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<h4>Background</h4>The factors determining why less than 10% of heavy drinkers develop advanced alcoholic liver disease (ALD) remain elusive, although genetic factors may be important. Interleukin 10 (IL-10) is an important cytokine with anti-inflammatory, anti-immune, and antifibrotic functions. Several polymorphisms have been identified in the IL-10 promoter and recent evidence suggests that some of these may have functional effects on IL-10 secretion.<h4>Aims</h4>To test the hypothesis that IL-10 promoter region polymorphisms are associated with susceptibility to ALD.<h4>Methods</h4>The allele frequencies for the two single base pair substitutions at positions -627 (C-->A) and -1117 (A-->G) in the IL-10 promoter were determined in 287 heavy drinkers with biopsy proved advanced ALD, 107 heavy drinkers with no evidence of liver disease or steatosis only on biopsy, and 227 local healthy volunteers.<h4>Results</h4>At position -627, 50% of patients with advanced ALD had a least one A allele compared with 33% of controls (p<0.0001) and 34% of drinkers with no or mild disease (p=0.017). At position -1117, the slight excess of the A allele in drinkers with advanced disease was because of linkage disequilibrium between the A alleles at the two sites.<h4>Conclusions</h4>Among heavy drinkers, possession of the A allele at position -627 in the IL-10 promoter is associated with an increased risk of advanced liver disease. This is consistent with recent functional data that the -627*A allele is associated with low IL-10 expression which will favour inflammatory, immune mediated, and profibrotic mechanisms of alcohol related liver injury.

Loss of heterozygosity (LOH) of chromosome arm 18q is frequent in gastrointestinal cancers. Over 90% of pancreatic carcinomas have 18q LOH. Bi-allelic inactivation of the MADH4/DPC4/SMAD4 gene at 18q21.1 is seen in about half of pancreatic carcinomas with 18q LOH. In the remaining tumors with 18q LOH, MADH4 is not mutated and its expression is unaffected, and no alterations in MADH2/SMAD2, a MADH4-related gene at 18q12.3, have been found. A controversial candidate tumor-suppressor gene at 18q21.2 is DCC (deleted in colorectal carcinoma), which encodes a netrin-1 receptor component with functions in cell migration and apoptosis. Reduced or absent DCC expression has been observed in many cancers, but few somatic mutations that would clearly inactivate DCC function have been reported. We studied a panel of 115 pancreatic and 14 biliary cancers for homozygous deletions of DCC exons and flanking 18q regions. Seven homozygous deletions were seen in the region that includes the DCC gene. In two tumors, the deletions inactivate DCC but not MADH4. A physical and transcript map of the deleted regions was constructed, and DCC was the only known gene affected by all seven deletions. These data are the strongest mutational evidence presented yet in support of the hypothesis that DCC or another gene in the region distal to MADH4 is inactivated, playing a causal role in cancer development.

Huntington's disease is a devastating progressive neurodegenerative illness characterized by massive neuronal loss in the striatum. It is caused by the presence of an expanded CAG repeat in the gene encoding huntingtin, a protein of unknown function. We have examined the expression of neurotransmitters and other antigens present in striatal neurons with immunohistochemistry, and the level of expression of mRNAs encoding enkephalin, substance P, and glutamic acid decarboxylases with quantitative in situ hybridization histochemistry, in the striatum of two mouse models of Huntington's disease: transgenic animals expressing exon 1 of the human huntingtin gene with 144 CAG repeats and "knock-in" mice containing a chimeric mouse/human exon 1 with 71 or 94 CAG repeats inserted by homologous targeting. Although the transgenic (but not the knock-in) mice were previously shown to display prominent huntingtin- and ubiquitin-containing nuclear inclusions in striatal neurons, in situ nick translation followed by emulsion autoradiography did not reveal any DNA damage in striatum or cortex in these mice. Immunolabeling for calbindin D 28K, enkephalin, substance P, glutamic acid decarboxylases (M(r) 65,000 or 67,000, GAD65 and GAD67), somatostatin, choline acetyltransferase, parvalbumin, and glial fibrillary acidic protein were remarkably similar in transgenic, knock-in, and wild-type mice. Both transgenic and knock-in mice, however, showed a marked decrease in the level of expression of enkephalin mRNA in striatal neurons without significant decreases in mRNAs encoding substance P, GAD65, or GAD67. The data indicate that decreased expression of enkephalin mRNA may be an early sign of neuronal dysfunction due to the Huntington's disease mutation.

