Equine herpesvirus-2 E10 gene product, but not its cellular homologue, activates NF-kappaB transcription factor and c-Jun N-terminal kinase.

We have previously reported on the death effector domain containing E8 gene product from equine herpesvirus-2, designated FLICE inhibitory protein (v-FLIP), and on its cellular homologue, c-FLIP, which inhibit the activation of caspase-8 by death receptors. Here we report on the structure and function of the E10 gene product of equine herpesvirus-2, designated v-CARMEN, and on its cellular homologue, c-CARMEN, which contain a caspase-recruiting domain (CARD) motif. c-CARMEN is highly homologous to the viral protein in its N-terminal CARD motif but differs in its C-terminal extension. v-CARMEN and c-CARMEN interact directly in a CARD-dependent manner yet reveal different binding specificities toward members of the tumor necrosis factor receptor-associated factor (TRAF) family. v-CARMEN binds to TRAF6 and weakly to TRAF3 and, upon overexpression, potently induces the c-Jun N-terminal kinase (JNK), p38, and nuclear factor (NF)-kappaB transcriptional pathways. c-CARMEN or truncated versions thereof do not appear to induce JNK and NF-kappaB activation by themselves, nor do they affect the JNK and NF-kappaB activating potential of v-CARMEN. Thus, in contrast to the cellular homologue, v-CARMEN may have additional properties in its unique C terminus that allow for an autonomous activator effect on NF-kappaB and JNK. Through activation of NF-kappaB, v-CARMEN may regulate the expression of the cellular and viral genes important for viral replication.

Ghrelin-induced orexigenic effect in rats depends on the metabolic status and is counteracted by peripheral CB1 receptor antagonism.

Ghrelin is an endogenous regulator of energy homeostasis synthesized by the stomach to stimulate appetite and positive energy balance. Similarly, the endocannabinoid system is part of our internal machinery controlling food intake and energy expenditure. Both peripheral and central mechanisms regulate CB1-mediated control of food intake and a functional relationship between hypothalamic ghrelin and cannabinoid CB1 receptor has been proposed. First of all, we investigated brain ghrelin actions on food intake in rats with different metabolic status (negative or equilibrate energy balance). Secondly, we tested a sub-anxiogenic ultra-low dose of the CB1 antagonist SR141716A (Rimonabant) and the peripheral-acting CB1 antagonist LH-21 on ghrelin orexigenic actions. We found that: 1) central administration of ghrelin promotes food intake in free feeding animals but not in 24 h food-deprived or chronically food-restricted animals; 2) an ultra-low dose of SR141716A (a subthreshold dose 75 folds lower than the EC50 for induction of anxiety) completely counteracts the orexigenic actions of central ghrelin in free feeding animals; 3) the peripheral-restricted CB1 antagonist LH-21 blocks ghrelin-induced hyperphagia in free feeding animals. Our study highlights the importance of the animaĺs metabolic status for the effectiveness of ghrelin in promoting feeding, and suggests that the peripheral endocannabinoid system may interact with ghrelińs signal in the control of food intake under equilibrate energy balance conditions.

Peroxisome proliferator-activated receptor-beta signaling contributes to enhanced proliferation of hepatic stellate cells.

BACKGROUND & AIMS: The peroxisome proliferator-activated nuclear receptors (PPAR-alpha, PPAR-beta, and PPAR-gamma), which modulate the expression of genes involved in energy homeostasis, cell cycle, and immune function, may play a role in hepatic stellate cell activation. Previous studies focused on the decreased expression of PPAR-gamma in hepatic stellate cell activation but did not investigate the expression and role of the PPAR-alpha and -beta isotypes. The aim of this study was to evaluate the expression of the different PPARs during hepatic stellate cell activation in vitro and in situ and to analyze possible factors that might contribute to their expression. In a second part of the study, the effect of a PPAR-beta agonist on acute liver injury was evaluated. METHODS: The effects of PPAR isotype-specific ligands on hepatic stellate cell transition were evaluated by bromodeoxyuridine incorporation, gel shifts, immunoprecipitation, and use of antisense PPAR-beta RNA-expressing adenoviruses. Tumor necrosis factor alpha-induced PPAR-beta phosphorylation and expression was evaluated by metabolic labeling and by using specific P38 inhibitors. RESULTS: Hepatic stellate cells constitutively express high levels of PPAR-beta, which become further induced during culture activation and in vivo fibrogenesis. No significant expression of PPAR-alpha or -gamma was found. Stimulation of the P38 mitogen-activated protein kinase pathway modulated the expression of PPAR-beta. Transcriptional activation of PPAR-beta by L165041 enhanced hepatic stellate cell proliferation. Treatment of rats with a single bolus of CCl(4) in combination with L165041 further enhanced the expression of fibrotic markers. CONCLUSIONS: PPAR-beta is an important signal-transducing factor contributing to hepatic stellate cell proliferation during acute and chronic liver inflammation.

