IrO2 as a promoter of Pt-Ru for methanol electro-oxidation.

A multifunctional catalyst may represent a valid route to enhance methanol electro-oxidation. Ternary catalysts based on Pt modified with both Ru and Ir oxides show better performance for methanol electro-oxidation than bi-metallic Pt-Ru catalysts.

Evaluation of in vitro and in vivo antimicrobial activity of a new topical antiinfective agent.

ST 1103 (Undecyl [4-N,N,N-trimethylammonium-(R)-3- isovaleroyloxy]-butanoate methanesulfonate) is a novel compound endowed with a broad antimicrobial spectrum. ST 1103 is able to inhibit the in vitro growth of Gram-positive bacteria (mean MIC value of 2.60 micrograms/ml), Gram-negative bacteria (mean MIC value of 27.00 micrograms/ml), yeasts and yeast-like fungi (mean MIC value of 3.76 micrograms/ml), filamentous and dermatophytic fungi (mean MIC value of 18.33 micrograms/ml). Since indirect evidence indicates a poor oral absorbtion, ST 1103 was topically administered to mice with skin infections caused by mixed inocula. In these conditions, ST 1103 was able to cure mice infected with T. quinckeanum, S. aureus as well as immunodepressed mice infected with T. quinckeanum, S. aureus and C. albicans. Conversely, miconazole (reference compound) appeared inadequate, in our experimental conditions, for a definitive therapy of the skin mycosis superinfected by staphylococcus. By using an in vitro 3D-human skin model, ST 1103 was fairly well tolerated in terms of both cell viability and release of inflammatory mediators. In a dermal tolerance study in mice, ST 1103 at a concentration of 1% did not show any sign of local irritation on both intact and abraded skin after an 8-day topical treatment. In conclusion, ST 1103 appears to be a promising candidate for treatment of cutaneous infections caused by mixed microbial pathogens.

L-carnitine and some of its analogs delay the onset of apoptotic cell death initiated in murine C2.8 hepatocytic cells after hepatocyte growth factor deprivation.

Addition of L-carnitine and some of its analogs to low-serum incubation medium of murine hepatocytic C2.8 cells prolonged maintenance of life and enhanced cell growth, as compared to controls. The drug acted synergistically with hepatocyte growth factor (HGF). Addition of L-carnitine to cells that had grown confluently in medium supplemented with HGF, significantly delayed the onset of cell death (apoptosis) initiated after HGF deprivation. Protection by L-carnitine was dose-dependent and stereospecific. Similar findings were obtained with three analogs of L-carnitine (i.e. isovaleryl-L-carnitine-HCl, isovaleryl-L-carnitine acid fumarate and butyryl L-carnitine taurine amide). In contrast, four different analogs (i.e. isovaleryl-L-carnitine-eptyl-ester-HCl, isovaleryl-L-carnitine-idroxy-butyric-HCl, L-threonyl-L-carnitine-HCl and L-paramethyl-cinnamoil-carnitine-HCl) were inactive. Although the mechanism of cytoprotection stimulated by L-carnitine remains unresolved, the data suggest that this compound serves as a co-factor that influences C2.8 cells to become less susceptible to damaging actions of noxious agents or conditions initiated after HGF withdrawal.

High molecular weight hepatotrophic factors released in serum-free medium by the cultured embryo rat fibroblastic cell line FRL.

Two hepatotrophic protein factors (HTF) were partially purified from the serum-free conditioned medium of the embryo rat fibroblastic cell line FRL. HTF induced cell adherence to the culture plate, promoted life maintenance, scattering and enhanced 3H-Thymidine uptake into DNA and cell proliferation in two sensitive murine non-transformed, epithelial hepatocytic cell lines (C6 and C2.8). In this bio-assay, cells were plated at low density in Dulbecco's Modified Eagle's Medium, in the absence of serum and other exogenous growth factors. HTF did not synergize with epidermal growth factor. Biological activity was associated to two major acidic proteins: a non-heparin-binding protein (M(r) approximately 70 kDa, pI 4.6-4.5) and a heparin-binding protein (M(r) approximately 200 KDa, pI 4.3-4.1).

