Biomedical subjects
R Silva
Publications and source records attributed to R Silva.
Characterisation of the lpdA gene from Neisseria meningitidis by polymerase chain reaction, restriction fragment length polymorphism and sequencing.
P64k protein from Neisseria meningitidis is well recognised in sera from individuals convalescent from meningococcal disease or vaccinated with the Cuban antimeningococcal vaccine VA-MENGOC-BC. The presence of the protein in more than 80 meningococcal strains has also been verified. It is immunogenic in animal models and the antibodies elicited show bactericidal activity against meningococci. To further investigate at the molecular level whether lpdA, the gene coding for P64k protein, is conserved among different N. meningitidis strains, a total of 20 strains isolated from different geographic areas were differentiated on the basis of restriction fragment length polymorphism (RFLP) patterns after polymerase chain reaction (PCR) amplification of the lpdA gene and restriction endonuclease digestion with HpaII. Although a total of five different PCR-RFLP patterns were present, nucleotide sequence determination showed that identity levels were as high as 93-99% among the N. meningitidis strains analysed.
Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and pH.
The molecular basis of bilirubin toxicity to nerve cell function is still unclear. Since astrocytes are the main transporters of synaptically released glutamate and impaired glutamate uptake results in neuronal death, we investigated the effect of unconjugated bilirubin (UCB) on [(3)H]glutamate uptake in cultured rat astrocytes and the role of bilirubin ionization on toxicity. Astrocytes were incubated for 5-15 min, with UCB concentrations from 17 to 342 microM and UCB/albumin molar ratios of 0.2-3.0, at pH 7.0, 7.4, and 8.0. Exposure of astrocytes for 15 min to 85.5 microM UCB and 28.5 microM albumin resulted in a 63.1% decrease of glutamate uptake (p < 0.01). Interestingly, the effect demonstrated to be correlated with the UCB/albumin molar ratio (r = -0.986, p < 0.01) and a significant decrease was observed for a UCB/albumin molar ratio as low as 0.8. Inhibition of glutamate transport was also pH-dependent as it occurred at 7.4 (p < 0.05) and 8.0 (p < 0.01), but not at 7.0, suggesting that the monoanionic species of UCB accounted for the inhibition. These findings indicate that UCB, and more precisely the monoanionic species, impairs a crucial function of astrocytes such as glutamate transport and support a potential role of astrocyte function in the pathogenesis of UCB-related brain damage (kernicterus).
Microbiology of sinusitis in patients undergoing bone marrow transplantation.
As a result of increasing use of bone marrow transplantation and new cytotoxic chemotherapy, more patients have become susceptible to sinus disease caused by unusual organisms. Sinusitis caused by fungi and gram-negative bacteria can be difficult to treat, may lead to severe complications, and should be managed promptly in the bone marrow transplant patient. Here we present the results of 41 cultures of the paranasal sinuses obtained from 18 bone marrow transplant patients in whom sinusitis developed. The most common agents were gram-negative bacteria (56.7%), followed by gram-positive bacteria (26.7%) and fungi (16.6%). In 13 samples the cultures were negative. Nasal cultures were performed ipsilateral to the sinus drained in 28 cases. Concordance was obtained in only 5 (17.8%) samples. The antibiogram of the isolated agents from the maxillary sinuses in this series revealed that the most efficient antibiotics were those that covered gram-negative bacteria. Treatment was usually prolonged in these patients, and different antibiotics were necessary to clear infections from the sinuses. In conclusion, treating sinusitis in bone marrow transplant patients may be challenging. Considerations about the microbiology and antibiogram susceptibilities of this specific population should be kept in mind when dealing with such cases.
Very large (CAG)(n) DNA repeat expansions in the sperm of two spinocerebellar ataxia type 7 males.
Genetic anticipation, i.e. increasing disease severity and decreasing age of onset from one generation to the next, is observed in a number of diseases, including myotonic dystrophy type 1, Huntington's disease and several of the spinocerebellar ataxias. All of these disorders are associated with the expansion of a trinucleotide repeat and array length is positively correlated with disease severity and inversely correlated with the age of onset. The expanded repeat is highly unstable and continues to expand from one generation to the next, providing a molecular explanation for anticipation. Spinocerebellar ataxia type 7 (SCA7) is one of the latest additions to the list of triplet repeat diseases and is distinct from the other SCAs in that it is accompanied by retinal degeneration. Pedigree analyses have previously revealed that the SCA7 repeat is highly unstable and liable to expand, in particular when transmitted by a male. Surprisingly, though, an under-representation of male transmission has also been reported. We now demonstrate directly by single molecule analyses that the expanded repeat is extraordinarily unstable in the male germline and biased toward massive increases. Nearly all of the mutant sperm of two SCA7 males contain alleles that are so large that most of the affected offspring would at best have a severe infantile form of the disease. Indeed, the gross under-representation of such very large expanded alleles in patients suggests that a significant proportion of such alleles might be associated with embryonic lethality or dysfunctional sperm.
