Eyal Gur

Gracilis muscle transposition for fistulas between the rectum and urethra or vagina.

PurposeThis study was designed to assess the efficacy of gracilis muscle transposition in repairing rectovaginal and rectourethral fistulas.MethodsData were retrieved from a retrospective chart review of patients who underwent gracilis muscle transposition for fistulas between the rectum and urethra/vagina. All patients had fecal diversion as a preliminary or concurrent step to fistula repair. Follow-up data were gathered from outpatient clinic visits. Success was defined as a healed fistula after stoma closure.ResultsSix females and three males, aged 30 to 64 years, underwent gracilis muscle transpositions from 1999 to 2005. One pouch-vaginal, three rectourethral, and five rectovaginal fistulas were repaired. The etiologies were Crohns disease (n = 2), iatrogenic injury to the rectum during radical prostatectomy (n = 2), previous pelvic irradiation for rectal cancer (n = 2) or for cervical cancer (n = 1), recurrent perianal abscesses with fistulas (n = 1), and obstetric tear (n = 1). Seven patients underwent previous medical and surgical repair attempts. There were no intraoperative complications. Postoperative complications included perineal wound infection (n = 1) and at the colostomy closure (n = 2). There were no long-term sequelae. At a median follow-up period of 14 (range, 1–66) months since stoma closure, the fistula healed in seven patients. One patient refused ileostomy closure. One patient with severe Crohns proctitis has a persistent rectovaginal fistula.ConclusionsGracilis muscle transposition is a viable option for repairing fistulas between the urethra, vagina, and the rectum, especially after failed perineal or transanal repairs. It is associated with low morbidity and a good success rate. Underlying Crohns disease and previous radiation are associated with poor prognosis.

Immature Leukemic CD34+CXCR4+ Cells from CML Patients Have Lower Integrin‐Dependent Migration and Adhesion in Response to the Chemokine SDF‐1

Chronic myelogenous leukemia (CML), a malignant myeloproliferative disorder originating from a pluripotent stem cell expressing the bcr‐abl oncogene, is characterized by abnormal release of the expanded, malignant stem cell clone from the bone marrow (BM) into the circulation. Moreover, immature CD34+ CML cells have lower adhesion to stromal cells and fibronectin as well as lower engraftment potential in severe combined immunedeficient (SCID) and nonobese diabetic (NOD)/SCID mice than normal CD34+ cells. We report in this study that leukemic Philadelphia chromosome‐positive (Ph+)CD34+ cells from newly diagnosed CML patients that express the chemokine receptor CXCR4 migrate in response to stromal‐derived factor‐1 (SDF‐1). However, normal Ph‐CD34+CXCR4+ cells derived from the same patient have significantly higher migration levels toward SDF‐1. In contrast to their transwell migration potential, the SDF‐1‐mediated integrin‐dependent polarization and migration of the Ph+CD34+CXCR4+ cells through extracellular matrix‐like gels were significantly lower than for normal cells. Concomitantly, binding of these cells to vascular cell adhesion molecule‐1 or fibronectin, in the presence of SDF‐1, was also substantially lower. These findings suggest a major role for SDF‐1‐mediated, integrin‐dependent BM retention of Ph+CD34+ cells.

Control of methionine biosynthesis in Escherichia coli by proteolysis

Most bacterial proteins are stable, with half‐lives considerably longer than the generation time. In Escherichia coli, the few exceptions are unstable regulatory proteins. The results presented here indicate that the first enzyme in methionine biosynthesis – homoserine trans‐succinylase (HTS) – is unstable and subject to energy‐dependent proteolysis. The enzyme is stable in triple mutants defective in Lon‐, HslVU‐ and ClpP‐dependent proteases. The instability of the protein is determined by the amino‐terminal part of the protein, and its removal or substitution by the N‐terminal part of β‐galactosidase confers stability. The effect of the amino‐terminal segment is not caused by the N‐end rule, as substitution of the first amino acid does not affect the stability of the protein. HTS is the first biosynthetic E. coli enzyme shown to have a short half‐life and may represent a group of biosynthetic enzymes whose expression is controlled by proteolysis. Alternatively, the proteolytic processing of HTS may be unique to this enzyme and could reflect its central role in regulating bacterial growth, especially at elevated temperatures.

