Iva Brčić

Inhibition of methyldigoxin-induced arrhythmias by pentadecapeptide BPC 157: A relation with NO-system

Pentadecapeptide BPC 157 (GEPPPGKPADDAGLV, MW 1419) reversed congestive heart failure and various arrhythmias, influenced the NO-system and showed no proarrhythmic effect. In therapy analogy, we challenged rats with digitalis, to show attenuation by BPC 157 and the relation between the NO-system and digitalis toxicity. (i). BPC 157 prophylactic effect. Development of cumulative intravenous digitalis toxicity, BPC 157 (50 microg, 10 microg, 10 ng/kg applied intravenously immediately before a methyldigoxin increment regimen (2.0/1.5/1.5/1.0 mg/kg at 15 min-intervals, total dose 6.0 mg/kg/45 min)) reduced the number of ventricular premature beats, prolonged the time before onset of ventricular tachycardia, reduced ventricular tachycardia and AV-block duration (microg-regimes) or reduced mainly the AV-block duration (ng-regimen). (ii). BPC 157 therapy. Advanced methyldigoxin toxicity (6.0 mg/kg i.v. bolus). BPC 157 applied at the 20th second of the grade 3 AV-block shortened AV-blocks, mitigated a further digitalis toxicity course. Ventricular tachycardias were either avoided (50 microg), or markedly reduced (10 microg, 10 ng). Fatal outcome was either avoided (50 microg), reduced (10 microg), or only delayed (10 ng) (iii) BPC 157, L-NAME, l-arginine, L-NAME+l-arginine application. L-NAME-application (5 mg/kg i.p.) aggravated methyldigoxin-arrhythmias. l-arginine (200 mg/kg i.p.) alone had no effect but blunted L-NAME-exaggeration (L-NAME+l-arginine). In this respect, BPC 157 (50 microg/kg i.p.) was prophylactically and therapeutically more effective: the antagonism of L-NAME with BPC 157 produced an effect similar to BPC 157 alone. In conclusion, digitalis-induced arrhythmias in rats could be prevented and counteracted by pentadecapeptide BPC 157, mainly through an interaction with the NO-system.

Gastric pentadecapeptide BPC 157 as an effective therapy for muscle crush injury in the rat

PurposeStable gastric pentadecapeptide BPC 157 accelerates the healing of a transected Achilles tendon and a transected quadriceps muscle. It may also be of clinical relevance as a systemic and local peptide treatment for crush injury of a major muscle, such as gastrocnemius muscle complex. BPC 157 is effective without a carrier, and it is presently undergoing trials for inflammatory bowel disease, and no toxicity has so far been reported.MethodsIn crushed rats (force delivered 0.727 Ns/cm2), BPC 157 was applied either intraperitoneally or locally, as a thin cream layer, immediately after injury (sacrifice at 2 h), and once a day for 14 days.ResultsBPC 157 improved muscle healing, macroscopically (less hematoma and edema, no post-injury leg contracture), microscopically, functionally, and also based on enzyme activity (creatine kinase, lactate dehydrogenase, aspartate aminotransferase, alanine aminotransferase).ConclusionBPC 157, at all investigated intervals, given locally or intraperitoneally, accelerated post-injury muscle healing and also helped to restore the full function.

Prognostic values of ETS-1, MMP-2 and MMP-9 expression and co-expression in breast cancer patients.