Fragile X trinucleotide repeats from a normal population in Rio de Janeiro, Brazil.
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Calcium and endoplasmic reticulum dynamics during oocyte maturation and fertilization in the marine worm Cerebratulus lacteus.
To monitor calcium and endoplasmic reticulum (ER) dynamics during oocyte maturation and fertilization, oocytes of the marine worm Cerebratulus lacteus were injected with the calcium-sensitive indicator calcium green dextran and/or the ER-specific probe "DiI." Based on time-lapse confocal imaging of such specimens, prophase-arrested immature oocytes failed to develop normally after insemination and typically produced non-wave-like calcium transients that were lower in amplitude and less persistent than the wave-like oscillations observed during fertilizations of mature oocytes. Accordingly, the ER of DiI-loaded immature oocytes lacked an obvious substructure, whereas ER clusters, or "microdomains," began to form in maturing specimens at about the time that these oocytes became competent to undergo normal fertilization-induced calcium dynamics and cleavage. The ER microdomains of mature oocytes typically reached widths of 1-8 micrometer and disappeared approximately 1 h after fertilization, which in turn coincided with the termination of the calcium oscillations. Collectively, these findings indicate: (i) changes in ER structure are temporally correlated with the onset and cessation of the calcium oscillations required for subsequent cleavage, and (ii) such ER reorganizations may play an important role in early development by enabling mature oocytes to generate a normal calcium response.
Elution of unmodified oligodeoxynucleotides from zinc-imidazole negatively stained polyacrylamide gels.
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Allelic frequency distribution for three VNTR markers--D6S132, D7S467, D17S26--in Rio de Janeiro population, Brazil.
A sample population of Rio de Janeiro, Brazil, was tested for D6S132, D7S467, and D17S26 VNTR loci to determine the fixed-bin frequencies of alleles. RFLP analysis was obtained by HaeIII-digested genomic DNA. The three VNTR loci meet Hardy-Weinberg expectations, and there is no evidence for association of alleles between the VNTR loci. The frequency data can be used in forensic analysis, in paternity tests and to estimate the frequency of a DNA profile in the general Brazilian population.
Relevance of serum bile acid profile in the diagnosis of intrahepatic cholestasis of pregnancy in an high incidence area: Portugal.
OBJECTIVE(S): The present work was conducted to clarify the relevance of usual liver function tests, and define the most predictive serum bile acid profile for diagnosis of intrahepatic cholestasis of pregnancy (ICP). STUDY DESIGN: This study comprised 20 healthy nonpregnant women and 77 pregnant women in the last trimester of pregnancy, from which 38 were normal pregnancies, and 39 suffered from ICP. Liver function tests were evaluated by routine laboratory techniques, conjugated bile acids were analysed by high-performance liquid chromatography, and unconjugated forms were measured by an enzymatic-fluorimetric assay. RESULTS: During the third trimester in normal pregnancy, increased concentration of conjugated species affected all primary bile acids, although only significantly for glycocholic acid. Moreover, deoxycholic acid proportion decreased when compared with healthy nonpregnant women. Important ICP-induced changes in serum profiles of amidated bile acids were observed, involving both a marked increase in cholic acid concentration and a shift towards a higher proportion of taurine-conjugated species. Among routine liver tests, alanine aminotransferase and conjugated bilirubin were the most common indicators of ICP. CONCLUSION(S): In the early diagnosis and follow-up of ICP, the most predictive and accurate markers (efficiency 100%) were: (i) TBA concentration in serum >11.0 micromol(-1): (ii) cholic/chenodeoxycholic acid ratio >1.5 and cholic acid percentage >42%: (iii) glycine/taurine bile acid ratio <1.0 or glycocholic acid concentration >2.0 micromol(-1). Accurate diagnosis based on sensitive biochemical markers followed by appropriate treatment may improve both pregnancy outcome and newborn prognosis.
Laparoscopic cholecystectomy in cirrhotic patients.
