PP078. Total antioxidant capacity in patients with pregnancy induced hypertension: Its relation to maternal and/or perinatal complications.

INTRODUCTION: Pregnancy-induced hypertension (PIH) is a major cause of maternal and perinatal mortality and morbidity, particularly in under-resourced countries, like Mexico. Studies on PIH have shown increased oxidative stress products such as malondialdehyde and decrease of total antioxidant capacity (TAC). In this research we measured one marker of oxidative stress (OS) the TAC in patients with PIH and we associated it with the development of maternal and/or fetal complications. OBJECTIVES: Determine whether the plasma level of total antioxidant capacity (as a marker of oxidative stress) influences the development of maternal and/or perinatal complications in patients with PIH. METHODS: A observational, analytical, clinical study was conducted in patients with gestational hypertension (GH), mild preeclampsia (MP), severe preeclampsia (SP) and normal pregnancy (NP) > or = 28weeks gestational age. Serum samples were collected and stored at -70°C until use for the determination of total antioxidant capacity. It was associated with the development of maternal and/or perinatal complications. RESULTS: TAC level in normotensive patients (NP) was mean of 2679 +/- 2014mEq/L while in hypertensive patients (GH, MP, SP) was on mean of 1502 +/-1340mEq/L (p<0.05), in the GH group was 1620 +/-1042mEq/L, in the MP group was 1977 +/-1865mEq/L, in the SP was 819 +/-305meq/L The mean TAC level in the 29 patients who had maternal and/or perinatal complications was 1521mEq/L, while in the 38 patients who showed no maternal and/or perinatal complications the mean was 2355mEq/L (p<0.05). Of the 29 patients who had complications 15 (52%) had greatly diminished TAC levels (less than 1000mEq/L), 9 (31%) had between 1000 and 2500mEq/L and only 5 (17%)>2500mEq/L. 72% (28/39) of PIH group had one or more maternal and/or perinatal complications, while only 1 patient (3.6%) of 28 patients with NP had one or more maternal and/or perinatal complications (p<0.05). CONCLUSION: Patients with decreased TAC level had a higher percentage of maternal and/or perinatal complications. Patients with PIH classified as mild preeclampsia, showing reduced TAC level should be in close observation as they have the risk of developing life-threatening complications since management is usually as outpatient.

Cirrhotic rat livers with extensive fibrosis can be safely transduced with clinical-grade adenoviral vectors. Evidence of cirrhosis reversion.

Adenoviral vectors efficiently target normal liver cells; however, a clear-cut description of the safety boundaries for using adenovectors in hepatic cirrhosis has not been settled. With this in mind, we used a first-generation, replication-deficient adenoviral vector carrying the E. coli lacZ gene (Ad5betaGal) to monitor therapeutic range, biodistribution, toxicity and transduction efficiency in Wistar rats made cirrhotic by two different experimental approaches resembling alcoholic cirrhosis and biliary cirrhosis in humans. Further, we show proof of concept on fibrosis reversion by a 'therapeutic' Ad-vector (AdMMP8) carrying a gene coding for a collagen-degrading enzyme. Dose-response experiments with Ad5betaGal ranging from 1 x 10(8)-3 x 10(12) viral particles (vp) per rat (250 g), demonstrated that adenovirus-mediated gene transfer via iliac vein at 3 x 10(11 )vp/rat, resulted in an approximately 40% transduction in livers of rats made cirrhotic by chronic intoxication with carbon tetrachloride, compared with approximately 80% in control non-cirrhotic livers. In rats made cirrhotic by bile-duct obstruction only, 10% efficiency of transduction was observed. Biodistribution analyses showed that vector expression was detected primarily in liver and at a low level in spleen and kidney. Although there was an important increase in liver enzymes between the first 48 h after adenovirus injection in cirrhotic animals compared to non-transduced cirrhotic rats, this hepatic damage was resolved after 72-96 h. Then, the cDNA for neutrophil collagenase, also known as Matrix Metalloproteinase 8 (MMP8), was cloned in an Ad-vector and delivered to cirrhotic rat livers being able to reverse fibrosis in 44%. This study demonstrates the potential use of adenoviral vectors in safe transient gene therapy strategies for human liver cirrhosis.