The aim of this study was to analyse expression of ETS-1 protein and two gelatinases (MMP-2 and MMP-9) and their possible prognostic value in breast carcinoma patients, as well as correlation of their expression with other known prognostic factors such as tumor size, grade, vascular invasion, steroid receptor values, HER2 values and proliferative index. The expression of MMP-2, MMP-9 and ETS-1 was immunohistochemicaly analysed in 121 consecutive primary breast carcinoma patients who underwent surgery at the Clinical Hospital Centre Zagreb during 2002. Three representative areas from each tumor paraffin blocks were taken and arranged on a recipient paraffin block with predefined coordinates for simultaneous analyses of multiple tissue samples (TMA). ETS-1, MMP-2 and MMP-9 expression and co-expression were correlated with other clinico-pathological parameters and based on the available clinical follow up data survival analysis was performed. The ETS-1 protein is found to be expressed in tumor cell nuclei and cytoplasm as well as in stromal lymphocytes, fibroblasts and endothelial cells. MMP-2 and MMP-9 were found to be expressed in cytoplasm of both, tumor and stromal cells. For our analysis only tumor cell expression was used for statistical analysis. We found 56,2% ETS-1 positive tumors, 77,7% were MMP-2 positive, and MMP-9 was expressed in 90% of primary breast carcinomas. There were no significant correlations between MMP-s expression and other patohistological prognostic factors, but expression of ETS-1 was significantly correlated with higher tumor size and grade, as well as with negative steroid receptors. Co-expression of MMP-2, MMP-9 and ETS-1 was found in 40,5 % of tumors, and more commonly was found in tumors larger than 2 cm, high grade tumors, and steroid receptor negative tumors. In univariate analysis, statistically significant negative impact on overall survival (OS) had tumor size, nuclear and tumor grade, ETS-1 expression in tumor cells, co-expression of ETS-1 either with MMP-2 or MMP-9, as well as co-expression of ETS-1, MMP-2 and MMP-3. Disease free survival (DFS) was significantly shorter in patients with tumors greater than 2 cm, ETS-1 positive tumors, ETS-1 and MMP-2 or MMP-9 co-expressed tumors, and additionally in tumors with ETS-1, MMP-2 and MMP-9 co-expression. These results suggest that expression of ETS-1 as well as MMP-2, MMP-9 and ETS-1 co-expression might be used as a poor prognostic factor in breast cancer patients.

Expression of Toll-like receptor 4 and beta 1 integrin in breast cancer

Toll-like receptor (TLR) 4 signaling pathway has been shown to support tumor cell growth in vitro and in vivo. Its stimulation on breast cancer cell lines induces β1 integrin and promotes tumor invasiveness. However, its role in predicting clinical behavior of tumor is not yet clarified. Therefore, we investigated TLR4 and β1 integrin expression on 133 primary breast cancer samples by immunohistochemistry and correlated it with overall survival and disease-free survival of patients as well as with clinicopathological characteristics of the tumor. We found higher β1 integrin expression in invasive lobular cancer in comparison with other tumor types. No significant association of TLR4 and β1 integrin expression with overall survival or disease-free survival was seen. Therefore, we conclude that expression of these markers is of biological interest but appears to be of little additional use as predictive clinical marker.

Fibronectin glomerulopathy in a 34-year-old man: a case report.

Fibronectin glomerulopathy is an inherited non-imune-mediated glomerulopathy associated with the massive deposition of fibronectin. It presents with proteinuria, microscopic hematuria, and hypertension that lead to end-stage renal failure in the second to sixth decade of life. A 34-year-old male was referred with proteinuria (6 g/day), microscopic hematuria and hypertension (220/130 mmHg). Renal biopsy specimen showed massive deposits of fibronectin in the mesangium and subendothelial spaces. After 20 months of multidrug treatment his renal function is stable.

Terminal deoxynucleotidyl transferase negative T-cell lymphoblastic lymphoma in aleukemic patient

Precursor lymphoblastic leukemia/lymphoblastic lymphoma (ALL/LBL) is a malignant neoplasm of precursor lymphocytes of T- or B-cell phenotype. We describe the unusual features of an ALL/LBL in an adolescent man in whom the disease presented with involvement of lymph nodes, but without bone marrow and peripheral blood involvement. Immunohistochemical studies revealed that the tumor cells were positive for CD3, CD34 class II, CD10, CD79a and CD99 but negative for TdT. Even though TdT was negative, he received ALL-therapy and is now in remission.

