Systemic administration of propranolol reduces bone resorption and inflammation in apical periodontitis of chronically stressed rats.

AIM: To evaluate the effect of systemic administration of propranolol on the severity of apical periodontitis (AP) in chronically stressed rats. METHODOLOGY: Twenty-four 70-day-old male Wistar rats (Rattus norvegicus, albinus) were distributed into three groups (n = 8): rats with AP without stressful conditions (AP-Control), rats with AP and submitted to a chronic unpredictable stress (CUS) protocol (AP + S) and rats with AP and submitted to a CUS protocol treated with propranolol (AP + S + PRO). Stress procedures were applied daily until the end of the experiment. After 3 weeks of CUS, AP was induced in all groups by exposing the pulpal tissue of mandibular and maxillary first molars to the oral environment. Propranolol treatment was administered orally once a day for the entire period of the experiment. Rats were sacrificed at 42 days, and the blood was collected for stress biomarkers serum dosage by multiplex assay. Mandibles were removed and submitted to microtomography and histopathological analyses. Periapical tissue surrounding the upper first molar was homogenized and subjected to RT-PCR analysis to evaluate the mRNA expression of RANKL, TRAP and OPG. Parametric data were assessed using one-way ANOVA followed by Tukey's test while the nonparametric data were analysed by the Kruskal-Wallis followed by Dunn's test. Significance level was set at 5% (p < .05) for all assessed parameters. RESULTS: Micro-CT revealed statistically significant differences in bone resorption which was greater in the AP + S group (p < .05), but no differences were observed between the Control and AP + S + PRO groups (p > .05). The AP + S + PRO group had a lower intensity and extent of inflammatory infiltrate compared to the AP + S group with smaller areas of bone loss (p < 0.05). The gene expression of RANKL and TRAP was significantly higher in the stressed group AP + S compared to the control group (p < .05), and a significantly higher OPG expression was observed in AP + S + PRO compared to the AP + S group (p < .05). CONCLUSIONS: Oral administration of propranolol had a significant effect on the AP severity in stressed rats, suggesting an anti-inflammatory effect and a protective role on bone resorption of AP in stressed animals. Further research is necessary to fully comprehend the underlying mechanisms.

Repurposing HIV Protease Inhibitors Atazanavir and Darunavir as Antifungal Treatments against Candida albicans Infections: An In Vitro and In Vivo Study.

Candida albicans is the chief etiological agent of candidiasis, a mycosis prevalent in individuals with acquired immunodeficiency syndrome (AIDS). In recent years, the introduction of human immunodeficiency virus (HIV) protease inhibitors (HIV-PI) has reduced the prevalence of candidiasis in these patients. Seeking new therapeutic strategies based on the perspective of drug repositioning, we evaluated the effects of two second-generation HIV-PIs, atazanavir (ATV) and darunavir (DRV), on virulence factors of C. albicans and experimental candidiasis. For this, clinical strains of C. albicans were subjected to in vitro and in vivo treatments with ATV or DRV. As a result, ATV and DRV exhibited antifungal activity against fungal cells at 512 µg/mL, reduced the viability and biomass of biofilms, and inhibited filamentation of C. albicans. In addition, these HIV-PIs downregulated the expression of SAP2 and BRC1 genes of C. albicans. In an in vivo study, prophylactic use of ATV and DRV prolonged the survival rate of Galleria mellonella larvae infected with C. albicans. Therefore, ATV and DRV showed activity against C. albicans by reducing cell growth, biofilm formation, filamentation, and expression of virulence genes. Furthermore, ATV and DRV decreased experimental candidiasis, suggesting the repurposing of HIV-PIs as antifungal treatments for C. albicans infections.

Essentials of oral manifestations of leukemia for the dental practitioner.

