BackgroundVaginal epithelial cells have receptors, signal transduction mechanisms, and cytokine secretion capabilities to recruit host defenses against Candida albicans infections. This research evaluates how probiotic lactobacilli affect the defensive epithelial response.MethodsThis study used quantitative reverse transcription-polymerase chain reaction assay (qRT-PCR), flow cytometry, and a multiplex immunoassay to observe changes in the regulation of gene expression related to cytokine responses in the VK2 (E6/E7) vaginal epithelial cell line treated with 17β-estradiol, exposed to probiotic Lactobacillus rhamnosus GR-1® and Lactobacillus reuteri RC-14® and challenged with C. albicans. Data were statistically evaluated by repeated measures analysis of variance and paired t-tests where appropriate.ResultsC. albicans induced mRNA expression of genes related to inflammatory cytokine responses associated with nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinase (MAPK) signal transduction pathways. 17β-estradiol suppressed expression of interleukin-1α (IL-1α), IL-6, IL-8, and tumor necrosis factor alpha (TNFα) mRNA. Probiotic lactobacilli suppressed C. albicans-induced nuclear factor-kappa B inhibitor kinase kinase alpha (Iκκα), Toll-like receptor-2 (TLR2), TLR6, IL-8, and TNFα, also suggesting inhibition of NF-κB signaling. The lactobacilli induced expression of IL-1α, and IL-1β mRNA, which was not inhibited by curcumin, suggesting that they induce an alternate inflammatory signal transduction pathway to NF-κB, such as the mitogen activated protein kinase and activator protein-1 (MAPK/AP-1) signal transduction pathway. Curcumin inhibited IL-13 secretion, suggesting that expression of this cytokine is mainly regulated by NF-κB signaling in VK2 cells.ConclusionsThe results suggest that C. albicans infection induces pro-inflammatory responses in vaginal epithelial cells, and estrogen and lactobacilli suppress expression of NF-κB-related inflammatory genes. Probiotic lactobacilli may induce IL-1α and IL-1β expression by an alternate signal transduction pathway, such as MAPK/AP-1. Activation of alternate signaling mechanisms by lactobacilli to modify epithelial cell cytokine production may be a mechanism for probiotic modulation of morbidity in vulvovaginal candidiasis.

A defined human microbiota-associated (HMA) mouse model in BALB/c and immunodeficient Tgepsilon26 mice was used to assess the ability of probiotic lactobacilli and bifidobacteria to enhance colonization resistance to gastrointestinal (GI) tract pathogens. Probiotic bacteria (1x10(8) colony forming unit (CFU)/mL) successfully excluded Campylobacter jejuni from both strains of mice 7 days after challenge. The probiotic bacteria also reduced the number of Salmonella in the large intestines of both mouse strains. The nylon wool fractionated spleen lymphocyte populations were incubated with Salmonella or C. jejuni antigens. The probiotic treatments did not affect lymphocyte proliferation to C. jejuni antigens, but significantly increased proliferation of lymphocytes to Salmonella antigens by 68 and 55%, respectively, over untreated mice. Caspase 3/7 activation was significantly reduced 33 and 38% in the T and B lymphocyte fractions, respectively, of probiotic-treated, Salmonella-challenged HMA BALB/c mice, suggesting that lymphocyte rescue from apoptosis was occurring as a result of probiotic bacteria activity. These results revealed an immunosuppressive activity by Salmonella that was inhibited by the presence of probiotic bacteria. In summary, lactobacilli and bifidobacteria competitively excluded C. jejuni from immunocompetent and immunodeficient mice and antagonized an observable Salmonella-induced immunosuppression in immunocompetent mice.

The veterinary cephalosporin drug ceftiofur is rapidly degraded in the bovine intestinal tract. A cylinderplate assay was used to detect microbiologically active ceftiofur, and high-performance liquid chromatographymass spectrometry analysis was used to quantify the amount of ceftiofur remaining after incubation with bovine intestinal anaerobic bacteria, which were isolated from colon contents or feces from 8 cattle. Ninety-six percent of the isolates were able to inactivate ceftiofur to some degree, and 54% actually degraded the drug. None of 9 fungal isolates inactivated or degraded ceftiofur. Facultative and obligate anaerobic bacterial species that inactivated or degraded ceftiofur were identified with Vitek and Biolog systems, respectively. A subset of ceftiofur degraders also degraded the chemically similar drug ceftriaxone. Most of the species of bacteria that degraded ceftiofur belonged to the genera Bacillus and Bacteroides. PCR analysis of bacterial DNA detected specific ␤-lactamase genes. Bacillus cereus and B. mycoides isolates produced extended-spectrum ␤-lactamases and metallo-␤-lactamases. Seven isolates of Bacteroides spp. produced multiple ␤-lactamases, including possibly CepA, and metallo-␤-lactamases. Isolates of Eubacterium biforme, Bifidobacterium breve, and several Clostridium spp. also produced ceftiofur-degrading ␤-lactamases. An agar gel overlay technique on isoelectric focusing separations of bacterial lysates showed that ␤-lactamase enzymes were sufficient to degrade ceftiofur. These results suggest that ceftiofur is inactivated nonenzymatically and degraded enzymatically by multiple ␤-lactamases from bacteria in the large intestines of cattle.

Mucous-penetrating nanoparticles consisting of poly lactic acid-co-glycolic acid (PLGA)-polyethylene glycol (PEG) could improve targeting of microbicidal drugs for sexually transmitted diseases by intravaginal inoculation. Nanoparticles can induce inflammatory responses, which may exacerbate the inflammation that occurs in the vaginal tracts of women with yeast infections. This study evaluated the effects of these drug-delivery nanoparticles on VK2(E6/E7) vaginal epithelial cell proinflammatory responses to Candida albicans yeast infections. Vaginal epithelial cell monolayers were infected with C. albicans and exposed to 100 μg/ml 49.5 nm PLGA-PEG nanospheres or 20 μg/ml 1.1 x 500 nm PEG-functionalized graphene oxide (GO-PEG) sheets. The cells were assessed for changes in mRNA and protein expression of inflammation-related genes by RT-qPCR and physiological markers of cell stress using high content analysis and flow cytometry. C. albicans exposure suppressed apoptotic gene expression, but induced oxidative stress in the cells. The nanomaterials induced cytotoxicity and programmed cell death responses alone and with C. albicans. PLGA-PEG nanoparticles induced mRNA expression of apoptosis-related genes and induced poly (ADP-ribose) polymerase (PARP) cleavage, increased BAX/BCL2 ratios, and chromatin condensation indicative of apoptosis. They also induced autophagy, endoplasmic reticulum stress, and DNA damage. They caused the cells to excrete inflammatory recruitment molecules chemokine (C-X-C motif) ligand 1 (CXCL1), interleukin-1α (IL1A), interleukin-1β (IL1B), calprotectin (S100A8), and tumor necrosis factor α (TNF). GO-PEG nanoparticles induced expression of necrosis-related genes and cytotoxicity. They reduced autophagy and endoplasmic reticulum stress, and apoptotic gene expression responses. The results show that stealth nanoparticle drug-delivery vehicles may cause intracellular damage to vaginal epithelial cells by several mechanisms and that their use for intravaginal drug delivery may exacerbate inflammation in active yeast infections by increased inflammatory recruitment.