Rarity among benign gastric tumors: Plexiform fibromyxoma - Report of two cases

Plexiform fibromyxoma is a very rare mesenchymal tumor of the stomach, found almost exclusively in the antrum/pylorus region. The most common presenting symptoms are anemia, hematemesis, nausea and unintentional weight loss, without sex or age predilection. We describe here two cases of plexiform fibromyxoma, involving a 16-year-old female and a 34-year-old male. Both patients underwent complete resection (R0) by distal gastrectomy and retrocolic gastrojejunostomy (according to Billroth 2); for both, the postoperative course was uneventful. Histology showed multiple intramural and subserosal nodules with characteristic plexiform growth, featuring bland spindle cells situated in an abundant myxoid stroma with low mitotic activity. Immunohistochemistry showed α-smooth muscle actin-positive spindle cells, focal positivity for CD10, and negative staining for KIT, DOG1, CD34, S100, β-catenin, STAT-6 and anaplastic lymphoma kinase. One of the cases showed focal positivity for h-caldesmon and desmin. Upon follow-up, no sign of disease was found. In the differential diagnosis of plexiform fibromyxoma, it is important to exclude the more common gastrointestinal stromal tumors as they have greater potential for aggressive behavior. Other lesions, like neuronal and vascular tumors, inflammatory fibroid polyps, abdominal desmoid-type fibromatosis, solitary fibrous tumors and smooth muscle tumors, must also be excluded.

Microsatellite instability in metaplasia-dysplasia-adenocarcinoma sequence of Barrett esophagus: a retrospective study

Aim To analyze the loss of mismatch repair (MMR) system protein expression in metaplasia-dysplasia-adenocarcinoma sequence of Barrett esophagus (BE). Methods This study retrospectively analyzed the data from 70 patients with pathohistological diagnosis of BE or esophageal adenocarcinoma (EAC) treated at the Clinical Department of Pathology and Cytology, University Hospital Center Zagreb, from January 2009 to January 2011. Patients were divided into three groups: BE without dysplasia (22 patients), BE with dysplasia (37 patients), and EAC (11 patients). Immunohistochemical expression of MutL homologue 1 (MLH1), MutS homologue 2 (MSH2), postmeiotic segregation increased 2 (PMS2), and MutS homologue 6 (MSH6) of DNA MMR system was measured and compared with tumor protein p53 expression. Results A total of 81.8% and 81.8% patients with EAC, 32.4% and 35.1% patients with dysplasia, and 50% and 54.5% patients without dysplasia had loss of MLH1 and PMS2 expression, respectively. Patients with EAC and patients with dysplasia did not have loss of MSH2 and MSH6 expression, and 18.2% patients without dysplasia had loss of MSH2 and MSH6 expression. There was a strong positive correlation between MLH1 and PMS2 expression (Spearman ρ 0.97; P < 0.001) and between MSH2 and MSH6 expression (Spearman ρ 0.90, P < 0.001) in the entire sample and in all BE groups. No significant correlations of MLH1 and PMS2 with p53 expression were found, except in dysplasia group (φ 0.402, P = 0.030 for MSH1; φ 0.371, P = 0.042 for PMS2). Conclusion Although we demonstrated considerable loss of MLH1 and PMS2 expression in BE-associated carcinoma sequence, due to the retrospective study design and low number of patients we cannot conclude that MLH1 and PMS2 can be used as biomarkers for patient surveillance and therapy-making decisions. Oxford Centre for Evidence-based Medicine level of evidence: 3

W1339 Safe Anti-Ulcer Peptide, in Trial for Inflammatory Bowel Desease, Stable Gastric Pentadecapeptide BPC 157 (PL14736) Can Cure Rats With Short Bowel Syndrome Complicated With Ulcerative Colitis