The enzymes chlorocatechol-1,2-dioxygenase, chloromuconate cycloisomerase, dienelactone hydrolase, and maleylacetate reductase allow Ralstonia eutropha JMP134(pJP4) to degrade chlorocatechols formed during growth in 2,4-dichlorophenoxyacetate or 3-chlorobenzoate (3-CB). There are two gene modules located in plasmid pJP4, tfdC(I)D(I)E(I)F(I) (module I) and tfdD(II)C(II)E(II)F(II) (module II), putatively encoding these enzymes. To assess the role of both tfd modules in the degradation of chloroaromatics, each module was cloned into the medium-copy-number plasmid vector pBBR1MCS-2 under the control of the tfdR regulatory gene. These constructs were introduced into R. eutropha JMP222 (a JMP134 derivative lacking pJP4) and Pseudomonas putida KT2442, two strains able to transform 3-CB into chlorocatechols. Specific activities in cell extracts of chlorocatechol-1,2-dioxygenase (tfdC), chloromuconate cycloisomerase (tfdD), and dienelactone hydrolase (tfdE) were 2 to 50 times higher for microorganisms containing module I compared to those containing module II. In contrast, a significantly (50-fold) higher activity of maleylacetate reductase (tfdF) was observed in cell extracts of microorganisms containing module II compared to module I. The R. eutropha JMP222 derivative containing tfdR-tfdC(I)D(I)E(I)F(I) grew four times faster in liquid cultures with 3-CB as a sole carbon and energy source than in cultures containing tfdR-tfdD(II)C(II)E(II)F(II). In the case of P. putida KT2442, only the derivative containing module I was able to grow in liquid cultures of 3-CB. These results indicate that efficient degradation of 3-CB by R. eutropha JMP134(pJP4) requires the two tfd modules such that TfdCDE is likely supplied primarily by module I, while TfdF is likely supplied by module II.

<h4>Background</h4>Genetic variation of the promoter for the serotonin transporter (5-HTT) gene has been associated with its functional capacity. In vitro, carriers of a short allele (s-carriers) of the 5-HTT promoter display significant reduction in 5-HTT capacity. Dysfunction of 5-HTT has been observed in alcoholic individuals. We assessed whether the allelic constitution of the 5-HTT gene is associated with reduced serotonin transporter availability among alcoholic individuals.<h4>Methods</h4>We genotyped the 5-HTT promoter region and measured the availability of serotonin transporter protein with [I-123]beta-CIT SPECT in the raphe area in 14 abstinent male alcoholic subjects and 8 age-matched control subjects of European American descent.<h4>Results</h4>Among control subjects, the ratio of in vivo 5-HTT availability for ll-homozygous individuals relative to s-carriers was comparable to serotonin uptake ratios measured in vitro. There was a significant interaction of diagnosis and 5-HTT promoter genotype on 5-HTT availability (p <.01). Among controls, ll-homozygous individuals displayed a significant increase as compared with s-carriers. The availability of raphe 5-HTT was significantly reduced in ll-homozygous alcoholic individuals and was negatively correlated with their amount of alcohol consumption. Among s-carriers, 5-HTT availability did not differ significantly between control and alcoholic subjects.<h4>Conclusions</h4>Our preliminary findings suggest an association between 5-HTT allelic constitution and in vivo measurements of human serotonin transporter availability, and a potentially selective susceptibility of ll-homozygous individuals to the neurotoxic effects of chronic excessive alcohol consumption.

In this study we present evidence about the cellular functions of KIF4. Using subcellular fractionation techniques and immunoisolation, we have now identified a type of vesicle that associates with KIF4, an NH(2)-terminal globular motor domain kinesin-like protein. This vesicle is highly concentrated in growth cones and contains L1, a cell adhesion molecule implicated in axonal elongation. It lacks synaptic vesicle markers, receptors for neurotrophins, and membrane proteins involved in growth cone guidance. In cultured neurons, KIF4 and L1 predominantly localize to the axonal shaft and its growth cone. Suppression of KIF4 with antisense oligonucleotides results in the accumulation of L1 within the cell body and in its complete disappearance from axonal tips. In addition, KIF4 suppression prevents L1-enhanced axonal elongation. Taken collectively, our results suggest an important role for KIF4 during neuronal development, a phenomenon which may be related to the anterograde transport of L1-containing vesicles.

