Antitumoral effects of intratumoral injection of two adenoviruses, one encoding IL-12 and another encodig MIP-3A in a murine model of liver metastatic pancreatic cancer

Category 10: Genetics, genetic disease, gene therapy adhesion. Its effect was potentiated when combined with P-selectin mAb in E-selectin knockout mice. E-selectin knockout on its own did not significantly affect the leukocyte rolling and adhesion. CD18 mAb or knockout did not alter this leukocyte rolling and adhesion event. Anti-neutrophil antibody depleted neutrophils and blocked their recruitment. Condusions: Adenoviral vector induced early and vigorous leukocytemediated hepatic inflammation. These results suggest a P-selectinand alpha4-integrin-dependent, late E-selectin-dependent, and CD18independent pathway in the neutrophil-induced inflammation.

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ANTITUMORAL EFFECTS OF INTRATUMORAL INJECTION OF TWO ADENOVlRUSES, ONE ENCODING IL-12 AND ANOTHER ENCODIG MIP-3A IN A MURINE MODEL OF LIVER METASTATIC PANCREATIC CANCER

Guillermo Mazzolini, Inigo Narvaiza, Miguel Barajas, Cheng Qian, Ignacio Melero, Jesus Prieto. Division De Hepatologia Y Terapia Genica,

Universidad De Navarra, Pamplona, Spain Introduction: Interleukin-12 (IL-12) is a cytokine with potent immunostimulatory activity. Macrophage inflammatory protein-3a (MIP-3 a) is chemokine kwon to attract immature dendritic cells, macrophages and monocytes to the inflammatory sites. According with these properties, we design a strategy consisting in the co-administration of two adenoviruses, one encoding for IL-12 and another encoding for MIP-3a for the treatment of a murine model of metastatic pancreatic adenocarcinoma into the liver. Materials and Methods: We developed two tumoral models, one consisting in subcutaneous inoculation of pancreatic adenocarcinoma cells (PanC02) in C57BL/6 mice, and a second model generated by direct injection of PanC02 cells into the liver of C57BL/6 mice. When the tumor nodules reach 4-5 mm in diameter, the animals were treated with AdlL-12, AdMIP3a or AdlL-12 + AdMip3a. We evaluated tumor growth, animal survival and the mechanisms involved in antitumor response. Results: I) We observed that PanC02 cell line is a very aggressive tumor for the animals. II) Intratumoral administration of single adenovirus (AdlL-12 or AdMip-3a) lack of antitumoral effects. However, combined gene transfer of both adenoviruses (AdlL-12+AdMip-3a) resulted in transitory but potent inhibition of tumoral growth. III) 90% of the animals that received intratumoral administration of combined treatment death in the hepatic model but not in the subcutaneous model. Conclusions: I) Intratumoral co-injection of AdlL-12 and AdMIP-3a had potent synergistic antitumoral effects in the subcutaneous model. II) This combination result in severe toxicity leading to the death of every animal treated in the hepatic model.

say (Gaucher Disease StripAssay) for the simultaneous detection of eight point mutations (84GG, IVS2(+I), 1226G, 1297T, 1342C, 1448C, 1504T and 1604A) and two multiply mutated alleles derived from rearrangements between the structural gene and the 16-kb downstream pseudogene (RecNciI, RecTL). The test is based on a single, multiplex DNA amplification reaction and ready-to-use test strips presenting a parallel array of oligonucleotide probes for each wild-type and mutated allele. The Gaucher Disease StripAssay was used to screen a cohort of 100 English Gaucher Disease patients previously genotyped by RFLE Data obtained with both assays will be presented and discussed with respect to specificity, sensitivity, design and throughput.