Strain-transcending Fc-dependent killing of Plasmodium falciparum by merozoite surface protein 2 allele-specific human antibodies.

It is widely accepted that antibody responses against the human parasitic pathogen Plasmodium falciparum protect the host from the rigors of severe malaria and death. However, there is a continuing need for the development of in vitro correlate assays of immune protection. To this end, the capacity of human monoclonal and polyclonal antibodies in eliciting phagocytosis and parasite growth inhibition via Fcγ receptor-dependent mechanisms was explored. In examining the extent to which sequence diversity in merozoite surface protein 2 (MSP2) results in the evasion of antibody responses, an unexpectedly high level of heterologous function was measured for allele-specific human antibodies. The dependence on Fcγ receptors for opsonic phagocytosis and monocyte-mediated antibody-dependent parasite inhibition was demonstrated by the mutation of the Fc domain of monoclonal antibodies against both MSP2 and a novel vaccine candidate, peptide 27 from the gene PFF0165c. The described flow cytometry-based functional assays are expected to be useful for assessing immunity in naturally infected and vaccinated individuals and for prioritizing among blood-stage antigens for inclusion in blood-stage vaccines.

Stringent V beta requirement for the development of NK1.1+ T cell receptor-alpha/beta+ cells in mouse liver.

The liver of C57BL/6 mice contains a major subset of CD4+8- and CD4-8- T cell receptor (TCR)-alpha/beta+ cells expressing the polymorphic natural killer NK1.1 surface marker. Liver NK1.1+TCR-alpha/beta+ (NK1+ T) cells require interaction with beta2-microglobulin-associated, major histocompatibility complex I-like molecules on hematopoietic cells for their development and have a TCR repertoire that is highly skewed to Vbeta8.2, Vbeta7, and Vbeta2. We show here that congenic C57BL/6.Vbeta(a) mice, which lack Vbeta8- expressing T cells owing to a genomic deletion at the Vbeta locus, maintain normal levels of liver NK1+ T cells owing to a dramatic increase in the proportion of cells expressing Vbeta7 and Vbeta2 (but not other Vbetas). Moreover, in C57BL/6 congenic TCR-V Vbeta3 and -Vbeta8.1 transgenic mice (which in theory should not express other Vbeta, owing to allelic exclusion at the TCR-beta locus), endogenous TCR-Vbeta8.2, Vbeta7, and Vbeta2 (but not other Vbetas) are frequently expressed on liver NK1+T cells but absent on lymph node T cells. Finally, when endogenous V beta expression is prevented in TCR-Vbeta3 and Vbeta8.1 transgenic mice (by introduction of a null allele at the C beta locus), the development of liver NK1+T cells is totally abrogated. Collectively, our data indicate that liver NK1+T cells have a stringent requirement for expression of TCR-Vbeta8.2, Vbeta7, or Vbeta2 for their development.

Selectively impaired development of intestinal T cell receptor gamma delta+ cells and liver CD4+ NK1+ T cell receptor alpha beta+ cells in T cell factor-1-deficient mice.