To determine whether an additional loss of the coagulation factor VII (FVII) gene influenced the coagulopathy observed in protein C gene-deficient (PC(-/-)) embryos and neonates, we crossed mice doubly heterozygous for the factor VII (FVII(+/-)) and protein C (PC(+/-)) genes to produce offspring possessing the 9 predicted genotypic combinations. FVII(-/-)/PC(-/-) embryos, although present at their expected Mendelian frequency, displayed a phenotype that had not been observed in either the FVII or PC singly deficient embryos. At E12.5 days postcoitum (dpc), FVII(-/-)/PC(-/-) embryos demonstrated an intra- and extravascular coagulopathy that progressed with substantial concomitant hemorrhage and peripheral edema by E17.5dpc, resulting in mortality immediately after birth. FVII(+/-)/PC(-/-) embryos showed a less severe phenotype, suggesting a gene dosage effect. The lack of rescue of PC(-/-) embryos and neonates and augmented coagulopathy resulting from an additional heterozygous or homozygous FVII deficiency are probably due to increased factor Xa and thrombin generation, resulting from loss of FVIIa-dependent tissue factor pathway inhibitor function and the absence of control at the levels of factors Va and VIIIa. The presence of fibrin in embryos in the absence of fetal FVII suggests that significant clot-generating potential exists outside of the embryonic factor VII-dependent pathway.

The intestinal absorption of the nephrotoxic environmental pollutant cadmium increases markedly when iron stores are depleted. This may be mediated by an up regulation of the recently identified mucosal transporter DMT1 (Nramp2 or DCT1) for divalent cations. We tested whether the highly increased iron absorption in hereditary hemochromatosis (HH) was accompanied by an enhanced absorption of cadmium and lead. Cadmium and lead in blood and iron status markers were determined in 21 nonsmoking subjects with HH genetically tested for the HFE mutations and in 21 nonsmoking controls matched for age and sex. In subjects with HH on maintenance phlebotomy treatment, blood concentrations of cadmium, but not lead, were significantly higher than in paired controls. There was a strong age-independent positive association between blood cadmium and the number of years of phlebotomy treatment. Blood lead showed a similar but less pronounced consequence of treatment. All HH subjects with lower blood cadmium than the corresponding controls had either no mutation in the HFE gene, were not phlebotomized, or were phlebotomized for only a limited time. Our findings indicate that the treatment rather than the disease increased the cadmium uptake in homozygous HH. Further studies are needed to confirm whether the disease decreased cadmium absorption and whether the absorption was dependent on the genotype.

The colorectal adenoma-carcinoma sequence represents a well-known paradigm for the sequential development of cancer driven by the accumulation of genomic defects. Although the colorectal adenoma-carcinoma sequence is well investigated, studies about tumours of different dignity co-existent in the same patient are seldom. In order to address the distribution of genetic alterations in different lesions of the same patient, we coincidently investigated carcinomas, adenomas and aberrant crypt foci in patients with sporadic colon cancer. By utilizing polymerase chain reaction, single-strand conformation polymorphism, heteroduplex-analysis, restriction fragment length polymorphism, protein truncation test and sequencing techniques we looked for mutations and microsatellite instability of APC, H-ras, K-ras, p53, DCC and the DNA repair genes hMLH1/hMSH2. In accordance with the suggested adenoma-carcinoma sequence of the colon, four patients reflected the progressive accumulation of genetic defects in synchronously appearing tumours during carcinogenesis. However, two patients with non-hereditary malignomas presented different genetic instabilities in different but synchronously appearing tumours suggesting non-clonal growth under almost identical conditions of the environment. Thus, sporadically manifesting multiple lesions of the colon were not necessarily driven by similar genetic mechanisms. Premalignant lesions may transform into malignant tumours starting from different types of genetic instability, which indicates independent and simultaneous tumorigenesis within the same organ.