Stable gastric pentadecapeptide BPC 157 recovered short bowel sydrome in rats after massive small bowel resection, improved intestinal adaptation, villus height, crypt depth, and muscle thickness (and inner (circular) and/or outer (longitudinal) muscular layer)), induced weight gain in weight of normal healthy rats (Dig Dis Sci, 2008 in press). BPC 157 is a safe anti-ulcer peptide (PL-14736, Pliva) in trial for IBD, and wound therapy, no toxicology reported (Gastroenterology, 2005) that healed intestinal anastomosis and fistulas (Dig Dis Sci, 2008, J Pharm Sci, 2008). BPC 157 also prevented and reversed ulcerative colitis induced by cysteamine enema (J Physiol, 1999). However, BPC 157 was not tested after small bowel massive resection in rats with ulcerative colitis. Small bowel resection. Throughtout a 4 week period we tested rats with escalating bowel syndrome and progressive weight loss that had only 20% of small bowel (Dig Dis Sci, 2008, in press). Ulcerative colitis. Cysteamine enema 400 mg/kg i.r., 1ml/rat. Ulcerative colitis+small bowel resection. Enema was applied at 10 min before surgery. BPC 157 medication. BPC 157 (10 μg, 10ng/kg i.p. or in drinking water) first application 30 min following surgery, last 24 h before sacrifice (7, 14, 21, 28 days). Small bowel resection. The rats had an escalating bowel syndrome and progressive weight loss despite the fourfold muscle thickness increase and twofold villus height and crypt depth increase during the first week. BPC 157 groups recovery showed improved intestinal adaptation, villus height, crypt depth, and muscle thickness (and inner (circular) and/or outer (longitudinal) muscular layer))additionally increased. The rats immediately gained weight, ultimately to the weight of normal healthy rats. Ulcerative colitis. Controls exhibited pertinent ulceration, initial weight loss, and then decreased weight gain. BPC 157 rats immediately gained weight, ultimately to the weight of normal healthy rats, and had consistently less ulcerations. Ulcerative colitis+small bowel resection. Ulcerative colitis significantly aggravated all these parameters. BPC 157 retained the same beneficial effects, decreased ulcerative colitis, improved intestinal adaptation, villus height, crypt depth, and muscle thickness (and inner (circular) and/or outer (longitudinal) muscular layer)) additionally increased, early weight gain, ultimately to the weight of normal healthy rats. In addition to the recovery of the rats with short bowel syndrome or ulcerative colitis, BPC 157 can cure rats with short bowel syndrome complicated with ulcerative colitis.

W1851 BPC 157 Reduced Postoperative Adhesion Formation in Rats

Background: Intestinal scarring is a major cause of morbidity in Crohns disease. Current therapies treat inflammation, but do not alter the progression of fibrosis and bowel obstruction. Given the observation that late use of potent anti-inflammatory therapies does not reduce stenosis or obstruction, we hypothesized that intestinal fibrosis becomes self-propagating, despite removal of inflammatory stimuli. Methods: The Salmonella typhimurium murine model of inflammation and fibrosis in which clearance of commensal microbiota by streptomycin, followed by infection with S. typhimurium, produces chronic inflammation, culminating with fibrosis by day 21 post infection was used. The inflammatory stimulus (S. typhimurium) was removed with the oral antibiotic levofloxacin at day 2, 4, or 8 post-infection. Eradication of S.typhimurium was confirmed by stool plating and T-RFLP analysis. Results: By day 2 post-infection, the mouse cecae infected with S.typhimurium developed an inflammatory phenotype, characterized by a shrunken yet heavier cecum, expansion of the cecal submucosa, and induction of inflammatory genes (IL-1b, TNFa, IL-6, IL-17, IL12p40). However, fibrotic gene expression (CTGF, IGF-1, TGFb) was indistinguishable from uninfected controls. In addition, aSMA protein expression was not induced until day 8 postinfection. Early intervention (day 2 & 4) repressed inflammation as determined by gross pathology, histopathology, and repression of inflammatory genes. Fibrotic gene expression was partially repressed by day 2 levofloxacin treatment. However, day 8 levofloxacin treatment did not prevent induction of aSMA protein or pro-fibrotic genes (CTGF, IGF-1, TGFb) by day 8 and which continued to increase after levofloxacin treatment to day 21, remaining significantly higher than uninfected controls or matched S.typhimurium infected mice harvested at day 4 and 8 post-infection (p < 0.03) Conclusions: Early removal of the inflammatory stimulus reduces fibrosis, but fibrosis after initiation continues to propagate in the absence of an inflammatory stimulus. Since many Crohns patients develop complications of fibrosis, understanding the mechanisms of auto-propagation of fibrosis and developing anti-fibrotic therapies is critical to improving outcomes in Crohns disease.