Oral lesions may be the initial or only manifestation of leukemia and can be the key to early diagnosis. The varied nature of presenting signs and dentists' general lack of familiarity with oral presentations makes diagnosis challenging. This retrospective review reports a series of cases of leukemia to familiarize dentists with the oral manifestations and facilitate earlier diagnosis or recognition of relapse of this life-threatening disease. Following institutional review board approval, the University of Florida Oral Pathology Biopsy Service archive from 1994 to 2018 was queried for all oral biopsies resulting in a diagnosis of leukemia. Cases with insufficient diagnostic information or extraoral manifestations were excluded. Demographic, clinical, and histologic findings were tabulated. Ten cases with 12 biopsy sites were identified. Men (n = 6) were affected more commonly. The mean age of the patients was 58.4 years (range of 17 to 88 years). The gingiva was the most frequently biopsied site (n = 6; 50%). Importantly, 40% of the patients (n = 4) had no prior diagnosis of leukemia. A wide spectrum of clinical impressions was rendered, pyogenic granuloma being the most common, and the reported duration of lesions ranged from several weeks to 6 months. The rarity of patients presenting with leukemia may lead to low levels of clinical suspicion, misdiagnosis, and delays in treatment. However, oral lesions may be the first and only manifestation of leukemia, and clinicians should be aware of the clinical characteristics of these oral presentations to ensure early diagnosis and treatment, thereby helping to reduce disease-related morbidity and mortality.

A SYK/SHC1 pathway regulates the amount of CFTR in the plasma membrane.

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause the recessive genetic disease cystic fibrosis, where the chloride transport across the apical membrane of epithelial cells mediated by the CFTR protein is impaired. CFTR protein trafficking to the plasma membrane (PM) is the result of a complex interplay between the secretory and membrane recycling pathways that control the number of channels present at the membrane. In addition, the ion transport activity of CFTR at the PM is modulated through post-translational protein modifications. Previously we described that spleen tyrosine kinase (SYK) phosphorylates a specific tyrosine residue in the nucleotide-binding domain 1 domain and this modification can regulate the PM abundance of CFTR. Here we identified the underlying biochemical mechanism using peptide pull-down assays followed by mass spectrometry. We identified in bronchial epithelial cells that the adaptor protein SHC1 recognizes tyrosine-phosphorylated CFTR through its phosphotyrosine-binding domain and that the formation of a complex between SHC1 and CFTR is induced at the PM in the presence of activated SYK. The depletion of endogenous SHC1 expression was sufficient to promote an increase in CFTR at the PM of these cells. The results identify a SYK/SHC1 pathway that regulates the PM levels of CFTR channels, contributing to a better understanding of how CFTR-mediated chloride secretion is regulated.

Inhibition of calpain 1 restores plasma membrane stability to pharmacologically rescued Phe508del-CFTR variant.

Cystic fibrosis (CF) is a genetic disease caused by mutations in the gene encoding CF transmembrane conductance regulator (CFTR), a chloride channel normally expressed at the surface of epithelial cells. The most frequent mutation, resulting in Phe-508 deletion, causes CFTR misfolding and its premature degradation. Low temperature or pharmacological correctors can partly rescue the Phe508del-CFTR processing defect and enhance trafficking of this channel variant to the plasma membrane (PM). Nevertheless, the rescued channels have an increased endocytosis rate, being quickly removed from the PM by the peripheral protein quality-control pathway. We previously reported that rescued Phe508del-CFTR (rPhe508del) can be retained at the cell surface by stimulating signaling pathways that coax the adaptor molecule ezrin (EZR) to tether rPhe508del-Na+/H+-exchange regulatory factor-1 complexes to the actin cytoskeleton, thereby averting the rapid internalization of this channel variant. However, the molecular basis for why rPhe508del fails to recruit active EZR to the PM remains elusive. Here, using a proteomics approach, we characterized and compared the core components of wt-CFTR- or rPhe508del-containing macromolecular complexes at the surface of human bronchial epithelial cells. We identified calpain 1 (CAPN1) as an exclusive rPhe508del interactor that prevents active EZR recruitment, impairs rPhe508del anchoring to actin, and reduces its stability in the PM. We show that either CAPN1 down-regulation or its chemical inhibition dramatically improves the functional rescue of Phe508del-CFTR in airway cells. These observations suggest that CAPN1 constitutes an appealing target for pharmacological intervention, as part of CF combination therapies restoring Phe508del-CFTR function.

Development of Probiotic Formulations for Oral Candidiasis Prevention: Gellan Gum as a Carrier To Deliver Lactobacillus paracasei 28.4.

