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Long-term potentiation (LTP) is the most prominent model for the molecular and cellular mechanisms of learning and memory. Two main forms of LTP have been distinguished. The N-methyl-D-aspartate-receptor-dependent forms of LTP have been studied most extensively, whereas much less is known about N-methyl-D-aspartate-receptor-independent forms of LTP. This latter type of LTP was first described at the mossy fiber synapses in the hippocampus and subsequently at parallel fiber synapses in the cerebellum as well as at corticothalamic synapses. These presynaptic forms of LTP require a rise in the intraterminal calcium concentration, but the channel through which calcium passes has not been identified. By using pharmacological tools as well as genetic deletion, we demonstrate here that alpha1E-containing voltage-dependent calcium channels (VDCCs) shift the threshold for mossy fiber LTP. The channel is not involved in the expression mechanism, but it contributes to the calcium influx during the induction phase. Indeed, optical recordings directly show the presence and the function of alpha1E-containing VDCCs at mossy fiber terminals. Hence, a previously undescribed role for alpha1E-containing VDCCs is suggested by these results.

Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high-density oligonucleotide arrays, representing approximately 22000 genes. Notably, the genes significantly up- and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up- and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL-8, IL-6, IL-11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr-1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb-EGF, FGF16, heuregulin beta 1, c-kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin-1 and flt-1 were also upregulated. At 24 h, a clear 'cell cycle' signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c-met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.

We previously reported a genomewide scan to identify autism-susceptibility loci in 110 multiplex families, showing suggestive evidence (P <.01) for linkage to autism-spectrum disorders (ASD) on chromosomes 5, 8, 16, 19, and X and showing nominal evidence (P <.05) on several additional chromosomes (2, 3, 4, 10, 11, 12, 15, 18, and 20). In this follow-up analysis we have increased the sample size threefold, while holding the study design constant, so that we now report 345 multiplex families, each with at least two siblings affected with autism or ASD phenotype. Along with 235 new multiplex families, 73 new microsatellite markers were also added in 10 regions, thereby increasing the marker density at these strategic locations from 10 cM to approximately 2 cM and bringing the total number of markers to 408 over the entire genome. Multipoint maximum LOD scores (MLS) obtained from affected-sib-pair analysis of all 345 families yielded suggestive evidence for linkage on chromosomes 17, 5, 11, 4, and 8 (listed in order by MLS) (P <.01). The most significant findings were an MLS of 2.83 (P =.00029) on chromosome 17q, near the serotonin transporter (5-hydroxytryptamine transporter [5-HTT]), and an MLS of 2.54 (P =.00059) on 5p. The present follow-up genome scan, which used a consistent research design across studies and examined the largest ASD sample collection reported to date, gave either equivalent or marginally increased evidence for linkage at several chromosomal regions implicated in our previous scan but eliminated evidence for linkage at other regions.

Nervous system-specific eve mutants were created by removing regulatory elements from a 16 kb transgene capable of complete rescue of normal eve function. When transgenes lacking the regulatory element for either RP2+a/pCC, EL or U/CQ neurons were placed in an eve-null background, eve expression was completely eliminated in the corresponding neurons, without affecting other aspects of eve expression. Many of these transgenic flies were able to survive to fertile adulthood. In the RP2+a/pCC mutant flies: (1) both RP2 and aCC showed abnormal axonal projection patterns, failing to innervate their normal target muscles; (2) the cell bodies of these neurons were positioned abnormally; and (3) in contrast to the wild type, pCC axons often crossed the midline. The Eve HD alone was able to provide a weak, partial rescue of the mutant phenotype, while both the Groucho-dependent and -independent repressor domains contributed equally to full rescue of each aspect of the mutant phenotype. Complete rescue was also obtained with a chimeric protein containing the Eve HD and the Engrailed repressor domain. Consistent with the apparent sufficiency of repressor function, a fusion protein between the Gal4 DNA-binding domain and Eve repressor domains was capable of actively repressing UAS target genes in these neurons. A key target of the repressor function of Eve was Drosophila Hb9, the derepression of which correlated with the mutant phenotype in individual eve-mutant neurons. Finally, homologues of Eve from diverse species were able to rescue the eve mutant phenotype, indicating conservation of both targeting and repression functions in the nervous system.

Somatic loss of heterozygosity (LOH) has been widely reported in breast cancer as a means of identifying putative tumor-suppressor genes. However, individual studies have rarely spanned more than a single chromosome, and the varying criteria used to declare LOH complicate efforts to formally differentiate regions of consistent versus sporadic (random) loss. We report here the compilation of an extensive database from 151 published LOH studies of breast cancer, with summary data from >15,000 tumors and primary allelotypes from >4,300 tumors. Allelic loss was evaluated at 1,168 marker loci, with large variation in the density of informative observations across the genome. Using studies in which primary allelotype information was available, we employed a likelihood-based approach with a formal chromosomal instability and selection model. The approach seeks direct evidence for preferential loss at each locus compared with nearby loci, accounts for heterogeneity across studies, and enables the direct comparison of candidate regions across the genome. Striking preferential loss was observed (in descending order of significance) in specific regions of chromosomes 7q, 16q, 13q, 17p, 8p, 21q, 3p, 18q, 2q, and 19p, as well as other regions, in many cases coinciding with previously identified candidate genes or known fragile sites. Many of these observations were not possible from any single LOH study, and our results suggest that many previously reported LOH results are not systematic or reproducible. Our approach provides a comparative framework for further investigation of regions exhibiting LOH and identifies broad genomic regions for which there exist few data.

A large-scale effort, termed the Secreted Protein Discovery Initiative (SPDI), was undertaken to identify novel secreted and transmembrane proteins. In the first of several approaches, a biological signal sequence trap in yeast cells was utilized to identify cDNA clones encoding putative secreted proteins. A second strategy utilized various algorithms that recognize features such as the hydrophobic properties of signal sequences to identify putative proteins encoded by expressed sequence tags (ESTs) from human cDNA libraries. A third approach surveyed ESTs for protein sequence similarity to a set of known receptors and their ligands with the BLAST algorithm. Finally, both signal-sequence prediction algorithms and BLAST were used to identify single exons of potential genes from within human genomic sequence. The isolation of full-length cDNA clones for each of these candidate genes resulted in the identification of >1000 novel proteins. A total of 256 of these cDNAs are still novel, including variants and novel genes, per the most recent GenBank release version. The success of this large-scale effort was assessed by a bioinformatics analysis of the proteins through predictions of protein domains, subcellular localizations, and possible functional roles. The SPDI collection should facilitate efforts to better understand intercellular communication, may lead to new understandings of human diseases, and provides potential opportunities for the development of therapeutics.

The structural maintenance of chromosomes (SMC) family of proteins play essential roles in genomic stability. SMC heterodimers are required for sister-chromatid cohesion (Cohesin: Smc1 & Smc3), chromatin condensation (Condensin: Smc2 & Smc4), and DNA repair (Smc5 & Smc6). The SMC heterodimers do not function alone and must associate with essential non-SMC subunits. To gain further insight into the essential and DNA repair roles of the Smc5-6 complex, we have purified fission yeast Smc5 and identified by mass spectrometry the co-precipitating proteins, Nse1 and Nse2. We show that both Nse1 and Nse2 interact with Smc5 in vivo, as part of the Smc5-6 complex. Nse1 and Nse2 are essential proteins and conserved from yeast to man. Loss of Nse1 and Nse2 function leads to strikingly similar terminal phenotypes to those observed for Smc5-6 inactivation. In addition, cells expressing hypomorphic alleles of Nse1 and Nse2 are, like Smc5-6 mutants, hypersensitive to DNA damage. Epistasis analysis suggests that like Smc5-6, Nse1, and Nse2 function together with Rhp51 in the homologous recombination repair of DNA double strand breaks. The results of this study strongly suggest that Nse1 and Nse2 are novel non-SMC subunits of the fission yeast Smc5-6 DNA repair complex.

