Multiplex PCR Assay for Identifi cation and Differentiation of Campylobacter jejuni and Campylobacter coli Isolates.

INTRODUCTION: Campylobacter spp. are important causative agents of gastrointestinal infections in humans. The most frequently isolated strains of this bacterial genus are Campylobacter jejuni and Campylobacter coli. To date, genetic methods for bacterial identification have not been used in Bulgaria. We optimized the multiplex PSR assay to identify Campylobacter spp. and differentiate C. jejuni from C. coli in clinical isolates. We also compared this method with the routinely used biochemical methods. AIM: To identify Campylobacter spp. and discriminate C. coli from C. jejuni in clinical isolates using multiplex PCR assay. MATERIALS AND METHODS: Between February 2014 and January 2015 we studied 93 stool samples taken from patients with diarrheal syndrome and identified 40 species of Campylobacter spp. in them. The clinical material was cultured in microaerophilic atmosphere, the isolated strains being biochemically diff erentiated (hydrolysis of sodium hippurate for C. jejuni, and hydrolysis of indoxyl acetate for C. coli). DNA was isolated from the strains using QiaAmp MiniKit (QIAGEN, Germany). Twenty strains were tested with multiplex PCR for the presence of these genes: cadF, characteristic for Campylobacter spp., hipO for C. jejuni and asp for C. coli. RESULTS AND DISCUSSION: The biochemical tests identified 16 strains of C. jejuni, 3 strains of C. coli, and 1 strain of C. upsaliensis. After the multiplex PCR assay the capillary gel electrophoresis confirmed 16 strains of C. jejuni, 2 strains of C. coli and 2 strains of Campylobacter spp. - because of the presence of the gene cadF. C. jejuni has the gene hipO, and it is possible that this gene may not be expressed in the biochemical differentiation yielding a negative reaction as a result. In comparison, we can conclude that the genetic differentiation is a more accurate method than the biochemical tests. CONCLUSION: The multiplex PCR assay is a fast, accurate method for identifi cation of Campylobacter spp. which makes it quite necessary in the clinical diagnostic practice.

Synchronous adenocarcinoma and echinococcosis in the same ovary: a rare clinical entity.

BACKGROUND: Serous adenocarcinoma is the most common subtype of ovarian carcinoma, while ovarian echinococcosis is a rare disease of the female genital system. The simultaneous presence of adenocarcinoma and echinococcosis in the same ovary is extremely rare and little data exist regarding diagnosis and treatment approach. MATERIALS AND METHODS: In the present paper, we report a rare case of a 66-year-old Greek Caucasian woman of rural origin, with an adnexal mass, ascitic and pleural fluid. The patient underwent exploratory laparotomy following neoadjuvant chemotherapy, based on positive for malignancy pleural and ascitic fluid cytology results. RESULTS: Histological examination of the surgical specimen indicated the synchronous presence of echinococcosis and adenocarcinoma in the same ovary. CONCLUSIONS: Ovarian hydatid cyst is extremely rare, but should be suspected in patients with echinococcosis in other organs and those coming from endemic areas. Moreover, the disease could mimic ovarian cancer, as there are no specific symptoms and clinical signs. The coexistence of these two diseases in the same ovary, although rare, is possible. It seems that the presence of the hydatid cyst does not change the treatment approach of the malignancy so that surgery with neoadjuvant or adjuvant chemotherapy, according to the present guidelines, should be administered. Hippokratia 2015, 19 (1): 89-91.

Selenomethionine treatment in patients with autoimmune thyroiditis: a prospective, quasi-randomised trial.