In vivo aggregation of a single enzyme limits growth of Escherichia coli at elevated temperatures

The formation of protein aggregates is associated with unfolding and denaturation of proteins. Recent studies have indicated that, in Escherichia coli, cellular proteins tend to aggregate when the bacteria are exposed to thermal stress. Here, we show that the aggregation of one single E. coli cytoplasmic protein limits growth at elevated temperatures in minimal media. Homoserine trans‐succinylase (HTS), the first enzyme in the methionine biosynthetic pathway, aggregates at temperatures higher than 44°C in vitro. Above this temperature, we can also observe in vivo aggregation that results in the complete disappearance of the enzyme from the soluble fraction. Moreover, reducing the in vivo level of HTS aggregation enables growth at non‐permissive temperatures. This is the first demonstration of the physiological role of aggregation of a specific protein in the growth of wild‐type bacteria.

The Escherichia coli DjlA and CbpA Proteins Can Substitute for DnaJ in DnaK-Mediated Protein Disaggregation

The DnaJ (Hsp40) protein of Escherichia coli serves as a cochaperone of DnaK (Hsp70), whose activity is involved in protein folding, protein targeting for degradation, and rescue of proteins from aggregates. Two other E. coli proteins, CbpA and DjlA, which exhibit homology with DnaJ, are known to interact with DnaK and to stimulate its chaperone activity. Although it has been shown that in dnaJ mutants both CbpA and DjlA are essential for growth at temperatures above 37 degrees C, their in vivo role is poorly understood. Here we show that in a dnaJ mutant both CbpA and DjlA are required for efficient protein dissaggregation at 42 degrees C.

Malignant melanoma and pregnancy: second thoughts.

Malignant melanoma (MM) was considered a hormone-sensitive tumour, and pregnancy was thought to increase its risk and cause faster progression and earlier metastasis. Several controlled studies demonstrated similar survival rates between pregnant and non-pregnant patients and concluded that early reports of advanced MM of pregnancy were probably due to late diagnosis. We retrieved information from our database between 1997 and 2006 on all patients diagnosed as having MM during and up to 6 months after pregnancy (n=11) and compared them to age-matched, non-pregnant, MM patients (n=65, controls) treated by us during that period. The mean Breslow thickness was 4.28mm for the pregnant patients and 1.69mm for the controls (p=0.15). The sentinel nodes were metastatic in five pregnant patients compared to four controls (p<0.0001). Two patients in the pregnancy group and one control died of MM (p=0.0532). Our results indicate a negative effect of pregnancy on the course of MM.

The Establishment of a Pseudomonas aeruginosa-Infected Burn-Wound Sepsis Model and the Effect of Imipenem Treatment

Introduction:We present a standardized Pseudomonas aeruginosa (PA) infected burn-wound model in mice for evaluating new antimicrobials and therapy strategies for PA infections and demonstrate the effect of the antibiotic imipenem in this model. Methods:A 6%–8% total body surface area, full-thickness, scald-burn wound was induced in anesthetized mice. Two study groups (PA-infected burn) were compared with 1 treatment group (systemic imipenem) and 3 control groups (noninfected burn, infected nonburned, and burn with distant infection). Seven-day mortality, quantitative culture from eschars and from spleens, weight loss, and time to healing were compared. Results:The 25%–100% mortality rate in the nontreated PA-infected burn group was directly related to the infecting inoculum. Imipenem treatment reduced the mortality rate to 0–17%. No control animal died. Systemic bacterial dissemination at 48 hours was significantly higher in the study group. Morbidity paralleled survival results. Wound healing was quicker in the imipenem-treated group and control groups compared with the infected nontreated group. Conclusions:The mice model is a useful tool for evaluating new antibacterial agents and strategies for treating PA-infected burn injuries. Imipenem was found to be efficacious in the treatment of severe PA sepsis.