<h4>Background and objective</h4>Most patients with hereditary hemochromatosis are homozygous for a Cys282AETyr mutation in the HFE gene. This mutation has been shown to impair the association of the HFE gene product with b(2)-microglobulin and to prevent its cell surface presentation in transfected COS-7 and 293 cells. This study was performed to examine the expression of HFE protein in epithelial cells, macrophages, and circulating leukocytes obtained from normal subjects and patients with hereditary hemochromatosis.<h4>Design and methods</h4>Antisera against two different peptides of the HFE protein were used to immunostain tissue sections and isolate granulocytes, lymphocytes and monocytes.<h4>Results</h4>Immunocytochemical staining showed that the HFE protein is expressed in gastric epithelial cells, tissue macrophages, and circulating monocytes and granulocytes. The cell surface associated signal, which was seen in normal gastric epithelial cells, monocytes and macrophages, was also present in C282Y mutant cells from patients with hereditary hemochromatosis, although at apparently reduced amounts in these cells.<h4>Interpretation and conclusions</h4>From these studies, it is clear that the C282Y mutation reduces but does not completely prevent presentation of the HFE protein on the cell surface of human monocytes, tissue macrophages, and gastric epithelial cells.

<h4>Background and objective</h4>HFE is a class-I MHC related protein which carries the C282Y mutation in most patients with hereditary hemochromatosis, an iron overload disease. HFE protein is expected to have a relevant role in the regulation of duodenal iron absorption, and HFE protein was immunohistochemically identified in the crypt cells. The aim of the work was to analyze whether the C282Y mutation affects HFE accumulation in the duodenum.<h4>Design and methods</h4>We developed antisera for the extracellular portion of recombinant human HFE protein expressed in E. coli. The antisera were specific for HFE protein and the C282Y mutant in immunoblotting, immunoprecipitation and immunocytochemistry experiments of transfected cells, and they did not cross react with HLA antigens in various analyses. The antisera gave positive results in the staining of paraffin-fixed sections of duodenal slices of subjects with hemochromatosis.<h4>Results</h4>The antisera stained evident supranuclear granules in all enterocytes of 7 C282Y homozygous subjects, and a dark area in the same region in 3 other C282Y homozygotes. Granular bodies were absent from the duodenal sections of 8 C282Y negative subjects, from 2 C282Y heterozygotes and 3 C282Y homozygotes, with or without hemochromatosis.<h4>Interpretation and conclusions</h4>The detection of HFE-protein in granular bodies in the enterocytes of the large majority (77%) of C282Y homozygotes and not in other subjects suggests that the mutation facilitates protein accumulation in the duodenum.

The mouse p locus encodes a gene that functions in normal pigmentation. We have characterized a radiation-induced mutant allele of the mouse p locus that is associated with a failure-to-thrive syndrome, in addition to diminished pigmentation. Mice homozygous for this mutant allele, p(100H), show delayed growth and die within 2 wk after birth. We have discovered that the mutant mice develop progressive atrioventricular heart block and significant ultrastructural changes in both cardiac and skeletal muscle cells. These observations are common characteristics described in human myopathies. The karyotype of p(100H) chromosomes indicated that the mutation is associated with a chromosome 7 inversion. We demonstrate here that the p(100H) chromosomal inversion disrupts both the p gene and the Sox6 gene. Normal Sox6 gene expression has been examined by Northern blot analysis and was found most abundantly expressed in skeletal muscle in adult mouse tissues, suggesting an involvement of Sox6 in muscle maintenance. The p(100H) mutant is thus a useful animal model in the elucidation of myopathies at the molecular level.

Histologically detectable iron (HDI) and HFE mutations were searched for in liver biopsy specimens from 58 Italian patients with chronic hepatitis C, and morphologic features were compared to examine their reciprocal relation and their contribution to disease progression. HDI was evident in 48% of cases with features of nonhemochromatosis iron overload. Total, sinusoidal, and portal HDI increased with stage; grade was related to all iron scores because of the contribution of portal inflammation and interface hepatitis. HFE mutations were seen in 47% of patients with chronic hepatitis C and in 28% of control subjects; they were related to stage and the His63Asp mutation to portal HDI. On multivariate analysis, grade but not stage or HFE mutations was associated with HDI in all sites. Interface hepatitis with its sequelae (sinusoidal capillarization and microshunting) represents a major factor in iron deposition in chronic hepatitis C and justifies the features of HDI. HFE mutations are not responsible for HDI deposition but could favor the progression of virus-induced damage independently from interference with iron metabolism.