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Gernot Kriegshaeuser 1 David Haisall 2, Anne Moritz I , Fritz Kury 1, Christian Oberkanins 1.1 ViennaLab Labordiagnostika GmbH, A - 1110

Vienna, Austria; 2Dept. Clinical Biochemistry, Addenbrook's Hospital, Cambridge, UK Gaucher disease (GD), the most frequent lysosomal storage disorder, is an autosomal recessive disease characterized by deficiency of glucocerebrosidase (GBA). Mutations in the GBA gene cause the disease and enzyme deficiency results in accumulation of glucocerebroside, mainly within cells of the monocyte/macrophage lineage, which may lead to splenomegaly, hepatomegaly, thromhocytopenia, bone marrow suppression, and bone lesions. The disease is panethnic and has been divided into three major types on the basis of the absence (type 1) or the presence and severity of neurologic manifestations (type 2 and type 3). The most common variant of the disease is type 1, which is particularly frequent in the Ashkenazi Jewish population with an estimated disease frequency of 1 in 850 and a carrier rate to approximate 1 in 15. We have developed a reverse-hybridization as-

UNREGISTERED INNATE DISEASE: 'HEPATIC TIGHT JUNCTION ABSENCE DISEASE'

Igor Morozov l, Vera Sherbina 2, Olga Mironova 2. 1Central Institute of

Gastroenterology, Moscow; 2Moscow's Area Clinical Institute, Moscow, Russia In our pathological observation during the last four years intrahepatic cholestasis of the unknown etiology has been documented at two cases of the persistent jaundice of the new born. These children did not have the innate cholangioatresia that was usually responsible for a hyperbilirubinaemia. Under the light microscope investigation of the hepatic biopsy section obtained from these children, the well-defined portal tract fibrosis has been shown to be complicated by the numerous conjunctive tissue portal-portal septae, false lobules, and proliferating cholangioles in the portal tracts. Electron microscope analysis of the biopsy has revealed the sinusoidal and vena porta branch biliary thrombosis. Hepatocyte bilic side was found to be covered by a few in numbers of microvilli and to form a continuous channels opening into a sinusoids. The interhepatocyte fight junctions as well as desmosomes participating in the bile capillary tube formation were found to be absent completely followed by a primary hepatic cholepoietic system disruption. The fight junction is structural elements, which establish a line of demarcation between hepatic blood and bile systems. Their absence results in an unrestricted bile penetration into the sinusoids and then into the vena porta. The indicated disease unique etiological reason may be connected with the innate modified synthesis of the specific integral protein regulating the fight junction formation. The possible confirmation of the herediary origin of the 'hepatic fight junction absence disease' may be found to study the analogous cell surface disruption discovered in the growing hepatoma cell line 60.

[-~ ] RAPID GENETIC TESTING FOR GAUCHER DISEASE: FROM RESTRICTION FRAGMENT LENGTH POLYMORPHISM (RFLP) TO REVERSE-HYBRIDIZATION

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TA-INSERTION MUTATION IN BILIRUBIN-UDP GLUCURONOSYLTRANSFERASE GENE (UGTIA1) PROMOTER IN INDIAN PATIENTS WITH GILBERT'S SYNDROME

M.K. Parvez l , A. Goyal I , N. Kazim 1, S.E. Hasnain 2, S.K. Satin I .

~Department of Gastroenterology, G.B. Pant Hospital, New Delhi, India; 2Centre for DNA Fingerprinting and Diagnostics, Hyderabad, India

Background: Gilbert's Syndrome (GS) is a common, mild and inherited form of unconjugated hyperbilirnbinaemia occurring in the absence of any liver disease or overt haemolysis. The molecular genetic basis of GS has been shown due to a dinucleotide (TA) insertion or deletion in the TATAbox [A(TA)6TAA] promoter of the gene UGT1A1 leading to a reduced hepatic bilirubin glucuronidation. There is no data available on the molecular profile of GS from the Indian subcontinent. Aim: To access the frequency and types of TA-mutation in the UGT1A1 promoter and its association with GS in Indian Patients and healthy controis. Patients and methods: Eight patients with clinically diagnosed GS (unconjugated hyperbilimbinaemia >2 times from baseline on 200 cal. diet for 24 hrs.) with no evidence of any liver disease, alcoholism or viral hepatitis