AIMS: To test whether selenium administration affects autoantibodies to thyroid peroxidase (anti-TPO) and thyroglobulin (anti-TG) titres in chronic autoimmune (Hashimoto's - HT) thyroiditis. METHODS: A prospective, open-label, quasi-randomised study in 86 HT patients (n = 86) assigned to either selenomethionine (Seme) 200µg daily for 3 months (Se3, n = 15) or 6 months (Se6, n = 46) or placebo (Control, n = 25). Serum Se, anti-TPO, anti-TG and thyroid hormones were measured in all patients at baseline, 3 and 6 months. A subgroup of 18 patients (twelve on Se6 and six controls) were subjected in thyroid fine-needle biopsy at baseline and 6 months to detect changes in lymphocyte infiltration. RESULTS: No significant difference in anti-TPO levels was recorded after 3 (p = 0.88) or 6 months (p = 0.62) on Seme. Anti-TG levels decreased both at 3 months (p = 0.001) and 6 months (p = 0.001). No significant changes in thyroid stimulating hormone, free thyroxine and free triiodothyronine levels or in the lymphocytes' number in thyroid cytology specimens were detected. Age, gender, duration of disease, baseline anti-TPO levels and per cent change in Se levels could not predict the response of anti-TPO levels to Seme administration. CONCLUSION: Our data suggest that Seme administration in pharmacological doses for a period of 6 months seems to have no significant effect on serum thyroid auto-antibodies' levels or lymphocyte infiltration of the thyroid gland.

Successful treatment of a spontaneous haemothorax with recombinant factor VIIa in a haemophilic child with inhibitors.

Haemophilia A is an X-linked disorder caused by a deficiency of factor VIII. Haemorrhage in various sites may occur spontaneously or secondary to trauma depending on the severity of the deficiency. Common manifestations include haemarthrosis, epistaxis, gastrointestinal haemorrhage and haematuria. Spontaneous haemothorax has rarely been reported both in children and adults1,2. We report the case of a haemophiliac child presenting with spontaneous haemothorax due to the rarity of this clinical presentation in order to raise the awareness among clinicians.

Acute respiratory admissions in Thessaloniki, Greece: 14-year follow-up.

BACKGROUND: Acute exacerbations of respiratory diseases are a common cause of hospitalization among infants and children. AIM: To assess the epidemiological characteristics of asthma, bronchiolitis and croup among hospitalized children in Thessaloniki, from 1990 to 2003 included. METHODS: Data from the patient registry with discharge diagnosis "asthma", "bronchiolitis" and "croup" were analyzed retrospectively, in five Paediatric Departments of Thessaloniki. Age and sex of the patients, as well as the month of admission were taken into consideration. RESULTS: A total of 8762 admissions of children (aged 3 months-14 years) with the diagnosis of asthma, "bronchiolitis" and "croup", were identified. Sex distribution was 65.86% males (64.86% bronchiolitis, 65.26% asthma and 70.31% croup). Asthma admissions decreased by 53.65%, croup admissions decreased by 4.73%, while bronchiolitis admissions increased by 25.03%, during the study period. A clear seasonal variation was found in all the three diseases, with the lowest incidence during summer months. Moreover there were two peaks for asthma (one during spring and a second during autumn), one peak for bronchiolitis (during winter early spring) and one peak for croup (during autumn). CONCLUSIONS: Paediatric asthma and croup admissions have declined during the last 14 years, in contrast with bronchiolitis admissions, which showed an increased tendency. More frequent use of inhaled steroids and induction of asthma education programs may have contributed to decreasing asthma admission rates.

[Laparoscopic repair of incisional and umbilical hernias. Our experience]. / Riparazione laparoscopica dei laparoceli e delle ernie ombelicali. Nostra esperienza.

BACKGROUND: Incisional hernia is a frequent complication of abdominal surgery (2-20% of all cases). Recurrence rate after simple repair without mesh is very high. The use of prosthetic materials has reduced the recurrences. Over the past decade, laparoscopic repair of incisional and umbilical hernias has become an interesting alternative to open procedure. PATIENTS AND METHODS: The aim of this retrospective study was to evaluate efficacy, safety and advantages of laparoscopic approach in the treatment of incisional and umbilical hernias. From February 2000 through June 2006, a total of 127 incisional hernias (primary and recurrent), 21 umbilical and 19 epigastric hernias, were treated by laparoscopic approach. The exclusions for laparoscopy were: defect size less than 2 cm or more 20 cm, anesthesiologic problems, hemocoagulative disorders. The females/males ratio was 0/7, with a mean age of 59 years (range 24-83). The abdominal associated disease treated were 26. Three cases were treated as urgencies (strangulated hernias). RESULTS: The conversion rate was 2 cases (1.2%). Mean operative time was 78 min (range 25-170). The mean postoperative hospital stay was 2.1 days (range: 1-5). The intra- and postoperative complication rate was 5% (above all seromas). The recurrence rate was 1.8% (3 cases). CONCLUSIONS: More studies with long-term follow-up are necessary to prove the advantages of laparoscopic technique, but this large experience of more than 5 years, demonstrates that in selected cases the laparoscopic approach may be an interesting and effective alternative to open technique.

