Genetic and immunological comparison of anti-botulinum type A antibodies from immune and non-immune human phage libraries

Understanding the antibody response in botulinum intoxication is important for vaccine design and passive prophylaxis. To investigate this activity, we have studied the immune response to BoNT/A (botulinum neurotoxin serotype A) binding domain (HC) at the molecular level using phage display. The scFv antibodies were isolated from V-gene repertoires prepared from (a) human volunteer immunized with pentavalent botulinum toxoid and (b) non-immune human peripheral blood lymphocytes and spleenocytes. A large panel of serotype specific phage expressing botulinum binding scFv could be selected from both libraries. Epitope mapping of immune scFv binders towards BoNT/A HC revealed surprisingly a limited number of scFv recognizing conformational epitopes that corresponded to two distinct groups, clusters I and II. Only scFv from cluster I exhibited neutralizing activity in the mouse hemidiaphragm assay. Anti- BoNT/A HC clones derived from a non-immune library could be conveniently grouped into clusters III-XI and appeared to share no overlapping epitopes with cluster I or II. In addition they showed no neutralization of toxin at biologically significant concentrations. We therefore suggest that a vaccine based on the pentavalent botulinum toxoid directs the humoral immune response to a limited number of immunodominant epitopes exposed on the binding domain HC.     

Bleomycin induced flagellate pigmentation

Bleomycin frequently causes cutaneous toxicity in the form of pigmentary disturbances. We report 2 patients with testicular tumours who developed distinctive "flagellate" pigmentation on trunk and extremities during bleomycin therapy.     

Ursodeoxycholic acid and F(6)-D(3) inhibit aberrant crypt proliferation in the rat azoxymethane model of colon cancer: roles of cyclin D1 and E-cadherin.

We have previously demonstrated that ursodeoxycholic acid(UDCA) and a fluorinated analogue of vitamin D(3), F(6)-D(3),inhibited colonic carcinogenesis in the azoxymethane (AOM) model. Generalized colonic mucosal hyperproliferation and aberrant crypt foci (ACF) are intermediate biomarkers of colon cancer. Using these biomarkers, in this study we examined the anticarcinogenic mechanisms of these chemopreventive agents. Rats were maintained on AIN-76A chow or supplemented with 0.4% UDCA or F(6)-D(3) (2.5 nmol/kg chow) and treated weekly with AOM 20 mg i.p./kg wt or saline x 2 weeks. F(6)-D(3) was continued for an additional 2 weeks and UDCA for the duration of the study. At 40 weeks, animals received bromodeoxyuridine (BrdUrd) i.p. 2 h before sacrifice. A portion of each tumor was fixed in formalin and the remainder flash frozen. Colons were divided longitudinally and half-fixed in formalin and half in ethanol. The size and location of methylene blue-stained ACF were recorded. Cell proliferation (BrdUrd labeling) and apoptosis (terminal deoxynucleotidyl transferase-mediated nick end labeling assay) were measured in colonic crypts and tumors. Protein expression levels of several regulators of cell proliferation were analyzed by immunostaining and Western blotting. Colonic crypt cyclin D1 and E-cadherin mRNA levels were measured by real-time PCR. In saline injected controls, neither UDCA nor F(6)-D(3) alone had any effect on cytokinetic parameters or on the expression of mitogenic regulators. AOM significantly increased the proliferation (percentage of BrdUrd-positive cells) of both ACF (23.1 +/- 1.7%) and non-ACF crypts (17.6 +/- 1.6%), compared with normal colonic crypts (4.5 +/- 0.8%; P < 0.05). This hyperproliferation was accompanied by a 5-fold increase in cyclin D1 and >50% decrease in E-cadherin protein (P < 0.05) in ACF, both of which are predicted to be growth-enhancing alterations. UDCA and F(6)-D(3) significantly (P < 0.05) inhibited AOM-induced crypt cell hyperproliferation, ACF development, and tumor burden. These chemopreventive agents also significantly blocked AOM-induced alterations in cyclin D1 and E-cadherin protein in ACF and tumors. In ACF, changes in mRNA levels of cyclin D1, but not E-cadherin, paralleled alterations in protein expression. Cyclooxygenase-2 and inducible nitric oxide synthase were increased in AOM tumors but not in ACF, and these changes were blocked by UDCA and F(6)-D(3). UDCA and F(6)-D(3) significantly inhibited ACF development and hyperproliferation, in part, by preventing carcinogen-induced alterations in cyclin D1 and E-cadherin. In established tumors, UDCA and F(6)-D(3) also limited inductions of cyclooxygenase-2 and inducible nitric oxide synthase, which together with their effects on cyclin D1 and E-cadherin, contribute to their chemopreventive actions.     

Simultaneous administration of diethylphthalate and ethyl alcohol and its toxicity in male Sprague-Dawley rats

