Immunodominant Antigens of Leishmania chagasi Associated with Protection against Human Visceral Leishmaniasis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

INFLUENCE of DIFFERENT SURFACE CONDITIONING PROTOCOLS on MICROTENSILE BOND STRENGTH of SELF-ADHESIVE RESIN CEMENTS TO DENTIN

Statement of problem. According to manufacturers, bonding with self-adhesive resin cements can be achieved without any pretreatment steps such as etching, priming, or bonding. However, the benefit of saving time with these simplified luting systems may be realized at the expense of compromising the bonding capacity.Purpose. The purpose of this study was to assess whether different dentin conditioning protocols influence the bond performance of self-adhesive resin cements to dentin.Material and methods. Flat dentin surfaces from 48 human molars were divided into 4 groups (n=12): 1) control, no conditioning; 2) H(3)PO(4), etching with 37% H(3)PO(4) for 15 seconds; 3) SEBond, bonding with self-etching primer adhesive (Clearfil SE Bond); and 4) EDTA, etching with 0.1M EDTA for 60 seconds. The specimens from each dentin pre-treatment were bonded using the self-adhesive cements RelyX Unicem, Maxcem or Multilink Sprint (n=4). The resin-cement-dentin specimens were stored in water at 37 degrees C for 7 days, and serially sectioned to produce beam specimens of 1.0 mm(2) cross-sectional area. Microtensile bond strength (mu TBS) testing was performed at 1.0 mm/min. Data (MPa) were analyzed by 2-way ANOVA and Tukey multiple comparisons test (alpha=.05). Fractured specimens were examined with a stereomicroscope (x40) and classified as adhesive, mixed, or cohesive. Additional bonded interfaces were evaluated under a scanning electron microscope (SEM).Results. Cement-dentin mu TBS was affected by the dentin conditioning approach (P <.001). RelyX Unicem attained statistically similar bond strengths to all pre-treated dentin surfaces. H(3)PO(4)-etching prior to the application of Maxcem resulted in bond strength values that were significantly higher than the other groups. The lowest mu TBS were attained when luting Multilink Sprint per manufacturers' recommendations, while H(3)PO(4)-etching produced the highest values followed by Clearfil SE bonding and EDTA. SEM observations disclosed an enhanced potential of the self-adhesive cements to form a hybrid layer when applied following manufacturer's instructions.Conclusions. When evaluated self-adhesive resin cements are used, selectively etching dentin with H(3)PO(4) prior to luting results in the most effective bonding. (J Prosthet Dent 2011;105:227-235)

Resistance to compression of weakened roots subjected to different root reconstruction protocols

Objective: This study evaluated, in vitro, the fracture resistance of human non-vital teeth restored with different reconstruction protocols. Material and methods: Forty human anterior roots of similar shape and dimensions were assigned to four groups (n=10), according to the root reconstruction protocol: Group I (control): non-weakened roots with glass fiber post; Group II: roots with composite resin by incremental technique and glass fiber post; Group III: roots with accessory glass fiber posts and glass fiber post; and Group IV: roots with anatomic glass fiber post technique. Following post cementation and core reconstruction, the roots were embedded in chemically activated acrylic resin and submitted to fracture resistance testing, with a compressive load at an angle of 45 degrees in relation to the long axis of the root at a speed of 0.5 mm/min until fracture. All data were statistically analyzed with bilateral Dunnett's test (alpha=0.05). Results: Group I presented higher mean values of fracture resistance when compared with the three experimental groups, which, in turn, presented similar resistance to fracture among each other. None of the techniques of root reconstruction with intraradicular posts improved root strength, and the incremental technique was suggested as being the most recommendable, since the type of fracture that occurred allowed the remaining dental structure to be repaired. Conclusion: The results of this in vitro study suggest that the healthy remaining radicular dentin is more important to increase fracture resistance than the root reconstruction protocol.

