Silver nanoparticles coated with dodecanethiol used as fillers in non-cytotoxic and antifungal PBAT surface based on nanocomposites.

In the present study, we report the preparation of antifungal and non-cytotoxic polymer nanocomposites with potential application in biomedical materials. Dodecanethiol-protected silver nanoparticles (AgNPs-DDT) were synthesized by a reduction/precipitation method and dispersed in chloroform to obtain stable colloidal dispersions. PBAT-based nanocomposites containing 0.25, 0.5 and 2 wt% AgNPs-DDT were prepared by casting method. The incorporation of AgNPs-DDT in PBAT matrix resulted in nanocomposites which combine improved mechanical performance and antifungal properties with a non-cytotoxic characteristic.

Is the oral fungal pathogen Candida albicans a cariogen?

Pathobiology of dental caries is complex. Data from recent molecular microbiologic studies have further redefined the role of the oral microbiome in the etiology of dental caries. This new information challenges the conventional view on the hegemony of classic cariogenic prokaryotes such as Streptococcus mutans in caries etiology, and raises the intriguing possibility of the participation of the eukaryotic oral fungal pathogen Candida in the caries process. The virulence attributes of Candida species such as their acidogenicity and aciduric nature, the ability to develop profuse biofilms, ferment and assimilate dietary sugars, and produce collagenolytic proteinases are all indicative of their latent cariogenic potential. Based on the above, oral candidal counts have been used by some as a caries risk indicator. On the contrary, other studies suggest that Candida is merely a passenger extant in an acidic cariogenic milieu, and not a true pathogen. In this review, we critically examine the varying roles of Candida, and traditionally accepted cariogens such as the mutans group of streptococci in the pathobiology of dental caries. The weight of available data tends to imply that Candida may play a pivotal role as a secondary agent perpetuating the carious process, especially in dentinal caries.

Systematic screening of plant extracts from the Brazilian Pantanal with antimicrobial activity against bacteria with cariogenic relevance.

This study proposes a bioprospection methodology regarding the antimicrobial potential of plant extracts against bacteria with cariogenic relevance. Sixty extracts were obtained from ten plants--(1) Jatropha weddelliana, (2) Attalea phalerata, (3) Buchenavia tomentosa, (4) Croton doctoris, (5) Mouriri elliptica, (6) Mascagnia benthamiana, (7) Senna aculeata, (8) Unonopsis guatterioides, (9) Allagoptera leucocalyx and (10) Bactris glaucescens--using different extraction methods - (A) 70° ethanol 72 h/25°C, (B) water 5 min/100°C, (C) water 1 h/55°C, (D) water 72 h/25°C, (E) hexane 72 h/25°C and (F) 90° ethanol 72 h/25°C. The plants were screened for antibacterial activity at 50 mg/ml using the agar well diffusion test against Actinomyces naeslundii ATCC 19039, Lactobacillus acidophilus ATCC 4356, Streptococcus gordonii ATCC 10558, Streptococcus mutans ATCC 35688, Streptococcus sanguinis ATCC 10556, Streptococcus sobrinus ATCC 33478 and Streptococcus mitis ATCC 9811. The active extracts were tested to determine their minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), cytotoxicity and chemical characterization. Forty-seven extracts (78%) were active against at least one microorganism. Extract 4A demonstrated the lowest MIC and MBC for all microorganisms except S. gordonii and the extract at MIC concentration was non-cytotoxic. The concentrated extracts were slightly cytotoxic. Electrospray ionization with tandem mass spectrometry analyses demonstrated that the extract constituents coincided with the mass of the terpenoids and phenolics. Overall, the best results were obtained for extraction methods A, B and C. The present work proved the antimicrobial activity of several plants. Particularly, extracts from C. doctoris were the most active against bacteria involved in dental caries disease.

Hydroxyurea therapy in sickle cell anemia patients aids to maintain oral fungal colonization balance.

