Microglial cell-mediated anti-Candida activity: temperature, ions, protein kinase C as crucial elements.

An in vitro established microglial cell line, BV-2, constitutively exhibits high levels of anti-Candida activity. To elucidate the cascade of events leading to the accomplishment of such activity, we studied its dependence on temperature and ion availability. The role of protein kinases has also been studied by the specific inhibitors, 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H7) and N-(2-guanidinoethyl)-5-isoquinoline sulfonamide hydrochloride (HA 1004). We found that (a) the BV-2 cell/Candida conjugate formation is a discrete step, temperature-, ion- and protein kinase-independent; (b) the phagocytic event, which is protein kinase-independent, is significantly impaired by temperature decrease and ion deprivation; (c) the fulfillment of anti-Candida effects is strictly dependent upon temperature, ion availability and functional protein kinase. Functional protein kinase C, but not other kinases, is required for the accomplishment of anti-Candida activity, which, in fact, is selectively abrogated by H7 but not HA. Furthermore, protein kinase C activators, such as 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1-oleoyl-2-acetyl glycerol (OAG), consistently potentiate BV-2 cell-mediated anti-Candida activity, the phenomena being dose-dependent. These results indicate that the multistep events leading a microglial cell to express anti-Candida activity can be dissected and differentiated for biochemical and biological demands, the latest along the cascade being the most demanding steps

Effects of Cupral® on the formation and persistence of microbial biofilms in vitro

Introduction: endodontic biofilm is a microbial community, enclosed in a polymeric matrix of polysaccharide origin where are frequently found pathogenic microorganisms, such as Gram+, Gram- and opportunistic fungi, belonging to Candida spp, responsible for several endodontic pathologies. As clinical importance is the fact that biofilm is extremely resistant to common intra-canal irrigants, antimicrobial drugs and host immune defenses. The aim of this in vitro study was to evaluate the efficacy of Cupral® on planktonic forms of some pathogens, as well as to assess its ability to prevent and affect the formation/persistence of microbial biofilms. Materials and Methods: ATCC strains of S. aureus, P. aeruginosa and C. albicans were exposed to various concentrations of Cupral® (an antiseptic compound based on calcium and copper hydroxide, used in endodoncy) to investigate its antimicrobial efficacy. This activity has been evaluated in terms of microbial growth and cellular doubling time (optical density, colony forming units and doubling time assays), inhibition/persistence (crystal violet staining), viability of microbial cells embedded in the biofilms (live/dead stain) and pyoverdine production (fluorimetric assay). Finally, the morphology of Cupral®-treated biofilms was investigated by optical/confocal microscopy analysis. Results: the addition of Cupral® to microbial cultures, influences, in a significantly and dose-dependent manner, the doubling time and growth of microbial cultures. Cupral® antimicrobial activity was also assessed on biofilms formation and persistence with meaningful decreases of residual biomass (observed reductions of 47-94% for S. aureus, 28-95% for P. aeruginosa and 27-75 % for C. albicans). Cupral®-treated biofilms analyzed by optical and confocal microscopy revealed loss of typical sessile structure, with few scattered microbial cells and a reduced thickness. Finally, the addition of Cupral® reduced both the number of embedded alive cells in the biofilms and the levels of pyoverdine in the culture supernatants. Discussion and Conclusions: this pilot in vitro study provided the first evidences on Cupral® efficacy against microbial biofilms. The wide range of action (vs Gram+, Gram- and fungi) of Cupral® strongly suggests its use as compound in the prevention and treatment of main oral biofilm-associated infections

Candida albicans hyphal form enhances tumor necrosis factor mRNA levels and protein secretion in murine ANA-1 macrophages.