Probiotics might provide an alternative approach for the control of oral candidiasis. However, studies on the antifungal activity of probiotics in the oral cavity are based on the consumption of yogurt or other dietary products, and it is necessary to use appropriate biomaterials and specific strains to obtain probiotic formulations targeted for local oral administration. In this study, we impregnated gellan gum, a natural biopolymer used as a food additive, with a probiotic and investigated its antifungal activity against Candida albicansLactobacillus paracasei 28.4, a strain recently isolated from the oral cavity of a caries-free individual, was incorporated in several concentrations of gellan gum (0.6% to 1% [wt/vol]). All tested concentrations could incorporate L. paracasei cells while maintaining bacterial viability. Probiotic-gellan gum formulations were stable for 7 days when stored at room temperature or 4°C. Long-term storage of bacterium-impregnated gellan gum was achieved when L. paracasei 28.4 was lyophilized. The probiotic-gellan gum formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the growth of C. albicans, with effects dependent on the cell concentrations incorporated into gellan gum. The probiotic-gellan gum formulations also had inhibitory activity against Candida sp. biofilms by reducing the number of Candida sp. cells (P < 0.0001), decreasing the total biomass (P = 0.0003), and impairing hyphae formation (P = 0.0002), compared to the control group which received no treatment. Interestingly, a probiotic formulation of 1% (wt/vol) gellan gum provided an oral colonization of L. paracasei in mice with approximately 6 log CFU/ml after 10 days. This formulation inhibited C. albicans growth (P < 0.0001), prevented the development of candidiasis lesions (P = 0.0013), and suppressed inflammation (P = 0.0006) compared to the mice not treated in the microscopic analysis of the tongue dorsum. These results indicate that gellan gum is a promising biomaterial and can be used as a carrier system to promote oral colonization for probiotics that prevent oral candidiasis.

Candida Biofilms: An Update on Developmental Mechanisms and Therapeutic Challenges.

Fungi of the genus Candida are important etiological agents of superficial and life-threatening infections in individuals with a compromised immune system. One of the main characteristics of Candida is its ability to form highly drug tolerance biofilms in the human host. Biofilms are a dynamic community of multiple cell types whose formation over time is orchestrated by a network of transcription regulators. In this brief review, we provide an update of the processes involved in biofilm formation by Candida spp. (formation, treatment, and control), as well as the transcriptional circuitry that regulates its development and interactions with other microorganisms. Candida albicans is known to build mixed species biofilms with other Candida species and with various other bacterial species in different host niches. Taken together, these properties play a key role in Candida pathogenesis. In addition, this review gathers recent studies with new insights and perspectives for the treatment and control of Candida biofilms.

Oral Colonization by Candida Species in Patients with Multiple Sclerosis.

INTRODUCTION: Multiple sclerosis (MS) is a chronic inflammatory demyelinating autoimmune disease that affects the central nervous system. Since immune system plays a key role in this disease, patients with MS can present higher risk of infections. PURPOSE: This study aimed to investigate the prevalence of Candida spp. in the oral cavity of MS patients in relation to a control group METHODS: In total, 100 individuals were selected: 55 diagnosed with MS and 45 healthy individuals (control group). Saliva samples were collected and seeded in culture media selecting for Candida. Following an incubation period of 48 h, colony-forming units (CFU mL-1) were counted and colonies were isolated for Candida species identification by multiplex PCR. The results were analysed by chi-squared and Mann-Whitney U statistical tests considering a significance level of 5%. RESULTS: Candida spp. were confirmed in the oral cavity of 50.09% patients in the MS group and 35.55% individuals in the control group. In individuals positive for the growth of Candida spp., the median values of Candida colonies were 220 CFU mL-1 for the MS group and 120 CFU mL-1 for the control group. However, no statistically significant differences were observed between groups for both prevalence and CFU mL-1 count. Of the Candida species identified, 73.91% were C. albicans, 21.73% C. glabrata, 2.17% C. tropicalis, and 2.17% C. krusei. CONCLUSIONS: The colonization of Candida spp. in the oral cavity of individuals with multiple sclerosis was higher than in the control group; however these findings were not proven to be statistically significant.