BACKGROUND: The aim of this study was to evaluate the frequency of Candida species and presence of lesions in the oral cavity of patients with sickle cell anemia (SS). METHODS: The study included 30 patients diagnosed with sickle cell anemia and taking hydroxyurea for at least 90 days (SS/HU+); and 39 patients with sickle cell anemia and without hydroxyurea therapy (SS/HU-). Two control groups were constituted by healthy individuals matched to the test groups in age, gender, and oral conditions (C/HU+ for SS/HU+ and C/HU- for SS/HU-). Oral clinical examination and anamnesis were performed. Yeasts were collected by oral rinses and identified by API system. Antifungal susceptibility evaluation was performed according to the CLSI methodology. Data obtained for microorganisms counts were compared by Student's t test (SS/HU+ vs. C/HU+ and SS/HU- vs. C/HU-) using MINITAB for Windows 1.4. Significance level was set at 5%. RESULTS: No oral candidosis lesions were detected. Significant differences in yeasts counts were observed between SS/HU- group and the respective control, but there were no differences between SS/HU+ and C/HU+. Candida albicans was the most prevalent species in all groups. Candida famata was observed both in SS and control groups. Candida dubliniensis, Candida glabrata, Candida krusei, Candida tropicalis, Candida pelliculosa, and Candida parapsilosis were observed only in SS groups. Most strains were susceptible to all antifungal agents. CONCLUSION: Hydroxyurea therapy seems to decrease candidal counts and resistance rate in sickle cell anemia patients. However, further studies should be conducted in the future to confirm this finding. Hydroxyurea therapy in sickle cell anemia patients maintains fungal species balance in oral cavity.

Oral microbial colonization in patients with systemic lupus erythematous: correlation with treatment and disease activity.

Treating patients with systemic lupus erythematosus (SLE) with steroids and immunosuppressive drugs may interfere in the presence of potentially opportunistic microorganisms in the oral cavity. The aim of this study was to evaluate the presence of Candida spp., Staphylococcus spp., Enterobacteria and Pseudomonas spp. in the oral cavity of SLE patients, compared with healthy controls. A group of 40 patients who had received therapy for at least 60 days was selected (19-53 years). For the control group, 40 healthy individuals matched for age, gender and use of partial prosthesis were selected. Oral rinse samples were collected and plated on specific culture media. After incubation, the number of colony forming units (CFU) was obtained and the isolates were identified at species level. Microbial counts were compared between SLE and control by analysis of variance (ANOVA) and Mann-Whitney (p < 0.05 significant). Microorganism counts in patients with and without immunosuppressive drugs, as well with active and inactive disease (according to SLEDAI score) were also compared. No significant differences in CFU/mL between SLE and control patients were observed (yeasts, p = 0.55; Staphylococci, p = 0.24; Enterobacteria/Pseudomonas spp., p = 0.26). No differences in microbial counts were observed regarding clinical parameters tested. The most frequent species isolated in the SLE group were Candida albicans, Staphylococcus epidermidis and Klebsiella oxytoca. In conclusion, no differences in frequency and microorganism levels were found between SLE patients and healthy individuals.

Prevalence and antifungal resistance profile of Candida spp. oral isolates from patients with type 1 and 2 diabetes mellitus.

OBJECTIVE: The goal of the study was to measure the prevalence of Candida spp. in the oral cavity of patients with diabetes types 1 and 2 when compared to healthy individuals and to study antifungal resistance profile of the isolates. DESIGN: There were 162 subjects in the study: diabetes type 1 (n=39); control group 1 (n=50): healthy individuals matched in gender, age, and oral conditions to diabetes type 1 patients; diabetes type 2 (n=37); control group 2 (n=36) who were matched to each patient of the diabetes type 2 group. Stimulated saliva was collected and isolates were identified with phenotypic tests. The presence of C. dubliniensis was determined by multiplex PCR. RESULTS: There were no statistically significant differences in Candida spp. frequency between the diabetes 1 group and its control (p=0.443) nor between the diabetes 2 group and its control (p=0.429). C. albicans was the most frequently isolated yeast in all groups. In the diabetes groups, C. stellatoidea, C. parapsilosis, C. tropicalis, C. lipolytica, C. glabrata, and C. krusei were also identified. Additionally, in control groups, C. kefyr was also detected. None of the isolates were resistant to amphotericin B and flucytosine. A low percentage of the isolates were resistant to ketoconazole. CONCLUSIONS: No differences were detected in colonization of Candida spp. oral isolates from type 1 and type 2 diabetes when compared to matched controls. The antifungal resistance of Candida spp. isolates for ketoconazole from type 1 diabetes patients was significantly higher than that of its matched control.