T cell factor-1 (Tcf-1) is a transcription factor that binds to a sequence motif present in several T cell-specific enhancer elements. In Tcf-1-deficient (Tcf-1-/-) mice, thymocyte development is partially blocked at the transition from the CD4-8+ immature single-positive stage to the CD4+8+ double-positive stage, resulting in a marked decrease of mature peripheral T cells in lymph node and spleen. We report here that the development of most intestinal TCR gamma delta+ cells and liver CD4+ NK1.1+TCR alpha beta+ (NK1+T) cells, which are believed to be of extrathymic origin, is selectively impaired in Tcf-1-/- mice. In contrast, thymic and thymus-derived (splenic) TCR gamma delta+ cells are present in normal numbers in Tcf-1-/- mice, as are other T cell subsets in intestine and liver. Collectively, our data suggest that Tcf-1 is differentially required for the development of some extrathymic T cell subsets, including intestinal TCR gamma delta+ cells and liver CD4+ NK1+T cells.

Lack of association between the protein tyrosine phosphatase non-receptor type 22 R263Q and R620W functional genetic variants and endogenous non-anterior uveitis.

OBJECTIVE Endogenous uveitis is a major cause of visual loss mediated by the immune system. The protein tyrosine phosphatase non-receptor type 22 (PTPN22) gene encodes a lymphoid-specific phosphatase that plays a key role in T-cell receptor (TCR) signaling. Two independent functional missense single nucleotide polymorphisms (SNPs) located within the PTPN22 gene (R263Q and R620W) have been associated with different autoimmune disorders. We aimed to analyze for the first time the influence of these PTPN22 genetic variants on endogenous non-anterior uveitis susceptibility. METHODS We performed a case-control study of 217 patients with endogenous non-anterior uveitis and 718 healthy controls from a Spanish population. The PTPN22 polymorphisms (rs33996649 and rs2476601) were genotyped using TaqMan allelic discrimination assays. The allele, genotype, carriers, and allelic combination frequencies were compared between cases and controls with χ(2) analysis or Fisher's exact test. RESULTS Our results showed no influence of the studied SNPs in the global susceptibility analysis (rs33996649: allelic P- value=0.92, odds ratio=0.97, 95% confidence interval=0.54-1.75; rs2476601: allelic P- value=0.86, odds ratio=1.04, 95% confidence interval=0.68-1.59). Similarly, the allelic combination analysis did not provide additional information. CONCLUSIONS Our results suggest that the studied polymorphisms of the PTPN22 gene do not play an important role in the pathophysiology of endogenous non-anterior uveitis.

Combined simulation and mutagenesis analyses reveal the involvement of key residues for peroxisome proliferator-activated receptor alpha helix 12 dynamic behavior.

The dynamic properties of helix 12 in the ligand binding domain of nuclear receptors are a major determinant of AF-2 domain activity. We investigated the molecular and structural basis of helix 12 mobility, as well as the involvement of individual residues with regard to peroxisome proliferator-activated receptor alpha (PPARalpha) constitutive and ligand-dependent transcriptional activity. Functional assays of the activity of PPARalpha helix 12 mutants were combined with free energy molecular dynamics simulations. The agreement between the results from these approaches allows us to make robust claims concerning the mechanisms that govern helix 12 functions. Our data support a model in which PPARalpha helix 12 transiently adopts a relatively stable active conformation even in the absence of a ligand. This conformation provides the interface for the recruitment of a coactivator and results in constitutive activity. The receptor agonists stabilize this conformation and increase PPARalpha transcription activation potential. Finally, we disclose important functions of residues in PPARalpha AF-2, which determine the positioning of helix 12 in the active conformation in the absence of a ligand. Substitution of these residues suppresses PPARalpha constitutive activity, without changing PPARalpha ligand-dependent activation potential.

Imaging of human leukemic T-cell xenografts in nude mice by radiolabeled monoclonal antibodies and F(ab')2 fragments.