BACKGROUND: Reported mortality for open cholecystectomy in patients with cirrhosis ranges from 10% to 80%. Laparoscopic cholecystectomy has gained acceptance in the general population and has become the procedure of choice for symptomatic cholelithiasis. We reviewed our experience with the use of laparoscopic cholecystectomy in this group. STUDY DESIGN: We did a retrospective review of the records of 25 consecutive laparoscopic choleoystectomy procedures performed on cirrhotic patients from May 1992 to July 1996. RESULTS: There were no mortalities in our group. All procedures were completed laparoscopically. Mean length of stay was 1.7 days (range, 1 to 8 days). Morbidity consisted of wound hematomas, pneumonia, and ascites for a rate of 32%. Only patients with Child's Class A and Class B cirrhosis were operated on. CONCLUSIONS: Laparoscopic cholecystectomy can be performed safely in cirrhotic patients with well compensated liver function.
The kinetic profile of vancomycin in neonates.
The pharmacokinetic parameters of vancomycin in a neonatal population have been characterized to enable development of optimum dosage guidelines for neonatal intensive-care units and to examine the relationship between these pharmacokinetic parameters and various demographic, developmental and clinical factors which might be associated with changes in the kinetic profile of vancomycin. Forty-four infants (twenty-five males and nineteen females) with suspected or proven Gram-positive infection and who received intravenous vancomycin between October 1993 and December 1996 were included in this retrospective analysis. Gestational age ranged from 25 to 40 weeks and postconceptional age at the time of the study ranged from 28 to 45 weeks. Sixty case-studies were obtained from the forty-four patients, with one period of study corresponding to one week or one cycle of therapy. Vancomycin pharmacokinetic parameters were determined by use of a one-compartment model. By regression analysis the current weight (g) was shown to be the stronger covariate, and both vancomycin clearance (L h(-1)) and volume of distribution (L) had to be normalized. The vancomycin volume of distribution depended on the postconceptional age with a cut-off at 32 weeks, whereas vancomycin clearance depended on the presence or absence of concomitant treatment with indomethacin or of mechanical ventilation, or both. On the basis of the pharmacokinetic parameters obtained we suggest initial dosage guidelines for vancomycin ranging from 10 mg kg(-1) every 8 h to 10 mg kg(-1) every 12 h, depending on the demographic and clinical characteristics of the patients. The results obtained enabled application of better a priori and a posteriori dosage schedules to infants in neonatal intensive-care units by use of the Bayesian approach, although further prospective study is recommended before direct extrapolation to patients in other settings.
Kinins mediate the inhibition of atrial natriuretic peptide diuretic effect induced by pepsanurin.
Pepsanurin is a peptidic fraction resulting from pepsin digestion of plasma globulins, that inhibits ANP renal excretory actions. We studied whether kinin-like peptides mediate the anti-ANP effect by testing if pepsanurin: 1) was blocked by the kinin B2 receptor antagonist HOE-140, 2) was produced from kininogen, and 3) was mimicked by bradykinin. Anti-ANP activity was assessed in anesthetized female rats by comparing the excretory response to two ANP boluses (0.5 microgram i.v.) given before and after i.p. injection of test samples. Pepsanurin from human or rat plasma (1-5 mL/kg), and bradykinin (5-20 micrograms/kg), dose-relatedly inhibited ANP-induced water, sodium, potassium and cyclic GMP urinary excretion, without affecting arterial blood pressure. The same effect was exerted by pepsin hydrolysates of purified kininogen, whereas hydrolysates of kininogen-free plasma had no effect. HOE-140 (5 micrograms, i.v.) did not alter baseline, or ANP-induced excretion, but blocked the anti-ANP effects of pepsanurin. Histamine (15 micrograms/kg) plus seroalbumin hydrolysates did not affect ANP response, despite inducing larger peritoneal fluid accumulation as compared with pepsanurin or bradykinin. We concluded that kinins cleaved from kininogen mediate the anti-ANP effects of pepsanurin by activation of kinin B2 receptors, independently of changes in systemic arterial pressure or peritoneal fluid sequestration.
[Glucose in conjunction with peptides derived from kininogens might act from digestive tract as blockers of atrial natriuretic peptide mediated diuresis-natriuresis].
This paper describes long term research efforts which have lead: 1) to the identification of peptides present in pepsanurin, a peptidic fraction obtained by pepsin hydrolysis of plasma globulins that inhibits the renal excretory action of atrial natriuretic peptide (ANP) and 2) to the discovery of an unexpected role of glucose, as a requisite for these inhibitory effects. The active peptides identified in pepsanurin are derived from plasma kininogens, substrates of the kallikrein-kinin system. Pro-kinins of 15, 16 and 18 amino acids, and bradykinin itself, block ANP-induced diuresis and natriuresis when injected i.v., i.p. or into the duodenal lumen of anesthetized rats in picomol doses. Furthermore, a novel 20 amino acids fragment derived from kininogen dominium-1, named PU-D1, is the most potent and longer lasting blocker of ANP renal effects. The anti-ANP effects of those peptides are prevented by B2-kinin receptor antagonists. The inhibition of ANP by kinins and PU-D1 was evident only in rats infused with isotonic glucose; whereas the excretory effect of ANP was not affected in fasted rats not infused, or infused with saline. These findings provide evidence that glucose facilitates liquid retention through a kinin-mediated inhibition of ANP excretory action that may be related to the prandial cycle.