Antimicrobial analysis of chlorhexidine gel and intracanal medicaments against microorganisms inoculated in root canals.

AIM: The aim of this paper was to evaluate the antimicrobial activity of 2% chlorhexidine gel (CLX) associated with various intracanal medicaments against Candida albicans and Enterococcus faecalis inoculated in root canals. METHODS: Thirty six human single-rooted teeth were contaminated with C.albicans and E.faecalis. The canals were instrumented using 2% CLX gel and were divided into three groups according to the intracanal medicaments (ICM) used. Group 1: calcium hydroxide paste [Ca(OH)2], Group 2: 2% chlorhexidine gel (CLX) and Group 3: 2% CLX gel + Ca(OH)2. The root canal collections were performed after 21 days of contamination (control collection), after instrumentation (1st collection), after 14 days of intracanal medicament (2nd collection) and 7 days after medicament removal (3rd collection). The microbiological samples were plated in culture media and incubated for 48 hours. The results were submitted to Kruskal-Wallis test (P ≤ 0.05). RESULTS: It was verified that the instrumentation with CLX reduced the number of CFU/ml significantly when compared with the confirmation collection (control). However, the use of the ICM was only capable to eliminate completely the microorganisms in the root canals without difference statistics between them. CONCLUSION: Although the use of 2% chlorherixidine gel reduces the number of microorganisms significantly, only the ICM calcium hydroxide and calcium hydroxide associated with chlorhexidine are able to eliminate these microorganisms completely.

In vitro evaluation of the effectiveness of irrigants and intracanal medicaments on microorganisms within root canals.

AIM: To evaluate in vitro the effectiveness of sodium hypochlorite (NaOCl), chlorhexidine (CHX) and five intracanal medicaments on microorganisms within root canals. METHODOLOGY: Ninety-six human single-rooted extracted teeth were used. After removing the crowns, canal preparation was completed and the external root surfaces were coated with epoxy resin. Following sterilization, the teeth were contaminated with Candida albicans and Enterococcus faecalis, and were incubated at 37 +/- 1 degrees C for 7 days. The teeth were divided according to the irrigant solution or intracanal medicament: group 1, sterile physiologic solution (SPS) and calcium hydroxide (Ca(OH)2) paste; group 2, SPS and camphorated paramonochlorophenol (CPMC); group 3, SPS and tricresol formalin; group 4, SPS and CaOH2 + CPMC paste; group 5, SPS and PMC furacin; group 6, 2.5% NaOCl without intracanal medication; group 7, 2.0% CHX without intracanal medication and group 8, SPS without intracanal medication (control group). Microbiological samples were collected with sterile paper points, and bacterial growth was determined. The data were submitted to the analysis of variance (anova, P = 0.05). RESULTS: For C. albicans, groups 3 and 8 were statistically less effective than groups 1, 2, 4 and 5 (Kruskal-Wallis (K-W) = 65.241; gl = 7; P = 0.001). For E. faecalis, groups 6 and 8 were statistically less effective than groups 1-4 and 7 (K-W = 61.048; gl = 7; P = 0.001). CONCLUSIONS: Ca(OH)2 + CPMC paste was the most effective intracanal medicament for the elimination of the two microorganisms; 2.0% CHX solution was more effective than 2.5% NaOCl against E. faecalis.

Caries risk tests and salivary levels of immunoglobulins to Streptococcus mutans and Candida albicans in mouthbreathing syndrome patients.

The aim of this study was to compare microbiological and salivary variables possibly related to caries risk in treated and untreated mouthbreathing syndrome (MBS) children and control children. Thirty control children, 30 mouthbreathers and 25 treated mouthbreathers were studied for the numbers of lactobacilli, mutans streptococci and yeasts in their saliva. Snyder's test, salivary flow and buffering capacity were also evaluated. Levels of immunoglobulins to Candida albicans and Streptococcus mutans in the saliva were quantified using ELISA. Considering the results obtained for the microbiological and salivary caries risk tests, no significant differences were observed among the proportions of patients with small/negative and high/moderate caries risk in the studied groups. The level of IgG to S. mutans was significantly higher in the treated MBS group in relation to MBS patients. On the other hand, the median anti-S. mutans IgM level was lower in the treated MBS patients than in the other groups. For the studied anti-Candida immunoglobulins, IgM level was significantly lower in the treated MBS group than in the other groups. No differences were observed for anti-S. mutans and anti-Candida IgA levels among the groups. The findings suggest that mouthbreathing cannot be considered a risk factor for dental caries.