Expression of the plant protein centroradialis (CEN) leads to a morphological switch between shoot growth and the development of flower structures (inflorescence). We have determined the crystal structure of Antirrhinum CEN to 1.9 A resolution. This structure confirms the CEN proteins as a subset of the family of phosphatidylethanolamine-binding proteins (PEBP), as predicted from sequence homology. Mammalian forms of PEBP have been found to act as inhibitors of MAP kinase signalling, a central signalling cascade regulating cell differentiation. CEN and PEBP proteins share a similar topology dominated by a large central beta-sheet. The strong conservation of a binding pocket at one end of this sheet which is capable of binding phosphoryl ligands, suggests the biological effects of CEN, like PEBP, arise from the ability of this region to form complexes with phosphorylated ligands, hence interfering with kinases and their effectors.

Iron deficiency affects 30% of the world's population. Iron metabolism is tightly regulated, with both gut transport and storage being coordinated. Hereditary haemochromatosis due to mutations in the HFE gene leads to increased absorption of iron and multiple end-organ damage. Myelodysplastic disorders are acquired clonal stem-cell disorders that cause ineffective erythropoiesis. Aplastic anaemia is caused by an intrinsic defect of haemopoietic stem cells; both inherited and acquired forms occur. Primary polycythaemia is a myeloproliferative disorder, a non-malignant stem-cell disease.

HFE is a MHC class 1-like protein that is mutated in hereditary hemochromatosis. In order to elucidate the role of HFE protein on cellular iron metabolism, functional studies were carried out in human hepatoma cells (HLF) overexpressing a fusion gene of HFE and green fluorescent protein (GFP). The expression of HFE-GFP was found to be localized on cell membrane and perinuclear compartment by fluorescent microscopy. By co-immunoprecipitation and Western blotting, HFE-GFP protein formed a complex with endogenous transferrin receptor and beta(2)-microglobulin, suggesting that this fusion protein has the function of HFE reported previously. We then examined the (59)Fe uptake and release, and internalization and recycling of (125)I-labeled transferrin in order to elucidate the functional roles of HFE in the cell system. In the transfectants, HFE protein decreased the rate of transferrin receptor-dependent iron ((59)Fe) uptake by the cells, but did not change the rate of iron release, indicating that HFE protein decreased the rate of iron influx. Scatchard analysis of transferrin binding to HFE-transfected cells showed an elevation of the dissociation constant from 1.9 to 4. 3 nM transferrin, indicating that HFE protein decreased the affinity of transferrin receptor for transferrin, while the number of transferrin receptors decreased from 1.5x10(5)/cell to 1. 2x10(5)/cell. In addition, the rate of transferrin recycling, especially return from endosome to surface, was decreased in the HFE-transfected cells by pulse-chase study with (125)I-labeled transferrin. Our results strongly suggest an additional role of HFE on transferrin receptor recycling in addition to the decrease of receptor affinity, resulting in the reduced cellular iron.

Several studies have attempted to confirm an association between a deletion/insertion polymorphism within the promoter region of the serotonin transporter gene (5-HTT) and Alzheimer's disease independent from the apolipoprotein E (APOE) varepsilon4 status. We examined this deletion/insertion polymorphism of the serotonin transporter gene in a sample of 222 consecutively recruited gerontopsychiatric patients which was divided into four different diagnostic groups: Alzheimer's disease (N=84), mild cognitive impairment (N=29), subjective cognitive complaints (N=49), depression/other psychiatric disorders (N=56) and 118 healthy, non-demented controls. The aim of this approach was to test whether the investigated polymorphism has a high enough selectivity and specificity to distinguish between the different gerontopsychiatric disorders or to differentiate genetically AD from other forms of dementia, respectively. We could not detect any significant differences in the allelic distribution of the deletion/insertion polymorphism of the 5-HTT gene between the four patient subgroups and the control group. This finding indicates that the serotonin transporter does not appear to be a major susceptibility factor in the pathophysiology of Alzheimer's disease and other psychogeriatric disorders.

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.