Two sporulated Bacillus enhance immunity in Galleria mellonella protecting against Candida albicans.

The aim of this study was to evaluate the effects of Bacillus subtilis and Bacillus atrophaeus on Galleria mellonella immunity challenged by Candida albicans. Firstly, we analyzed the susceptibility of G. mellonella to bacilli (vegetative and sporulating forms). It was found that both vegetative and sporulating forms were not pathogenic to G. mellonella at a concentration of 1 × 104 cells/larva. Next, larvae were pretreated with two species of Bacillus, in the vegetative and sporulating forms, and then challenged with C. albicans. In addition, the gene expression of antimicrobial peptides (AMPs) such as Gallerimycin, Gloverin, Cecropin-D and Galiomicin was investigated. Survival rates increased in the Bacillus treated larvae compared with control larvae inoculated with C. albicans only. Cells and spores of Bacillus spp. upregulated Gloverin, Galiomicin and Gallerimycin genes in relation to the control group (PBS + PBS). When these larvae were infected with C. albicans, the group pretreated with spores of B. atrophaeus and B. subtilis showed a greater increase in expression of Galiomycin (49.08-fold and 13.50-fold) and Gallerimycin (27.88-fold and 68.15-fold), respectively, compared to the group infected with C. albicans only (p = 0.0001). After that, we investigated the effects of B. subtilis and B. atrophaeus on immune system of G. mellonella evaluating the number of hemocytes, quantification of melanization, cocoon formation and colony forming units (CFU) count. Hemocyte count increased in response to stimulation by Bacillus, and a higher increase was achieved when larvae were inoculated with B. subtilis spores (p = 0.0011). In the melanization assay, all groups tested demonstrated lower production of melanin compared to that in the phosphate-buffered saline (PBS) group. In addition, full cocoon formation was observed in all groups analyzed, which corresponded to a healthier wax worm. Hemolymph culture revealed higher growth of B. atrophaeus and B. subtilis in the groups inoculated with spores. We concluded that spores and cells of B. atrophaeus and B. subtilis stimulated the immune system of G. mellonella larvae and protected them of C. albicans infection.

Tyrosine phosphorylation modulates cell surface expression of chloride cotransporters NKCC2 and KCC3.

Cellular chloride transport has a fundamental role in cell volume regulation and renal salt handling. Cellular chloride entry or exit are mediated at the plasma membrane by cotransporter proteins of the solute carrier 12 family. For example, NKCC2 resorbs chloride with sodium and potassium ions at the apical membrane of epithelial cells in the kidney, whereas KCC3 releases chloride with potassium ions at the basolateral membrane. Their ion transport activity is regulated by protein phosphorylation in response to signaling pathways. An additional regulatory mechanism concerns the amount of cotransporter molecules inserted into the plasma membrane. Here we describe that tyrosine phosphorylation of NKCC2 and KCC3 regulates their plasma membrane expression levels. We identified that spleen tyrosine kinase (SYK) phosphorylates a specific N-terminal tyrosine residue in each cotransporter. Experimental depletion of endogenous SYK or pharmacological inhibition of its kinase activity increased the abundance of NKCC2 at the plasma membrane of human embryonic kidney cells. In contrast, overexpression of a constitutively active SYK mutant decreased NKCC2 membrane abundance. Intriguingly, the same experimental approaches revealed the opposite effect on KCC3 abundance at the plasma membrane, compatible with the known antagonistic roles of NKCC and KCC cotransporters in cell volume regulation. Thus, we identified a novel pathway modulating the cell surface expression of NKCC2 and KCC3 and show that this same pathway has opposite functional outcomes for these two cotransporters. The findings have several biomedical implications considering the role of these cotransporters in regulating blood pressure and cell volume.

Thiazole derivatives act on virulence factors of Cryptococcus spp.