Estrogens control cell growth and viability in target cells via an interplay of genomic and extragenomic pathways not yet elucidated. Here, we show evidence that cell proliferation and survival are differentially regulated by estrogen in rat pituitary tumor PR1 cells. Pico- to femtomolar concentrations of 17beta-estradiol (E2) are sufficient to foster PR1 cell proliferation, whereas nanomolar concentrations of the same are needed to prevent cell death that occurs at a high rate in these cells in the absence of hormone. Activation of endogenous (PRL) or transfected estrogen-responsive genes occurs at the same, higher concentrations of E2 required to promote cell survival, whereas stimulation of cyclin D3 expression and DNA synthesis occur at lower E2 concentrations. Similarly, the pure antiestrogen ICI 182,780 inhibits estrogen response element-dependent trans-activation and cell death more effectively than cyclin-cdk activity, G1-S transition, or DNA synthesis rate. In antiestrogen-treated and/or estrogen-deprived cells, death is due predominantly to apoptosis. Estrogen-induced cell survival, but not E2-dependent cell cycle progression, can be prevented by an inhibitor of c-Src kinase or by blockade of the mitogen-activated protein kinase kinase/extracellular signal-regulated kinase signaling pathway. These data indicate the coexistence of two distinguishable estrogen signaling pathways in PR1 cells, characterized by different functions and sensitivity to hormones and antihormones.

We report on a Spanish family in which three members of different generations were diagnosed with hereditary spherocytosis (HS). Additionally, one of them II-I (44-years-old), presented iron overload with hepatic deposit and needed treatment with periodic phlebotomies. The rest of the family members presented normal analytical values in iron metabolism. To investigate the presence of H63D and C282Y mutations in the HFE gene, patient II-I was found to be compound heterozygous and was the only family member presenting HS and this genetic condition in HFE. We propose a synergistic effect of HS and mutations in HFE as the cause of the iron deposits.

A2A adenosine receptors specifically found on striatal medium spiny neurons play a major role in sensory motor function and may also be involved in neuropsychiatric and neurodegenerative disorders. One hypothesis concerning Huntington's disease (HD) proposes that an imbalance of the cortico-striatal pathway, due to the mutation in the HD gene, leads to striatal vulnerability. An A2A receptor dysfunction has been previously demonstrated in striatal cells engineered to express mutant huntingtin. Here we tested whether a similar dysfunction (i.e., the binding and functional parameters of A2A adenosine receptors) is present in peripheral blood cells (platelets, lymphocytes, and neutrophils) of subjects carrying the mutant gene. This study involved 48 heterozygous and three homozygous patients compared with 58 healthy subjects. Moreover, we selected seven at-risk mutation carriers. A2A receptor density and function are substantially increased in peripheral blood cells from both patients and subjects at the presymptomatic stage. In the neutrophils of the three homozygous HD subjects receptor dysfunction was higher than in heterozygotes. These data indicate the existence of an aberrant A2A receptor phenotype in the peripheral blood cells of subjects carrying the HD mutation. Future studies will assess whether this parameter can be exploited as a peripheral biomarker of Huntington's disease.

This study was undertaken to investigate pharmacological variables that influence the reinforcing efficacy of psychostimulants. Rhesus monkeys (n = 9) responded under a within-session, exponentially increasing, progressive ratio schedule of cocaine reinforcement. Doses of cocaine, methylphenidate (MP), cocaine analogs [(+/-)-2beta-propanoyl-3beta-(2-naphthyl)-tropane (WF-23), HD-23; (+/-)-2beta-propanoyl-3beta-(2-isopropenyl)tropane (WF-60), HD-60; and 2beta-propanoyl-3beta-(4-tolyl)-tropane (HD-11, WF-11), and 2beta-propanoyl-3beta-(4-tolyl)-tropane (HD-11, WF-11), PTT], and MP analogs [(alphaR,2R)-alpha-(2-naphthalenyl)-2-piperidineacetic acid methyl ester, HDMP-28; and (alphaR,2S)-alpha-(2-naphthalenyl)-2-pyrrolideneacetic acid methyl ester, HDMP-29] that varied in their pharmacokinetic and pharmacodynamic properties were substituted for cocaine. These drugs were chosen according to their selectivity for dopamine transporters (DAT) and 5-hydroxytryptamine (serotonin) transporters (5-HTT) as assessed in rodents and their duration of action. In addition, data pertaining to the rate of onset at DAT were collected for the cocaine analogs using an ex vivo binding assay in rodent tissue. Finally, the pharmacodynamic profile of select drugs was confirmed in primate brain tissue. All drugs had reinforcing effects except HDMP-29. The rank ordering of the peak breaking points (BPs) was cocaine = MP = HDMP-28 >or= HD-60 >or= PTT >or= HD-23 > HDMP-29. The time to peak DAT occupancy for the cocaine analogs was greater than 30 min. The potency to maintain peak BP was significantly correlated with DAT affinity. There was not a linear relationship between monoamine transporter affinity and reinforcing efficacy, but it appeared that in nonhuman primates there is a range of DAT affinity under which maximal responding is maintained. Interestingly, the 5-HTT-selective cocaine analog HD-60 functioned robustly as a reinforcer at several doses in all monkeys tested. These data question the dogma regarding the role of pharmacokinetic factors and the relative influence of DAT and 5-HTT in stimulant reinforcement.

The ability to overexpress full-length huntingtin or large fragments represents an important challenge to mimic Huntington's pathology and reproduce all stages of the disease in a time frame compatible with rodent life span. In the present study, tetracycline-regulated lentiviral vectors leading to high expression levels were used to accelerate the pathological process. Rats were simultaneously injected with vectors coding for the transactivator and wild type (WT) or mutated huntingtin (TRE-853-19Q/82Q) in the left and right striatum, respectively, and analyzed in the 'on' and 'off' conditions. Overexpression of TRE-853-19Q protein or residual expression of TRE-853-82Q in 'off' condition did not cause any significant neuronal pathology. Overexpressed TRE-853-82Q protein led to proteolytic release of N-terminal htt fragments, nuclear aggregation, and a striatal dysfunction as revealed by decrease of DARPP-32 staining but absence of NeuN down-regulation. The differential effect on the DARPP-32/NeuN neuronal staining was observed as early as 1 month after injection and maintained at 3 months. In contrast, expression of a shorter htt form (htt171-82Q) did not require processing prior formation of nuclear aggregates and caused decrease of both DARPP-32 and NeuN neuronal markers at one month post-injection suggesting that polyQ pathology may be dependent on protein context. Finally, the reversibility of the pathology was assessed. Huntingtin expression was turn 'on' for 1 month and then shut 'off' for 2 months. Recovery of DARPP-32 immunoreactivity and clearance of huntingtin aggregates were observed in animals treated with doxycycline. These results suggest that a tetracycline-regulated system may be particularly attractive to model Huntington's disease and induce early and reversible striatal neuropathology in vivo.

Genetic factors contribute to the risk of psychopathology in many psychiatric conditions, but the specific genes are yet to be identified. Neurotransmitter alterations are implicated in the etiology of psychopathology based, in part, on studies of neurotransmitter receptors and their biosynthetic or degradative enzymes in postmortem tissue. Identification of the altered receptors and enzymes serves to identify candidate genes of potential etiological significance. Polymorphisms in these genes can contribute to alterations in protein function in vivo that are part of the neurochemical underpinnings of psychopathologies such as major depressive disorder, psychoses, alcoholism, personality disorders, aggressive-impulsive traits, or suicidal behavior. Altered serotonergic function is implicated in the etiology and pathogenesis of several major psychiatric conditions. In particular, there is much evidence for an association of lower serotonergic function and suicidal behavior. Thus genes related to the serotonergic system are candidate genes worthy of study as part of the genetic diathesis for suicidal behavior. This review examines the following polymorphisms in the serotonin biosynthetic enzyme tryptophan hydroxylase (TPH; A779C substitution), the serotonin transporter (5-HTT, 5-HTTLPR allele), the 5-HT(1B) receptor (G861C, C129T substitution) and the 5-HT(2A) receptor (T102C) for their relationship to suicidal behavior. For the TPH gene, we found the less common U or A allele variant of the A779C polymorphism was associated with suicide attempt. Other studies have found the U allele to be associated with aggression and lower serotonergic function in vivo. A 44 base pair insertion/deletion in the 5' flanking promoter region of the 5-HTT gene may result in less 5-HTT expression and 5-HTT binding. We examined 220 cases postmortem and found no association between the promoter genotype and 5-HTT binding. We also found no association with major depressive disorder (MDD), suicide or pathological aggression, despite finding significantly fewer 5-HTT sites in the prefrontal cortex of depressed and/or suicide cases. In genomic DNA samples from 178 unrelated subjects, we detected two polymorphisms for the 5-HT(1B) receptor at nucleotides 861 and 129. However, no association between either polymorphism and depression, suicide, aggression, or alcoholism was observed. There are two common polymorphisms for the 5-HT(2A) receptor gene in humans. The results of studies of 5-HT(2A) receptor gene polymorphisms do not indicate significant major associations with suicidal behavior. In contrast, the 5-HT(2A) receptor itself is reported to be increased in suicide. Functional polymorphisms involving the promoter region that affect gene expression may explain this finding. Studies of candidate genes related to serotonergic function in brain are increasingly used to establish genetic alterations contributing to psychiatric illness. The most meaningful studies combine the study of candidate genes with direct measures of related proteins as well as psychopathology.