Monoclonal antibodies (MoAb) that react with the T-lymphocyte markers called cluster of differentiation CD5 and CD2 were labeled with iodine 131 (131I) and were injected intravenously in nude mice bearing solid subcutaneous xenografts derived from the human T-cell leukemia line Ichikawa. Both MoAb anti-CD5 and anti-CD2 yielded favorable mean tumor to whole-body ratios of 3.8 and 5.1, respectively. These ratios were further increased up to 10.0 for MoAb anti-CD5 and 15.5 for MoAb anti-CD2 by using their F(ab')2 fragments. The tumors could be imaged clearly by external scanning after injection of F(ab')2 fragments from both MoAb. F(ab')2 fragments from MoAb anti-CD2 and of a third MoAb recognizing the clonotypic determinant (Ti) of the antigen receptor expressed by the human T-cell line Jurkat were injected in mice bearing intrasplenic Jurkat xenografts. A selective localization of both fragments in tumor tissue was demonstrated with mean tumor to whole-body ratios of 7.5 and 4.1 for MoAb anti-CD2 and anti-Ti, respectively. These in vivo experimental results may provide useful information for the potential use of radiolabeled MoAb and fragments in the diagnosis and treatment of patients with T-cell lymphoma and different other forms of T-cell malignancies.

Pore-forming pyocin S5 utilizes the FptA ferripyochelin receptor to kill Pseudomonas aeruginosa.

Pyocins are toxic proteins produced by some strains of Pseudomonas aeruginosa that are lethal for related strains of the same species. Some soluble pyocins (S2, S3 and S4) were previously shown to use the pyoverdine siderophore receptors to enter the cell. The P. aeruginosa PAO1 pore-forming pyocin S5 encoding gene (PAO985) was cloned into the expression vector pET15b, and the affinity-purified protein product tested for its killing activity against different P. aeruginosa strains. The results, however, did not show any correlation with a specific ferripyoverdine receptor. To further identify the S5 receptor, transposon mutants were generated. Pooled mutants were exposed to pyocin S5 and the resistant colonies growing in the killing zone were selected. The majority of S5-resistant mutants had an insertion in the fptA gene encoding the receptor for the siderophore pyochelin. Complementation of an fptA transposon mutant with the P. aeruginosa fptA gene in trans restored the sensitivity to S5. In order to define the receptor-binding domain of pyocin S5, two hybrid pyocins were constructed containing different regions from pyocin S5 fused to the C-terminal translocation and DNase killing domains of pyocin S2. Only the protein containing amino acid residues 151 to 300 from S5 showed toxicity, indicating that the pyocin S5 receptor-binding domain is not at the N-terminus of the protein as in other S-type pyocins. Pyocin S5 was, however, unable to kill Burkholderia cenocepacia strains producing a ferripyochelin FptA receptor, nor was the B. cenocepacia fptA gene able to restore the sensitivity of the resistant fptA mutant P. aeruginosa strain.

In vivo therapeutic synergism of anti-epidermal growth factor receptor and anti-HER2 monoclonal antibodies against pancreatic carcinomas.

PURPOSE: Pancreatic carcinoma is highly resistant to therapy. Epidermal growth factor receptor (EGFR) and HER2 have been reported to be both dysregulated in this cancer. To evaluate the in vivo effect of binding both EGFR and HER2 with two therapeutic humanized monoclonal antibodies (mAb), we treated human pancreatic carcinoma xenografts, expressing high EGFR and low HER2 levels. EXPERIMENTAL DESIGN: Nude mice, bearing xenografts of BxPC-3 or MiaPaCa-2 human pancreatic carcinoma cell lines, were injected twice weekly for 4 weeks with different doses of anti-EGFR (matuzumab) and anti-HER2 (trastuzumab) mAbs either alone or in combination. The effect of the two mAbs, on HER receptor phosphorylation, was also studied in vitro by Western blot analysis. RESULTS: The combined mAb treatment significantly inhibited tumor progression of the BxPC-3 xenografts compared with single mAb injection (P = 0.006) or no treatment (P = 0.0004) and specifically induced some complete remissions. The two mAbs had more antitumor effect than 4-fold greater doses of each mAb. The significant synergistic effect of the two mAbs was confirmed on the MiaPaCa-2 xenograft and on another type of carcinoma, SK-OV-3 ovarian carcinoma xenografts. In vitro, the cooperative effect of the two mAbs was associated with a decrease in EGFR and HER2 receptor phosphorylation. CONCLUSIONS: Anti-HER2 mAb has a synergistic therapeutic effect when combined with an anti-EGFR mAb on pancreatic carcinomas with low HER2 expression. These observations may open the way to the use of these two mAbs in a large panel of carcinomas expressing different levels of the two HER receptors.