We have demonstrated that Candida albicans in its hyphal form (H-Candida) acts as a stimulating agent in the cloned macrophage population ANA-1. Both tumor necrosis factor (TNF) mRNA levels and secreted biological activity augment in ANA-1 macrophages exposed to H-Candida. Such effects are observed at an effector-to-target cell ratio of 1:1 and occur after 1 and 3 hr of coincubation, respectively. The phenomenon is independent of the metabolic status of the fungus, since viable as well as heat-killed H-Candida are comparable in inducing TNF mRNA levels. The extent and kinetics of H-Candida-mediated effects are similar to those observed following exposure of ANA-1 macrophages to lipopolysaccharide (LPS). This implies that C. albicans in its hyphal form is a potent macrophage modulator; whether it acts through the same mechanism(s) as LPS remains to be elucidate

Interaction between <i>Mycobacterium tuberculosis</i>, <i>Mycobacterium bovis</i>, <i>Mycobacterium avium</i> subspecies <i>paratuberculosis</i> with the enteric glia and microglial cells

Background We investigated the interaction of Mycobacterium avium subspecies paratuberculosis, M. bovis and M. tuberculosis and different glial cells (enteric glial and microglial cells) in order to evaluate the infecting ability of these microorganisms and the effects produced on these cells, such as the evaluation of cytokines expression. Results Our experiments demonstrated the adhesion of M. paratuberculosis to the enteroglial cells and the induction of IL-1A and IL-6 expression; M. tuberculosis and M. bovis showed a good adhesive capability to the enteric cell line with the expression of the following cytokines: IL-1A and IL-1B, TNF-α, G-CSF and GM-CSF; M. bovis induced the expression of IL-6 too. The experiment performed with the microglial cells confirmed the results obtained with the enteroglial cells after the infection with M. tuberculosis and M. bovis, whereas M. paratuberculosis stimulated the production of IL-1A and IL-1B. Conclusion Enteroglial and microglial cells, could be the target of pathogenic mycobacteria and, even if present in different locations (Enteric Nervous System and Central Nervous System), show to have similar mechanism of immunomodulation

The Propionibacterium spp. extract reduces Candida albicans-induced damage to vaginal epithelial cells and increases mitochondrial response to Candida albicans infection in vitro

Introduction. Bacterial lysates are prepared by inactivated microorganisms and are extensively employed in clinical settings as immunomodulants and to improve mucosal immunity. However, despite their extensive clinical use, their effects on the host are only partially known. The Propionibacterium spp. extract (PE) is a bacterial lysate included as an active compound in a gel formulation used to treat the symptoms of vulvovaginal candidiasis. Here, we analyzed its possible beneficial effects in an in vitro model of vaginal epithelial cells infected with Candida. Materials and Methods. Initially, we analyzed the PE effects on C. albicans and C. parapsilosis growth by the microdilution method. We then assessed the capacity of PE to reduce C. albicans-induced damage of vaginal epithelial cells through the quantification of lactate-dehydrogenase released by damaged cells in the growth medium. Moreover, in order to test the capacity of the PE to modulate epithelial mitochondrial activity, we evaluated Reactive-Oxygen-Species (ROS) production by the infected epithelial cells, stimulated or not with PE. This was kinetically monitored through the analysis of emitted fluorescence, after addition of the MitoSOX Red probe. Results. Our results show that PE did not affect directly microbial growth. In addition, the epithelial cells stimulation with PE reduced C. albicans-induced cell damage. Moreover, the treatment with PE increased the epithelial cells mitochondrial activity in response to C. albicans infection in vitro. Discussion and Conclusions. Taken together, our results show that PE increases ROS production by epithelial cells in response to C. albicans infection. Therefore, our results suggest that the increased mitochondrial activity induced by PE, could protect epithelial cells against the damage induced by C. albicans infection

In vitro and ex vivo studies on the antibacterial efficacy of sodium hypochlorite and two new generation endodontic irrigants, Tetraclean® and MTAD, in comparison with sodium hypochlorite.

The aim of this work was to compare the efficacy of two endodontic iorrigants of new generation, Tetraclean and MTAD. Their antimicrobial effectiveness was assessed by in vitro and in vivo studies. Sodium hypochlorite was included as standard reference irrigant

Compounds released from Lactobacillus (L.) acidophilus, L. plantarum, L. rhamnosus and L. reuteri inhibit Candida parapsilosis pathogenic potential after infection of vaginal epithelial cells in vitro.