<h4>Background</h4>Despite heavy alcohol consumption, only a low percentage of heavy drinkers develop liver disease. Imbalances in T-cell subsets and iron metabolism parameters are common findings in heavy drinkers, yet the possible role played by discrete T-lymphocyte subsets under heavy alcohol consumption remains unclear.<h4>Methods</h4>To gain new insights into the possible role played by T lymphocytes during alcohol consumption, characterization of CD28 expression and TcR repertoire in peripheral blood CD4+ and CD8+ T cells by two and three-color flow cytometry was performed. A group of heavy alcohol drinkers (AHD, n = 71) and a group of age-matched controls (n = 81), both HLA-phenotyped and HFE-genotyped, constituted the groups under study.<h4>Results</h4>Marked expansions of CD28- T cells within the CD8+ but not the CD4+ T-cell pool were observed in AHD compared with controls. These CD8+CD28- expansions were paralleled by expansions of CD8+ T cells bearing specific TcR Valpha/beta chains, namely VP5.2. Moreover, AHD, but not controls, carrying the H63D mutation in the HFE gene showed significantly higher percentages of CD28- T cells within the CD8+ T-cell pool than AHD carrying the normal HFE gene. Finally, high numbers of CD8+CD28- T cells in AHD were associated with lower levels of the liver-related enzymes ALT and GGT.<h4>Conclusions</h4>This study showed that under active ethanol consumption, expansions of discrete CD8+ T-cell subsets occur within the CD8+ T-cell pool, that molecules of the MHC-class I locus seem to influence the extent of the expansions, and that high numbers of CD8+CD28- T cells are associated with low levels of liver enzymes in AHD.

Hereditary hemochromatosis (HFE), which affects 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals in Western population, results in multiple organ damage caused by iron deposition, and is treatable if detected early. C282Y mutation in HFE gene has been known to be responsible for the most hereditary hemochromatosis cases and 5-10% of white subjects are heterozygous for this mutation. However, the prevalence of hemochromatosis in the Asian population was reported to be very low and ethnic heterogeneity has been suspected. The aim of our study was to determine the prevalence of heterozygosity and homozygosity for the C282Y HFE gene mutations in 502 unrelated Koreans. Results revealed that none of them had the mutant gene, suggesting a significant ethnic difference when compared with Caucasians. Our study excluded underlying possibility of hereditary hemochromatosis in Korean which could mimic the findings of alcoholic liver disease with iron overload or liver cirrhosis with chronic hepatitis C.

No abstract available.

Prenatal exposure of pregnant rats to methylazoxymethanol acetate (MAM) induces microencephaly in the offspring. In the present study of these microencephalic rats (MAM rats) we used quantitative autoradiography to investigate [3H] paroxetine binding sites, which are a selective marker of serotonin (5-HT) transporters (5-HTT). The binding in the accumbens, cortex, hippocampus, and dorsolateral thalamus was significantly increased in MAM rats, compared to the control rats, while there was a significant decrease in the dorsal raphe nucleus of the MAM rats. The levels of 5-HTT mRNA in the dorsal raphe nuclei were analyzed by in situ hybridization, which revealed a significant decrease in 5-HTT mRNA-positive neurons in the MAM rats compared to the control rats. The results imply serotonergic hyperinnervation in the cerebral hemispheres of MAM rats, while a target-dependent secondary degeneration of 5-HT neurons might be induced in the dorsal raphe nuclei of MAM rats.

Cell kinetics holds a prominent role among biological factors in predicting clinical outcome and response to treatment in neoplastic patients. Different cell kinetic variables are often considered as valid alternatives to each other, but the limited size of case series analysed in several studies and the lack of simultaneous determinations of all the variables on the same tumours do not justify this conclusion. In the present study, the correlation between [3H]thymidine labelling index ([3H]dT LI), flow cytometric S phase cell fraction (FCM-S) and Ki-67 immunoreactivity (Ki-67/MIB-1) was verified and the type of correlation with the most important clinical, pathological and biological patient and tumour characteristics was investigated in a very large series of breast cancer patients. Ki-67/MIB-1, FCM-S and [3H]dT LI were determined in 609, 526 and 485 patients, respectively, and all three cell proliferation indices were evaluated in parallel on the same tumour in a series of 330 breast cancer patients. All the cell kinetic determinations were performed within the context of National Quality Control Programmes. Very poor correlation coefficients (ranging from 0.37 to 0.18) were observed between the different cell kinetic variables determined in parallel on the same series of breast cancers. Moreover, Ki-67/MIB-1 and FCM-S showed a significant relationship with histological type, grade and tumour size, whereas statistically significant correlations were not observed for [3H]dT LI. In conclusion, the results show that the different cell kinetic variables provide different biological information and cannot be considered as alternatives to each other.