Phthalate esters have been implicated as xenoestrogens. One among them is di-ethylphthalate (DEP), which is used as plasticizer, detergent base, and binder in incense sticks and after-shave lotions. DEP is one of the contaminants of freshwater and marine ecosystems. Incense stick workers are occupationally exposed to DEP and some workers are chronic alcoholics. Therefore, a study was undertaken to evaluate the interactive toxicity of DEP with ethyl alcohol (EtOH) in young male Sprague-Dawley rats. The rats were given 50 ppm DEP (w/v), 5% EtOH (v/v) and a combined dose of 50 ppm DEP (w/v)+EtOH (5% v/v) in water ad libitum for a period of 120 days and were maintained on normal diet. Control animals received normal diet and plain water. During the treatment rats were weighed every week and water consumption per day was measured. After the completion of treatment, liver weight/body weight, liver weight, body weight, serum enzymes and other biochemical parameters were assessed. It was found that there was no significant change observed in body weight, liver weight, liver weight/body weight and water consumption. It was observed that there was a significant decrease in liver aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels in EtOH, DEP and EtOH+DEP treated rats in the order of EtOH>DEP>EtOH+DEP as compared with control. Serum AST, ALT, acid phosphatase (ACP), alkaline phosphatase (ALP), succinate dehydrogenase (SDH) and liver ACP showed significant increase in DEP and EtOH+DEP treated rats in the order of DEP>EtOH+DEP as compared with control and EtOH treated rats. On the contrary, there was no significant change in liver ALP levels in treated rats. There was significant increase in liver SDH, glycogen, total triglyceride, total cholesterol and lipid peroxidation in DEP and EtOH+DEP treated rats, but no significant changes in the serum SDH, glucose and total triglyceride levels. Serum total cholesterol levels in DEP and EtOH+DEP treated rats were significantly high as compared to control and EtOH treated rats. These results show that there is no interaction of DEP with EtOH but DEP alone leads to severe impairment of lipid metabolism coupled with toxic injury to the liver as evident from significantly altered lipid and enzyme levels in the liver and serum. Long term simultaneous exposure to DEP and EtOH may have severe implications for humans who are occupationally exposed to these two xenobiotics.     

Aortobronchial fistula in a pediatric patient with massive hemoptysis: treatment by means of an aortic endograft

We present an 11-year-old girl with acute myelogenous leukemia and hemoptysis from abscess erosion into the descending thoracic aorta. We report a pediatric case of an aortobronchial fistula treated with an aortic endograft and discuss the technical limitations and potential complications of this procedure.     

External pancreatic fistula as a sequel to management of acute severe necrotizing pancreatitis

BACKGROUND/AIMS: External pancreatic fistula (EPF) is a common sequel to surgical or percutaneous intervention for infective complications of acute severe pancreatitis. The present study was aimed at studying the clinical profile, course and outcome of patients with EPF following surgical or percutaneous management of these infective complications. METHODS: A retrospective analysis of clinical data of patients with EPF following intervention (surgical or percutaneous) for acute severe pancreatitis managed between January 1989 and April 2002 recorded on a prospective database was done. Univariate analysis of various factors (etiology, imaging findings prior to intervention, fistula characteristics and management) that could predict early closure of fistula was performed. RESULTS: Of 210 patients with acute severe pancreatitis, 43 (20%) patients developed EPF (mean age 38 (range 16-78) years, M:F ratio 5:1) following intervention for infected pancreatic necrosis (n=23) and pancreatic abscess (n=20) and constituted the study group. The fistula output was categorized as low (<200 ml), moderate (200-500 ml) and high (>500 ml) in 29 (67%), 11 (26%) and 3 (7%) patients, respectively. Fifteen patients (35%) had morbidity in the form of abscess (n=5), bleeding (n=1), pseudoaneurysm (n=2) and fever with no other focus of infection (n=7). Spontaneous closure of the fistula occurred in 38 (88%) patients. The average time to closure of fistula was 109+/- 26 (median 70) days. Fistula closed after intervention in 5 patients (2 after endoscopic papillotomy, 1 after fistulojejunostomy and 2 after downsizing the drains). Of the 38 patients with spontaneous closure, 9 (24%) patients developed a pseudocyst after a mean interval of 123 days of which 7 underwent surgical drainage of the cyst. Univariate analysis of various factors (etiology, imaging findings prior to intervention, fistula characteristics and management) failed to identify any factors that could predict early closure of fistula. CONCLUSIONS: EPF is a common sequel following intervention in acute severe pancreatitis. The majority of these are low output fistulae and close spontaneously with conservative management. One-fourth of patients with spontaneous closure develop a pseudocyst as a sequel, requiring surgical management.     

Bilateral lacrimal sac mucocele with punctal and canalicular atresia

Congenital absence of lacrimal puncta may be an isolated finding or associated with other developmental abnormality. Nasolacrirnal ducts can be absent thus predisposing to the formation of a congenital lacrimal mucocele. Punctal and canalicular agenesis is very rare. Four percent of new patients attending the lacrimal clinic at Moorfields Eye Hospital, London, UK. from 1981 to 1990 inclusive were diagnosed to have this condition. We describe a case of bilateral congenital absence of lacrimal puncta with lacrimal mucocele. Combined surgery was carried out by Ophthalmologist and Otolaryngologist with successful results.     

Safety of oral iron chelator deferiprone in young thalassaemics

Deferiprone is now widely used for iron chelation in patients with thalassaemia. Studies on its efficacy and safety have largely included older children and adults. OBJECTIVE: To assess the safety of deferiprone in children <6 yr of age. METHODS: The study is based on scrutiny of follow-up data of 44 patients of age <6 yr receiving deferiprone for a variable period of time. Occurrence of various side effects including gastrointestinal, osteoarticular were noticed and complete blood counts were performed every 2-4 wk. RESULTS: Nausea and vomiting were noticed in 12 (27.2%), joint symptoms were reported by four (9.1%) and neutropenia was observed in only two patients (4.5%). None of the patient had agranulocytosis. Thrombocytopenia was observed in 20 patients (45.45%), which occurred 3 months to 1 yr after deferiprone therapy. Interruption of deferiprone for 2-4 wk led to reversal of symptoms in all but two patients. CONCLUSION: Thrombocytopenia is one of the major side effects in young thalassaemics and necessitates frequent close monitoring of blood counts but its resolution after discontinuation and absence of clinical evidence of bleeding does not preclude its use.