A whole genome association study of neuroticism using DNA pooling.

We describe a multistage approach to identify single nucleotide polymorphisms (SNPs) associated with neuroticism, a personality trait that shares genetic determinants with major depression and anxiety disorders. Whole genome association with 452 574 SNPs was performed on DNA pools from approximately 2000 individuals selected on extremes of neuroticism scores from a cohort of 88 142 people from southwest England. The most significant SNPs were then genotyped on independent samples to replicate findings. We were able to replicate association of one SNP within the PDE4D gene in a second sample collected by our laboratory and in a family-based test in an independent sample; however, the SNP was not significantly associated with neuroticism in two other independent samples. We also observed an enrichment of low P-values in known regions of copy number variations. Simulation indicates that our study had approximately 80% power to identify neuroticism loci in the genome with odds ratio (OR)>2, and approximately 50% power to identify small effects (OR=1.5). Since we failed to find any loci accounting for more than 1% of the variance, the heritability of neuroticism probably arises from many loci each explaining much less than 1%. Our findings argue the need for much larger samples than anticipated in genetic association studies and that the biological basis of emotional disorders is extremely complex.

High prevalence of early-onset osteopenia/osteoporosis after allogeneic stem cell transplantation and improvement after bisphosphonate therapy.

Osteopenia/osteoporosis (O/O) has been associated with allogeneic stem cell transplantation (alloSCT). We retrospectively reviewed 102 patients undergoing a first alloSCT from 2000 to 2005 at our center to evaluate the prevalence of O/O < or =6 and >6 months post-alloSCT. Fifty-six patients did not have a dual energy X-ray absorptiometry (DXA) scan following alloSCT. Approximately half (n=13/27) of those with a first DXA scan < or =6 months post-alloSCT had O/O and a similar rate (n=9/19) was seen in those with a first DXA scan >6 months. There were no significant differences in patient characteristics between the normal and O/O groups. The dual femur (DF) appeared to be more vulnerable to alloSCT-induced bone mineral density (BMD) loss than the lumbar spine (LS), regardless of screening time. O/O patients were treated with bisphosphonates and 41% had a repeat DXA scan post-treatment. No patient developed jaw osteonecrosis and significant BMD improvement was seen at the LS (mean BMD, 1.03+/-0.13 vs 1.08+/-0.12, P=0.004) but not the DF (mean BMD, 0.84+/-0.06 vs 0.85+/-0.08, P=0.29), indicating BMD loss at the DF is more resistant than the LS to antiresorptive therapy. Our results demonstrate that O/O is an early and late complication post-alloSCT and bisphosphonate treatment reverses BMD loss at the LS.

Intensive conditioning regimen of etoposide (VP-16), cyclophosphamide and carmustine (VCB) followed by autologous hematopoietic stem cell transplantation for relapsed and refractory Hodgkin's lymphoma.

Several high-dose therapy regimens are used for autologous hematopoietic stem cell transplantation (auto-HSCT) for relapsed and refractory Hodgkin's lymphoma (HL) with variable disease response. An intensified regimen of etoposide (VP-16) 2,400 mg/m(2), cyclophosphamide 7,200 mg/m(2) and carmustine (BCNU) 600 mg/m(2) (VCB) pre-auto-HSCT was developed to overcome disease recurrence. A total of 43 relapsed and refractory HL patients underwent auto-HSCT between January 1992 and December 2004. At day 100 there were 37 (86%) complete responses. A total of 40 patients survived beyond day 100, 14 of whom subsequently relapsed/progressed. At a median follow-up of 4.9 years (range 1.5-11.4 years), 26 patients (60%) are alive and disease free. Five-year actuarial event-free survival (EFS) was 53% (95% CI 35-70%) and median EFS was 5.9 years. Median progression-free and overall survivals have not been reached. EFS was reduced with an increasing number of prognostic factors (Karnofsky performance status, KPS <90, chemotherapy-resistant disease and >or=3 chemotherapy regimens prior to transplant or=2; P=0.049). Grade III-IV regimen-related toxicity was 9% (n=4). The 1-year cumulative incidence of interstitial pneumonitis (IP) was 36%, however only two patients died of IP complications. Disease progression was the most common cause of death (n=10, 23%). Intensive VCB is an effective and well-tolerated preparative regimen for relapsed and refractory HL auto-HSCT.

Fibrinogen stimulates macrophage chemokine secretion through toll-like receptor 4.