The gut and the liver are the key organs in nutrient absorption and metabolism. Bile acids, drugs, and toxins undergo extensive enterohepatic circulation. Bile acids play a major role in several hepatic and intestinal diseases. Endotoxins deriving from intestinal Gram-negative bacteria are important in the pathogenesis of liver and systemic diseases. Chronic liver diseases can influence gastrointestinal motility, which together with other factors may contribute to bacterial overgrowth and in patients with ascites to an increased risk of spontaneous bacterial peritonitis. Patients with end-stage liver disease frequently develop portal hypertension leading to varices, gastric vascular ectasia, and portal hypertensive gastroenteropathy. Several liver and biliary abnormalities are observed in patients with inflammatory bowel disease (primary sclerosing cholangitis, autoimmune hepatitis, cholelithiasis). The primary defect in hemochromatosis is located in the intestine, causing an inappropriate increase in iron absorption, and the liver is the site of earliest and heaviest iron deposition. Elevated transaminases are observed in many patients with celiac disease, and steatohepatitis frequently develops in patients with jejunoileal bypass and short bowel syndrome. Furthermore, the liver is the primary organ for metastasis of intestinal cancer. Many viral, bacterial, fungal, and parasitic diseases affect the intestine as well as the liver and the biliary tract.

Hepatocellular carcinoma (HCC) is one of the most common tumors in humans today, and its incidence has been rising over recent decades in Western countries. The main risk factors for the development of HCC are viral infections (hepatitis B and C), alcohol use, and the intake of mycotoxins in some areas of the world. In addition to these risk factors, genetic disorders such as hemochromatosis or alpha1-antitrypsin deficiency play a role. Regardless of the etiology, HCC generally develops on the basis of liver cirrhosis. Various pathomechanisms are effective during the neoplastic transformation of the hepatocyte. In chemical experimental models, hepatocarcinogenesis occurs in three stages: initiation, progression, and promotion. Molecular mechanisms of HCC development include the alteration in growth factor and tumor suppressor gene expression, dysregulation of apoptosis, and occurrence of reactive oxygen species generated during inflammation.

The identification of the HFE gene involved in hemochromatosis allows genetic tests based on mutation analysis to be performed. However, discrepancies in the correlation between HFE genotypes and iron-loading status have arisen. We investigated 708 patients with various signs or symptoms suggesting a putative iron overload that, nevertheless, did not reach the current criteria for hemochromatosis diagnosis. Most of the patients (91.4%) included in our study displayed one of three classical iron marker values above the threshold defined for iron overloading. HFE mutation analysis allowed us to identify 45.7% of carrier chromosomes in the studied group of patients that showed higher frequencies of HFE mutations compared with controls. In addition, the frequencies of compound C282Y/H63D heterozygous, H63D/H63D homozygous, and C282Y heterozygous genotypes were higher than those in HH probands and controls; they accounted for 16, 5.6, and 22.5% of the patients, respectively. All genotypic groups had a significantly higher value of serum ferritin concentration compared to the normal value; only the C282Y homozygotes and compound heterozygotes with H63D had a transferrin saturation significantly higher than the normal value. On the whole the H63D homozygous and compound heterozygous patients constitute an intermediate phenotypic group between HH and controls. Some of them may reach the critical overloading defined for HH diagnosis along with a potential risk of developing complications, whereas others only show a partial phenotypic expression.

The serotonin transporter (5-HTT) regulates serotonergic neurotransmission and is thought to influence emotion. A 5-HTT-linked polymorphic region (5-HTTLPR) has two common variants, short (s) and long (l). We previously found population and within-family associations between the lower-expressing s allele and neuroticism, a trait related to anxiety, hostility, and depression, on a standard measure (the NEO Personality Inventory, Revised [NEO-PI-R]) in a primarily male population (n=505), and that the s allele was dominant. We investigated this association in a new sample (n=397, 84% female, primarily sib-pairs). The results robustly replicated the 5-HTTLPR neuroticism association, and the dominance of the s allele. Combined data from the two studies (n=902) showed a highly significant association between the s allele and higher NEO Neuroticism both across individuals and within families. Association between genotype and a related measure, Anxiety on the 16PF inventory, was replicated in the new population and within families in the combined sample. Association to another trait, estimated TPQ Harm Avoidance, was not replicated in the new sample but found only within the combined sibship group. Another association found in our original study, between the s allele and lower scores on NEO-PI-R Agreeableness, was also replicated and was more robust in the current and the combined samples. Associations between the functional 5-HTTLPR polymorphism were similar in women and men. These results help to define specific personality features reproducibly associated with 5-HTTLPR genotype. Such associations were strongest for traits defined by the NEO, enhancing the attractiveness of the five-factor personality model in genetic research on complex behavioral dimensions. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:202-216, 2000. Published 2000 Wiley-Liss, Inc.