Iron overload increases oxidative stress and may lead to neurodegenerative disease like Parkinson's disease (PD). We studied the role of mutations in the hemochromatosis gene HFE in PD and other parkinsonism (non-PD PS) in two population-based series. The first series consisted of 137 patients with PD and 47 with non-PD PS, and the second of 60 patients with PD and 25 with non-PD PS. In the first series, PD patients were significantly more often homozygous for the C282Y mutation than controls (P=0.03). Patients with non-PD PS in both series were more often carriers for the C282Y mutation than controls (P=0.009, P=0.006, respectively). Our data are hampered by small numbers, yet suggest that the C282Y mutation increases the risk of PD and non-PD PS. The rarity of this genotype requires a large series of patients to prove our hypothesis.

Mood, emotion and cognition are modulated by serotonergic neurotransmission, while the physiological function of serotonergic synapses depends on serotonin reuptake, which is mediated by the serotonin transporter (5-HTT). Allelic variation of 5-HTT expression in humans is caused by a functional gene-promoter polymorphism with two predominant variant alleles, which are associated with variations in anxiety measures as previously reported. Here we report that administration of dexamethasone, a potent glucocorticosteroid hormone, results in an increase in 5-HTT expression in immortalized human B-lymphoblastoid cells, which express the human 5-HTT. Functional reporter gene assays as well as 5-HT uptake and inhibitor binding measures revealed a genotype-dependent dose-response to glucocorticosteroid administration, which was antagonized by RU 38486, a non-specific glucocorticosteroid hormone antagonist. The allele-specific differences after administration of dexamethasone depended on the repetitive GC-rich sequence located approximately 1.4 kb upstream of the 5-HTT gene transcription site because of absence of a significant steroid effect after transfecting a deletional mutant reporter gene construct, which lacks this repetitive promoter sequence. Our findings may contribute to explain the vulnerability to stress-related disorders in susceptible individuals, in whom further clinical studies should follow up on these in vitro findings.

<h4>Objectives</h4>Excess iron stores have been postulated to enhance the risk of ischaemic heart disease. This study aims to determine whether the two major mutations of the haemochromatosis (HFE) gene (C282Y and H63D) are associated with ischaemic heart disease (IHD) or myocardial infarction (MI).<h4>Design</h4>Cross sectional case-control study.<h4>Setting</h4>The geographical area studied by the MONICA (monitoring trends and determinants in cardiovascular disease) heart attack register for North Glasgow in Scotland, UK.<h4>Patients</h4>1009 control subjects chosen at random from general practitioner registers were studied. Additionally, 924 subjects who had survived a first MI sustained between 1985 and 1992 were identified from the MONICA register.<h4>Main outcome measures</h4>C282Y and H63D mutations, previous MI, and presence or absence of IHD.<h4>Results</h4>Mutant gene prevalences in the whole control population were as follows: C282Y: homozygote 0.9%, heterozygote 17.7%; H63D: homozygote 2.1%, heterozygote 25.5%; and compound heterozygote: 2.4%. Analysis by chi(2) test and logistic regression analysis did not identify any significant difference in genotype prevalence between normal control, IHD control, and MI survivor groups.<h4>Conclusions</h4>The C282Y homozygote and heterozygote prevalences are among the highest reported worldwide. No association between IHD or MI and HFE genotype was identified. However, these results need to be interpreted in the light of the cross sectional case-control nature of the study.

<h4>Background/aims</h4>Very little is known about the HFE gene in the rat. The aim of the present study was to determine: (1) the structure of the rat HFE gene; and (2) the tissue expression of the HFE mRNA in the rat, with special emphasis on the liver.<h4>Methods</h4>Cloning of the rat HFE gene was performed using library screening and PCR. Exon-intron borders were assigned by DNA sequencing. Parenchymal and non-parenchymal liver cells were isolated by fractionation of normal rat liver. HFE mRNA levels were determined by Northern blot (tissues) and real-time PCR (isolated liver cells).<h4>Results</h4>The rat HFE gene contained six exons and five introns. The HFE gene is expressed in multiple tissues in the rat, including bone marrow, with the highest expression in the liver. We observed HFE transcripts in several categories of isolated rat liver cells. Unexpectedly, expression also occurred in rat hepatocytes.<h4>Conclusions</h4>The exon-intron pattern of the HFE gene is strongly conserved between rat and mouse. The pattern of tissue expression of the HFE gene is rather similar in humans and rodents. The finding of HFE gene expression in rat hepatocytes raises interesting questions regarding its role in the hepatocyte iron metabolism.

<h4>Background</h4>Peripheral neuropathies (PN) associated with monoclonal gammopathy (MG) are widely considered as autoimmune disorders, but the putative role of incriminated antigens is still not understood.<h4>Objective</h4>Fifty five patients with PN associated with MG were studied to investigate whether new antigens could be found, and to evaluate their relation to clinical manifestations.<h4>Methods</h4>An immunological study was conducted on patient sera to identify autoreactivities against nerve proteins by western blotting. Antigen proteins were purified and analysed by proteomic tools. Correlation with ultrastrucural and clinical features was then studied.<h4>Results</h4>Of the 55 patients suffering from PN associated with MG, 17 exhibited IgG autoantibodies directed against peripheral nerve proteins of 35, 58, and 60 kDa. N-terminal microsequencing and mass spectrometry analyses of the 35 kDa protein revealed perfect peptidic matching with 47% of the amino acid sequence of P0, whereas the 58 and 60 kDa proteins were identified as the reduced and non-reduced forms of a P0 dimer. Deglycosylation did not affect IgG binding to the 35 kDa P0 related protein, suggesting a peptidic epitope. In contrast, deglycosylation abolished IgG recognition of the P0 dimer protein, so that a carbohydrate moiety may be implicated in the epitope formation. This confirmed the existence of two different types of IgG, one recognising the 58 and 60 kDa proteins and one directed against the 35 kDa protein.<h4>Conclusions</h4>This is the first report of antibody activity directed against the dimeric association of P0. Although P0 oligomerisation and adhesion properties play a crucial part in the myelin sheath compaction, the pathogenic significance of these autoantibodies needs further investigations to be elucidated.

A manufacturing process for the production of Anti-thrombin IIII concentrate is described, which is based primarily on Heparin Sepharose affinity chromatography. The process includes two sequential viral inactivation/removal procedures, applied to the fraction eluted from the column, the first by heating in aqueous solution at 60 degrees C for 10 h and the second by nanofiltration. Using viral validation on a scaled-down process both treatments proved to be effective steps; able to inactivate or remove more than 4 logs of virus, and their combined effect (>8 logs) assured the safety of the final product. Viral validation studies of the Heparin Sepharose chromatographic step demonstrated a consistency of the affinity of the resin for viruses over repeated use (16 runs), thus providing evidence of absence of cross-contamination from one batch to the next. It was concluded that the process of ATIII manufacturing provides a high level of confidence that the product will not transmit viruses.

The role of OX40-OX40 ligand (OX40L) interaction in Th cell differentiation remains contentious. In vitro studies have revealed a Th2-biased effect by OX40 signals in T cells. However, in vivo studies demonstrated that OX40-OX40L interaction is involved in responses either Th1 or Th2, or both, which appears to be dependent on the experimental conditions used. We document in our report Th cell differentiation in OX40L-deficient and OX40L-transgenic (Tg) mice in response to protein antigens (Ag) and to Leishmania major (L. major) infection. Upon immunization with protein Ag, we demonstrate the adjuvant effect of OX40 signals during in vivo Th2 responses. However, adjuvant treatment to mice ameliorates the Th2-specific effect of OX40-OX40L interaction and rather induces concurrent promotion of both Th1 and Th2 responses via OX40 signals. Thus, previous reports showing promotion of Th1 response by OX40-OX40L interaction may in actual fact be affected by the adjuvant effects mediated by the various experimental conditions. Indeed, constitutive OX40-OX40L interactions in OX40L-Tg mice converted the normally resistant C57BL/6 strain, into a susceptible status following L. major infection due to an extraordinary elevated Th2 response. These results provide convincing evidence demonstrating that the OX40-OX40L interaction is paramount in the development of Th2 responses in vivo.