Extravascular fibrin deposition is an early and persistent hallmark of inflammatory responses. Fibrin is generated from plasma-derived fibrinogen, which escapes the vasculature in response to endothelial cell retraction at sites of inflammation. Our ongoing efforts to define the physiologic functions of extravasated fibrin(ogen) have led to the discovery, reported here, that fibrinogen stimulates macrophage chemokine secretion. Differential mRNA expression analysis and RNase protection assays revealed that macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, MIP-2, and monocyte chemoattractant protein-1 are fibrinogen inducible in the RAW264.7 mouse macrophage-like cell line, and ELISA confirmed that both RAW264.7 cells and primary murine thioglycolate-elicited peritoneal macrophages up-regulate the secretion of monocyte chemoattractant protein-1 >100-fold upon exposure to fibrinogen. Human U937 and THP-1 precursor-1 (THP-1) monocytic cell lines also secreted chemokines in response to fibrinogen, upon activation with IFN-gamma and differentiation with vitamin D(3), respectively. LPS contamination could not account for our observations, as fibrinogen-induced chemokine secretion was sensitive to heat denaturation and was unaffected by the pharmacologic LPS antagonist polymyxin B. Nevertheless, fibrinogen- and LPS-induced chemokine secretion both apparently required expression of functional Toll-like receptor 4, as each was diminished in macrophages derived from C3H/HeJ mice. Thus, innate responses to fibrinogen and bacterial endotoxin may converge at the evolutionarily conserved Toll-like recognition molecules. Our data suggest that extravascular fibrin(ogen) induces macrophage chemokine expression, thereby promoting immune surveillance at sites of inflammation.

Differential effects of cyclosporine A, methylprednisolone, mycophenolate, and rapamycin on CD154 induction and requirement for NFkappaB: implications for tolerance induction.

BACKGROUND: Recent experimental data indicate that the targeting of the costimulatory molecule CD40-ligand (CD154) may well offer an opportunity for tolerance induction in transplant recipients and patients with autoimmune diseases, although the optimal therapeutic strategy for clinical application of CD154 monoclonal antibody (mAb) is unclear. METHODS: We undertook vascularized heterotopic cardiac allograft transplantation in completely MHC-mismatched mice, treated recipients with CD154 mAb plus various immunosuppressive agents, and performed flow cytometric analysis of CD154 expression by T cells activated in vitro in the presence of corresponding immunosuppressive agents. We also tested the extent to which CD154 induction was NFkappaB-dependent by using NFkappaB/p50-deficient mice as allograft recipients and as source of cells for in vitro studies of CD154 induction, and through use of proteasome inhibitors to block IkappaBalpha degradation and NFKB activation in wild-type mice. RESULTS: Concomitant use of cyclosporin A or methylprednisolone, but not rapamycin or mycophenolate, inhibited CD154 mAb-induced allograft survival. The differential effects of these agents on CD154 mAb-induced tolerance correlated with their capacity to inhibit activation-induced CD154 expression on CD4+ T cells. Full expression of CD154 expression was found to require NF-kappaB activation, and CD154 mAb was ineffective in NF-kappaB/p50 deficient allograft recipients or control mice in which NF-kappaB activation was blocked by proteasome inhibition. CONCLUSIONS: Strategies to use CD154 mAb clinically must take into account the effects of immunosuppressive agents on CD154 induction, which seems to be at least partially NF-kappaB dependent. Our data suggest that ligation of surface-expressed CD154 provides an important signal that modulates T cell activation and thereby contributes to the effects of CD154 mAb, in addition to previously recognized actions involving blockade of CD40/CD154-dependent cell activation and activation-induced cell death.

Avian polyomavirus major capsid protein VP1 interacts with the minor capsid proteins and is transported into the cell nucleus but does not assemble into capsid-like particles when expressed in the baculovirus system.

The baculovirus system was used to construct and isolate AcMNPV-VP1, AcMNPV-VP2 and AcMNPV-VP3 recombinant viruses which express the respective avian polyomavirus (APV) structural proteins in Sf9 insect cells. These recombinant AcMNPVs containing APV structural protein genes were utilized to investigate protein-protein interactions between the structural proteins. Immunofluorescence studies utilizing Sf9 cells infected with the AcMNPV-VP1 revealed that the VP1 protein was expressed and localized in the cytoplasm and not transported into the nucleus. When the cells were co-infected with the VP1 and either VP2 or VP3 recombinant viruses, immunofluorescence of the VP1 protein was localized in the nucleus, indicating that the VP1 protein was transported to the nucleus by both the VP2 and VP3 minor proteins. This observation was suggestive of a protein-protein interaction between the expressed proteins. This protein-protein interaction was substantiated by laser scanning confocal microscopy of Sf9 cells that were co-infected with VP1, VP2 and VP3 recombinant viruses. However, the minor proteins could not be co-isolated with VP1 protein by immunoaffinity chromatography using a monoclonal anti-VP1 serum. In addition, capsid-like particles could not be purified either by CsC1 density gradient centrifugation or by immunoaffinity chromatography. VP1 capsomeres were isolated by immunoaffinity chromatography from Sf9 cells infected with AcMNPV-VP1, with or without the minor protein(s), and these capsomeres could assemble in vitro into capsid-like particles. Electron microscopic observation of thin-sectioned Sf9 cells, which were co-infected with VP1, VP2 and VP3 recombinant viruses, demonstrated capsomere-like structures in the nucleus, but capsid-like particles were not observed.