Adenoviral gene transfer into dendritic cells efficiently amplifies the immune response to LMP2A antigen: a potential treatment strategy for Epstein-Barr virus--positive Hodgkin's lymphoma.

The EBV-encoded LMP2A protein is consistently expressed in EBV(+) Hodgkin's lymphoma and can be targeted by CTLs. CTLs stimulated conventionally by LCLs have little activity against LMP2A(+) target cells. Here, we describe an alternative approach, based on the in vitro stimulation of CTLs with DCs genetically modified with 2 E1/E3-deleted recombinant adenoviruses, AdGFPLMP2A, encoding a fusion gene of GFP and LMP2A, and AdLMP2A, encoding LMP2A only. Transduction of DCs with AdGFPLMP2A at MOI 1,000 resulted in LMP2A expression in up to 88% of DCs. LMP2A protein was expressed in 40% of DCs transduced with AdLMP2A at an MOI of 100. Higher MOI resulted in DC death. CTL lines activated by transduced DCs had a higher frequency of LMP2A tetramer-specific CTLs than CTL lines activated by LCLs. CTLs stimulated with transduced DCs lysed both autologous fibroblasts infected with vaccinia virus LMP2A (FBvaccLMP2A) and autologous LCLs, which express LMP2A at lower levels. In contrast, CTLs generated from the same donors by stimulation with autologous LCLs showed minimal lysis of FBvaccLMP2A. Moreover, 1 donor who did not respond to LMP2A when CTLs were stimulated with LCLs became a responder when LMP2A was expressed by transduced DCs. Hence, recombinant adenoviruses encoding LMP2A effectively transduce DCs and direct the generation of LMP2A-specific CTLs. This approach will be a potent strategy in Hodgkin's lymphoma immunotherapy.

Truncated active matrix metalloproteinase-8 gene expression in HepG2 cells is active against native type I collagen.

BACKGROUND/AIMS: Excess type I collagen accumulation is a major feature of fibrotic diseases such as liver cirrhosis. Reversion of this disease has not been fully accomplished. Physiologically, collagen is degraded by interstitial collagenases, neutrophil collagenase (MMP-8) being the most active against type I collagen. Introduction of MMP-8 gene into liver cells could be an advantageous tool to potentiate fibrosis degradation. METHODS: We cloned latent and active MMP-8 genes in prokaryotic and eukaryotic expression vectors and an adenoviral vector. Transfection of MMP-8 in HepG2 was effectuated by CaPO4, polylysine-lactose (P-L) and adenoviral transduction, and cells and culture supernatant were harvested 72 h after transfection for analysis of MMP-8 expression by reverse transcription-polymerase chain reaction and collagenolytic activity. RESULTS AND CONCLUSIONS: We show that a truncated neutrophil collagenase (tMMP-8) lacking a portion of the carboxy terminus and with an intact aminoterminus (latent; l-tMMP-8) or a truncated amino terminus (active; a-tMMP-8) has enzymatic activity against native type I collagen, and the activity was inhibited by EDTA, 1,10-phenanthroline and TIMP-1. Both MMP-8 mRNA (latent and active) were detected by polymerase chain reaction in cells transfected with CaPO4, P-L and adenoviral transduction; however, relative expression of MMP-8 was enhanced when the plasmid was delivered as a P-L complex and increased by adenoviral infection. Finally, a-tMMP-8 cDNA was cloned in a vector under transcriptional control of a regulated promoter (PEPCK-a-tMMP-8). HepG2 cells transfected with the PEPCK-a-tMMP-8 plasmid DNA up-regulated expression of a-tMMP-8 after incubation of the cells with butyryl-cAMP and glucagon, while stimulation with insulin slightly down-regulated its expression. Recombinant MMP-8 expressed by HepG2-transduced cells can efficiently degrade soluble type I collagen, which is potentially useful for gene transfer therapies.