Efficacy of antibodies against the N-terminal of Pseudomonas aeruginosa flagellin for treating infections in a murine burn wound model.

Background: In an era of increasing drug resistance, immunotherapy is a desirable treatment against Pseudomonas aeruginosa infections. The flagellum, which is an important pseudomonal virulence factor, was targeted for immunotherapy. The aim of the study was to evaluate the efficacy of polyclonal immunotherapy targeted against the N-terminal of flagellin (anti-N′-fla-b) for treating severe P. aeruginosa infection in a murine burn wound model. Methods: Groups of 12 mice were infected (subeschar) with P. aeruginosa strain PA01, and were treated either with systemic anti-N′-fla-b immunoglobulin G (IgG), nonspecific IgG, or imipenem. The control groups included mice with burn alone, mice with untreated infected burn, and mice without burn infected with P. aeruginosa. Three separate regimens were examined: prophylaxis (preinfection), therapeutic (postinfection), and combined. The efficacy of anti-N′-fla-b was evaluated by monitoring the mortality and morbidity (relative weight loss) during a period of 2 weeks. Results: Anti-N′-fla-b IgG immunotherapy significantly decreased the mortality rate of infected burned mice followed by severe P. aeruginosa infection. The mortality rate in the anti-N′-fla-b–treated groups ranged from 0 to 17 percent compared with 58 to 83 percent in nontreated groups infected with 2 to 5 × 106 colony-forming units of P. aeruginosa (p < 0.05). The mortality rate in the anti-N′-fla-b–treated groups was similar to that of groups treated with imipenem. The three tested regimens yielded similar results. Morbidity paralleled survival results. Histopathologic examination revealed an earlier reepithelialization of the infected wound in the anti-N′-fla-b–treated mice compared with untreated mice. Conclusion: Immunotherapy with anti-N′-fla-b IgG, given either as prophylaxis or therapeutically, effectively reduced mortality and morbidity and improved wound healing in a severely P. aeruginosa–infected murine burn model.

All three J-domain proteins of the Escherichia coli DnaK chaperone machinery are DNA binding proteins.

DnaJ, DjlA and CbpA are the J‐domain proteins of DnaK, the major Hsp70 of Escherichia coli. CbpA was originally discovered as a DNA binding protein. Here, we show that DNA binding is a property of DnaJ and DjlA as well. Of special interest in this respect is DjlA, as this cytoplasmic protein is membrane bound and, as shown here, its affinity for DNA is extremely high. The finding that all the three J‐proteins of DnaK are DNA binding proteins sheds new light on the cellular activity of these proteins.

Simultaneous contralateral breast adjustment in unilateral deep inferior epigastric perforator breast reconstruction.

BACKGROUND Breast symmetry is a key factor in deep inferior epigastric perforator (DIEP) flap breast reconstruction, which necessitates in many cases contralateral breast adjustment, traditionally done at a second stage. We present our experience with simultaneous contralateral breast adjustment in unilateral DIEP breast reconstruction. METHODS We retrospectively reviewed all consecutive unilateral DIEP breast reconstructions done in our institution. The patients were divided into three groups according to contralateral breast surgery performed: simultaneous adjustment, late adjustment, and no contralateral breast adjustment surgery. The groups were compared by aesthetic outcome and patient satisfaction using the BREAST-Q questionnaire. RESULTS A total of 77 unilateral breast reconstructions were performed using the DIEP flap. Fifty-one eligible patients agreed to respond to the questionnaire by telephone and were enrolled in the study; 33 underwent simultaneous contralateral breast adjustment, eight underwent late adjustment procedure, and 10 had no contralateral surgery performed. Aesthetic outcome and patient satisfaction was comparable in the simultaneous and late adjustment groups, but was reduced during the latent period. CONCLUSION Simultaneous contralateral breast adjustment in unilateral DIEP breast reconstruction is a safe and a worthwhile procedure that should be offered to the patient when appropriate.