles by using typing methods, indicating that most of the binary toxin-positive strains were unrelated.     

Impaired responses to toll-like receptor 4 and toll-like receptor 3 ligands in human cord blood

Toll-like receptor (TLR)-4 signaling pathway plays an essential role in host defense against gram-negative bacteria while TLR-3-mediated signaling is critically involved in anti-viral immunity. To gain insight into the defects responsible for impaired Th1 responses in human newborns, we investigated the responses of human cord blood cells to lipopolysaccharide, LPS, and to polyinosinic-polycytidylic acid, Poly (I:C), ligands of TLR-4 and TLR-3, respectively. Measurement of cytokine levels revealed a profound defect in IL-12 (p70) synthesis and an increased release of IL-10 in cord blood exposed to LPS or Poly (I:C), as compared to adult blood. Moreover, Poly (I:C)-induced IFN-alpha production was found to be significantly impaired in cord blood. Phenotypic maturation of myeloid DC in response to LPS or Poly (I:C) was next compared in cord and adult blood. We observed that neonatal myeloid DC displayed decreased upregulation of CD40, CD80 whereas CD86 and HLA-DR upregulation did not differ significantly between adults and neonates. Taken together, these findings might be relevant to the increased vulnerability of human newborns to intracellular pathogens and to their inability to develop efficient Th1-type responses.     

Acquisition of vimentin in astrocytes cultured from postnatal rat brain

Summary Vimentin and glial fibrillary acidic protein (GFAP) represent the principal constituents of intermediate filaments found in astrocytes. In contrast to vimentin—GFAP transition which occurs during glial developmentin situ, vimentin coexists with GFAP in cortical astrocytes allowed to differentiate in culture. To examine whether culture conditions or proliferative activity of the cells is responsible for the expression of vimentin, we generated cultures of GFAP-positive, vimentin-negative astrocytes isolated from 26-day postnatal rat brain cortices. Isolated astrocytes are characterized by a very thin rim of perinuclear cytoplasm and by numerous processes. Antiserum to GFAP labelled major processes and cell somata of some astrocytes, especially those with relatively short and large processes. Within 3 days in culture, all astrocytes accumulated GFAP in hypertrophic cell bodies and many began to express vimentin. Vimentin appeared primarily close to nuclei, and filaments of vimentin extended into proximal segments of the cell processes. In some astrocytes, however, vimentin was always absent. Combined double immunolabelling and histoautoradiography experiments demonstrated that the acquisition of vimentin was independent of the ability of astrocytes to incorporate tritiated thymidine. The results indicate that astrocytes isolated from 26-day postnatal rat brain are heterogeneous with respect to their ability to express vimentin and that vimentin synthesis is not correlated with the growth state of the cells as had been previously suspected.     

Haplotype analysis of the BRCA2 9254delATCAT recurrent mutation in breast/ovarian cancer families from Spain

A frame-shift 9254del5 mutation was independently identified in 12 families, eleven of them with Spanish ancestors, in a BRCA2 screening performed in 841 breast and/or ovarian cancer families and in 339 women with breast cancer diagnosed before the age of 40 at different centers in France and Spain. We sought to analyze in detail the haplotype and founder effects of the 9254del5 and to estimate the time of origin of the mutation. Eight polymorphic microsatellite markers and two BRCA2 polymorphisms were used for the haplotype analyses. The markers were located flanking the BRCA2 gene spanning a region of 6.1 cM. Our results suggest that these families shared a common ancestry with BRCA2 9254del5, which is a founder mutation originating in the Northeast Spanish, with an estimated age of 92 (95% CI 56-141) generations.     

Lipoteichoic acids from Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 antagonize the responsiveness of human intestinal epithelial HT29 cells to lipopolysaccharide and gram-negative bacteria

Intestinal epithelial cells (IECs) respond to lipopolysaccharide (LPS) from gram-negative bacteria in the presence of the soluble form of CD14 (sCD14), a major endotoxin receptor. Since sCD14 is also known to interact with gram-positive bacteria and their components, we looked at whether sCD14 could mediate their effects on human IECs. To this end, we examined the production of proinflammatory cytokines following exposure of the IECs to specific gram-positive bacteria or their lipoteichoic acids (LTAs) in the absence and presence of human milk as a source of sCD14. In contrast to LPS from Escherichia coli or Salmonella enteritidis, neither the gram-positive bacteria Lactobacillus johnsonii strain La1 and Lactobacillus acidophilus strain La10 nor their LTAs stimulated IECs, even in the presence of sCD14. However, both LTAs inhibited the sCD14-mediated LPS responsiveness of IECs. We have previously hypothesized that sCD14 in human milk is a means by which the neonate gauges the bacterial load in the intestinal lumen and liberates protective proinflammatory cytokines from IECs. The present observations suggest that gram-positive organisms, via their LTAs, temper this response and prevent an exaggerated inflammatory response.     

Effects of long-term lamivudine therapy in renal-transplant patients.

BACKGROUND: Following renal transplantation (RT), chronic immunosuppression is associated in hepatitis B virus (HBV) (+) patients with a flare-up of the disease, which might be harmful in the long term. OBJECTIVES: We report on the effect of long-term lamivudine therapy given at an initial daily dose of 100mg in 18 HBV (+) RT patients. RESULTS: When lamivudine therapy was commenced, 14 patients (77%) had an increase in their aspartate (AST) and alanine (ALT) aminotransferase levels. During a mean follow-up, under treatment, of 36.5 +/- 3.5 months (up to 66 months), 10 patients (55%) had a sustained partial (HBV DNA < 4 x 10(5)copies/ml) (n = 4) or complete (HBV DNA < 400 copies/ml) (n = 6) virological response. Overall, 12 virological breakthroughs were observed. Of those who were HBe Ag(+) prior to lamivudine therapy (n = 4), one seroconverted to HBe Ab during therapy. At the last follow-up, AST and ALT levels were normal in 13 patients. When liver biopsy was repeated during treatment (n = 15), the virological responders showed a significant decrease in total Knodell score from 10 +/- 0.6 to 7 +/- 1 (P = 0.04), but no significant change in the stage of fibrosis. Conversely, in those patients with high HBV DNA titers, there were no significant changes in the total Knodell score or in the grade of fibrosis. CONCLUSION: In conclusion, lamivudine therapy is safe in HBV(+)ve renal-transplant patients. However, even if the full and partial virological response rates are still high (55%) in the long term, relapse or primary non-responses occur. The implementation of alternative efficient strategies is warranted.     

Treatment of acute lymphoblastic leukaemia.

In acute lymphoblastic leukaemia the combination prednisone-vincristine induces more than 85% complete remissions. L-asparaginase which was used in complete remissions, seemed to increase their duration. Actually the best maintenance treatment consists in the combination of 6-mercaptopurine and methotrexate interrupted by reinductions. In other respects C.N.S. prophylaxis with intrathecal methotrexate and craniospinal irradiation is necessary. The well-known prognostic factors are: age, leucocytosis, tumoral syndrome, and cytological type: 216 cases of long remission have been observed. One group of these patients was treated by old methods: this represents 0.8 to 1% of the material, while 20% were treated by recent protocols with reinductions (20%).