Intracellular survival of persistent group A streptococci in cultured epithelial cells

Group A streptococcus (GAS) is the principle etiologic agent of bacterial pharyngotonsillitis and a wide range of other diseases. Failure to eradicate GAS from patients has been documented in 5-30% of patients with pharyngotonsillitis, in spite of the continued sensitivity of GAS to penicillin and other beta-lactams. It was recently proposed that eradication failure might be attributed to the ability of GAS to maintain an intracellular reservoir during antibiotic treatment. We have previously shown that strains derived from patients with bacterial eradication failure, despite antibiotic treatment (persistent strains), adhered to and were internalized by cultured epithelial cells more efficiently than strains that were successfully eradicated. Since, penicillin and other beta-lactams do not penetrate well into mammalian cells, intracellular survival of GAS is crucial in order to persist during prolonged antibiotic treatment. In this study, we compared the survival of GAS strains from cases of eradication failure and eradication success, using an epithelial cell culture model. We found that persistent strains show significantly increased intracellular survival, compared to the 'eradication success' strains. This finding supports the idea that an intracellular reservoir of GAS plays a role in the etiology of antibiotic eradication failure.     

Molecular mechanism of kNBC1–carbonic anhydrase II interaction in proximal tubule cells

We have recently shown that carbonic anhydrase II (CAII) binds in vitro to the C-terminus of the electrogenic sodium bicarbonate cotransporter kNBC1 (kNBC1-ct). In the present study we determined the molecular mechanisms for the interaction between the two proteins and whether kNBC1 and CAII form a transport metabolon in vivo wherein bicarbonate is transferred from CAII directly to the cotransporter. Various residues in the C-terminus of kNBC1 were mutated and the effect of these mutations on both the magnitude of CAII binding and the function of kNBC1 expressed in mPCT cells was determined. Two clusters of acidic amino acids, L⁹⁵⁸DDV and D⁹⁸⁶NDD in the wild-type kNBC1-ct involved in CAII binding were identified. In both acidic clusters, the first aspartate residue played a more important role in CAII binding than others. A significant correlation between the magnitude of CAII binding and kNBC1-mediated flux was shown. The results indicated that CAII activity enhances flux through the cotransporter when the enzyme is bound to kNBC1. These data are the first direct evidence that a complex of an electrogenic sodium bicarbonate cotransporter with CAII functions as a transport metabolon.     

Genetic association of the PERIOD3 (PER3) Clock gene with extreme obesity

BackgroundObesity has reached epidemic proportions worldwide, affecting life quality and span. Susceptibility to obesity is partly mediated by genetic differences. Indeed, several genes from the clock gene family have already been shown to be intimately associated with obesity in diverse ethnic groups. In the present study, an association between BMI and the rs707467, rs228697 and rs228729 PER3 (Period Circadian Clock 3) polymorphisms in subjects with class II (BMI ≥ 35.0–39.9 kg/m²) and class III obesity (>40 kg/m², extreme obesity) were carried out using TaqMan real-time PCR. Overall, 259 Brazilian adults were genotyped, of whom 122 had class II or III obesity (BMI ≥ 35.0 kg/m²) and 137 were controls having normal weight (BMI > 18.5 and <24.9 kg/m²).ResultsPER3 tag SNP (rs228729) shows a significant association with extreme obesity (1000 permutation p = 0.03 and p = 0.04), for genotype and allele frequency respectively) and a haplotype among the three assessed SNPs (alleles G/T/A, rs228697, rs228729, and rs707467, respectively, 1000 permutation p = 0.03) was significantly more prevalent in the group with obesity.ConclusionThis exploratory association study suggests that PER3 rs228729 may be associated with extreme obesity in Brazilian adults, however, replication is needed.     

The impact of patient delay and physician delay on the outcome of laparoscopic cholecystectomy for acute cholecystitis

BACKGROUND: Laparoscopic cholecystectomy is now used in the management of acute cholecystitis. Under these circumstances unfavorable conditions may result in conversion and complications. Information about these conditions may help in planning the laparoscopic approach or in proceeding directly to open cholecystectomy. This study was initiated to evaluate perioperative factors associated with conversion and complications of laparoscopic cholecystectomy in acute cholecystitis. Special attention was paid to the duration of complaints until surgery, to the delay on the part of the patient, and to the delay on the part of the physician. METHODS: Between January 1994 and December 1997, we attempted to perform laparoscopic cholecystectomy on 348 patients with acute cholecystitis. All perioperative data were collected on standardized forms. RESULTS: There were 182 cases (52%) of acute uncomplicated cholecystitis, 90 (26%) of gangrenous cholecystitis, 33 of hydrops (9.5%), and 43 of empyema of the gallbladder (12.5%). Seventy six patients (22%) needed conversion to open cholecystectomy and complications occurred in 57 cases. Advanced cholecystitis was associated with significant patient delay (P = 0.01), and it had a significantly higher conversion rate (39%) compared with early cholecystitis (14.5%); (P <0.00001). Conversion rates were also associated with male gender (P = 0.0017), a history of biliary disease (P = 0.0085), and a patient delay of >48 hours (P = 0.028). The total and infectious complication rates were associated with an age older than 60 years (P = 0.023 and 0.007, respectively) and male gender (P = 0.026 and 0.014, respectively). CONCLUSIONS: In acute cholecystitis, patient delay is associated with a high conversion rate. Early timing of laparoscopic cholecystectomy tends to reduce the conversion rate, as well as the total and the infectious complication rates. Male gender, a history of biliary disease, and advanced cholecystitis are associated with conversion. Male and older patients are associated with a high total and infectious complication rates.     

