Lack of cilia and differentiation defects in the liver of human foetuses with the Meckel syndrome

Background/Aims: Meckel syndrome is an autosomal‐recessive disease characterized by a combination of renal cysts, anomalies of the central nervous system, polydactyly and ductal plate malformations (DPM), which are hepatic anomalies consisting of excessive and abnormal foetal biliary structures. Among the genomic loci associated with Meckel syndrome, mutations in four genes were recently identified. These genes code for proteins associated with primary cilia and are possibly involved in cell differentiation. The aim of the present work was to investigate the formation of the primary cilia and the differentiation of the hepatic cells in foetuses with Meckel syndrome. Methods: Sections of livers from human foetuses with Meckel syndrome were analysed by immunofluorescence, immunohistochemistry and electron microscopy. Results: The primary cilia of the biliary cells were absent in some Meckel foetuses, but were present in others. In addition, defects in hepatic differentiation were observed in Meckel livers, as evidenced by the presence of hybrid cells co‐expressing hepatocytic and biliary markers. Conclusions: Defects in cilia formation occur in some Meckel livers, and most cases show DPM associated with abnormal hepatic cell differentiation. Because differentiation precedes the formation of the cilia during liver development, we propose that defective differentiation may constitute the initial defect in the liver of Meckel syndrome foetuses.     

The effect of the thromboxane A2 synthesis inhibitor OKY-046 on renal function in rabbits following release of unilateral ureteral obstruction

A marked decrease in renal blood flow (RBF) and glomerular filtration rate (GFR) was found after 24, 48 and 72 hours of total unilateral ureteral obstruction (UUO) in the rabbit. Contralateral GFR showed a modest increase consistent with compensatory hypertrophy. The urinary excretion of thromboxane B2 (TxB2), the stable metabolite of the vasoconstrictor prostaglandin, thromboxane A2 (TxA2) was significantly elevated in the urine obtained following release of the obstructed ureter when compared to the TxB2 level in the urine from the contralateral kidney. Continuous infusion of OKY-046 at 100 micrograms./kg./min. over 24 hours during UUO decreased TxB2 excretion by greater than 80 per cent. However there was no significant preservation of RBF or GFR of the obstructed kidney following ureteral release despite the selective inhibition of TxA2. Moreover the increase in contralateral GFR was also abolished. Taken together with other studies these results strongly suggest that the potent vasoconstrictor TxA2 is not responsible for the rise in renal resistance that follows acute UUO.     

Decrease in plasma levels of 3,4-dihydroxyphenylethyleneglycol in major depression

Plasma levels of free and sulfoconjugated 3,4-dihydroxyphenylethyleneglycol (DOPEG), the main deaminated metabolite of norepinephrine, were measured in a group of 45 hospitalized patients presenting a major depression and a group of 45 healthy subjects, matched for sex and age. Compared to healthy subjects, depressed patients had significantly lower plasma levels of free and sulfoconjugated DOPEG. The ratio of free over conjugated DOPEG was not statistically different in the two groups. The reduction of plasma DOPEG levels in the depressed patients did not appear to be related to the duration of drug-free period and was similar in males and females. There was no statistically significant correlation between plasma DOPEG levels and total score on the Hamilton Rating Scale for Depression. Finally, plasma DOPEG levels did not differ in unior bipolar patients. The present data provides further evidence for a reduced CNS noradrenergic transmission in major depression.     

An inflammation-inducible adenoviral expression system for local treatment of the arthritic joint

To achieve a disease-regulated transgene expression for physiologically responsive gene therapy of arthritis, a hybrid promoter was constructed. The human IL-1 beta enhancer region (-3690 to -2720) upstream of the human IL-6 promoter region (-163 to +12) was essential in mounting a robust response in HIG-82 synovial fibroblasts and in RAW 264,7 macrophages. A replication-deficient adenovirus was engineered with luciferase (Luc) controlled by the IL-1/IL-6 promoter (Ad5.IL-1/IL-6-Luc). LPS caused a 23- and 4.6-fold induction of Luc. activity in RAW cells infected with Ad5.IL-1/IL-6-Luc or the conventional Ad5.CMV-Luc construct, respectively. Next, adenoviruses (10(6) ffu) were injected into the knees of C57Bl/6 mice. An intra-articular injection of zymosan, 3 days after Ad5.IL-1/IL-6-Luc, increased Luc. activity by 39-fold but had no effect in the Ad5.CMV-Luc joints. The constitutive CMV promoter was rapidly silenced and could not be reactivated in vivo. In contrast, the IL-1/IL-6 promoter could be reactivated by Streptococcal cell wall (SCW)-induced arthritis up to 21 days after infection. Next the IL-1/IL-6 promoter was compared to the C3-Tat/HIV-LTR two-component system in wild-type, IL-6(-/-) and IL-1(-/-) gene knockout mice. Both systems responded well to LPS-, zymosan- and SCW-induced arthritis. However, the basal activity of the IL-1/IL-6 promoter was lower and IL-6 independent. This study showed that the IL-1/IL-6 promoter is feasible to achieve disease-regulated transgene expression for treatment of arthritis.     

A multi-domain protein for beta1 integrin-targeted DNA delivery

The development of effective receptor-targeted nonviral vectors for use in vivo is complicated by a number of technical problems. One of these is the low efficiency of the conjugation procedures used to couple protein ligands to the DNA condensing carrier molecules. We have made and characterized a multi-domain protein (SPKR)4inv, that is designed to target plasmid DNA to beta1 integrins in remodeling tissue. It contains a nonspecific DNA-binding domain (SPKR)4, a rigid alpha-helical linker, and the C-terminal beta1 integrin binding domain (aa 793-987) of the Yersinia pseudotuberculosis invasin protein. (SPKR)4inv could be purified at high yields using a bacterial expression system. We show that (SPKR)4inv binds with high affinity to both plasmid DNA and beta1 integrins. In a cell attachment assay, the apparent affinity of (SPKR)4inv for beta1 integrins is three orders of magnitude higher than that of the synthetic peptide integrin ligand RGDS. (SPKR)4inv-plasmid complexes are not active in an in vitro transfection assay. However, transfection efficiencies of plasmid complexes with a cationic lipid micelle (DOTAP/Tween-20) or a cationic polymer (polyethylenimine), are significantly increased in combination with (SPKR)4inv. (SPKR)4inv-mediated transfection can be inhibited by a soluble form of beta1 integrin, which is evidence for its receptor specificity. In conclusion, (SPKR)4inv allows beta1 integrin-specific targeting of plasmid-carrier complexes, while avoiding inefficient and cumbersome coupling chemistry. The modular design of the expression vector allows production of similar multi-domain proteins with a different affinity. The further development of such complexes for use in vivo is discussed.