Comparing normal primary endocervical adenoepithelial cells to uninfected and influenza B virus infected human cervical adenocarcinoma HeLa cells.

Human adenocarcinoma HeLa cells surviving infection with low (10(-9) units), medium (10(-6) units), and high (10(-2) units) influenza B titers were compared to their uninfected precursors and to normal endocervical adenoepithelial and metaplastic cells using Papanikolaou-staining method and immunocytochemistry. Normal primary endocervical and infected HeLa cells surviving infection shared similar morphologic, phenotypic, and divisional patterns that differed drastically from those of uninfected HeLa cells. The number of infected hosts surviving 6-7 days of viral exposure did not change during 3-week follow-up period, and their cyclin E levels suggested that they had been arrested to the G1 phase of the cell cycle by viral stress. Our findings suggest that in addition to apoptosis, nononcogenic viral stress activated the expression of endocervical metaplastic-like motifs in surviving hosts. A mechanism of cell response to nononcogenic viral stress was proposed to explain these findings. We conclude that nononcogenic respiratory viruses specifically target and eliminate abnormal cells ectopically overexpressing appropriate receptors and may complement current treatments of cervical cancer.

The biliopancreatic diversion. A comparison of laparoscopic and laparotomic techniques.

AIM: Morbid obesity has increased its frequency in the last 20 years in association with the increase of a country's richness. Bariatric surgery has developed a role which is becoming more and more important. The aim of this study, after 10 years of experience with the biliopancreatic diversion, is to compare the laparoscopic versus open technique METHODS: From March 1993 to December 2004, 150 patients were operated by biliopancreatic diversion. We divided our experience into 2 groups: laparotomic and laparoscopic techniques. We compared the following variables in the 2 groups: total operating time, intestinal functions, postoperative pain, patient's discharge and recovery time, major postoperative complications, postoperative mortality, late complications, incisional hernia incidence and anastomotic ulceration. RESULTS: We found a significant difference in both the reduction of the postoperative pain and the recanalization time in the laparoscopic group. Equally, we found a reduction in the incidence of abdominal wall complications, especially the reduction of incisional hernia and infections. The mean operative time was longer in the laparoscopic group, in particular due to the hard learning curve. CONCLUSIONS: The laparoscopic biliopancreatic diversion is a feasible and safe operation with good results: less postoperative discomfort, shorter recanalization and ospedalization time, less incidence of abdominal wall morbidity (incisional hernia). However, the procedure may prove difficult and it needs a highly experienced surgeon in laparoscopic technique.

[Laparoscopic colon-sigmoid resection with mesenteric artery preservation for diverticular disease]. / Resezione laparoscopica di colon-sigma con conservazione della mesenterica per malattia diverticolare.

AIM: About 2/3 of the Western population over the age of 80 years are affected by colic diverticulosis; 25% will develop diverticular disease with or without complications: fistula, obstruction, pericolic abscess, free perforation or hemorrhage. Laparoscopic approach for benign diseases of the colon such as diverticulosis, Chrohn's disease, etc. is unanimously considered as a very effective procedure. We have performed a retrospective analysis of 9 years with laparoscopic approach of the diverticular disease. The purpose of this study was to determine the feasibility, safety and benefits of laparoscopic approach. METHODS: From May 1994 to November 2002, 69 patients affected by non-complicated diverticular disease, were treated laparoscopically, at the Mininvasive Surgery Department of Spoleto. In the same period other 213 patients suffering from cancer of the colon-rectum were operated through laparoscopic surgery. A colosigmoid resection with mesenteric inferior artery preservation was performed in 46 cases (67%). RESULTS: We have performed only a laparoscopic colectomy technique (no hand-assisted procedures); the convertion rate was of 7.2%, the mean operative time was 145 min, no intraoperative complications were observed and finally the mean hospital stay was of 7.4 days (6-9). The postoperative complications were 1 case of infection of the umbilical scar and 1 case of pleural effusion with bronchopneumonia. CONCLUSIONS: Elective laparoscopic colectomy for diverticular disease is feasible, safe and gives very good results: minimum postoperative discomfort, rapid recovery, low mobility and postoperative stay, rapid return to normal activities. However, in some cases, this procedure can be extremely difficult because of previous inflammatory complications (adhesions, fistulas or stenosis), therefore, high surgical skills in laparoscopic-colic surgery are required.