Expression in Escherichia coli of the lpdA gene, protein sequence analysis and immunological characterization of the P64k protein from Neisseria meningitidis.
By making use of recombinant DNA technology it is possible to characterize meningococcal outer membrane proteins (OMPs) capable of stimulating a host immune response. The lpdA gene, which codes for an OMP (P64k) from Neisseria meningitidis, was cloned in Escherichia coli. The recombinant protein was recognized by sera from patients convalescing from meningococcal disease. The monoclonal antibodies obtained against the recombinant protein recognized the natural protein on a Western blot, and monoclonal antibody 114 was assayed in ELISA with a panel of 85 N. meningitidis strains. The protein was recognized in 81 strains (95.3%); the strains that were not recognized were neither epidemic nor isolated from systemic disease. The complete amino acid sequence of P64k was obtained by automatic sequencing and MS.
Vulnerability to stress: self-criticism and stress-induced changes in biochemistry.
It has been hypothesized that individuals who are high on the attribute of self-criticism are particularly vulnerable to failure stress. To test this hypothesis, we examined the relationship between self-criticism and changes in plasma homovanillic acid (HVA; the metabolite of dopamine) and emotion during exposure to an induced-failure task. Participants consisted of 21 women. Plasma HVA and emotion were assessed at three time points: baseline (T1), during stress exposure (T2), and 40 minutes after cessation of the stressor (T3). We found that self-criticism was significantly and positively related to changes in plasma HVA during stress exposure. In addition, the personality attribute was significantly and positively related to subjective ratings of stress and changes in scores on the Confusion-Bewilderment scale of the Profile of Mood States during the task. To our knowledge, this is the first study to report that self-criticism is related to stress-induced changes in biochemistry.
Contact allergy to gold sodium thiosulfate. A comparative study.
During 1995, we added gold sodium thiosulfate and potassium dicyanoaurate to the patch test standard series. Our purpose was to compare the reactivity of the gold salts, to determine the frequency of contact allergy to gold in our country, to compare our results with those of the Malmö group and, finally to try to clarify the clinical relevance 2853 patients were routinely tested and 168 patients answered a questionnaire similar to that used by the Malmö group. Contact allergy to gold was found in 23 patients, all women: 22 to gold sodium thiosulfate (0.78%), only 1 to potassium dicyanoaurate and 2 to both salts. All reactors had their ears pierced and were exposed to gold jewelery (mainly earrings). Simultaneous allergies to nickel and cobalt were statistically significant in patients with positive patch tests to gold sodium thiosulfate. Atopy was no more common among these patients.
Adjuvant modulation of immune responses to tuberculosis subunit vaccines.
Mice were immunized with experimental subunit vaccines based on secreted antigens from Mycobacterium tuberculosis in a series of adjuvants, comprising incomplete Freund's adjuvant (IFA), dimethyl dioctadecyl ammoniumbromide (DDA), RIBI adjuvant, Quil-A saponin, and aluminum hydroxide. Immune responses induced by these vaccines were characterized by in vitro culture of primed cells, PCR analysis for cytokine mRNA, detection of specific immunoglobulin G isotypes induced, and monitoring of protective immunity to tuberculosis (TB). The study demonstrated marked differences in the immune responses induced by the different adjuvants and identified both IFA and DDA as efficient adjuvants for a TB subunit vaccine. Aluminum hydroxide, on the other hand, induced a Th2 response which increased the susceptibility of the animals to a subsequent TB challenge. DDA was further coadjuvanted with either the Th1-stimulating polymer poly(I-C) or the cytokines gamma interferon, interleukin 2 (IL-2), and IL-12. The addition of IL-12 was found to amplify a Th1 response in a dose-dependent manner and promoted a protective immune response against a virulent challenge. However, if the initial priming in the presence of IL-12 was followed by two booster injections of vaccine without IL-12, no improvement in long-term efficacy was found. This demonstrates the efficacy of DDA to promote an efficient immune response and suggests that IL-12 may accelerate this development, but not change the final outcome of a full vaccination regime.