Cryptococcosis is an opportunistic or primary fungal infection considered to be the most prevalent fatal fungal disease worldwide. Owing to the limited number of available drugs, it is necessary to search for novel antifungal compounds. In the present work, we assessed the antifungal efficacy of three thiazole derivatives (1, 2, and 3). We conducted in vitro and in vivo assays to investigate their effects on important virulence factors, such as capsule and biofilm formation. In addition, the phagocytosis index of murine macrophages exposed to compounds 1, 2, and 3 and the in vivo efficacy of 1, 2, and 3 in Galleria mellonella infected with Cryptococcus spp. were evaluated. All compounds exhibited antifungal activity against biofilms and demonstrated a reduction in biofilm metabolic activity by 43-50% for C. gattii and 26-42% for C. neoformans. Thiazole compounds promoted significant changes in the capsule thickness of C. gattii compared to that of C. neoformans. Further examination of these compounds suggests that they can improve the phagocytosis process of peritoneal murine macrophages in vitro, causing an increase in the phagocytosis rate. Survival percentage was examined in the invertebrate model Galleria mellonella larvae, and only compound 3 could increase the survival at doses of 5 mg/kg after infection with C. gattii (P = .0001) and C. neoformans (P = .0007), similar to fluconazole at 10 mg/kg. The results demonstrated that thiazole compounds, mainly compound 3, have potential to be used for future studies in the search for new therapeutics for cryptococcosis.

Effects of surface pre-reacted glass-ionomer (S-PRG) eluate on Candida spp.: antifungal activity, anti-biofilm properties, and protective effects on Galleria mellonella against C. albicans infection.

Surface pre-reacted glass-ionomer (S-PRG) is a bioactive filler produced by PRG technology, which is applied to various dental materials. The inhibitory effects of S-PRG eluate against Candida, the most common fungal oral pathogen, were investigated. Minimum inhibitory concentrations (MIC) and anti-biofilm activities were tested against Candida albicans, Candida glabrata, Candida krusei, and Candida tropicalis. For the in vivo study, Galleria mellonella was used as a model to evaluate the effects of S-PRG on toxicity, hemocyte counts and candidiasis. The MIC of S-PRG ranged from 5 to 40% (v/v). S-PRG eluate exhibited anti-biofilm activity for all the Candida species tested. Furthermore, injection of S-PRG eluate into G. mellonella was not toxic to the larvae and protected G. mellonella against experimental candidiasis. In addition, S-PRG eluate inhibited biofilm formation by C. albicans, C. glabrata, C. krusei, and C. tropicalis and exerted protective effects on G. mellonella against experimental candidiasis in vivo.

Antimicrobial photodynamic therapy against clinical isolates of carbapenem-susceptible and carbapenem-resistant Acinetobacter baumannii.

Infections caused by Acinetobacter baumannii have become a challenge for healthcare professionals because of the rapid increase in Gram-negative bacteria resistant to carbapenem antibiotics. The objective of this study was to evaluate the effect of antimicrobial photodynamic therapy (aPDT) against different strains of A. baumannii isolated from patients with infectious process and hospitalized at the intensive care unit of the hospitals of São Jose dos Campos, São Paulo. These isolates were obtained from the Valeclin Clinical Analysis Laboratory (SP, Brazil) and were tested for susceptibility to the carbapenems imipenem and meropenem by determination of the minimal inhibitory concentration (MIC) using the broth microdilution method. The strains susceptible and resistant to these antibiotics were submitted to aPDT using methylene blue and a low-level laser with a wavelength of 660 nm and fluence of 39.5 J/cm2 (energy of 15 J and time of 428 s). The number of colony-forming units (CFU/mL) was analyzed by ANOVA and the Tukey test. The laboratory of origin of the clinical isolates identified 1.54% of 13,715 strains tested over a period of 8 months as A. baumannii. Among the A. baumannii isolates, 58% were resistant to carbapenems by the disk diffusion test. Susceptible isolates exhibited MIC of 0.5 to 1 µg/mL and resistant isolates of 64 to > 128 µg/mL. PDT reduced the number of A. baumannii cells for all isolates tested, with this reduction ranging from 63 to 88% for susceptible isolates and from 26 to 97% for resistant isolates. The percentage of viability was dependent on the strain analyzed. In conclusion, these data indicate that PDT could be an alternative strategy for the control of infections caused by carbapenem-resistant A. baumannii.

Lactobacillus paracasei 28.4 reduces in vitro hyphae formation of Candida albicans and prevents the filamentation in an experimental model of Caenorhabditis elegans.