Basaloid squamous cell carcinoma of the uterine cervix with an uncommon metastasis to the left iliac region--case report.

We describe a rare and interesting case of a basaloid squamous cell carcinoma (BSC) of the uterine cervix with metastasis in the left iliac region diagnosed by fine needle aspiration (FNA). A 54-year-old woman underwent FNA because of a mass in the left iliac region. The material was processed by conventional liquid based cytology (ThinPrep), and by the cell-block technique and diagnosis was based on the cytomorphologic and immunochemical characteristics as well as the patient's history. The cytologic diagnosis concerned a poorly differentiated squamous cell carcinoma. After a laborious search, we found out that the patient had undergone a total hysterectomy almost 15 years before. The histological diagnosis then was an "infiltrative squamous cell carcinoma of basaloid type" of the uterine cervix. Our case is of particular interest because it is a rare type of neoplasm with an unusual site of metastasis after a long disease-free period.

[Non palpable lesions of the breast: the Mammotome-biopsy in the preoperative management of breast cancer]. / Lesioni non palpabili della mammella: la Mammotome-biopsy nella gestione preoperatoria del cancro della mammella.

BACKGROUND: Breast tumour takes first place for frequency in women in Western Countries and is in constant increase. The diagnosis of the so-called non palpable lesions is increased remarkably above all due to the diffusion of mammographic screening and to a greater awareness of the problem. Furthermore it is helped by an important development of mininvasive diagnostic methods: the traditonal cytology with fine needle is supported by various trans-skin bioptic procedures (micro-histological examination). This methods almost always replaces the surgical excisional biopsy and frozen intraoperative examination, still used but reserved for particular cases. PATIENTS AND METHODS: In our Department of General and Mininvasive Surgery, from December 1999 to September 2004, we carried out 214 biopsies, with the collaboration of the radiological Service, under echographic guidance using vacuum--assisted biopsy (Mammotome) with 11-Gauge needle. The results are examined and discussed here in this report with regard to diagnostic accuracy, quantity and quality of information, significant for subsequent surgical management. RESULTS: Of 214 biopsies carried out with Mammotome technique, 89.3% of the cases are clinically non palpable lesions, with a average diameter of 8 mm. The average age of patients was 57.6 years (range 31-88). There are 90 cases of positive malignant pathology (42%). In the atypical ductal iperplasia and radial scar cases (6%) surgical removal of lesion was carried out which confirmed the previous bioptic diagnosis in 100% of cases. The 19% of patients submitted to a Mammotome biopsy was subject previously to cytology with fine needle. Comparing the results of both methods the diagnostic reliability of Mammotome was significantly superior (p < 0.05) as also the amount of information obtained (histotype, invasivity, grading, estrogen receptor, etc.); discomfort linked to the procedure, valued as pain (VAS), resulted inferior to the discomfort of biopsy with fine needle. The only complication of Mammotome biopsy is represented by haematoma in the biopsy site (8% of cases). The number of false negatives was one case due to incorrect targeting. CONCLUSION: In the present situation, the choice of method is conditioned by the degree of radiological suspicion, taking into account the information obtained thereby, in order to ensure the appropriate surgise management. Mammotome biopsy of non palpable lesions of the breast, in our experience, is preferable if suspicion of malignancy is high. In this way a correct preoperative strategy can be prepared. Including the sentinel lymphnode method. Consequently a decision regarding the type of surgery can be taken (generally conservative), as well as making easier the intraoperative localisation of lesion by positioning the metallic clips during biopsy.

[Neoplastic residues in the trocar tract in oncologic laparoscopic surgery]. / Recidive neoplastiche in sede di trocar nella chirurgia oncologica laparoscopica.