INTRODUCTION. Lactobacillus spp. are the most represented microorganisms in the vaginal microbiota of healthy women, where they provide a shelter against infections from several pathogens, such as the yeasts belonging to the genus Candida. The latter are responsible for the vulvovaginal candidiasis (VVC), a condition affecting up to 75% of women during their child-bearing age at least once in their lifetime. Moreover, 5-8% of such women develop the recurrent form of the disease (RVVC), consisting of at least 5 VVC episodes per year. Notwithstanding C. albicans is the main responsible of VVC cases, in the last decades, the incidence of VVC cases by non-albicans Candida (NAC) species has become prevalent, especially in some geographical areas. C. parapsilosis, in particular, has been reported to be second species most commonly isolated from women affected by VVC. However, little is known on this species, and on its role in the pathogenesis of VVC. MATERIALS AND METHODS. Cell-free supernatants (CFS) were obtained following an overnight culture of 4 different Lactobacilli species (L. acidophilus, L. plantarum, L. rhamnosus, L. reuteri). Lactobacilli-released compounds, contained in CFS, were assessed for their effect on several virulence factors of C. parapsilosis (strain CLIB214), such as growth rate, capacity to form pseudohyphae, capacity to adhere to a vaginal epithelium in vitro (A-431 cells monolayer) and to induce cell damage. The latter was evaluated by measuring lactate dehydrogenase (LDH) release from A431 cells. RESULTS. C. parapsilosis growth inhibition by L. acidophilus, L. plantarum and L. reuteri CFS was 47%, 55% and 52% respectively, whereas L. rhamnosus CFS effect was weaker (33% inhibition growth). All the Lactobacilli significantly inhibited C. parapsilosis adhesion to vaginal epithelial cells: upon incubation with CFS, only 5-7% of fungal cells adhered to epithelial cells, after 90 minutes incubation; differently, the adhesion of the control reached 19%. Interestingly, no effect on pseudohyphae formation by any of the CSF was ever observed. Finally, the C. parapsilosis-induced damage on A-431 cells was significantly reduced by the addition of the CSF. DISCUSSION AND CONCLUSIONS. Our results show that the investigated species of Lactobacilli release compounds capable to impair several C. parapsilosis virulence factors, such as growth rate and adhesion to vaginal epithelial cells; interestingly, while not affecting fungal capacity to form pseudohyphae, such compounds significantly reduce Candida-mediated epithelial damage.. These data suggest that, in the context of vaginal microbiota, these Lactobacilli species may play an important role in counteracting the onset of mucosal Candida infections

Prep1.1 has essential genetic functions in hindbrain development and cranial neural crest cell differentiation

In this study we analysed the function of the Meinox gene prep1.1 during zebrafish development. Meinox proteins form heterotrimeric complexes with Hox and Pbx members, increasing the DNA binding specificity of Hox proteins in vitro and in vivo. However, a role for a specific Meinox protein in the regulation of Hox activity in vivo has not been demonstrated. In situ hybridization showed that prep1.1 is expressed maternally and ubiquitously up to 24 hours post-fertilization (hpf), and restricted to the head from 48 hpf onwards. Morpholino-induced prep1.1 loss-of-function caused significant apoptosis in the CNS. Hindbrain segmentation and patterning was affected severely, as revealed by either loss or defective expression of several hindbrain markers ( foxb1.2/mariposa , krox20 , pax2.1 and pax6.1 ), including anteriorly expressed Hox genes ( hoxb1a , hoxa2 and hoxb2 ), the impaired migration of facial nerve motor neurons, and the lack of reticulospinal neurons (RSNs) except Mauthner cells. Furthermore, the heads of prep1.1 morphants lacked all pharyngeal cartilages. This was not caused by the absence of neural crest cells or their impaired migration into the pharyngeal arches, as shown by expression of dlx2 and snail1 , but by the inability of these cells to differentiate into chondroblasts. Our results indicate that prep1.1 has a unique genetic function in craniofacial chondrogenesis and, acting as a member of Meinox-Pbc-Hox trimers, it plays an essential role in hindbrain development

In vitro analysis of epithelial tolerability and anti-Candida effect of a new lactic acid-based vaginal gel formulation