Oxytocin receptors (OTR) have been described in a number of tumours of different origin, and represent a new target for specific radiolabelled oxytocin (OT) analogues in cancer diagnosis and therapy. By linking the DOTA chelating agent to position 8 of the deamino derivative of Lys(8)-vasotocin (dLVT), we obtained a new compound (DOTA-dLVT) with the following characteristics: (1) it forms a monomeric and stable compound that binds to OTR with an affinity comparable to that of the endogenous OT ligand; (2) it is characterised by a very good selectivity profile for the human OTR, with a low affinity binding to the closely related V1a, V1b and V2 vasopressin receptor subtypes; (3) it induces rapid and persistent receptor internalisation and (4) when radiolabelled, [(111)In]-DOTA-dLVT is efficiently and selectively taken up by OTR-positive tumours grown in mice. These features makes radiolabelled DOTA-dLVT a very good candidate for the radiotargeting of OTR-expressing tumours.

<h4>Background & aims</h4>An increased duodenal expression of the iron transporters, divalent-metal-transporter-1, and ferroportin is observed in patients with iron deficiency or hereditary hemochromatosis. Two oxidoreductases, termed duodenal cytochrome b and hephaestin, are proposed to co-operate with divalent-metal-transporter-1 and FPN1, respectively, to transfer iron from the duodenal lumen to the circulation.<h4>Methods</h4>In the present study, we investigated the mRNA and protein expression of Dcytb and hephaestin in duodenal biopsies from patients with iron deficiency, HFE, and non-HFE-associated hemochromatosis and in control subjects by means of real-time polymerase chain reaction, Western blot, and immunofluorescence.<h4>Results</h4>In iron deficiency a coordinated upregulation of the iron transporters divalent-metal-transporter-1 and ferroportin and of duodenal-cytochrome b and hephaestin was found, whereas in patients with HFE and non-HFE-associated hemochromatosis duodenal-cytochrome b and hephaestin protein and mRNA expression were not significantly different from control subjects. However, HFE but not non-HFE hemochromatosis patients presented with an increased duodenal ferric reductase activity. Spearman rank correlations showed that Dcytb, hephaestin, FPN1, and DMT1 mRNA expression are positively related to each other independently of the underlying disease, which ensures an efficient transepithelial transport of absorbed iron.<h4>Conclusions</h4>Our data show that duodenal-cytochrome b activity in iron deficiency is stimulated via enhanced protein expression, whereas in HFE hemochromatosis it is up-regulated post-translationally. This points to different kinetics of intestinal iron uptake between iron deficiency and HFE hemochromatosis and also indicates that duodenal iron accumulation in HFE and non-HFE hemochromatosis is pathophysiologically different.

We have demonstrated previously that the transcription factor activator protein-1 (AP-1) complex is translocated into mitochondria into the nucleus in murine hippocampus after systemic kainate injection (Ogita et al. [2002] J. Neurosci. 22:2561-2570). The present study investigates whether the mitochondrial AP-1 complex translocated in response to kainate treatment binds to AP-1-like sites located at the non-coding region of the mitochondrial genome in mouse hippocampus. There are 10 sites with sequences similar to the nuclear AP-1 site in the non-coding region. Of 10 pieces (MT-1-MT-10) of synthesized double-stranded oligonucleotides, each containing a mitochondrial AP-1-like site, MT-3, MT-4, and MT-9 were effective in inhibiting mitochondrial AP-1 DNA binding enhanced by kainate. Electrophoresis mobility shift analysis using radiolabeled MT-3 and MT-9 probes demonstrated marked enhancement with binding of these 2 probes in hippocampal mitochondrial extracts prepared 2-6 hr after kainate treatment. Unlabeled AP-1 probe was more potent than unlabeled MT-9 probe in inhibiting the mitochondrial MT-9 binding. Supershift analysis revealed participation of particular Fos/Jun family proteins, such as c-Fos, Fos-B, c-Jun, Jun-B, and Jun-D, in MT-9 binding in hippocampal mitochondrial extracts prepared 4 hr after kainate treatment. Immunoprecipitation analysis using anti-c-Fos antibody demonstrated that c-Fos associated with the mitochondrial genome in hippocampal mitochondria prepared from kainate-treated animals. These results suggest that the AP-1 complex expressed by in vivo kainate treatment would bind to AP-1-like sites in the non-coding region of the mitochondrial genome after translocation into mitochondria from murine hippocampus.

Commissural axons generally cross the midline only once. In the Drosophila nerve cord and mouse spinal cord, commissural axons are guided by Slit only after they cross the midline, where Slit prevents these axons from recrossing the midline. In the developing corpus callosum, Slit2 expressed by the glial wedge guides callosal axons before they cross the midline, as they approach the corticoseptal boundary. These data highlighted a potential difference between the role of Slit2 in guiding commissural axons in the brain compared with the spinal cord. Here, we investigate whether Slit2 also guides callosal axons after they cross the midline. Because such questions cannot be addressed in conventional gene knock-out animals, we used in utero injections of antisense oligonucleotides to specifically deplete Slit2 on only one side of the brain. We used this technique together with a novel in vitro assay of hemisected brain slices to specifically analyze postcrossing callosal axons. We find that in the brain, unlike the spinal cord, Slit2 mediates both precrossing and postcrossing axonal guidance. Depletion of Slit2 on one side of the brain causes axons to defasciculate and, in some cases, to aberrantly enter the septum. Because these axons do not recross the midline, we conclude that the principle function of Slit2 at the cortical midline may be to channel the axons along the correct path and possibly repel them away from the midline. We find no evidence that Slit2 prevents axons from recrossing the midline in the brain.

Loss of huntingtin-mediated brain-derived neurotrophic factor (BDNF) gene transcription has been described in Huntington's disease (HD) [Zuccato et al. (2001) Science, 293, 493-498]. It has been shown that BDNF is synthesized in the pyramidal layer of cerebral cortex and released in the striatum [Altar et al. (1997) Nature, 389, 856-860; Conner et al. (1997) J. Neurosci., 17, 2295-2313]. Here we show the cellular localization of BDNF in huntingtin-containing neurons in normal rat brain; our double-label immunofluorescence study shows that huntingtin and BDNF are co-contained in approximately 99% of pyramidal neurons of motor cortex. In the striatum, huntingtin is expressed in 75% of neurons containing BDNF. In normal striatum we also show that BDNF is contained in cholinergic and in NOS-containing interneurons, which are relatively resistant to HD degeneration. Furthermore, we show a reduction in huntingtin and in BDNF immunoreactivity in cortical neurons after striatal excitotoxic lesion. Our data are confirmed by an ELISA study of BDNF and by a Western blot analysis of huntingtin in cortex of quinolic acid (QUIN)-lesioned hemispheres. In the lesioned striatum we describe that the striatal subpopulation of cholinergic neurons, surviving degeneration, contain BDNF. The finding that BDNF is contained in nearly all neurons that contain huntingtin in the normal cortex, along with the reduced expression of BDNF after QUIN injection of the striatum, shows that huntingtin may be required for BDNF production in cortex.

The gene defect in Huntington's disease (HD) causes a polyglutamine expansion in the N-terminal region of huntingtin (N-htt). In vitro studies suggest that mutant N-htt fragments can aggregate and cause cell death in HD. The physiological and pathological conditions that affect htt proteolysis in the brain are unclear. We examined htt expression by Western blot in the rat brain after transient ischemic injury, which causes striatal neurodegeneration similar to that seen in HD and activates proteases including calcium-dependent calpains. Focal brain ischemia reduced levels of full-length htt in the infarcted cortex and striatum and increased expression of a 55-kDa N-htt fragment that was also produced by treating control brain extracts with calpain. N-htt fragments between 65 and 80 kDa also rose after injury, but these fragments were not as long-lived as the 55-kDa N-htt fragment. The results suggest that after ischemic injury full-length htt is degraded in degenerating neurons and an N-htt fragment accumulates.