AIM: The laparoscopic approach to malignant diseases runs up against both old and new problems: respect for the principles of radicality, operating times, the postoperative course and surgical complications, long-term oncological results in terms of survival and recurrence of the disease. One of the problems which has received most attention regards the onset of a metastasis on a trocar scar or a mini-laparotomy recurrence. Trocar site tumor recurrences have been described in the literature following laparoscopic surgery in almost all abdominal malignant pathologies (colorectal, gynaecological, pancreatic, etc.) and even after thoracoscopy. The real frequency is currently of the order of 1% (0-2%) in colic surgery and of 14% (10-17%) after cholecystectomy for occult gallbladder carcinoma. METHODS: A retrospective survey was carried out of our laparoscopic experience; between 1994 and 2002 213 colic resections were carried out for cancer; we also observed 18 occult carcinomas of the gallbladder in 2386 laparoscopic cholecystectomies for lithiasis. RESULTS: Respectively 2 cases (11%) of trocar site neoplastic recurrences in gallbladder carcinoma and 2 cases (0.9%) from colon carcinoma were observed. CONCLUSIONS: The real extent of the problem would appear to be on a much lesser scale at the moment than was initially thought, especially as regards colic surgery. The multifactorial aetiology of the problem explains the importance of their prevention, on the basis above all of rigorous respect for the rules and protocols of laparoscopic technique.

[Laparoscopic right hemicolectomy with intracorporeal anastomosis. Technical aspects and personal experience]. / Emicolectomia destra laparoscopica con anastomosi intracorporea. Aspetti tecnici e nostra esperienza.

BACKGROUND: Laparoscopic colo-rectal surgery has gained wide acceptance as a treatment in a variety of benign and malignant diseases. The reproducibility and safety of all the principal colo-rectal procedures has been demonstrated, but some indications are still controversial. A great part of surgeons perform right hemicolectomy by laparo-assisted technique and consider it more difficult than left hemicolectomy. METHODS: A retrospective analysis of a series of 180 laparoscopic colon resections performed between 1994 and 2001 is presented. Surgical indications were: 46 cases of benign disorders and 134 colonic malignancy. The right hemicolectomy were 27 (15%): 2 benign and 25 malignant diseases. The technical aspects of completely laparoscopic right hemicolectomy is described in details. A completely mechanical intra-corporeal anastomosis is always performed. To remove the bowel specimen from the abdominal cavity it is put it in a large bag and pull it out of a 3-4 cm enlargement of a trocar-site (in the umbilical scar). RESULTS: Data of the 27 laparoscopic right hemicolectomy were analysed: there was 1 conversion to open surgery; no intra-operative complications were observed; the postoperative period was complicated by 1 anastomotic fistula. The median operative time was of 150 minutes. No case of port-site recurrence was observed. CONCLUSIONS: The laparoscopic colo-rectal surgery can reproduce in selected patients, the techniques performed in open surgery with minimally invasive treatment. It is possible to perform a completely laparoscopic right hemicolectomy after an adequate training in advanced laparoscopy, anyway there are many advantages: less postoperative pain, short-term postoperative ileus, earlier return to daily activity.

Operative evaluation of cardiac tamponade in a patient with multiple myeloma.

Multiple myeloma presents with various clinical manifestations depending on the mode and the extent of organ involvement. Pericardial involvement by myeloma and subsequent cardiac tamponade is extremely rare. We report on the case of a patient with multiple myeloma who presented with cardiac tamponade and was evaluated surgically with thoracotomy and minimal debulking pericardiectomy (fenestration).

Clinical evaluation and changes of the respiratory epithelium function after administration of Pidotimod in Greek children with recurrent respiratory tract infections.