A simplified laparoscopic technique for mesh placement in ventral hernia repair

The use of a large synthetic mesh for laparoscopic repair of significant ventral abdominal wall defects may be accompanied by technical difficulties resulting from improper orientation and positioning of the mesh over the defect. We suggest a technique based on initial fixation of the mesh center to the central point of the defect, and subsequent centrifugal attachment of the mesh to the abdominal wall. This technique is advantageous because it leads to precise orientation and positioning of the synthetic patch and to significant reduction of the time needed for its reinforcement over and around the defect. Received: 25 September 1998/Accepted: 27 November 1998     

Screening for mutations in candidate genes for hypospadias

Hypospadias, a condition with a frontally placed urethral orifice on the penis, is the most common malformation in males. During fetal development several components are necessary for normal male genital development. Testosterone and dihydrotestosterone act via the androgen receptor but a defective receptor function results in different degrees of genital malformations. Testosterone-5α-reductase converts testosterone to dihydrotestosterone, which is crucial for normal differentiation, and a total lack of this enzyme results, in syndromes with hypospadias. The Wilms' tumour 1 (WT1) gene is expressed in the fetal gonad and genital malformations can occur due to WT1 gene mutations. These genes are therefore strong candidate genes for hypospadias. We have analysed 35 boys with hypopadias and one girl diagnosed as with complete androgen insensitivity syndrome, using exon by exon polymerace chain reaction (PCR) amplification of the AR, WT1 and 5α-reductase genes and screened for point mutations and performed subsequent DNA sequencing. No mutations in any of these genes were found in the 26 patients with isolated hypospadias. Two patients with severe hypospadias with cryptorchidism were found to carry mutations in the androgen receptor gene. Also the girl with clinically diagnosed complete androgen insensitivity was found to be homozygous for a splice mutation in the 5α-reductase gene. In summary, mutations in the WT1, AR and 5α-reductase genes are not common causes of isolated hypospadias. Received: 1 October 1997 / Accepted: 4 May 1998     

Surveillance of antimicrobial prophylaxis for surgical procedures.

OBJECTIVE: To assess the practice of antimicrobial prophylaxis for surgical procedures in eight surgical departments in a 550-bed teaching hospital. METHODS: A list of all major procedures performed in our hospital, with recommendations for prophylaxis based upon the literature, has been distributed since 1993 and is updated periodically. The practice of surgical prophylaxis between January 1 and March 31, 1996, was examined by assessing four variables: (1) Did the particular procedure justify prophylaxis, and was it provided? (2) Was timing optimal, ie, within 1 hour prior to surgery? (3) Was the appropriate antimicrobial selected? (4) Was duration optimal, ie, < or =24 hours? RESULTS: During the study period, 2,117 operations were performed, of which 1,631 (77%) were reviewed. Sixty-six percent were clean surgery, 28% clean-contaminated, and 6% contaminated; 72% of procedures were elective, 28% emergencies. Of 1,631 operations requiring prophylaxis, 1,142 (70%) received it, 489 (30%) did not. Of 1,631 patients, 1,392 (85%) received appropriate care: 929 (67%) appropriately received prophylaxis, and 463 (33%) appropriately did not receive prophylaxis. Of 955 patients who received prophylaxis, 26 (3%) did so inappropriately. Of 1,142 patients who should have received prophylaxis, 213 (19%) did not receive it. Female gender, clean surgery, elective operations, and infrequently performed procedures were all significant indicators of inappropriately withheld prophylaxis (P<.001). In addition, the rate of appropriately provided prophylaxis varied between departments from 71% to 97% (P<.001). Assessment of the 929 procedures for which prophylaxis was justified and given revealed that 100% of patients received it on time, the choice of antimicrobial was appropriate in 95% of cases, and duration was < or =24 hours in 91%. CONCLUSIONS: Audits of surgical prophylaxis are expected to detect different errors in different institutions. Conducting audits of surgical prophylaxis probably should be part of the routine activity of infection control teams. Feeding the information back to surgeons could improve adherence to recommended guidelines and might contribute to reduced wound infection rates.     

Anticholinergic and antiglutamatergic agents protect against soman-induced brain damage and cognitive dysfunction.

Soman, a powerful inhibitor of acetylcholinesterase, causes an array of toxic effects in the central nervous system including convulsions, learning and memory impairments, and, ultimately, death. We report on the protection afforded by postexposure antidotal treatments, combined with pyridostigmine (0.1 mg/kg) pretreatment, against these consequences associated with soman poisoning. Scopolamine (0.1 mg/kg) or caramiphen (10 mg/kg) were administered 5 min after soman (1.2 LD50), whereas TAB (i.e., TMB4, atropine, and benactyzine, 7.5, 3, and 1 mg/kg, respectively) was injected in rats concomitant with the development of toxic signs. Atropine (4 mg/kg) was given to the two former groups at the onset of toxic symptoms. Caramiphen and TAB completely abolished electrographic seizure activity while scopolamine treatment exhibited only partial protection. Additionally, no significant alteration in the density of peripheral benzodiazepine receptors was noted following caramiphen or TAB administration, while scopolamine application resulted in a complex outcome: a portion of the animals demonstrated no change in the number of these sites whereas the others exhibited markedly higher densities. Cognitive functions (i.e., learning and memory processes) evaluated using the Morris water maze improved considerably by the three treatments when compared to soman-injected animals; the following rank order was observed: caramiphen > TAB > scopolamine. Additionally, statistically significant correlations (r = 0.72, r = 0.73) were demonstrated between two learning parameters and [3H]Ro5-4864 binding to brain membrane. These results show that drugs with a pharmacological profile consisting of anticholinergic and antiglutamatergic properties such as caramiphen and TAB, have a substantial potential as postexposure therapies against intoxication by organophosphates.