Extracellular activity in Cryptococcus neoformans strains isolated from AIDS patients and from environmental sources.

Nineteen Cryptococcus neoformans strains isolated from AIDS patients and 16 from bird droppings were tested for their extracellular activity. Typical enzymatic activity that was different from other medically important yeasts was found. The results obtained may indicate that there are new extracellular enzymatic activities that imply a relationship between C. neoformans and its virulence. A correlation among the different enzymatic activities was also investigated and according to the results obtained no relationship was observed among any of the recorded extracellular enzymatic activities. Research on C. neoformans extracellular enzymatic activity is useful not only to better understand its metabolism but in particular to establish a possible relationship between its virulence and pathogenicity.

Characterization and retroviral transduction of an early human lymphomyeloid precursor assayed in nonswitched long-term culture on murine stroma.

In the hierarchy of human hematopoietic progenitors, long-term culture-initiating cells (LTC-IC) and extended LTC-IC belong to the earliest cell populations that can be assayed in vitro. We report the identification of a multipotential lymphomyeloid progenitor detected in a nonswitch culture system. We observed the emergence of CD33+ myeloid and CD19+ B-lymphoid cells following plating of lineage-depleted (Lin-) CD34 -enriched or purified CD34+ CD38- cord blood cells on MS-5 stroma in the absence of exogenous cytokines. Both CD19+ CD20- pro-B and CD19+ CD20+ pre-B lymphocytes coexist with myeloid cells in long-term culture. A limiting dilution approach was used to show that a single CD34+ CD38- cell can generate lymphomyeloid progeny in conventional (5-week) and extended (10-week) cultures. Most of the clones in long-term culture or extended long-term culture contained not only lymphoid and myeloid cells, but also myeloid clonogenic progenitors. A high proportion of CD34+ CD38- cells gave rise to lymphomyeloid clones after 5 and 10 weeks of culturing (up to 48% and 16%, respectively), which distinguishes the assay reported here from those using switch culture conditions. We performed retroviral gene transfer experiments involving 1-3 days of exposure of Lin CD34+ -enriched cells to virus encoding enhanced green fluorescent protein. Monitoring of gene transfer efficiency into LTC-IC by enhanced green fluorescent protein fluorescence showed that it is possible to achieve marking of lymphomyeloid LTC-IC, albeit to a lesser extent than myeloid-restricted LTC-IC.

Retroviral transduction of TLS-ERG initiates a leukemogenic program in normal human hematopoietic cells.

Many chimeric oncogenes have been identified by virtue of the association between chromosomal translocation and specific human leukemias. However, the biological mechanism by which these oncogenes disrupt the developmental program of normal human hematopoietic cells during the initiation of the leukemogenic process is poorly understood due to the absence of an appropriate experimental system to study their function. Here, we report that retroviral transduction of TLS-ERG, a myeloid leukemia-associated fusion gene, to human cord blood cells results in altered myeloid and arrested erythroid differentiation and a dramatic increase in the proliferative and self-renewal capacity of transduced myeloid progenitors. Thus, TLS-ERG expression alone induced a leukemogenic program that exhibited similarities to the human disease associated with this translocation. These results provide an experimental examination of the early stages of the human leukemogenic process induced by a single oncogene and establish a paradigm to functionally assay putative leukemogenic genes in normal human hematopoietic cells.

Structure and evolution of the human IKBA gene.

IkappaBalpha belongs to a gene family whose members are characterized by their 6-7 Ankyrin repeats, which allow them to interact with members of the Rel family of transcription factors. We have sequenced a human IkappaBalpha genomic clone to determine its gene structure. The human IkappaBalpha gene (IKBA) has six exons and five introns that span approximately 3.5 kb. This genomic organization is similar to that of other members of the Ankyrin gene family. The human IKBA gene shares similar intron/exon boundaries with the human BCL3 and NFKB2 genes, which is consistent with their conserved Ankyrin repeats. To examine further the evolutionary relationship between human IkappaBalpha and other members of its gene family, we performed a phylogenetic analysis. Although the resulting phylogenetic tree does not identify a common ancestor of the IkappaBalpha and other members of its gene family, it indicates that this family diverges into two groups based on structure and function.