Three types of cytolethal distending toxin (CDT), namely, CDT-I, CDT-II, and CDT-III, have been described in Escherichia coli. Using primers designed for the detection of sequences common to the cdtB genes, we analyzed by PCR a set of 21 CDT-producing E. coli strains of intestinal and extraintestinal origins isolated from human and different animal species in several European countries and in the United States. On the basis of the existing differences in the cdtB genes, cdt-I-, cdt-II-, and cdt-III-specific primer pairs were designed and used for cdt typing. These new primers successfully differentiated all of the previously described cdt genes. Six strains proved to be cdt-I; eight strains proved to be cdt-III. However, none of the type I-, II-, and III-specific primers generated amplicons from six CDT(+) strains, suggesting the existence of a new cdt variant. Sequence analysis of the amplicons from two untypeable genes confirmed the existence of a new cdt variant that we called cdt-IV. Using the new specific primers, cdt-IV was detected in human, porcine, and poultry strains of intestinal and extraintestinal origins. To validate all sets of cdt specific primers, a group of 353 human E. coli strains isolated in Hungary was then investigated for the presence of cdt genes. This included 190 strains isolated from patients with urinary tract infections (UTI), 51 strains isolated from other (nonurinary) extraintestinal infections, and 112 intestinal strains isolated from healthy individuals. Of 190 UTI strains, 15 (7.9%) had cdt genes. Of 51 non-UTI extraintestinal strains 3 (5.9%) contained the cdt gene, and 1 (0.9%) of 112 healthy intestinal strains was PCR positive. Five strains proved to be cdt-I, and fourteen strains proved to be cdt-IV. The CDT-producing extraintestinal strains belonged to a wide variety of serogroups, including O2, O6, O75, and O170. In conclusion, we have developed a new PCR typing system for CDT able to detect a new CDT variant present in pathogenic E. coli strains obtained from animals and humans.

Uterine tumor resembling ovarian sex cord tumor (UTROSCT) is a rare tumor of reproductive-age and postmenopausal women. We present the first case of UTROSCT with cytogenetic analysis. The tumor occurred in a 34-year-old woman who presented with menorrhagia and a uterine mass. Histologic examination showed tumor with features of sex cord-like epithelium and abundant fibromuscular stroma without an endometrial stromal sarcoma component. The tumor cells expressed cytokeratin, CD99, vimentin, desmin, smooth muscle actin, and estrogen and progesterone receptors. The majority of the cells analyzed by cytogenetic studies showed two balanced chromosomal translocations: t(X;6)(p22.3;q23.1) and t(4;18)(q21.1;q21.3). Several known tumor-related genes (bcl-2, MALT-1, FVT1, SCCA1, SCCA2, and DCC at 18q21; RAP1 at 4q21; and STL at 6q23) and a gonadal-development related gene (H-Y regulator gene at Xp22.3) are located at or near the translocation breakpoints. The tumor cells of sex cord-like elements were strongly and diffusely immunoreactive for bcl-2 antibody. These cytogenetic and immunohistochemical data may suggest potential molecular mechanisms of tumorigenesis of UTROSCT.

<h4>Aim</h4>Recent clinical epidemiological studies have demonstrated the preventive effect of non-steroidal anti-inflammatory drugs (NSAIDs) against colorectal cancer. The underlying mechanism might be the inhibition of rate-limiting enzyme cyclooxygenase-2 (COX-2) in metabolism of arachidonic acid. The role of COX-2 in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients' prognosis still remain unclear. This study was to investigate the role of COX-2 expression in carcinogenesis of colorectal cancer and its relationship with tumor biological characteristics and patients' prognosis.<h4>Methods</h4>A total of 139 colorectal cancers and 19 adenomas surgically treated in School of Oncology, Peking University, from January 1993 to September 2001 were retrospectively studied. COX-2 expression was detected with tissue microarray (TMA) and immunohistochemistry (IHC) procedure. The association between COX-2 expression and clinicopathological features and its influence on patients' prognosis were studied.<h4>Results</h4>COX-2 expression was strong in colorectal cancer, moderate in adenoma and weak in normal mucosa, which demonstrated statistically significant difference (chi(2)=46.997, P<0.001). COX-2 expression had no association with clinicopathological features such as gross type, differentiation, invasion depth, vessel emboli and TNM staging. Cox proportional hazards modeling analysis and Log rank test revealed no prognostic role of COX-2 expression in colorectal cancer patients.<h4>Conclusion</h4>COX-2 may play an important role in the early stage of carcinogenesis, and its expression in colorectal cancer is not associated with clinicopathological features and patients' prognosis.

<h4>Objective</h4>To establish the possible relationship between some coagulation factors and the onset of acute cerebral infarction (ACI).<h4>Methods</h4>The study population consisted of 71 patients with ACI confirmed by CT and 50 age-matched healthy volunteers. Blood samples were obtained during the onset period of ACI. Tissue factor (TF) and tissue factor pathway inhibitor (TFPI) activity in plasma were assayed with the chromogenic assay. Plasma TF and TFPI antigen were measured with enzyme linked immunoadsorbent assay (ELISA). Plasma F VII coagulation activity (F VII: C) and F VIII coagulation activity (F VIII: C) were developed in the one-stage system. Plasma prothrombin (FII) was determined with Ecarin assay. Plasma fibrinogen (Fbg) was measured with thrombin assay. Plasma antithrombin III activity (ATIII) was determined using heparin cofactor activity assay.<h4>Results</h4>Compared with the control, plasma TF activity and antigen in patients with ACI were significantly higher (both P<0.05). But plasma TFPI activity and antigen were remarkably lower in the ACI group (both P<0.05). Plasma F VII: C was significantly higher (P<0.01), and F VIII: C was markedly lower (P<0.05). Plasma FII was remarkably higher (P<0.01). Similarly the Fbg was significantly higher in the ACI than that in the control group (P<0.01), whereas ATIII was significantly lower (P<0.01).<h4>Conclusion</h4>The initiation of TF pathway is contributed to the onset of ACI and the blood is in hypercoagulable state during the early period of ACI.

Most individuals diagnosed with hereditary hemochromatosis have mutations in both copies of the HFE gene, with such mutations being common in populations of north European origin. The number of individuals currently diagnosed and treated for hemochromatosis is small relative to the number carrying two HFE mutations. Studies searching for undiagnosed hemochromatosis cases among disease cohorts have generally failed to find the number of cases that would be expected if disease were the commonest outcome for individuals with two C282Y HFE mutations. Our aim was to test the hypothesis that individuals with two HFE mutations would be under-represented in an elderly population because many would have died from disease caused by hemochromatosis before they reached old age. This is a cross-sectional study of elderly patients referred for full blood counts at the Norfolk and Norwich University Hospital. We screened blood samples from 1,000 elderly men (aged 85 and over) and women (aged 89 and over) for the C282Y, H63D, and S65C mutations of the HFE gene. We also analyzed any recent laboratory data relevant to signs of hemochromatosis. None of the ten possible genotypes was significantly under- or over-represented compared to the expected frequency calculated from the Hardy-Weinberg equation. Four C282Y homozygotes were found. There were few significant differences in the laboratory findings between the genotypes. Our data suggest that most people with HFE mutations survive to old age and do not suffer from signs of iron overload and hemochromatosis.

Iron-regulated transporter protein 1 (IREG1 or ferroportin 1) is a transmembrane iron transporter that has been described in macrophages and hepatocytes. Ferroportin mutations have been described to result in hepatic iron overload in human pedigrees. The role of hepatic ferroportin in the pathogenesis of C282Y-linked hemochromatosis has not been clearly established. The objective was to study the expression of ferroportin mRNA and protein in C282Y-linked hemochromatosis liver and in controls. Human liver biopsies were stained with an anti-ferroportin antibody and quantitation of ferroportin at 62 kDa was done by Western blotting. mRNA was studied by real time RT-PCR. Ferroportin protein expression was increased in C282Y homozygotes (n = 23) compared to wild-type patients (n = 37) (P < 0.003). There was no significant correlation between ferroportin protein or mRNA expression (n = 25) and liver iron concentration or serum ferritin. Immunohistochemical staining demonstrated ferroportin in hepatocytes and macrophages. In conclusion, ferroportin protein is increased in iron-loaded hemochromatosis liver. The increase in ferroportin protein without an increase in mRNA is consistent with iron-mediated translational regulation through the 5'IRE in the mRNA.

