Intragenic integration in DLC1 sustains factor VIII expression in primary human cells without insertional oncogenicity.

Techniques enabling precise genome modifications enhance the safety of gene-based therapy. DLC1 is a hot spot for phiC31 integrase-mediated transgene integration in vitro and in vivo. Here we show that integration of a coagulation factor VIII transgene into intron 7 of DLC1 supports durable expression of factor VIII in primary human umbilical cord-lining epithelial cells. Oligoclonal cells with factor VIII transgene integrated in DLC1 did not have altered expression of DLC1 or neighbouring genes within a 1-Mb interval. Only 1.9% of all expressed genes were transcriptionally altered; most were downregulated and mapped to cell cycle and DNA repair pathways. DLC1-integrated cells were not tumourigenic in vivo and were normal by high-resolution genomic DNA copy number analysis. Our data identify DLC1 as a locus for durable transgene expression that does not incur features of insertional oncogenesis, thus expanding options for developing ex vivo cell therapy mediated by site-specific integration methods.

Plasmid-electroporated primary hepatocytes acquire quasi-physiological secretion of human insulin and restore euglycemia in diabetic mice.

We describe the durable correction of streptozotocin-induced murine diabetes by in vivo implantation of primary mouse hepatocytes electroporated ex vivo with a human proinsulin cDNA plasmid construct controlled by glucose and zinc regulatory elements. Transfected hepatocytes increased insulin transgene transcription and secretion within 10-20 min of exposure to 25 mM glucose or 60 microM zinc. Insulin release did not occur from secretory granules. Electroporated Rosa26 hepatocytes ( approximately 8 x 10(5) viable cells) were implanted in C57BL/6J diabetic mice in one of three sites: unresected liver, regenerating liver or mesentery. Control diabetic mice were implanted with untransfected hepatocytes. At 30 days after implantation, 8/15 control mice were alive, while 19/19 treated mice were alive. The ratio of body weight on day 30/nadir body weight was significantly higher for all treated groups compared with controls. All eight surviving control mice were hyperglycemic 30 days post-implantation, while 16/19 treated diabetic mice remained normoglycemic. Treated mice had lower mean glucose values (P< or =0.001) without fasting hypoglycemia and better glucose tolerance (P< or =0.0003) than untreated controls. All (6/6) diabetic mice implanted in regenerating liver and 71% (5/7) implanted in unresected liver were alive 77 days after implantation. Engrafted hepatocytes were identified, mainly around central veins, by staining for beta-galactosidase activity and with anti-human insulin antibody.

Actinomycosis--an unusual case of an uncommon disease.

Actinomycosis is an uncommon disease caused by organisms of the Actinomyces genus. These organisms are commonly found in the mucous membranes but do not cause infection unless there is disruption of the membranes, as occurs, for example, during dental trauma or abdominal surgery. Use of an IUD is also a risk factor for pelvic actinomycosis. The disease is usually insidious and is often mistaken for other conditions. Treatment of the infection, once diagnosed, is a regimen of long-term antibiotics such as penicillin, clindamycin, and others. Our patient had pelvic and sacral actinomycosis without any of the traditional risk factors for infection.

Candida pericarditis: clinical profile and treatment.

BACKGROUND: Candida pericarditis is a rare medical and surgical emergency which, unless treated, leads to impaired cardiac function and death. To facilitate early diagnosis, the clinical features of this condition should be identified. METHODS: Twenty-five cases of Candida pericarditis reported in the last 30 years along with 1 new case were reviewed with regard to demographics, precipitating factors, diagnosis, treatment, and outcome. RESULTS: The syndrome occurred in immunocompromised (73%), antibiotic-treated (62%), or postpericardiotomy (54%) patients. The clinical presentation was frequently subtle and nonspecific. Nevertheless, unexplained fever, an increasing cardiac shadow on chest roentgenogram, or the development of cardiac tamponade may be suggestive. Positive culture for Candida in pericardial fluid or histologic evidence of yeast forms in pericardial tissue establishes the diagnosis. A combination of pericardiocentesis followed by operative drainage and antifungal agents is the usual treatment. Untreated, Candida pericarditis is 100% lethal, whereas prompt diagnosis and treatment lead to cure (mean follow-up, 19 months). CONCLUSIONS: Fever and evolving cardiac tamponade in immunocompromised or postpericardiotomy patients may be suggestive of Candida pericarditis; the presence of organisms in pericardial fluid is diagnostic. Pericardiocentesis followed by operative drainage and antifungal agents appears to be the treatment that is most likely to be curative.

Listeria monocytogenes peritonitis: case report and literature review.

Listeria monocytogenes is a Gram-positive bacillus that is pathogenic in both the normal and compromised host. We describe Listeria peritonitis and cerebritis in a patient with cirrhosis due to non-A, non-B hepatitis, and review the 11 other cases of Listeria peritonitis reported in the English-language literature. Listeria is a rare cause of peritonitis in debilitated, older patients, with two-thirds of the cases occurring in patients with chronic liver disease. Listeria peritonitis may also occur in patients undergoing peritoneal dialysis, or in those with malignancy. Peritonitis due to Listeria is clinically similar to spontaneous bacterial peritonitis, and is associated with fever, variable abdominal pain, and neutrocytic ascites; bacteremia commonly accompanies Listeria peritonitis. This syndrome can be successfully treated with antimicrobial drugs, although the third-generation cephalosporins commonly used in the therapy of spontaneous bacterial peritonitis are not recommended. Ampicillin may be the drug of choice, with combination therapy with an aminoglycoside reserved for cases that do not respond to ampicillin alone.