LPS-induced serum TNF production and lethality in mice: effect of L-carnitine and some acyl-derivatives.

The effect of L-carnitine and some of its acyl derivatives on serum TNF production and lethality in a murine experimental endotoxin shock model was investigated. In some instances, serum IL-6 production was also evaluated. In this experimental model, C57BL/6 mice received 30 mg/kg LPS (E. cell 055:B5) injected intraperitoneally, while L-carnitine and its derivatives were administered according to different schedules. Serum levels of TNF and IL-6 were evaluated 1 h following LPS injection. The treated animals were also monitored daily for differences in body temperature, feeding, and survival for 10 days after LPS injection. Although some derivatives were able to significantly affect TNF production, the marked decrease in serum TNF levels of LPS-treated mice was not paralleled by a substantial increase in survival.

Apoptosis of serum-free C2.8 mouse embryo hepatocytic cells caused by hepatocyte growth factor deprivation.

C2.8 mouse embryo hepatocytic cells, acutely required exogenous hepatocyte growth factor (HGF) to survive and proliferate in serum-free Dulbecco's modified Eagle's medium supplemented with insulin, transferrin and Na-selenite. Greater than 90% of cultured C2.8 cells died within 48 hours from plating in the absence of HGF. Conversely, HGF prolonged maintenance of life and stimulated cell proliferation. Removal of HGF from the medium of cultures that had grown to confluency, also resulted in a rapid decreased cell survival. In the last circumstance, light microscopic observations revealed, with high frequency, morphological features characteristic of apoptosis. DNA within the affected cells underwent rapid fragmentation, revealed as a ladder of DNA fragments in multiples of about 200 base pairs. HGF prevented loss of cell viability, morphological damages and retarded DNA fragmentation in confluent C2.8 cells. Cycloheximide delayed cell death caused by HGF deprivation.

Differential susceptibility of cultured human melanoma cell lines to enhancement by retinoic acid of intercellular adhesion molecule 1 expression.

The potential role of intercellular adhesion molecule 1 (ICAM-1) in the biology of human melanoma cells has stimulated interest in the characterization of its modulation. The present study has shown that the differentiating agent retinoic acid (RA) up-regulates ICAM-1 expression by melanoma cells in a dose- and time-dependent fashion. The enhancement of ICAM-1 cell surface expression is paralleled by an increase in ICAM-1 mRNA. Therefore, ICAM-1 represents an additional gene which may be transcriptionally regulated by RA. The five melanoma cell lines tested displayed a differential susceptibility to the modulation of ICAM-1 expression by RA, since the cell line MeWo did not change in its ICAM-1 expression following incubation with RA. Nevertheless, RA-insensitive as well as RA-sensitive melanoma cell lines displayed a higher increase in ICAM-1 expression following incubation with RA and cytokines than following incubation with each of them. Analysis of the distribution in the melanoma cell lines of retinoic acid receptors (RARs) showed a relationship between susceptibility to a RA-mediated increase of ICAM-1 expression and RAR beta expression, suggesting that the latter receptor may play a role in the phenomenon. RAR alpha and RAR gamma were present in RA-sensitive and -insensitive melanoma cell lines, suggesting that they play a role in the enhancement by RA of cytokine-mediated up-regulation of ICAM-1 expression. The melanoma cell lines we have described may represent a useful system for investigating the role of RAR in the regulation of gene expression and the mechanism(s) which underlie this effect.

Molecular abnormalities in the expression of HLA class-I antigens by melanoma cells.

Testing of a panel of cultured human melanoma cells with radiolabelled anti-HLA-class-I monoclonal antibodies (MAbs) in a binding assay has shown lack of reactivity of FO-I and SK-MEL-33 cells and low reactivity of SK-MEK-19 cells. SDS-PAGE analysis of the components immunoprecipitated from the 3 intrinsically radiolabelled melanoma cell lines by antibodies to the 2 subunits of HLA-class-I antigens has not detected beta 2-mu in the immunoprecipitates from melanoma cells FO-I and SK-MEL-33 and only a low level of HLA-class-I heavy chain in the immunoprecipitate from SK-MEL-19 cells. Northern blotting analysis with probes specific for HLA-class-I heavy chain and for beta 2-mu indicates that the abnormalities in HLA-class-I-antigen expression reflects a defect at the transcriptional level in FO-I cells and at the post-transcriptional level in SK-MEL-19 and in SK-MEL-33 cells. FO-I, SK-MEL-19 and SK-MEL-33 cells represent useful models to analyze the molecular mechanisms underlying the loss of HLA-class-I-antigen expression which is often associated with malignant transformation of melanocytes and to characterize the role of HLA-class-I antigens in the biology of melanoma cells and in their interactions with effector cells.