Three NF-kappa B sites in the I kappa B-alpha promoter are required for induction of gene expression by TNF alpha.

NF-kappa B was first identified as a postive regulator which bound to a 10 bp sequence in the first intron of the Ig kappa light chain gene. Further characterization of this transcription factor has revealed that NF-kappa B is kept from binding to its consensus sequence by its inhibitor, IkB-alpha, which retains NF-kappa B in the cytoplasm. Upon receiving various extra- and intracellular signals, I kappa B-alpha is rapidly degraded and NF-kappa B is induced to translocate into the nucleus. This process precedes the rapid induction of I kappa B-alpha mRNA and protein. To understand how I kappa B-alpha is replenished, we have cloned and sequenced the 5' flanking region of the I kappa B-alpha gene and have identified the transcription start site and three NF-kappa B sites in this region. Further characterization of these NF-kappa B sites show that they have different affinities for three specific protein complexes which we identify here to consist of various members of the Rel family. In transient assays, cotransfection with a p65 expression vector is able to activate an I kappa B-alpha promoter-CAT reporter construct and all three NF-kappa B sites are required for full activation of the I kappa B-alpha gene following stimulation with TNF-alpha. Our data confirm a transcriptional autoregulatory loop involved in maintaining appropriate NF-kappa B and I kappa B-alpha levels in the cell.

Mapping and characterization of 129 cosmids on human chromosome 11p.

We constructed cosmid libraries from human-hamster somatic cell hybrids that possess all or part of the short arm of chromosome 11 as their only human complement and isolated 129 human 11p clones. These cosmids map to 22 of 25 intervals distinguished by a hybrid panel for chromosome 11p. Forty-eight single-copy sequences were subcloned from 25 cosmids. Six of 17 (35%) single-copy sequences tested identify 11 new polymorphisms. Restriction endonuclease analysis identified CpG islands in 16 of 68 cosmids (23.5%). Analysis of the distribution of restriction endonuclease sites recognizing CpG dinucleotides showed that clusters of these sites, including those associated with the 5' region of an 11p13 Wilms' tumor gene, WT1, can span greater distances than generally recognized. The cosmids reported here should contribute to the construction of long-range physical maps and the isolation of additional genes on the short arm of chromosome 11.

Isolation and characterization of a zinc finger polypeptide gene at the human chromosome 11 Wilms' tumor locus.

We have isolated a series of genomic and cDNA clones mapping within the boundaries of constitutional and tumor deletions that define the Wilms' tumor locus on human chromosome 11 (band p13). The transcription unit corresponding to these clones spans approximately 50 kb and encodes an mRNA approximately 3 kb long. This mRNA is expressed in a limited range of cell types, predominantly in the kidney and a subset of hematopoietic cells. The polypeptide encoded by this locus has a number of features suggesting a potential role in transcriptional regulation. These include the presence of four zinc finger domains and a region rich in proline and glutamine. The amino acid sequence of the predicted polypeptide shows significant homology to two growth regulated mammalian polypeptides, EGR1 and EGR2. The genetic localization of this gene, its tissue-specific expression, and the function predicted from its sequence lead us to suggest that it represents the 11p13 Wilms' tumor gene.

A genetic linkage map of 27 loci from PND to FY on the short arm of human chromosome I.

A genetic linkage map of 27 loci on the short arm of human chromosome 1 has been developed by analysis of the 40 families in the Centre d'Etude du Polymorphisme Humain (CEPH) reference panel. Probes that recognize 14 novel RFLPs at loci designated D1S9-D1S22 were isolated from a flow-sorted chromosome 1 library. A linkage map of chromosome 1p was constructed from the genotypic data at these 14 loci, RFLPs at eight cloned genes (PND, ALPL, FUCA1, SRC2, MYCL, GLUT, TSHB, and NGFB), two previously identified RFLPs (D1S2 and D1S57), two blood group antigens (RH and FY), and the isozyme PGM1. All 27 loci form a continuous linkage group, from FY to PND, of 102 cM in males and 230 cM in females. This map provides a basis for highly informative multipoint mapping studies for most of the short arm of chromosome 1.