<h4>Background</h4>The molecular pathogenesis of poorly differentiated endocrine carcinomas of the gastrointestinal tract (GI PDECs) remains unclear. It has been suggested that these lesions either originate from multipotent stem cells that also can serve as the origin of nonendocrine adenocarcinomas or arise due to the dedifferentiation of well-differentiated endocrine carcinomas (WDECs).<h4>Methods</h4>Ten gastric and 9 colorectal PDECs, 9 gastric WDECs, and 12 colorectal carcinomas (CRCs) were analyzed for loss of heterozygosity (LOH) at 11q13 (MEN1), 17p13.1 (p53), 3p14.2 (FHIT), 3p21.3 (RASSF1A), and 18q23 (DCC/DPC4/Smad2), and for immunohistochemical expression of p53, FHIT, Rb, and p16.<h4>Results</h4>PDECs exhibited high fractional allelic loss (FAL; 0.49), with frequent (> 40%) alterations in p53, Rb, MEN1, FHIT, and 18q. No significant differences were found between gastric and colorectal PDECs. Gastric WDECs also exhibited high FAL (0.44), with frequent alterations in Rb and/or p16, MEN1, and 3p21. CRCs exhibited a low level of FAL (0.23), with frequent (> 50%) p16 and p53 alterations. When gastric PDECs and WDECs were compared, substantial similarities were found with respect to FAL (0.42 vs. 0.44) and with respect to individual gene alterations, except in p53, which was consistently altered only in PDECs. CRCs, which were characterized by a lower FAL (0.56 vs. 0.23) and which lacked alterations in both 3p and Rb, were found to be significantly different from colorectal PDECs.<h4>Conclusions</h4>GI PDECs demonstrated a high level of chromosomal instability; consistent inactivation of both the p53 and p16/Rb pathways; and frequent LOH at 3p (possibly involving FHIT), the MEN1 locus, and 18q. The profile of genetic alterations in PDECs was more consistent with the profile in WDECs than with the profile in CRCs.

The aim of this study was to evaluate the efficacy of and tolerance for radiofrequency thermoablation (RFTA) in patients with hepatocellular carcinoma (HCC). From March 1999 to September 2001, a total of 56 patients (46 men and 10 women) whose mean age was 67.8 years (range 51 to 76 years) underwent RFTA for 71 HCCs at our institution. RFTA was carried out in 45 patients with one lesion less than 6 cm in diameter, in seven patients with two lesions less than 4 cm in diameter each, and in four patients with three lesions less than 3 cm in diameter each. The mean diameter of the lesions was 4.1 cm (range 0.8 to 6.0 cm). The etiology of the cirrhosis was alcoholism in 31 patients, post-hepatitis C in 19 patients, post-hepatitis B in four patients, and hemochromatosis in two patients. Forty-five patients were classified as Child stage A and 11 were Child stage B. No ascites, prothrombin time >60%, and platelet count <60,000/mm(3) were needed. Two types of cooled needles were used depending on the size of the lesion (a needle 15 cm in length was used for 2 or 3 cm tumors, and a cluster of needles was used for tumors larger than 4 cm). Helical computed tomography was performed 8 weeks after treatment. The main criterion for a complete response was the presence of a hypodense lesion without contrast enhancement. Mean follow-up was 14 months. Complete tumor destruction was achieved in 50 (89.2%) of 56 patients after one session and in 52 (92.8%) of 56 after two sessions. Twelve months later, a complete response was confirmed in 45 patients (80.3%), four patients had a local recurrence and new liver nodules, and three patients had died (one of bone metastasis, one of acute alcoholic hepatitis, and one of bronchial carcinoma). Thirty-nine patients (69.6%) were still in complete remission 36 months later, and a new HCC had developed in six patients. At 36 months 49 of 56 patients were alive and 39 of 56 were free of disease. Patients with HCCs that developed following viral cirrhosis had a worse prognosis than those with HCCs that occurred after alcoholic cirrhosis (2-year survival, 57.7% vs. 77.7%; P=0.0241). It was concluded that radiofrequency ablation is an effective treatment for HCC, although the prognosis is better in patients who develop HCC after alcoholic cirrhosis compared to those in whom HCC occurs after viral cirrhosis.

The Wistar Kyoto (WKY) rat has long been proposed as an animal model of depressive behavior. Exposure to stress produces symptoms such as anhedonia, psychomotor retardation, ambivalence, and negative memory bias. Autoradiographic studies have revealed significant differences in the density of norepinephrine transporter (NET) and serotonin transporter (5-HTT) sites in several brain regions in WKY rats compared to Sprague-Dawley (S-D) rats. Since the mesolimbic dopamine (DA) system is involved in cognitive, emotional, and motivational behaviors, this study examined the distribution of dopamine transporter (DAT) sites in the brains of WKY compared to Wistar (WIS) and S-D rats. DAT sites were labeled with [3H]-GBR12935 (1 nM), and mazindol (50 microM) was used to define nonspecific binding. Quantitative analysis of the specific binding indicated that WKY rats exhibited significant differences in DAT binding sites in the cell body as well as mesolimbic areas in comparison to WIS and S-D rats. While the binding of [3H]-GBR to DAT sites was significantly decreased in the nucleus accumbens (NAc), the amygdala, the ventral tegmental area (VTA), and the reticular part of the substantia nigra (P<.05), the binding was significantly increased in the hippocampal subregions and the hypothalamus (P<.05) in WKY rats compared to the other two strains. In contrast, no strain differences were found in the caudate-putamen. The observed differences in the density and distribution of DAT sites in WKY rats may lead to altered modulation of synaptic DA levels in the cell body and mesolimbic regions, thereby contributing to the noted depression-like behaviors reported in this rat strain.

<h4>Background</h4>Hereditary haemochromatosis (HH) is one of the commonest genetic disorders in European populations. Transferrin saturation (TFS) measurement has been advocated as a phenotypic screening test to improve detection. We undertook a prospective study to examine the value of routine TFS measurement in detecting new cases of HH in unselected liver clinic attenders.<h4>Methods</h4>Non-fasting TFS was measured in new patients. HH mutations were determined in those with elevated TFS (>45%) and all who underwent liver biopsy. Liver biopsy was performed in 349 patients, including all found to be C282Y homozygotes or compound heterozygotes.<h4>Results</h4>Of 667 new patients attending over 5 years, 156 had TFS >45% and 18 had significant mutations (12 C282Y homozygotes and six compound heterozygotes). Eleven of the 12 C282Y homozygotes identified had an elevated TFS and 10 had significant hepatic siderosis. Only two of the six compound heterozygotes had an elevated TFS and hepatic siderosis.<h4>Conclusions</h4>The prevalence of new HH cases in patients of European origin attending a liver clinic, detected by phenotypic screening over a 5-year period, was 2.8%. All were identified by a TFS cut off >45%, but TFS >60% provided the best combination of sensitivity and specificity for detecting C282Y homozygosity.

Although non-myeloablative stem cell transplantation(NST) was originally developed to induce graft-versus-malignancy effect, the new transplant modality is now being applied for treatment of non-malignant disorders such as aplastic anemia(AA) and paroxysmal nocturnal hemoglobinuria due to its low toxicities. NST may be preferable than conventional transplantation when an AA patient has complications due to hemochromatosis such as heart failure and diabetes mellitus, and severe infections. We treated three aplastic anemia patients complicated by various organ failures, with a conditioning regimen consisting of fludarabine (Flu), cyclophosphamide(CY), and antithymocyte globulin (ATG). Engraftment promptly occurred in all patients without accompanying severe graft-versus-host disease or organ damages. Successful induction of complete chimerism and a low incidence of toxicities warrant a further clinical trial using the Flu + CY + ATG regimen.

The pathogenesis of pulmonary arterial hypertension (PAH) remains uncertain. Both the serotonin and endothelin (ET) systems are believed to be involved. Recent studies pointed to the importance of the serotonin 2B receptor as a limiting step. The current authors investigated the lung tissue expression of serotonin receptors and of the serotonin transporter (5-HTT) by real-time-quantitative polymerase chain reaction in chronic overcirculation-induced PAH in growing piglets, with and without treatment with the dual ET receptor blocker bosentan. Pulmonary haemodynamic changes were described by pulmonary arterial impedance spectra. Three months after the surgical anastomosis of the left subclavian artery to the pulmonary arterial trunk, there was a shift of the impedance spectra to higher ratios of pressure and flow moduli, with increases in both 0 Hz impedance and characteristic impedance, and these changes were completely prevented by bosentan therapy. There was an increase in the expression of the serotonin 1B receptor. There was no change in the expression of the 5-HTT, and of the serotonin 2B, 1D, and 4 receptors. The overexpression of the serotonin 1B receptor was partially prevented by bosentan therapy. The present authors conclude that this early pulmonary arterial hypertension model is characterised by an endothelin receptor-dependent increased expression of the serotonin 1B receptor.

