Polyphenols journey through blood-brain barrier towards neuronal protection.

Age-related complications such as neurodegenerative disorders are increasing and remain cureless. The possibility of altering the progression or the development of these multifactorial diseases through diet is an emerging and attractive approach with increasing experimental support. We examined the potential of known bioavailable phenolic sulfates, arising from colonic metabolism of berries, to influence hallmarks of neurodegenerative processes. In silico predictions and in vitro transport studies across blood-brain barrier (BBB) endothelial cells, at circulating concentrations, provided evidence for differential transport, likely related to chemical structure. Moreover, endothelial metabolism of these phenolic sulfates produced a plethora of novel chemical entities with further potential bioactivies. Pre-conditioning with phenolic sulfates improved cellular responses to oxidative, excitotoxicity and inflammatory injuries and this attenuation of neuroinflammation was achieved via modulation of NF-κB pathway. Our results support the hypothesis that these small molecules, derived from dietary (poly)phenols may cross the BBB, reach brain cells, modulate microglia-mediated inflammation and exert neuroprotective effects, with potential for alleviation of neurodegenerative diseases.

Blocking the receptor for IL-10 improves antimycobacterial chemotherapy and vaccination.

Novel approaches are required for the prevention and therapy of mycobacterial infections since the only vaccine in use, bacillus Calmette-Guérin, is poorly effective and chemotherapy is long and often ineffective in sterilizing the infection. We used a mouse model of Mycobacterium avium infection to address the usefulness of a mAb able to block IL-10R both in treatment of primary infections and in conventional multidrug therapy and subunit vaccination. Treatment of infected mice with this mAb during the entire period of experimental infection had little impact on the course of M. avium infection, with a slight improvement in the resistance of infected mice observed in the liver and spleen at day 30 of infection, which was associated with increased macrophage activation and priming of CD4(+) T cells for IFN-gamma production. Administration of this mAb later in infection had no effect on its course, but improved the effectiveness of chemotherapy when the latter was started in a chronic phase of infection. Also, the anti-IL-10R mAb acted as an adjuvant in the induction of protective immunity upon vaccination with a mycobacterial subunit preparation.

Effects of interleukin-12 in the long-term protection conferred by a Mycobacterium avium subunit vaccine.

The effects of the addition of recombinant interleukin (IL)-12 to a mycobacterial subunit vaccine were analyzed in terms of the longevity of the protective immunity generated. BALB/c mice were immunized with culture filtrate proteins from Mycobacterium avium with dimethyl-dioctadecilammonium bromide (DDA) as an adjuvant. This subunit vaccine induced protection against a challenge by M. avium which lasted for at least 6 months while waning with time until 1 year postvaccination. Whereas the addition of IL-12 enhanced the initial protective efficacy of this subunit vaccine during the first 6 months, it accelerated the loss of protective efficacy observed at 1 year postvaccination. These data confirm the adjuvant properties of IL-12 in vaccines against mycobacteria and raise the possibility of late counter-protective untoward effects.

Antigen specificity of T-cell response to Mycobacterium avium infection in mice.

T cells from Mycobacterium avium-infected C57BL/6 mice reacted to culture filtrate, envelope, and cytosol proteins and to fractions obtained from these proteins. Multiple targets were recognized, such as 29- to 45-kDa and <21-kDa antigens of the culture filtrate, antigens of around 30 kDa in the envelope and cytosol, and 45- to 116-kDa proteins in the envelope.

Differences in resistance of C57BL/6 and C57BL/10 mice to infection by Mycobacterium avium are independent of gamma interferon.

After infection with a low-virulence strain of Mycobacterium avium, C57BL/6 and C57BL/10 mice had clear differences in the control of the infection in their livers and spleens. This difference in susceptibility was not associated with differences in the H-2 complex. It was dependent on the activity of CD4(+) T cells but unrelated to the ability of these cells to secrete gamma interferon or to the development of delayed-type hypersensitivity responses at 3 weeks of infection. It was associated with lower total numbers of CD4(+) cells present in infected spleens and was related to an earlier induction of protective T cells, as measured by adoptive-transfer assays. These data further strengthen the notion of gamma-interferon-independent mechanisms of protection against mycobacteria.

Macrophage control of mycobacterial growth induced by picolinic acid is dependent on host cell apoptosis.