The objective of this study was to evaluate the influence of microbe-microbe interactions to identify a strain of Lactobacillus that could reduce the filamentation of Candida albicans ATCC 18804 using in vitro and in vivo models. Thus presenting a probiotic effect against the fungal pathogen. First, we analyzed the ability of 25 clinical isolates of Lactobacillus to reduce filamentation in C. albicans in vitro. We found that L. paracasei isolate 28.4 exhibited the greatest reduction of C. albicans hyphae (p = 0.0109). This reduction was confirmed by scanning electron microscopy analysis. The influence of C. albicans filamentation was found to be contributed through reduced gene expression of filament associated genes (TEC1 and UME6). In an in vivo study, prophylactic provisions with L. paracasei increased the survival of Caenorhabditis elegans worms infected with C. albicans (p = 0.0001) by 29%. Prolonged survival was accompanied by the prevention of cuticle rupture of 27% of the worms by filamentation of C. albicans, a phenotype that is characteristic of C. albicans killing of nematodes, compared to the control group. Lactobacillus paracasei isolate 28.4 reduced the filamentation of C. albicans in vitro by negatively regulating the TEC1 and UME6 genes that are essential for the production of hyphae. Prophylactic provision of Lactobacillus paracasei 28.4 protected C. elegans against candidiasis in vivo. L. paracasei 28.4 has the potential to be employed as an alternative method to control candidiasis.

Inhibitory effect of probiotic Lactobacillus supernatants from the oral cavity on Streptococcus mutans biofilms.

Probiotics can release bioactive substances that can inhibit the growth and biofilm formation of pathogenic microorganisms such as Streptococcus mutans. In this context, we evaluated whether the supernatants of Lactobacillus strains isolated from caries-free subjects can inhibit S. mutans, one of the most important bacteria for dental caries. First, the supernatants of 22 Lactobacillus strains were screened for antibacterial activity against S. mutans in planktonic cultures. All 22 Lactobacillus strains studied (100%) showed antibacterial activity. Thereafter, the Lactobacillus strains with the greatest reductions in the planktonic S. mutans cultures were tested on biofilms. The L. fermentum 20.4, L. paracasei 11.6, L. paracasei 20.3 and L. paracasei 25.4 strains could significantly reduce the number of S. mutans cells in biofilms formed in hydroxyapatite (p < 0.05). This reduction was also confirmed by scanning electron microscopy analysis and was not caused by the decreased pH value in the medium (p > 0.05). In addition, the supernatants of these probiotic strains could also reduce the total biomass of S. mutans biofilms (p < 0.05). In conclusion, most of the Lactobacillus strains tested have some antibacterial activity against S. mutans. L. fermentum 20.4, L. paracasei 11.6, L. paracasei 20.3 and L. paracasei 25.4 produce bioactive substances that caused a significant reduction in S. mutans biofilms.

Antifungal activity of clinical Lactobacillus strains against Candida albicans biofilms: identification of potential probiotic candidates to prevent oral candidiasis.

This study isolated Lactobacillus strains from caries-free subjects and evaluated the inhibitory effects directly on three strains of C. albicans, two clinical strains and one reference strain. Thirty Lactobacillus strains were isolated and evaluated for antimicrobial activity against in vitro C. albicans biofilms. L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 isolates exhibited the most significant inhibitory activity against C. albicans. Co-incubation between these microorganisms resulted in deterrence of biofilm development and retardation of hyphal formation. The hindrance of biofilm development was characterized by the downregulated expression of C. albicans biofilm-specific genes (ALS3, HWP1, EFG1 and CPH1). L. paracasei 28.4, L. rhamnosus 5.2 and L. fermentum 20.4 demonstrated the ability to exert antifungal activity through the inhibition of C. albicans biofilms.

Photodynamic inactivation in the expression of the Candida albicans genes ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 in biofilms.