Medium-term protocols for in vivo evaluation of chemical modifiers of carcinogenesis

Cancer development is a long-term multistep process which allows interventional measure before the clincial disease emerges. the detection of natural substances which can block the process of carcinogenesis is a important as the identification of anti-tumoral drugs since they might be used in chemoprevention of cancer in high-risk groups. In vivo rodent models of chemical caecinogenesis have been used to study plant-derived inhibitors of carcinofenesis such as indols, coumarins, isothiocyanates, flavones, phenols and allyl-sulfides. Since the standard in vivo rodent bioassay is prolonged and expensive, shorter reliable protocols are needed. Two in vivo medium-term protocols for evaluation of modifiers of carcinogenesis are presented, one related to liver and the other to bladder cancer. Both protocols use rats, last 8 and 36 weeks and are based on the two-step concept of carcinogenesis: initiation and promotion. The protocols use respectively the development of altered foci of hepatocytes expressing immunochistochemically the placental form of gluthation S-transferase and the appearence of pre-neoplastic urothelium and papillomas as the end-points. the use of these protocols for detection of plantpderived inhibitors of carcinogenesis appear warranted.

Protocols for hyperlactatemia induction in the lactate minimum test adapted to swimming rats

The lactate minimum test (LACmin) has been considered an important indicator of endurance exercise capacity and a single session protocol can predict the maximal steady state lactate (MLSS). The objective of this study was to determine the best swimming protocol to induce hyperlactatemia in order to assure the LACmin in rats (Rattus norvegicus), standardized to four different protocols (P) of lactate elevation. The protocols were PI: 6 min of intermittent jumping exercise in water (load of 50% of the body weight - bw); P2: two 13% bw load swimming bouts until exhaustion (thin); P3: one thin 13% bw load swimming bout; and P4: two 13% bw load swimming bouts (1st 30 s, 2nd to thin), separated by a 30 s interval. The incremental phase of LACmin beginning with initial loads of 4% bw, increased in 0.5% at each 5 min. Peak lactate concentration was collected after 5, 7 and 9 min (mmol L-1) and differed among the protocols P 1 (15.2 +/- 0.4, 14.9 +/- 0.7, 14.8 +/- 0.6) and P2 (14.0 +/- 0.4, 14.9 +/- 0.4, 15.5 +/- 0.5) compared to P3 (5.1 +/- 0.1, 5.6 +/- 0.3, 5.6 +/- 0.3) and P4 (4.7 +/- 0.2, 6.8 +/- 0.2, 7.1 +/- 0.2). The LACmin determination success rates were 58%, 55%, 80% and 91% in P1, P2, P3 and P4 protocols, respectively. The MLSS did not differ from LACmin in any protocol. The LACmin obtained from P4 protocol showed better assurance for the MLSS identification in most of the tested rats. (c) 2007 Elsevier B.V. All rights reserved.

Crystallization and X-ray diffraction data analysis of human deoxyhaemoglobin A(0) fully stripped of any anions

In this work, initial crystallographic studies of human haemoglobin (Hb) crystallized in isoionic and oxygen-free PEG solution are presented. Under these conditions, functional measurements of the O-2-linked binding of water molecules and release of protons have evidenced that Hb assumes an unforeseen new allosteric conformation. The determination of the high-resolution structure of the crystal of human deoxy-Hb fully stripped of anions may provide a structural explanation for the role of anions in the allosteric properties of Hb and, particularly, for the influence of chloride on the Bohr effect, the mechanism by which Hb oxygen affinity is regulated by pH. X-ray diffraction data were collected to 1.87 Angstrom resolution using a synchrotron-radiation source. Crystals belong to the space group P2(1)2(1)2 and preliminary analysis revealed the presence of one tetramer in the asymmetric unit. The structure is currently being refined using maximum-likelihood protocols.

Escherichia coli phylogenetic group determination and its application in the identification of the major animal source of fecal contamination

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Primary cutaneous amyloidosis: Clinical, laboratorial and histopathological study of 25 cases. Identification of gammaglobulins and C3 in the lesions by immunofluorescence

25 cases of primary cutaneous amyloidosis are studied. 16 patients had macular amyloidosis (MPA) and 9 lichen amyloidosus (LPA). γ-Globulins were increased in 50% of the patients. IgG and IgA were increased in the serum of 5 and 3 patients with MPA and 4 and 2 patients with LPA, respectively. Volume of amyloid deposits was similar in both forms. By direct immunofluorescence we demonstrated IgG in the amyloid deposits of 21 of the 25 cases and C3 in 13; IgM was present in 9 cases of MPA and in 3 cases of LPA. MPA was more frequent than LPA; histologically, it was impossible to distinguish MPA from LPA; correlation between serum levels of γ-globulins and their presence in amyloid deposits was weak; MPA and LPA seem to be distinct clinical manifestations of the same disease and itching does not cause transformation of MPA in LPA.