The effects of picolinic acid (PA) on the intramacrophagic growth of Mycobacterium avium were studied. PA reduced M. avium growth inside mouse macrophages and led to a complete control of mycobacterial growth when added together with IFN-gamma. The mechanism involved did not require TNF-alpha, NO, or the respiratory burst, and was not dependent on either iron or zinc withholding. The mycobacteriostatic activity of the macrophages was associated with the induction of morphological changes that culminated in apoptosis at day 4 of treatment. PA alone induced apoptosis in macrophages, and this effect was increased by IFN-gamma treatment. Apoptosis at day 4 of infection was reduced by inhibiting macrophage activation with the prostaglandin 15 deoxy-prostaglandin J2 or by treating the cells with the antioxidant N-acetylcysteine. Mycobacterial growth was partially restored in macrophages treated with PA and IFN-gamma when 15 deoxy-prostaglandin J2 was added, concomitant with a delay in apoptosis. N-Acetylcysteine or glutathione could also completely revert the mycobacteriostatic effects of PA or PA plus IFN-gamma.

Improved clearance of Mycobacterium avium upon disruption of the inducible nitric oxide synthase gene.

Mice genetically deficient in the inducible NO synthase gene (iNOS-/-) were used to study the role played by NO during infection by Mycobacterium avium. iNOS-/- macrophages were equally able to restrict M. avium growth in vitro following stimulation by IFN-gamma and TNF-alpha as macrophages from wild-type mice. In vivo, the infection progressed at similar rates in wild-type and NO-deficient mice during the first 2 mo of infection, but the latter mice were subsequently more efficient in clearing the mycobacteria than the former. The increased resistance of iNOS-/- mice was associated with higher IFN-gamma levels in the serum and following in vitro restimulation of spleen cells with specific Ag, increased formation of granulomas and increased survival of CD4+ T cells. We show that NO is not involved in the antimycobacterial mechanisms of M. avium-infected macrophages and, furthermore, that it exacerbates the infection by causing the suppression of the immune response to the pathogen.

Evaluation of IL-12 in immunotherapy and vaccine design in experimental Mycobacterium avium infections.

IL-12 is a pivotal cytokine in the induction of IFN-gamma-mediated protective immune responses. We tested the effects of rIL-12 administration to Mycobacterium avium-infected mice and found a limited ability to induce protection against the infection; this ability varied according to the mycobacterial strain studied. IL-12 accelerated the expression and production of IFN-gamma in both immunocompetent and immunodeficient SCID or CD4-depleted mice. Evidence of NK cell activation was found as well as an enhancement of the ability to adoptively transfer resistance with T cell-enriched spleen cell populations and an increase in inflammatory cell recruitment in the liver. The protective ability of IL-12 was dependent upon the endogenous production of IFN-gamma as evaluated by the use of specific neutralizing Abs or IFN-gamma gene-disrupted mice. IL-12 potentiated the protective immunity conferred by a subunit vaccine containing M. avium culture filtrate proteins and dimethyl dioctadecyl ammonium chloride as an adjuvant. Thus, we show limited immunotherapeutic benefits from IL-12 administration in M. avium infections and promising results in its use as a coadjuvant in vaccine design.

Analysis of T cells recruited during delayed-type hypersensitivity to purified protein derivative (PPD) versus challenge with tuberculosis infection.

The delayed-type hypersensitivity (DTH) to purified protein derivative (PPD) test has been used to infer about protective immunity to Mycobacterium tuberculosis and to diagnose tuberculosis. We showed that in memory tuberculosis-immune mice both DTH to PPD and resistance to M. tuberculosis could be effectively elicited in the footpad and both reactions led to the accumulation of reactive T cells in the regional lymph nodes with a CD4+ phenotype and characterized by the secretion of high levels of interleukin-2 (IL-2) and interferon-gamma (IFN-gamma) and no IL-4. By adoptive transfer into nude mice of highly purified CD4+ T cells harvested during the recall of protective immunity it was confirmed that this population mediated both manifestations. However, the specificity of the T cells recruited during these processes were found to differ markedly; T cells involved in protection to a challenge with live tuberculosis bacilli recognized predominantly low-mass culture filtrate antigens below 15 000 MW, while cells recruited during DTH to PPD were directed to molecular mass fractions between 15 000 and 31 000. Using single purified antigens we showed that the latter cells recognized the secreted mycobacterial protein Ag85B and the heat-shock proteins, DnaK and GroEL. Protective T cells, in contrast, were characterized by a very high frequency of T cells directed to the ESAT-6 peptide 1-20.