BACKGROUND: Several studies have been conducted on young children with recurrent respiratory infections using several compounds (synthetic derivates or lyophilized bacterial extracts) causing improvement in the clinical process. METHODS: We conducted a prospective, randomized study comparing the clinical results and the changes of the respiratory epithelium function after the administration of immunostimulating drug (Pidotimod) to children with respiratory infections over a 9 month period. A total of 32 children (group A) were randomly assigned to receive Pidotimod therapy while a second group of 18 children (group B) weren't. All the children in group A received Pidotimod (400 mg x 2 daily) for fifteen days and 400 mg daily for the next twenty days. The proper function of the ciliary respiratory epithelium in all children was checked, using the Edicol Orange and CaH PO4 2H2O, coloring method before the therapeutic intervention and after the first and the sixth month. RESULTS: 87.5% of group A, responded exceptionally well to treatment presenting two or less infections in the nine month period, whereas only 33.3% of group B showed improvement (p<0.001). In group A, the clearance of the respiratory epithelium, from a primary 37 minutes decreased to 32 minutes in the first month and 19'5" six months after the therapy. In group B, the corresponding time was decreased from a primary 36'4" to 34'2" and 31' respectively (p=0.01). CONCLUSIONS: Our results suggest that Pidotimod therapy is a reliable, simple and safe approach to treat children with recurrent respiratory infections and it can reduce the frequency of such infections as a result of improvement of the ciliary respiratory epithelium.

Subglottic hemangioma treated with interferon alpha 2A.

Infantile subglottic hemagioma is a rare vascular malformation involving the subglottic larynx and although present from birth, symptoms will not be noted until later in infancy (due to tendency to enlarge). Typically presents with a progressive crouplike illness that begins a few weeks after birth and the infant develops inspiratory stridor, which becomes expiratory as the obstruction increases. Although benign lesions which involute spontaneously, they may demand the attention of an ENT surgeon to maintain a secure airway. We report a case of a 3-month-old female patient (full term, normal delivery) who was referred to us for investigation of respiratory distress. Endoscopic examination revealed a hemangioma at the left lateral wall of the subglottic larynx while the rest of the airway was normal. The infant was treated with a combination of steroids (dexamethazone 0.5 mg x 3 per os) and interferon A-2a (650.000 IU subcutaneously, every other day, for 12 months) and had fast improvement of her symptoms. Repeated endoscopy 3 months after the diagnosis reveal impressive remission of the subglottic hemangioma. Throughout the years, a variety of treatments have been proposed and utilised for subglottic hemangioma. Interfron 2-alpha, drug acting by interference with angiogenesis, is very effective in treating subglottic hemangiomas without the need for tracheostomy. Its side effects are generally not serious. As congenital subglottic hemangiomas have tendency towards spontaneous regression, conservative treatment seems more appropriate compared to more aggressive treatment that carry substantial risks of long term complications.

[Laparoscopic approach versus laparotomy for suspected acute appendicitis]. / Approccio laparoscopico versus laparotomia per sospetta appendicite acuta.

Acute appendicitis is one of the most common surgical disease but, in spite of the progression diagnostic imaging, a definite diagnosis is frequently difficult and often is based in essentially clinical grounds. The Authors retrospectively analyze the results of conventional laparotomic appendectomy (CLA) and videolaparascopic appendectomy (VLA) as performed by two teams of their Department of Surgery. Between January 2000 and November 2001, 156 patients, age ranging from 3 to 67 yrs, underwent surgery because suspected acute appendicitis; 96 patients underwent VLA and 60 patients underwent CLA; a diagnosis of acute appendicitis was confirmed in 142 cases (91%). The patients who had a confirmed diagnosis of acute appendicitis were grouped according to clinical-pathologic criteria into follicular, gangrenous and complicated appendicitisi; operating time, complications and length of hospital stay were compared among the three groups. VLA resulted superior to CLA in terms of diagnostic performance: among the patients with suspected acute appendicitis, VLA allowed to establish a different diagnosis in 41% as compared to 13% with CLA. In two cases (2.08%) VLA was turned into CLA. There were non differences between VLA and CLA in terms of surgical operating time and hospital stay. A significantly higher incidence of surgical complications, such as wound infections and pelvic abscesses, was observed in the CLA group as compared to VLA. The Authors conclude that VLA allows a complete exploration of the abdomen, a better localization of the appendix and an easier diagnosis of unexpected conditions or pathologic processes associated with the clinical appendicitis; further, VLA is gravated by fewer complications.

Rapamycin impairs recovery from acute renal failure: role of cell-cycle arrest and apoptosis of tubular cells.