Lack of HLA class I antigen expression by cultured melanoma cells FO-1 due to a defect in B2m gene expression.

The melanoma cell line FO-1 does not express HLA class I antigens and does not acquire them on the cell surface after incubation with IFN-gamma. Immunochemical studies showed that FO-1 cells synthesize HLA class I heavy chain, but do not synthesize beta 2-microglobulin (beta 2-mu). The latter abnormality is associated with lack of beta 2-mu mRNA which remains undetectable in FO-1 cells incubated with IFN-gamma. The defect was identified as a genetic lesion in the B2m gene, since DNA hybridization analysis detected a deletion of the first exon of the 5'-flanking region, and of a segment of the first intron of the B2m gene. HLA class I antigen expression was reconstituted on melanoma cells FO-1 after transfection with the wild-type mouse B2m gene, thereby confirming the abnormality of the endogenous B2m gene. The defect identified in FO-1 cells is distinct from that underlying the lack of HLA class I antigen expression by lymphoblastoid cells Daudi, but is remarkably similar to that causing lack of H-2 class I antigen expression by mouse lymphoblastoid cells R1 (TL-). These results suggest that genetic recombination in the 5' region of the B2m gene is a recurrent mechanism in B2m gene defects. In addition to contributing to our understanding of molecular abnormalities in HLA class I antigen expression by melanoma cells, FO-1 cells represent a useful model for analyzing the role of HLA class I antigens in the biology of melanoma cells and in their interaction with cells of the immune system.

Analysis of Mycobacterium tuberculosis genoma and production of a recombinant protein containing specific B and T cell antigenic determinants--new approaches to second generation antituberculosis vaccines.

Tuberculosis is still a major health problem in almost all over the world. Thus, new directions in basic and applied research of tuberculosis are under investigation in several laboratories. In this paper, we provide recent data obtained in our laboratory with the recombinant DNA technology which allow a systematic survey of the microbial genome. Screening of the M. tuberculosis genomic DNA library in the phage lambda gt11 expression vector, using E. coli as surrogate host, has evidenced the possibility of producing recombinant M. tuberculosis proteins recognized by sera from tuberculosis patients and by specific monoclonal antibodies. Using this technology, we have isolated a recombinant protein (molecular weight 130 kilodaltons) which is identified by a murine monoclonal antibody recognizing a mycobacterial cell wall antigenic determinant present on a mycobacterial protein. This protein is only present on the mycobacterial species related to the tuberculosis complex (M. tuberculosis, M. bovis, M. africanum) and does not crossreact with nonpathogenic Mycobacteria. Since the immune response to mycobacterial infections is cell-mediated, the question arises about the use of M. tuberculosis-specific T lymphocytes to screen this gene bank. Thus, the recombinant mycobacterial protein isolated by antibodies has been then used to stimulate the proliferation of T lymphocytes from patients with tubercular pleuritis. This experiment indicates that the recombinant protein contains antigenic determinants recognized by T cells. Moreover, such protein is able to elicit delayed type hypersensitivity skin reaction in mice immunized or infected with M. tuberculosis and M. bovis. Finally, gene mapping and hybridization studies with native M. tuberculosis DNA confirme the mycobacterial nature of the recombinant DNA insert. Thus a good candidate for the prophylaxis and the immunodiagnosis of tuberculosis has been identified. The identification and selection of genes encoding antigenic determinants recognized by mycobacteria-specific T cells with protective functions will allow in the near future the construction by genetic engineering of recombinant vaccines effective in the control of this disease. This paper will briefly discuss the present strategy used in our laboratory to reach this goal.