Peroxisomes are essential subcellular organelles involved in a variety of metabolic processes. Their importance is underlined by the identification of a large group of inherited diseases in humans in which one or more of the peroxisomal functions are impaired. The yeast Saccharomyces cerevisiae has been used as a model organism to study the functions of peroxisomes. Efficient oxidation of fatty acids does not only require the participation of peroxisomal enzymes but also the active involvement of other gene products. One group of important gene products in this respect includes peroxisomal membrane proteins involved in metabolite transport. This overview discusses the various aspects of fatty acid beta-oxidation in S. cerevisiae. Addressed are the various enzymes and their particular functions as well as the various transport mechanisms to take up fatty acids into peroxisomes or to export the beta-oxidation products out of the peroxisome to mitochondria for full oxidation to CO2 and H2O.

Behçet's disease (BD) is a multisystemic inflammatory disorder. Although the cause and pathogenesis of BD are still unclear, there is evidence for genetic, immunologic and infectious factors at the onset or in the course of BD. This review focuses on the functional genomics and immunology of BD. HLA-B51 is the major disease susceptibility gene locus in BD. An increased number of gammadelta T cells in the peripheral blood and in the involved tissues have been reported. However, the T cells at the sites of inflammation appear to be a phenotypically distinct subset. There is also a significant gammadelta T cell proliferative response to mycobacterial 65-kDa heat shock protein peptides. Homologous peptides derived from the human 60-kDa heat shock protein were observed in BD patients. There is evidence that natural killer T cells may also play a role in BD.

The corpus callosum is the most important cerebral commissure allowing axonal fibres to cross the midline. Corpus callosum agenesis is an important condition in man that can reveal numerous genetic syndromes. The corpus callosum develops from the commissural plate, a dorsal region of the telencephalon. Then, axons growing from pyramidal neurons of cortical layer III extend and cross the midline. In experimental models, it is possible to decipher two conditions in which the development of the corpus callosum is impaired. The first condition is characterized by an impairment of the formation of the roof of the telencephalon (the primordium of the commissural plate). This condition can be explained by an abortive induction of this region by an impairment of BMP signaling. This can generate all the forms of holoprosencephaly. Other forms are due to a defective gene coding Hesx1, a transcription factor involved in the control of telencephalic morphogenesis. Such a genetic defect can be observed in human dominant forms of septo-optic dysplasia. The second condition is explained by an impairment of the molecular control of axon growth: such is the case for the couple netrin 1 and DCC or for the adhesion molecule L1.

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.

Huntington's and Kennedy's disease are autosomal dominant neurodegenerative diseases caused by pathogenic expansion of polyglutamine tracts. Expansion of glutamine repeats must in some way confer a gain of pathological function that disrupts an essential cellular process and leads to loss of affected neurons. Association of huntingtin with vesicular structures raised the possibility that axonal transport might be altered. Here we show that polypeptides containing expanded polyglutamine tracts, but not normal N-terminal huntingtin or androgen receptor, directly inhibit both fast axonal transport in isolated axoplasm and elongation of neuritic processes in intact cells. Effects were greater with truncated polypeptides and occurred without detectable morphological aggregates.

In this first article of a series of papers listing first case reports of animal diseases published since 2000, the following 19 cases of dog diseases are discussed: Blastomycotic granuloma involving the cranial vena cava. Congenital myocardial hamartoma. Discospondylitis: three cases caused respectively by Pseudomonas aeruginosa, Enterococcus faecalis and Staphylococcus epidermidis. Dystrophin deficient muscular dystrophy in a Labrador Retriever. Emphysematous prostatitis. Erythema multiforme major caused by a Parvovirus infection of keratinocytes. Hemochromatosis due to repeated blood transfusions. Intraspinal synovial cyst. Juvenile nephropathy in the Collie and the Irish Wolfhound. Primary cerebellar cortical degeneration (abiotrophy) in a Scottish terrier. Primary pulmonary artery chondrosarcoma. Renal dysplasia in a Bull Mastiff. Rhabdomyosarcoma (botryoid sarcoma) of the urinary bladder in a Maltese. Spinal mast cell tumor. Spongiform degeneration of the white matter in the central nervous system of Australian Cattle dog. Systemic pasteurellosis caused by Pasteurella canis. Thymic hemorrhage caused by dicumarol intoxication. Undimerized biclonal gammopathy with a single heavy chain class IgA in a dog with multiple myeloma. After a short introduction, the bibliographical data and the abstract of the author(s) and mostly some additional information derived from the article are given. The article will be regularly updated adding overlooked as well as new first reports.

A 67-year-old woman and a 22-year-old man, great aunt and great nephew, were examined because of hyperferritinaemia; both had been operated during adolescence for bilateral cataract. The clinical diagnosis 'hereditary hyperferritinaemia-cataract syndrome' (HHCS) was confirmed after DNA-analysis, which showed a point mutation in the L-ferritin gene on chromosome 19 (32G > A, the previously reported Pavia-1 mutation). The further supervision of the patients consisted of reassurance, providing an explanation about the background of HHCS and how it differs from HFE-gene related haemochromatosis, and informing other family members. Both patients were referred back to their general practitioners. HHCS is an autosomal dominant disorder that is characterised by elevated serum ferritin in the absence of iron overload. The dysregulation of ferritin production is caused by heterogenous mutations in the iron responsive element of the L-ferritin gene on chromosome 19, which reduce the binding of iron-regulatory proteins. This results in exaggerated L-ferritin mRNA translation, which is normally tightly controlled by the intracellular iron availability. The only relevant clinical symptom is early-onset, bilateral cataract, which is due to L-ferritin deposits in the stroma of the lens. Internists and haematologists should differentiate this syndrome from haemochromatosis in order to avoid invasive diagnostics and mistreatment. Ophthalmologists should consider this syndrome in patients with congenitial or juvenile cataract.

HIV-specific CD4+ T cell responses, in particular to the HIV envelope antigen gp120, are often undetectable in the peripheral blood of HIV-infected individuals. The failure to detect these cells poses a significant impediment to studying the T cell populations that are considered to be essential for controlling HIV infection and has led to speculation that these cells are entirely depleted during HIV infection. This study was designed to test whether gp120-specific CD4+ T cells exist in HIV-infected subjects and can be expanded from peripheral blood mononuclear cells by in vitro stimulation with the gp120 antigen, allowing better characterization of these cells. Although gp120-specific T cell responses were barely observed in patient cells ex vivo before antigenic stimulation, CD4+ T cells specific for gp120 were successfully propagated from the blood of each asymptomatic chronically HIV-infected subject studied. The dominant epitopes recognized by gp120-specific CD4+ T cells from these HIV-infected subjects were mapped to well-conserved sites in the C1 and C2 domains of gp120. Two CD4+ T cell lines recognizing these two regions were subsequently established. The CD4+ T cell lines proliferated and produced interferon gamma in response to the specific epitopes, and the responses were MHC class II restricted. These T cell lines also exhibited cross-reactivity with gp120 from T cell line-adapted HIV-1 strains IIIB and MN, as well as with gp120 from primary isolates SF33 (subtype B), CA1 (subtype A), and CA10 (subtype A/E). The data demonstrate that CD4+ T cells specific for gp120 are not entirely depleted from the peripheral blood of chronically HIV-infected subjects; these cells are present in low numbers but can be expanded after antigenic stimulation in vitro.

Budd-Chiari syndrome (BCS) is a disorder caused by occlusion of the hepatic vein or inferior vena cava. The clinical presentation include abdominal pain, hepatomegaly, ascites, leg edema, collateral venous dilatation of the body trunk, and portal hypertension. In addition, BCS can cause hepatocellular carcinoma (HCC) in some patients, although its pathogenesis is not yet completely understood. The average reported time lag from diagnosis of BCS to full-blown HCC ranges from several years to several decades. Hepatic carcinogenesis in patients with BCS perhaps reflects a prolonged and persistent liver injury in that it occurs in the primary inferior vena cava obstruction rather than the primary hepatic vein thrombosis. Among patients with BCS, membranous obstruction of the vena cava (MOVC) usually presents an insidious and chronic illness, whereas primary hepatic vein thrombosis presents an acute or subacute illness. We experienced a case of a patient with BCS, which progressed rapidly that HCC developed only nine months after the diagnosis of BCS. The factors causing this rapid progression are still unclear and remain to be investigated.