Tissue-specific segregation of CD1d-dependent and CD1d-independent NK T cells.

NKT cells, defined as T cells expressing the NK cell marker NK1.1, are involved in tumor rejection and regulation of autoimmunity via the production of cytokines. We show in this study that two types of NKT cells can be defined on the basis of their reactivity to the monomorphic MHC class I-like molecule CD1d. One type of NKT cell is positively selected by CD1d and expresses a biased TCR repertoire together with a phenotype found on activated T cells. A second type of NKT cell, in contrast, develops in the absence of CD1d, and expresses a diverse TCR repertoire and a phenotype found on naive T cells and NK cells. Importantly, the two types of NKT cells segregate in distinct tissues. Whereas thymus and liver contain primarily CD1d-dependent NKT cells, spleen and bone marrow are enriched in CD1d-independent NKT cells. Collectively, our data suggest that recognition of tissue-specific ligands by the TCR controls localization and activation of NKT cells.

Polyomavirus major capsid protein VP1 is capable of packaging cellular DNA when expressed in the baculovirus system.

Using the p2Bac dual multiple cloning site transfer vector, the polyomavirus major capsid protein gene VP1 was cloned for expression in the baculovirus-insect cell expression system. The 5-day-infected cellular lysate from this recombinant preparation was purified by cesium chloride density gradient centrifugation. Capsid-like particles were observed in the resulting preparation. The purified particle preparation was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and was shown to have accurately expressed the polyomavirus VP1 protein as cloned. It was found that the preparation revealed the presence of host histones in the stained gels, which is indicative of DNA packaging. To determine if cellular DNA was being packaged in the particles, Sf9 insect cells were prelabeled with [3H] thymidine. The label was removed, and the cells were subsequently infected with a recombinant Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) carrying the polyomavirus VP1 gene. Upon purification through three cesium chloride gradients and DNase I treatment, capsid-like particles, containing [3H]thymidine-labeled DNA, were isolated which were found to coincide with hemagglutination activity. Studies have indicated that the AcMNPV appears to have the ability to fragment Sf9 cellular DNA. When infected with the recombinant AcMNPV carrying the VP1 gene of polyomavirus, these host DNA fragments are being packaged by the VPI major capsid protein; further, these DNA fragments have been shown to be approximately 5 kb in size, which corresponds to the size of the native polyomavirus genome. These studies demonstrate that the recombinant polyomavirus VP1 protein has the ability to package DNA in the absence of the minor structural proteins VP2 and VP3 and independently of the polyomavirus T antigens.

The effect of space flight on monoclonal antibody synthesis in a hybridoma mouse cell line.

The hybridoma cell line, 3G10G5, producing a monoclonal antibody to the major capsid protein VP1 from the avian polyomavirus budgerigar fledgling disease virus, was produced from a Balb/C mouse. This cell line was used to test the effects of microgravity on cellular processes, specifically protein synthesis. A time course study utilizing incorporation of [35S]methionine into newly synthesized monoclonal antibody was performed on STS-77. After 5.5 days, it was observed that cell counts for the samples exposed to microgravity were lower than those of ground-based samples. However, radiolabel incorporation of the synthesized monoclonal antibody was similar in both orbiter and ground control samples. Overall, microgravity does not seem to have an effect on this cell line's ability to synthesize IgG protein.

Single amino acid mutations in the murine MHC class II A beta cytoplasmic domain abrogate antigen presentation.