INTRODUCTION. Vulvovaginal candidiasis (VVC) is the most prevalent vaginal infection in adult women. It is mainly caused by Candida albicans, and it affects 75% of healthy women at least once during their reproductive age; 5-10% of such women have recurrent episodes (RVVC), with more of 4 episodes of acute VVC per year. Symptoms of VVC include itching, burning, swelling and redness of the vaginal mucosa with white vaginal discharge. The urinary system can also be affected, with pain and burning when urinating. This condition seriously damages the well-being and the life quality of the affected women. Since Candida is a commensal fungus of the vaginal mucosa of healthy women, the main question is how the fungus can switch from harmless component of the vaginal microbiota to virulent pathogen. In this work we analyzed the capacity of lactic acid-based vaginal gel formulation Respecta® Balance Gel (RBG) to counteract C. albicans virulence after epithelial cells infection in vitro. MATERIALS AND METHODS. For the establishment of the in vitro infection model, we used a monolayer of the A-431 vaginal epithelial cell line and two different strains of C. albicans (strain SC5314 and the bioluminescent strain gLUC59). Dose-dependent experiments were performed to test the epithelial tolerability to RBG (IHS srl, Biofarma Group) by monitoring lactate-dehydrogenase (LDH) release from damaged cells. The capacity of RGB to counteract Candida-induced epithelial damage were analysed by monitoring LDH release from cells. Fungal growth and adhesion capacity during vaginal epithelial cells infection in the presence of RGB were evaluated by quantify the Relative Luminescent Units (RLU) and CFU counts, respectively. RESULTS. Our results show that, at dilution 1:150, RGB is well tolerated by the vaginal epithelium and consequently we used this dose for the subsequent experiments. RBG was able to significantly reduce (by 65%) C. albicans-induced damage of vaginal epithelial cells. This effect was accompanied with the capacity of RGB to significantly reduce Candida adhesion to the epithelium (adhesion reduction by 34%). Intriguingly, no inhibition of fungal growth was observed after 24h of infection in the presence of RGB in our experimental conditions. DISCUSSION AND CONCLUSIONS. Our results show that RGB significantly reduce C. albicans-induced damage of vaginal epithelial cells. One of the mechanisms underlying this effect is the inhibition of C. albicans adhesion to the vaginal epithelial cells, which may prevent Candida from penetrating and damaging epithelial cells, hence counteract Candida virulence. Collectively our preliminary results suggest that RBG can strengthen the VVC therapy favoring the establishment of an ecosystem that prevent Candida virulence

Herpes Simplex Virus-1 entrapped in Candida albicans biofilm displays decreased sensitivity to antivirals and UVA1 laser treatment

Abstract Background: Recently, we published data suggesting a mutualistic relationship between HSV-1 and Candida. albicans; in particular: (a) HSV-1 infected macrophages are inhibited in their anti-Candida effector function and (b) Candida biofilm protects HSV-1 from inactivation. The present in vitro study is aimed at testing the effects of Candida biofilm on HSV-1 sensitivity to pharmacological and physical stress, such as antiviral drugs (acyclovir and foscarnet) and laser UVA1 irradiation. We also investigated whether fungus growth pattern, either sessile or planktonic, influences HSV-1 sensitivity to antivirals. Methods: Mature Candida biofilms were exposed to HSV-1 and then irradiated with laser light (UVA1, 355 \u3bb). In another set of experiments, mature Candida biofilm were co-cultured with HSV-1 infected VERO cells in the presence of different concentrations of acyclovir or foscarnet. In both protocols, controls unexposed to laser or drugs were included. The viral yield of treated and untreated samples was evaluated by end-point titration. To evaluate whether this protective effect might occur in relation with a different growth pattern, HSV-1 infected cells were co-cultured with either sessile or planktonic forms of Candida and then assessed for susceptibility to antiviral drugs. Results: UVA1 irradiation caused a 2 Log reduction of virus yield in the control cultures whereas the reduction was only 1 Log with Candida biofilm, regardless to the laser dose applied to the experimental samples (50 or 100 J/cm2). The presence of biofilm increased the IC90 from 18.4\u201325.6 J/cm2. Acyclovir caused a 2.3 Log reduction of virus yield in the control cultures whereas with Candida biofilm the reduction was only 0.5 Log; foscarnet determined a reduction of 1.4 Log in the controls and 0.2 Log in biofilm cultures. Consequently, the ICs50 for acyclovir and foscarnet increased by 4- and 12-folds, respectively, compared to controls. When HSV-1 was exposed to either sessile or planktonic fungal cells, the antiviral treatments caused approximately the same weak reduction of virus yield. Conclusions: These data demonstrate that: (1) HSV-1 encompassed in Candida biofilm is protected from inactivation by physical (laser) and pharmacological (acyclovir or foscarnet) treatments; (2) the drug antiviral activity is reduced at a similar extent for both sessile or planktonic Candida