<h4>Unlabelled</h4>The objective of this study was to assess concentrations of selected markers of coagulation in children with relapse of idiopathic nephrotic syndrome during a 6-week therapy. Study groups: 22 subjects (32 relapses)--14 males, 8 females (mean age 7.15 +/- 1.5 y.) with no thrombotic complications were included into the study. All children were clinically steroid-sensitive.<h4>Methods</h4>Coagulation markers (platelet count, thrombin time, APTT, INR, fibrinogen 1 + 2 fragments (F1 + 2), thrombin-antithrombin complexes (TAT), serum levels of D-dimer (DD), fibrin monomers (FM) and antithrombin activity (ATIII)) were measured three times: on admission, after 2 and 6 weeks. The control group consisted of 13 healthy children.<h4>Results</h4>Serum concentration of TAT or F1 + 2 did not differ between 3 stages (p > 0.05). However, values at 0 and 2 weeks were significantly higher than in control group (p < 0.05). We found no correlation between TAT or F1 + 2 and FBG, ALB, TCH, TG levels. [table: see text]<h4>Conclusions</h4>The coagulation cascade in relapse of NS was activated during first 6 weeks of therapy whereas metabolic disturbances (low ALB, high FGB, TCH, TG, high platelets) normalized. It is speculative whether it was caused by active immunological process but definitely it resulted in "prothrombotic state" in INS patients.

The MHC class I-related HFE gene appears to be involved in iron metabolism, but its pathogenic mechanism in hemochromatosis remains unknown. Furthermore, very little is known about the regulation of its expression. Hybridization of human tissue Northern blots revealed five different HFE mRNAs, indicating that HFE gene transcription is subject to alternative processes. cDNA selection and RT-PCR performed on HeLa cells clearly demonstrated the occurrence of either differential termination or splicing in HFE transcription. Among the numerous molecules identified, two may have a genuine biological significance.

Hereditary hemochromatosis (HH) is common among Caucasians; reported disease frequencies vary from 0.3 to 0.8%. Identification of a candidate HFE gene in 1996 was soon followed by the description of two ancestral mutations, i.e., c.845G-->A (C282Y) and c.187C-->G (H63D). To these was recently added the mutation S65C, which may represent a simple polymorphism. The incidence of HH in Greece is unknown but clinical cases are rare. Also unknown is the carrier frequency of the two mutant alleles. A first estimate of the latter is given in the present report. It is based on data from the genetic analysis of 10 unrelated patients of Greek origin who were referred to our center for genotyping and 158 unselected male blood donors. The allele frequencies for the C282Y and H63D mutations were 0.003 and 0.145, respectively. The C282Y allele was detected in 50% of HH patients. This is considerably lower than the frequencies reported for HH patients in the U.S.A. (82%) and France (91 %) and closer to that reported in Italy (64%). Five patients did not carry any known HFE mutation; three may represent cases of juvenile hemochromatosis, given their early onset with iron overload, hypogonadism, and heart disease. We suggest that genetic heterogeneity is more prominent in Southern Europe. It is also possible that the penetrance of the responsible genes is different across the Mediterranean.

Increasing use of maintenance parenteral iron in the erythropoietin (EPO) era has been accompanied by growing concern about iron overload. This article attempts to put the issue of iron overload in hemodialysis patients into perspective. The condition is less common in all dialysis patients today than it was in the pre-EPO era, since fewer patients are being transfused and EPO therapy shifts iron into erythroid cells. Patients with end stage renal disease (ESRD) are less likely than patients with hemochromatosis to develop iron-induced organ dysfunction. Diagnosis of iron overload is best accomplished through liver biopsy. Clinically significant iron overload, which rarely occurs if ESRD patients are properly managed, can be treated in most EPO-treated renal failure patients by simply withholding parenteral iron therapy.

No abstract available.