We have previously reported that Fms-like tyrosine kinase-3 ligand (flt3-L) induced tumor stabilization and regression of palpable ectopic prostate tumors (TRAMP-C1). Although some mice remained "tumor free" for several months following termination of therapy, tumors invariably reappeared and grew progressively in all animals. The lack of a curative response suggests that TRAMP-C1 tumors may inhibit the development of a flt3-L-induced anti-tumor immune response. Consistent with this view, we demonstrate herein that TRAMP-C1 tumors isolated from flt3-L treated animals contained a marked dendritic cell (DC) infiltrate that was temporally correlated with tumor regression. However, tumor-associated DCs, especially in a flt3-L setting, progressively lost MHC class II antigen expression during tumor growth. Treatment with the DC maturation factor trimeric CD40 ligand (CD40-L) either alone or in combination with fl3-L neither prevented loss of DC class II antigens nor disease relapse. Because loss of class II antigens would prevent CD4+ helper T (Th) cell development, we treated tumor-bearing mice with agonistic anti-4-1BB antibody (Ab), which can promote cytotoxic T lymphocyte (CTL) development independent of Th cell function. However, anti-4-1BB Ab alone did not alter TRAMP-C1 growth kinetics, and, when used in combination, was no more effective than flt3-L alone. The inability of the 4-1BB co-stimulatory signal to promote tumor regression may have been related to two additional features of TRAMP-C1 tumors. First, tumor-associated T cells, but not splenic T cells from tumor-bearing animals, were profoundly deficient in expression of CD3-epsilon (CD3epsilon) and T cell receptor-beta chain (TCRbeta). Second, CTLs required 24 h to efficiently kill TRAMP-C1 target cells even after up-regulation of MHC class I antigens by interferon-gamma. This rate of tumor cell destruction by CTLs may not be sufficient to prevent tumor progression. Taken together, these data reveal several important immunosuppressive characteristics of the prostate tumor microenvironment (TME) that immunotherapeutic interventions must first overcome to achieve longterm cures. These data also highlight the importance of utilizing treatment versus vaccination models in the evaluation of immunotherapeutic modalities.

Following an information campaign through the media on the subject of 'andropause', 81 consecutive self-referred patients were evaluated. At presentation these patients had complaints possibly related to partial androgen deficiency in the aging male. Hormonal parameters were measured and specific aging male questionnaires, such as Androgen Deficiency in Aging Male (ADAM) and Aging Males' Symptoms (AMS) scale questionnaires were completed. The median age was 59 (range 53-66) and mean body mass index (BMI) was 26.4 +/- 3.3 kg/m2. Mean testosterone was 479.8 +/- 149.0 ng/dl and mean free testosterone was 10.0 +/- 3.0 ng/dl. Only 7.1% of all patients were diagnosed with 'idiopathic' ADAM. The majority of patients presented with erectile dysfunction. A wide variety of conditions were diagnosed, including hemochromatosis, micro- and macroprolactinoma, hypothyroidism, benign prostate hypertrophism and prostate carcinoma. The scores of AMS psychological, somatovegetative and sexual subscales were 12, 16 and 13, according to AMS classification corresponding to severe, moderate and severe complaints, respectively. There was no correlation between AMS (total and subscales) and testosterone levels. A positive correlation between the somatovegetative subscale of the AMS and both BMI and insulin was found. In conclusion, we observed that in a group of self-referred men andropause is not a frequent diagnosis, and other pathology needs to be addressed requiring a multi-disciplinary approach.

Since the 1980s, sputum induction by inhalation of hypertonic saline has been successfully used for diagnosing Pneumocystis carinii pneumonia in patients infected with HIV. In recent years, sputum induction and its subsequent processing has been refined as a noninvasive research tool providing important information about inflammatory events in the lower airways, and it has been used for studying various illnesses. In asthma, one application is to use sputum inflammatory indices to increase our understanding of complex relationships between inflammatory cells, mediators, and cytokine mechanisms. In chronic obstructive pulmonary disease, sputum assessment could be used as a screening test before deciding on long-term corticosteroid treatment. In tuberculosis, sputum induction is a valuable diagnostic tool for HIV-seropositive patients who do not produce sputum. Sputum induction appears to be a relatively safe, noninvasive means of obtaining airway secretions from subjects with cystic fibrosis, especially from those who do not normally produce sputum. Moreover, sputum induction can also be used in chronic cough and lung cancer. Generally, induction is performed through ultrasonic nebulizers, using hypertonic saline. It is recommended that sputum be processed as soon as possible, with complete homogenization by the use of dithiothreitol. We have also shown in this article an example of a protocol for inducing and processing sputum employing a nebulizer produced in Brazil.

Hereditary haemochromatosis is an autosomal recessive disorder, leading to progressive iron overload, which is very common among the Caucasian population. In the vast majority of the cases, the hereditary iron overload is caused by mutations in the HFE gene. Most prominently this is the homozygous Cys282Tyr mutation. We report two Dutch families in which both propositi were found to be heterozygous for Cys282Tyr in the work-up of hyperferritinaemia. Frequent phlebotomies had no effect on the ferritin level, but led to microcytic anaemia. Finally, the family history with bilateral cataracts was the clue for the correct diagnosis. Hereditary hyperferritinaemia-cataract syndrome (HHCS) is an autosomal dominant disease characterised by elevated serum ferritin levels and bilateral cataracts in the absence of iron overload. Several point mutations and deletions within the iron-responsive element (IRE) in the 5' noncoding region of the L-ferritin gene have been found in HHCS families. In the first Dutch family a G to C transition at position 32 was found and a G to A mutation at the same location was found in the second Dutch family. In individuals with an isolated hyperferritinaemia (normal transferrin saturation), the presence of early onset (familial) cataract should raise the possibility of HHCS, even when Cys282Tyr heterozygosity is found.

<h4>Background & aims</h4>The discovery of a genetic test for hereditary hemochromatosis has identified many individuals who are homozygous for the C282Y mutation of the HFE gene with a normal transferrin saturation and serum ferritin level. The long-term prognosis, rate of iron accumulation, and surveillance guidelines for these individuals are unknown.<h4>Methods</h4>To determine the degree of iron accumulation over time, an updated serum ferritin level was obtained in patients initially identified as homozygous for the C282Y mutation with a normal serum ferritin level.<h4>Results</h4>Twenty-two asymptomatic untreated C282Y homozygotes with a normal serum ferritin level were identified, 10 through population screening, 9 through pedigree analysis, and 3 through a general medical work-up. There were 18 women (4 postmenopausal) and 4 men with a median age of 46 years, range 28-76 years. The median follow-up interval was 4 years, range 2-23 years. The serum ferritin levels of 20 of 22 patients remained below the upper limit of normal during the follow-up period. A decline in serum ferritin level was observed in 13 of 22 patients. Three patients had an increase in serum ferritin level of greater than 50%, with only 1 male patient exceeding the upper limit of normal by having a serum ferritin level increase from 295 to 344 microg/L during a 3-year period.<h4>Conclusions</h4>In C282Y homozygotes with a normal ferritin level at the time of diagnosis, 20 of 22 patients failed to show any significant increase in serum ferritin level during a median follow-up of 4 years. This has clinical and economic implications for follow-up and surveillance of this selected population.

It has been conventional wisdom that early deprivation and trauma can lead unequivocally to later adult debilitation and disorder. That this is not in fact the case has become abundantly clear via a variety of recent new research. While early adversity can be a severe impediment, there is a myriad of accounts of people who have been born into lives of abject destitution, yet have grown into stable, productive, and generative adults. There are certainly personal and social factors that increase the risks of frailty and failing. By the same token, these same risk factors can contribute to the enhancement of one's life, and increase the chances of resilience and of leading fulfilling lives. There is now evidence that society has the knovi/ledge to implement prevention and early intervention programs that foster and enhance personal development; the question is, does it have the will and commitment to do so?

Much of the research on the neurobiology of human anxiety disorders has focused on psychopaihological abnormalities in patients with anxiety disorders. While this line of research is obviously important, more investigation is needed to elucidate the psychobiology of resilience to extreme stress. Study of the psychobiology of resilience has the potential to identify neurochemical, neuropeptide, and hormonal mediators of vulnerability and resilience to severe stress. In addition, the relevance of neural mechanisms of reward and motivation, fear responsiveness, and social behavior to character traits associated with risk and resistance to anxiety disorders may be clarified. These areas of investigation should lead to improved methods of diagnosis, novel approaches to prevention, and new targets for antianxiety drug discovery.

No abstract available.