Class II MHC molecules are heterodimeric transmembrane glycoproteins that function in the presentation of Ag to CD4+ T cells. Deletion of the cytoplasmic domains of the murine class II A alpha- and A beta-chains has previously been shown to diminish Ag presentation and abrogate rejection of class II-transfected tumor cells. To examine the contributions of individual amino acid residues of the A beta cytoplasmic domain to Ag presentation and tumor rejection, we have produced a series of cell lines expressing A beta class II molecules with site-directed mutations. An A beta(k) cDNA was constructed with mutations in the five conserved amino acid residues, Q224, K225, L235, L236, and Q237 (delta5). In addition, cDNA were produced in which alanine was individually substituted for A beta(k) cytoplasmic domain residues 224 through 237 or doubly substituted at residues G226 and P227 or L235 and L236. These mutant cDNAs were individually cotransfected with wild-type A alpha cDNA into the class II-negative M12.C3 B lymphoma and Sal sarcoma cell lines. As was previously reported for transfectants lacking the entire A beta(k) cytoplasmic domain, the delta5 M12.C3 transfectant could not effectively present Ag to an autoreactive Ak-restricted T cell hybrid, and the delta5 Sal transfectant was not rejected when inoculated into syngeneic hosts. A finer analysis revealed that alteration of the individual residue Q224 or the two residues G226 and P227 abrogated Ag presentation in vitro, while mutation of G226 diminished tumor rejection in vivo. Thus, the function of the A beta cytoplasmic domain in Ag presentation both in vitro and in vivo can be disturbed by mutation of single amino acid residues.

Truncation of the class II beta-chain cytoplasmic domain influences the level of class II/invariant chain-derived peptide complexes.

Previous studies have established that antigen presenting cells (APC) expressing major histocompatibility complex class II beta chains with truncated cytoplasmic domains are impaired in their capacity to activate T cells. While it had been widely accepted that this impairment is due to a defect in class II cytoplasmic domain-dependent signal transduction, we recently generated transgenic mice expressing only truncated class II beta chains, and functional analyses of APC from these mice revealed signaling-independent defects in antigen presentation. Here, we demonstrate that T cells primed on such transgenic APC respond better to stimulation by APC expressing truncated beta chains than by wild-type APC. This finding suggests that APC expressing truncated class II beta chains are not inherently defective in their antigen presenting capacity but, rather, may differ from wild-type APC in the peptide antigens that they present. Indeed, analysis of the peptides bound to class II molecules isolated from normal and transgenic spleen cells revealed clear differences. Most notably, the level of class II-associated invariant chain-derived peptides (CLIP) is significantly reduced in cells expressing only truncated beta chains. Prior studies have established that CLIP and antigenic peptides compete for binding to class II molecules. Thus, our results suggest that the cytoplasmic domain of the class II beta chain affects antigen presentation by influencing the level of CLIP/class II complexes.

Transgenic mice expressing MHC class II molecules with truncated A beta cytoplasmic domains reveal signaling-independent defects in antigen presentation.

The thymic development and peripheral activation of CD4+ T cells are critically dependent upon interactions with MHC class II molecules on the surface of antigen presenting cells (APC). In vitro studies involving transfection of cell lines with mutant MHC molecules have demonstrated that the cytoplasmic domains of class II molecules can be required for efficient antigen presentation. To address the role of class II cytoplasmic domains in physiological, non-transformed APC and in vivo immune responses, we have generated transgenic mice which express only truncated class II A beta molecules lacking the 13 membrane distal residues. In vivo, CD4+ T cell development and immune responses to conventional protein antigens, parasitic infections and skin grafts were indistinguishable between control and transgenic mice. Nevertheless, in vitro, APC from transgenic mice poorly stimulate T cell hybridomas and wild-type in vivo-primed T cells. Neither class II-mediated induction of B7-1 expression nor homotypic aggregation were diminished in transgenic B cells, suggesting that both cAMP and tyrosine kinase signaling pathways remain intact despite truncation of the A beta cytoplasmic domain. Furthermore, chemically-fixed cells from transgenic animals are impaired in their antigen presenting capacity. Thus, in contrast to previous studies with cell lines transfected with truncated class II molecules, these results suggest that signaling-independent mechanisms account for the defective in vitro antigen presenting capacity of physiological APC expressing truncated A beta proteins.

Developmental regulation of flavin-containing monooxygenase (FMO) isoforms 1 and 2 in pregnant rabbit.

Mammalian flavin-containing monooxygenase (FMO, EC 1.14.13.8) metabolizes a vast number of structurally diverse xenobiotics containing a soft-nucleophile, typically a nitrogen or sulfur. FMO is not inducible by the classical cytochrome P450 (CYP) inducers, such as phenobarbital, polycyclic aromatic hydrocarbons, ethanol or macrolide antibiotics. Evidence does exist from a number of laboratories, however, for developmental and hormonal regulation of FMO. Our laboratory has confirmed previous observations of enhanced FMO activity during mid- and late-gestation in maternal rabbit lung and have demonstrated that this response is due to increased protein and catalytic activity associated with FMO2. The time course of expression of FMO2 during mid- and late-gestation correlates to plasma peaks of progesterone or cortisol. FMO2 also peaks at parturition in maternal kidney, coincident with plasma cortisol levels. FMO2 is induced by s.c. administration of either progesterone or dexamethasone in lung, or by dexamethasone in kidney. Correlation of plasma progesterone or cortisol levels during gestation and postpartum support a role for progesterone, but not cortisol in regulation of FMO2 in maternal rabbit lung. The levels of FMO1 also appear to be increased during mid- and late-gestation in liver. FMO1 in liver may also be regulated during gestation by progesterone or glucocorticoids as administration of these steroids enhanced FMO1 mRNA levels 4-fold.

