Femtosecond spectral pulse shaping with holographic gratings recorded in photopolymerizable glasses.

The majority of the applications of ultrashort laser pulses require a control of its spectral bandwidth. In this paper we show the capability of volume phase holographic gratings recorded in photopolymerizable glasses for spectral pulse reshaping of ultrashort laser pulses originated in an Amplified Ti: Sapphire laser system and its second harmonic. Gratings with high laser induce damage threshold (LIDT) allowing wide spectral bandwidth operability satisfy these demands. We have performed LIDT testing in the photopolymerizable glass showing that the sample remains unaltered after more than 10 million pulses with 0,75 TW/cm2 at 1 KHz repetition rate. Furthermore, it has been developed a theoretical model, as an extension of the Kogelnik's theory, providing key gratings design for bandwidth operability. The main features of the diffracted beams are in agreement with the model, showing that non-linear effects are negligible in this material up to the fluence threshold for laser induced damage. The high versatility of the grating design along with the excellent LIDT indicates that this material is a promising candidate for ultrashort laser pulses manipulations.

Generation of femtosecond paraxial beams with arbitrary spatial distribution.

We present an approach to generate paraxial laser beams with arbitrary spatial distribution in the femtosecond time regime. The proposed technique is based upon a pair of volume phase holographic gratings working in parallel arrangement. It exploits the spatial coherence properties of the incoming laser beam in a compact and robust setup that mitigates angular and spatial chirp. The gratings were recorded in a photopolymerizable glass with a high optical damage threshold and a large optical throughput. Setup performance is studied and experimentally demonstrated by generating Laguerre-Gaussian femtosecond pulses.

Induction of apoptosis in bacillus Calmette-Guérin-activated T cells by transforming growth factor-beta.

In view of the critical role played by bacillus Calmette-Guérin (BCG) in the development and functional activation of protective T cells against tuberculosis, it has become important to understand the mechanisms by which cytokines regulate BCG-mediated immune responses. There is evidence that cytokine-mediated suppression of T cell function by mechanisms, including apoptosis, may reduce host resistance in tuberculosis. However, it is unclear whether cytokine-mediated suppression of antigen-responsive T cells through apoptotic mechanisms may be operating during human cellular activation induced by BCG. Here we present evidence, for the first time, that treatment of BCG-activated T cells with transforming growth factor-beta (TGF-beta) induces cellular apoptosis. These results were further supported by the fact that treatment of cells with a blocking mAb directed to TGF-beta significantly inhibited the percentage of apoptosis induced by TGF-beta. Interestingly, TGF-beta-mediated death of BCG-activated T cells in cultures containing interleukin (IL)-12 was observed. Moreover, our results demonstrated the induction of apoptosis by TGF-beta in BCG-activated T cells cultured in the presence of exogenous IL-12. In addition, our data indicated that TGF-beta significantly inhibited both BCG-induced cell growth determined by thymidine uptake and BCG-induced IFN-gamma secretion. Finally, TGF-beta-induced apoptosis in BCG-activated T cells correlated inversely with BCG-induced IFN-gamma secretion. Taken together, these findings indicate that TGF-beta induces apoptosis in human T cells activated with BCG and at the same time suggest that loss of BCG-reactive T cells through apoptotic mechanisms could contribute to an increased susceptibility to Mycobacterium tuberculosis infection.

Inhibition of Mycobacterium bovis BCG-induced tumor necrosis factor alpha secretion in human cells by transforming growth factor beta.

The effect of exogenous transforming growth factor beta (TGF-beta) on Mycobacterium bovis BCG-induced tumor necrosis factor alpha (TNF-alpha) production by human mononuclear cells was studied. It was found that TNF-alpha production by human cells stimulated with BCG was significantly inhibited by TGF-beta. The specificity of the observed inhibition was demonstrated, since the addition of an anti-TGF-beta neutralizing monoclonal antibody completely reversed the inhibitory effect. Furthermore, the suppressive effect of TGF-beta on TNF-alpha secretion in this system was not due to a direct cytotoxic effect, since cell viability was comparable in the presence or absence of TGF-beta. Interestingly, our results demonstrated comparative suppressive effects of TGF-beta and interleukin-10 on BCG-induced TNF-alpha secretion. Together, the data demonstrate, for the first time, that TGF-beta inhibits BCG-induced TNF-alpha secretion by human cells.

Depletion of endogenous interleukin-10 augments interleukin-1 beta secretion by Mycobacterium bovis BCG-reactive human cells.