The objective of this study was to evaluate the effects of photodynamic inactivation (PDI) on Candida albicans biofilms, evaluating its effects on gene expression of ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 by yeast. Three samples of C. albicans were used in this study: a clinical sample from a patient with HIV (39S), a clinical sample from a patient with denture stomatitis lesion (Ca30), and a standard strain ATCC 18804. The quantification of gene expression was related to the production of those genes in the samples referred above using quantitative polymerase chain reaction (qPCR) assay in real time. The photosensitizer methylene blue at 300 uM and erythrosine at 400 uM, sensitized with low-power laser (visible red, 660 nm) and green LED (532 nm), respectively, were used for PDI. Four groups of each sample and PDI protocol were evaluated: (a) P+L+: sensitization with the photosensitizer and irradiation with light, (b) P+L-: only treatment with the photosensitizer, (c) P-L+: only irradiation with light, and (d) P-L-: without sensitization with the dye and absence of light. The results were analyzed by t test, with a significance level of 5%. The photodynamic inactivation was able to reduce the expression of all genes for both treatments, laser and LED. The fold-decrease for the genes ALS3, HWP1, BCR1, TEC1, CPH1, and EFG1 were 0.73, 0.39, 0.77, 0.71, 0.67, and 0.60 for laser, respectively, and 0.66, 0.61, .050, 0.43, 0.54, and 0.66 for LED, respectively. It could be concluded that PDI showed a reduction in the expression of C. albicans genes, suggesting its virulence decrease.

Genotype and phenotype characterization in a Spanish cohort with isovaleric acidemia.

Isovaleric acidemia (IVA) is a rare disorder of leucine metabolism. We carried out a multicenter study of IVA patients diagnosed by newborn screening (NBS) or symptoms clinics over a period of 28 years in Spain. Evaluated at diagnosis, data included age, detection method, levels of C5 and IVG, enzymatic studies, clinical presentation parameters and genotype in 16 patients. Follow-up data included C5 levels, intellectual quotient and correlation genotype-phenotype. IVA was detected by NBS in 8 patients (prevalence of 1/326 629). Except 1, all the 8 patients identified by NBS were asymptomatic at diagnosis and had isovalerylcarnitine (C5) levels of 1.6-6.4 µM and isovalerylglycine (IVG) levels <1100 mmol per mol creatinine; they remained asymptomatic with a natural protein intake ⩾1.5 g kg-1 per day. Symptomatic patients with chronic intermittent or acute neonatal IVA had C5 levels of 3.9-16.3 µM and IVG levels >3400 mmol per mol creatinine. The percentage of isovalerate incorporation in fibroblasts was 64-80% in patients detected by NBS and 4.9-13% in symptomatic patients. Cognitive function was within normal ranges in all patients but was negatively correlated with IVG at detection (-0.592; P<0.05). The genetic analysis revealed nine novel mutations. The clinical/biochemical phenotype correlated fairly well with the phenotype predicted by the mutations found. In conclusion, although blood C5 levels have traditionally been considered the prognostic marker of choice, urine IVG levels would appear to be a better predictor, as they correlated well with severity of mutations and were associated with a lower incorporation rate of IVA in fibroblasts and a less favorable clinical course.

Immunomodulatory effects and anti-Candida activity of lactobacilli in macrophages and in invertebrate model of Galleria mellonella.

Due to the growing number of multi-resistant Candida spp., adjuvant treatments that may help combat these fungal pathogens are relevant and useful. This study evaluated the immunomodulation and anti-Candida activity of Lactobacillus rhamnosus (LR), Lactobacillus acidophilus and Lactobacillus paracasei suspensions, either single- or multiple-strain, in mouse macrophages (RAW 264.7) and Galleria mellonella (GM). Mouse macrophages were activated by different lactobacilli suspensions and challenged with C. albicans (CA). Tumor necrosis factor (TNF)-α, interleukin IL-1ß, IL-6 and IL-17 production and cell viability were investigated. LR was the best suspension for stimulating all evaluated cytokines and thus was used in subsequent in vivo assays. Two C. albicans clinical strains, CA21 and CA60, were then added to the GM assays to further confirm the results. LR suspension was injected into the larvae 24 h before challenging with CA. Survival curve, CFU per larva and hemocytes were counted. In the GM, the LR suspension increased the survival rate and hemocyte counts and decreased the CFU per larva counts for all groups. Lactobacilli suspensions presented strain-dependent immunomodulation; however, single suspensions showed better results. Anti-Candida activity was demonstrated by decreased Candida counts in the GM with the use of LR.