The immunosuppressive effect of rapamycin is mediated by inhibition of interleukin-2-stimulated T cell proliferation. We report for the first time that rapamycin also inhibits growth factor-induced proliferation of cultured mouse proximal tubular (MPT; IC(50) ~1 ng/ml) cells and promotes apoptosis of these cells by impairing the survival effects of the same growth factors. On the basis of these in vitro data, we tested the hypothesis that rapamycin would impair recovery of renal function after ischemic acute renal failure induced in vivo by renal artery occlusion (RAO). Rats given daily injections of rapamycin or vehicle were subjected to RAO or sham surgery. Rapamycin had no effect on the glomerular filtration rate (GFR) of sham-operated animals. In rats subjected to RAO, GFR fell to comparable levels 1 day later in vehicle- and rapamycin-treated rats (0.25 +/- 0.08 and 0.12 +/- 0.05 ml. min(-1). 300 g(-1), respectively) (P = not significant). In vehicle-treated rats subjected to RAO, GFR increased to 0.61 +/- 0.08 ml. min(-1). 300 g(-1) on day 3 (P < 0.02 vs. day 1) and then rose further to 0.99 +/- 0.09 ml. min(-1). 300 g(-1) on day 4 (P < 0.02 vs. day 3). By contrast, GFR did not improve in rapamycin-treated rats subjected to RAO over the same time period. Rapamycin also increased apoptosis of tubular cells while markedly reducing their proliferative response after RAO. Furthermore, rapamycin inhibited activation of 70-kDa S6 protein kinase (p70(S6k)) in cultured MPT cells as well as in the renal tissue of rats subjected to RAO. We conclude that rapamycin severely impairs the recovery of renal function after ischemia-reperfusion injury. This effect appears to be due to the combined effects of increased tubular cell loss (via apoptosis) and profound inhibition of the regenerative response of tubular cells. These effects are likely mediated by inhibition of p70(S6k).

Soluble P-selectin moderates complement dependent injury.

P-selectin is an adhesion molecule expressed on activated endothelial and platelet membranes containing 9 short consensus repeats (SCRs) similar to the composition of complement regulatory proteins. In our murine model of intestinal ischemia and reperfusion where local injury is mediated by the classical complement pathway we hypothesized the SCRs would moderate the complement response. Confirmatory data were sought following hindlimb ischemia and reperfusion where injury is both complement- and neutrophil-mediated. Mice deficient in P-selectin (P-/-) were found to have similar intestinal and hindlimb permeability compared to normal wild types mice (P+/+). When reconstituted with P+/+ platelets, but not P-/- platelets, P-/- mice subjected to intestinal ischemia had a significant 29% decrease in permeability (P < 0.05) and after hindlimb ischemia the decrease was 33% (P<0.05). Reperfusion after intestinal ischemia led to a 76% fall in CH50 in P-/- compared to sham animals (P < 0.05) indicating complement activation and consumption, but only a 36% fall in animals reconstituted with P+/+ platelets (P < 0.05). Full-length, soluble P-selectin (sPsel) derived from processed platelets, but not the truncated version of sPsel has been shown to adhere to a heat labile fraction of serum and sensitized red blood cells thereby reducing Clq adherence to the sensitized red cell. From these data we conclude that sPsel moderates complement activation by competing with C1q binding to antibody, thereby limiting activation of the classical pathway that mediates murine reperfusion injury.

Soluble P-selectin moderates complement-dependent reperfusion injury of ischemic skeletal muscle.

P-selectin is an adhesion molecule expressed on activated endothelial and platelet surfaces. The function of the short consensus repeats (SCRs) of P-selectin, homologous with the SCRs of complement regulatory proteins is largely unknown. In a model of murine hindlimb ischemia where local reperfusion injury is partly mediated by IgM natural antibody and classical complement pathway activation, we hypothesized that human soluble P-selectin (sP-sel) would moderate the complement component of the inflammatory response. Infusion of sP-sel supernatant or purified (p) sP-sel prepared from activated human platelets, reduced ischemic muscle vascular permeability by 48% and 43%, respectively, following reperfusion. Hindlimb immunohistochemistry demonstrated negligible C3 staining colocalized with IgM in these groups compared with intense staining in the untreated injured mice. In vitro studies of mouse serum complement hemolytic activity showed that psP-sel inhibited the classical but not alternative complement pathway. Flow cytometry demonstrated that psP-sel inhibited C1q adherence to sensitized red blood cells. From these data we conclude that sP-sel moderates skeletal muscle reperfusion injury by inhibition of the classical complement pathway.