In this study, we found evidence that the interleukin-10 (IL-10) protein is functionally relevant in Mycobacterium bovis BCG-induced cytokine synthesis, as neutralization of endogenously synthesized IL-10 in human cells activated with BCG resulted in a two- to threefold increase in the level of IL-1 beta. When exogenous recombinant human IL-10 was added to human mononuclear cells, a significant reduction of BCG-induced IL-1 beta secretion was observed. This inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in the presence of IL-10, and it was specific, as it was completely abolished in the presence of anti-IL-10 neutralizing monoclonal antibody while an irrelevant antibody used as a control had no effect. Taken together, these are the first studies that demonstrate that the depletion of endogenous IL-10 via anti-IL-10 antibody results in a very significantly enhanced BCG-induced IL-1 beta secretion and that the addition of exogenous IL-10 to human mononuclear cells stimulated with BCG inhibits IL-1 beta production. Further experimental work is needed to determine if the neutralization of IL-10 activity via anti-IL-10 antibody indeed enhances cytokine synthesis in vivo. However, the present results may be of importance, since the use of anti-IL-10 antibody could presumably contribute to the protective immunity induced by BCG against tuberculosis via an increase in cytokine synthesis that would amplify antimicrobial systems.

Interleukin-4 inhibits secretion of interleukin-1beta in the response of human cells to mycobacterial heat shock proteins.

Cellular activation induced by Mycobacterium bovis bacillus Calmette-Guérin (BCG) and heat shock proteins (HSP) leads to the production of proinflammatory cytokines such as interleukin-1beta (IL-1beta) and IL-6. In this study, we found that IL-4 significantly suppressed IL-1beta secretion induced by BCG and the 70- and 65-kDa HSP. When exogenous recombinant human IL-4 was added to human mononuclear cells, a dose- and time-related inhibition of the 70-kDa HSP- and BCG-induced IL-1beta secretion was observed. IL-1beta secretion was maximally inhibited at 24 h of culture, and this inhibitory effect was sustained at a later time point of culture (120 h). In addition, IL-2, another T-cell-derived cytokine acting on monocytes, had no effect on IL-1beta secretion induced by either BCG or the 70-kDa HSP, indicating that in these experiments not all cytokines could immunoregulate IL-1beta secretion. This inhibitory effect was not due to a cytotoxic effect of IL-4, since the viabilities of human mononuclear cells were comparable in the presence and absence of IL-4. IL-4 was also able to inhibit the secretion of IL-1beta by mycobacterium-stimulated cells from three rheumatoid arthritis patients. This inhibitory effect of IL-4 was reversed with a blocking anti-IL-4 antibody. Finally, IL-4 inhibited IL-6 secretion by mycobacterium-activated human cells. These results suggest that IL-4 may be important in the regulation of the immune response to mycobacterial antigens.

Cellular activation induced by BCG is a PTK-dependent event.

Mycobacterial antigens including BCG stimulate human peripheral blood mononuclear cells resulting in cellular proliferation and the release of inflammatory cytokines such as TNF-alpha. However, the signal transduction mechanisms responsible for the BCG-induced cell activation are not completely understood. In this study, we investigated the role of PTK as a signal transduction pathway in BCG-induced cell activation, with the use of two PTK inhibitors (genistein and tyrphostin). Our results indicated that genistein significantly inhibited BCG-induced cell growth determined by thymidine uptake in a dose-dependent manner. BCG-induced TNF-alpha secretion was completely suppressed by genistein in a dose-dependent manner, producing 92% inhibition at a concentration of 50 microM. In addition, strong inhibition (81%) of BCG-induced TNF-alpha secretion was observed with tyrphostin (30 microM), another specific protein tyrosine kinase with a different mechanism of action. These inhibitory effects were not attributed to an alteration in cell viability as judged by trypan blue staining, and were not due to LPS contamination. On the other hand, monoclonal antibodies directed against HLA-DR and DQ inhibited the BCG-induced secretion of TNF-alpha. Taken together, these findings suggest that PTK may play an essential role in BCG-induced cellular activation.

Down-modulation of mycobacterial-induced IL-1 beta production in human mononuclear cells by IL-4.

Tuberculosis is characterized by a cellular immune response mediated by various cytokines, including IL-1 beta released by stimulated mononuclear cells. It is now well established that IL-I beta plays an important role in local and systemic inflammatory response in tuberculosis. Here we have demonstrated, for the first time, that addition to IL-4 to human mononuclear cells obtained from 11 healthy bacille Calmette-Guérin (BCG)-vaccinated donors reduced BCG-induced production of IL-1 beta by 91.46 +/- 2.2%. This inhibitory effect was found highly significant (P less than 0.001) and was dose-dependent. The effect of IL-4 on the secretion of IL-1 beta was specific, since a complete reversion was obtained with a neutralizing MoAb to human IL-4. In addition, this inhibitory effect was not attributed to a cytotoxic effect, since trypan blue exclusion studies indicated no loss of cell viability in response to IL-4. Interestingly, the induction of IL-1 beta was regulated by IL-4, at least in part, by direct mechanism mediated through the extracellular domain of the 130-kD IL-4 receptor, as demonstrated by incubation of the mononuclear cells with the neutralizing anti-IL-4 receptor MoAb. Finally, a significant down-regulation of IL-1 beta secretion was observed in hsp70-stimulated mononuclear cells cultured with IL-4. Further experimental work is needed to establish the relevance of IL-4 in human mycobacterial infection in vivo. However, an understanding of the mechanisms that control IL-1 beta secretion in human mycobacterial infections is essential to understand the pathogenesis of tuberculosis.