The diagnostic value of Gram stain for initial identification of the etiologic agent of peritonitis in CAPD patients

Objective: To evaluate the effectiveness of the Gram stain in the initial diagnosis of the etiologic agent of peritonitis in continuous ambulatory peritoneal dialysis (CAPD). Design: Retrospective study analyzing the sensitivity (S), specificity (SS), positive predictive value (+PV), and negative predictive value (-PV) of the Gram stain relating to the results of cultures in 149 episodes of peritonitis in CAPD. The data were analyzed in two studies. In the first, only the cases with detection of a single agent by Gram stain were taken (Study 1). In the second, only the cases with two agents in Gram stain were evaluated (Study 2). Setting: Dialysis Unit and Laboratory of Microbiology of a tertiary medical center. Patients: Sixty-three patients on regular CAPD who presented one or more episodes of peritonitis from May 1992 to May 1995. Results: The positivity of Gram stain was 93.2% and the sensitivity was 95.7%. The values of S, SS, +PV, and -PV were respectively: 94.9%, 53.5%, 68.3%, and 90.9% for gram-positive cocci and 83.3%, 98.8%, 95.2%, and 95.6% for gram-negative bacilli. The association of gram-positive cocci plus gram-negative bacilli were predictive of growth of both in 6.8%, growth of gram-positive cocci in 13.7%, and growth of gram-negative bacilli in 72.5%. Conclusions: The Gram stain is a method of great value in the initial diagnosis of the etiologic agent of peritonitis in CAPD, especially for gram-negative bacilli.

A SequenceSpace analysis of Lys49 phospholipases A2: Clues towards identification of residues involved in a novel mechanism of membrane damage and in myotoxicity

'SequenceSpace' analysis is a novel approach which has been used to identify unique amino acids within a subfamily of phospholipases A2 (PLA2) in which the highly conserved active site residue Asp49 is substituted by Lys (Lys49-PLA2s). Although Lys49-PLA2s do not bind the catalytic co-factor Ca2+ and possess extremely low catalytic activity, they demonstrate a Ca2+-independent membrane damaging activity through a poorly understood mechanism, which does not involve lipid hydrolysis. Additionally, Lys49-PLA2s possess combined myotoxic, oedema forming and cardiotoxic pharmacological activities, however the structural basis of these varied functions is largely unknown. Using the 'SequenceSpace' analysis we have identified nine residues highly unique to the Lys49-PLA2 sub-family, which are grouped in three amino acid clusters in the active site, hydrophobic substrate binding channel and homodimer interface regions. These three highly specific residue clusters may have relevance for the Ca2+-independent membrane damaging activity. Of a further 15 less stringently conserved residues, nine are located in two additional clusters which are well isolated from the active site region. The less strictly conserved clusters have been used in predictive sequence searches to correlate amino acid patterns in other venom PLA2s with their pharmacological activities, and motifs for presynaptic and combined toxicities are proposed.

Cloning, overexpression, and purification of functional human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the phosphorolysis of the N-ribosidic bonds of purine nucleosides and deoxynucleosides. A genetic deficiency due to mutations in the gene encoding for human PNP causes T-cell deficiency as the major physiological defect. Inappropriate activation of T-cells has been implicated in several clinically relevant human conditions such as transplant tissue rejection, psoriasis, rheumatoid arthritis, lupus, and T-cell lymphomas. Human PNP is therefore a target for inhibitor development aiming at T-cell immune response modulation. In addition, bacterial PNP has been used as reactant in a fast and sensitive spectrophotometric method that allows both quantitation of inorganic phosphate (Pi) and continuous assay of reactions that generate P i such as those catalyzed by ATPases and GTPases. Human PNP may therefore be an important biotechnological tool for P i detection. However, low expression of human PNP in bacterial hosts, protein purification protocols involving many steps, and low protein yields represent technical obstacles to be overcome if human PNP is to be used in either high-throughput drug screening or as a reagent in an affordable P i detection method. Here, we describe PCR amplification of human PNP from a liver cDNA library, cloning, expression in Escherichia coli host, purification, and activity measurement of homogeneous enzyme. Human PNP represented approximately 42% of total soluble cell proteins with no induction being necessary to express the target protein. Enzyme activity measurements demonstrated a 707-fold increase in specific activity of cloned human PNP as compared to control. Purification of cloned human PNP was achieved by a two-step purification protocol, yielding 48 mg homogeneous enzyme from 1 L cell culture, with a specific activity value of 80 U mg -1. © 2002 Elsevier Science (USA). All rights reserved.