Cytotoxic effect of thiacarbocyanine dyes on human colon carcinoma cells and inhibition of bovine heart mitochondrial NADH-ubiquinone reductase activity via a rotenone-type mechanism by two of the dyes.

Five lipophilic-cationic thiacarbocyanine compounds differing in the side chains (methyl-S13, ethyl-S23, propyl-S33, butyl-S43, and pentyl-S53) and a related thiadicarbocyanine compound with ethyl side chains (S25) exhibited a selective cytotoxic effect on human colon carcinoma cells compared to green monkey kidney epithelial cells. The inhibitory concentration for 50% inhibition of growth (IC50) for the carcinoma cells ranged from 13 nM for S13 and S23 to 160 nM for S25. The carcinoma cells were 4- to 100-fold more sensitive than the normal cells. Two of the five compounds, S13 and S23, selectively inhibited NADH oxidase activity with bovine heart submitochondrial particles (SMP). There was no discernable inhibitory effect by the other three thiacarbocyanine compounds on electron transport chain activity. The primary site of inhibition within the respiratory chain for S13 and S23 appeared to be the NADH to coenzyme Q portion of the mitochondrial electron transport chain. Artificial electron acceptors for this segment of respiratory chain were used to localize the inhibitory site. Using SMP, both S13 and S23 inhibited reduction of menadione, duroquinone, and coenzyme Q. Using purified complex I (NADH-ubiquinone reductase) (EC 1.6.99.3), S13 slightly inhibited reduction of juglone, duroquinone, and coenzyme Q, whereas S23 had no effect on any of the substrates. When rotenone-saturated SMP were used, the inhibitory effects of S13, but not S23, on the reduction of menadione were abolished, as was the inhibitory effect of S13 on coenzyme Q reduction when rotenone-insensitive complex I was used as the source of the enzyme. These results suggest that (1) S13 and S23 inhibition of NADH-ubiquinone reductase activity is enhanced by the membrane environment of the enzyme, and (2) the inhibition appears to be in part akin to the inhibiting mode of rotenone.

Inhibition of rodent protein kinase C by the anticarcinoma agent dequalinium.

Dequalinium has previously been shown to be an anticarcinoma agent (M. J. Weiss et al., Proc. Natl. Acad. Sci. USA, 84: 5444-5448, 1987). The present study demonstrates that it can inhibit protein kinase C-beta 1 isolated from an overproducing cell line with a 50% inhibitory concentration of 8-15 microM. Further examination of the inhibition by using structural analogues of dequalinium reveals that the length of the methylene bridge between the two quinaldinium moieties, the presence of the ring substituents, and the bipartite character of the compound each contributes to the inhibitory potency. Related studies show that the analogues display the same rank order of inhibitory potency when tested with the trypsin-generated catalytic fragment of the enzyme, indicating that dequalinium inhibits kinase activity through an interaction with the catalytic subunit. Further studies argue that the ability of a given analogue to inhibit phosphotransferase activity correlates with its ability to compete with [3H]phorbol-12,13-dibutyrate binding on the intact enzyme (50% inhibitory concentration of 2-5 microM). This suggests that the inhibitor is either binding directly to the regulatory subunit as well, or that due to its interaction with the catalytic subunit, dequalinium produces an indirect effect on sites defined by phorbol ester binding. Kinetic analysis revealed that inhibition is noncompetitive with respect to ATP or phosphatidylserine. Studies conducted with types I, II, and III rat brain isozymes, resolved by hydroxylapatite chromatography, demonstrate that dequalinium inhibits each of them with similar potency (50% inhibitory concentration of 11 microM) and imply that the site of contact on the enzyme is a highly conserved region. Morphology studies with dequalinium in intact cells demonstrate that the inhibitor can protect control cells against phorbol ester-induced morphology changes but cannot protect protein kinase C-overproducing cells, suggesting that an elevation in protein kinase C levels alone is sufficient to overturn the protection conferred by dequalinium. On the basis of these results, we propose that protein kinase C could be a critical in vivo target of dequalinium.