Niche-specific association of Aeromonas ribotypes from human and environmental origin

A total of 88 Aeromonas isolates from distinct locations and sources (39 from extraintestinal infections, 31 from diarrhoeic, ten from non-diarrhoeic faeces, all human, and eight from fresh water) were subjected to phenospecies identification, serotyping, ribotyping and detection of some virulence markers. The strains belonged to four different phenospecies marked by 19 O serogroups and 38 ribotypes. No strong correlation between these parameters was found, and no group, as defined by the typing methods, could be characterized with a particular set of virulence markers. There was a clear association of ribotypes with the source of the strains. Cluster analysis allowed the identification of a complex of ribotypes belonging to distinct but related sources, including clinical and environmental isolates. These results suggest that ribotyping may be an epidemiological tool suitable for the study of Aeromonas infections.

rDNA-RFLP identification of Candida species in immunocompromised and seriously diseased patients

PCR was used to amplify a targeted region of the ribosomal DNA of 76 Candida spp. isolates from immunocompromised and seriously diseased patients. Thirty-seven strains isolated from different anatomical sites of 11 patients infected with HIV (Vitória, ES, Brazil), 26 isolates from patients under treatment at Odilon Behrens Hospital and 13 isolates from skin and urine samples from São Marcos Clinical Analysis Laboratory (Belo Horizonte, Brazil) were scored. Fragments of rDNA were amplified using primer pairs ITS1-ITS4, for the amplification of ITS1 and ITS2 regions, including the gene for the 5.8s subunit. Amplification resulted in fragments ranging in size from 350 to 950 bp. Amplicons were digested with eight restriction enzymes. A pattern of species-specificity among the different medically important Candida species could be identified following restriction digestion of the PCR products. Candida albicans was the species most frequently observed, except for the group of newborns under treatment at the Odilon Behrens Hospital and for the isolates from the clinical analysis laboratory. C. parapsilosis was the species most frequently observed in these two groups.

Comparative analysis of noncoding sequences of orthologous bovine and human gene pairs

Genomic sequence comparison across species has enabled the elucidation of important coding and regulatory sequences encoded within DNA. Of particular interest are the noncoding regulatory sequences, which influence gene transcriptional and posttranscriptional processes. A phylogenetic footprinting strategy was employed to identify noncoding conservation patterns of 39 human and bovine orthologous genes. Seventy-three conserved noncoding sequences were identified that shared greater than 70% identity over at least 100 bp. Thirteen of these conserved sequences were also identified in the mouse genome. Evolutionary conservation of noncoding sequences across diverse species may have functional significance, and these conserved sequences may be good candidates for regulatory elements.

Arbitrarily primed polymerase chain reaction for fingerprinting the genotype identification of mutans streptococci in children with Down syndrome

This study investigated the possible intrafamilial similarity of mutans streptococcal strains in some families with a child with Down syndrome using chromosomal DNA fingerprinting. The isolates were genotyped using arbitrarily primed polymerase chain reaction with the OPA 02 and OPA 03 primers. The results showed that five children with Down syndrome harbored mutans streptococci genotypes different from those of their mothers. A matching of genotypes was observed within the control pair (mother/child without Down syndrome). After six months, new samples were collected from all participants. Analysis showed that samples from children with Down syndrome were colonized by a new strain of Streptococcus mutans that did not match the previously collected one. The results suggest the S. mutans indigenous bacteria change more than once in children with Down syndrome.