Relationship between dopamine D2 receptor occupancy, clinical response, and drug and monoamine metabolites levels in plasma and cerebrospinal fluid. A pilot study in patients suffering from first-episode schizophrenia treated with quetiapine.

Combining measurements of the monoamine metabolites in the cerebrospinal fluid (CSF) and neuroimaging can increase efficiency of drug discovery for treatment of brain disorders. To address this question, we examined five drug-naïve patients suffering from schizophrenic disorder. Patients were assessed clinically, using the Positive and Negative Syndrome Scale (PANSS): at baseline and then at weekly intervals. Plasma and CSF levels of quetiapine and norquetiapine as well CSF 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), 5-hydroxyindole-acetic acid (5-HIAA) and 3-methoxy-4-hydroxyphenylglycol (MHPG) were obtained at baseline and again after at least a 4 week medication trail with 600 mg/day quetiapine. CSF monoamine metabolites levels were compared with dopamine D(2) receptor occupancy (DA-D(2)) using [(18)F]fallypride and positron emission tomography (PET). Quetiapine produced preferential occupancy of parietal cortex vs. putamenal DA-D(2), 41.4% (p<0.05, corrected for multiple comparisons). DA-D(2) receptor occupancies in the occipital and parietal cortex were correlated with CSF quetiapine and norquetiapine levels (p<0.01 and p<0.05, respectively). CSF monoamine metabolites were significantly increased after treatment and correlated with regional receptor occupancies in the putamen [DOPAC: (p<0.01) and HVA: (p<0.05)], caudate nucleus [HVA: (p<0.01)], thalamus [MHPG: (p<0.05)] and in the temporal cortex [HVA: (p<0.05) and 5-HIAA: (p<0.05)]. This suggests that CSF monoamine metabolites levels reflect the effects of quetiapine treatment on neurotransmitters in vivo and indicates that monitoring plasma and CSF quetiapine and norquetiapine levels may be of clinical relevance.

A constitutively active mutant of the alpha 1B-adrenergic receptor can cause greater agonist-dependent down-regulation of the G-proteins G9 alpha and G11 alpha than the wild-type receptor.

Rat 1 fibroblasts transfected to express either the wild-type hamster alpha 1B-adrenergic receptor or a constitutively active mutant (CAM) form of this receptor resulting from the alteration of amino acid residues 288-294 to encode the equivalent region of the human beta 2-adrenergic receptor were examined. The basal level of inositol phosphate generation in cells expressing the CAM alpha 1B-adrenergic receptor was greater than for the wild-type receptor, The addition of maximally effective concentrations of phenylephrine or noradrenaline resulted in substantially greater levels of inositol phosphate generation by the CAM alpha 1B-adrenergic receptor, although this receptor was expressed at lower steady-state levels than the wild-type receptor. The potency of both phenylephrine and noradrenaline to stimulate inositol phosphate production was approx. 200-fold greater at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. In contrast, endothelin 1, acting at the endogenously expressed endothelin ETA, receptor, displayed similar potency and maximal effects in the two cell lines. The sustained presence of phenylephrine resulted in down-regulation of the alpha subunits of the phosphoinositidase C-linked, pertussis toxin-insensitive, G-proteins G9 and G11 in cells expressing either the wild-type or the CAM alpha 1B-adrenergic receptor. The degree of down-regulation achieved was substantially greater in cells expressing the CAM alpha 1B-adrenergic receptor at all concentrations of the agonist. However, in this assay phenylephrine displayed only a slightly greater potency at the CAM alpha 1B-adrenergic receptor than at the wild-type receptor. There were no detectable differences in the basal rate of G9 alpha/G11 alpha degradation between cells expressing the wild-type or the CAMalpha 1B-adrenergic receptor. In both cell lines the addition of phenylephrine substantially increased the rate of degradation of these G-proteins, with a greater effect at the CAM alpha 1B-adrenergic receptor. The enhanced capacity of agonist both to stimulate second-messenger production at the CAM alpha 1B-adrenergic receptor and to regulate cellular levels of its associated G-proteins by stimulating their rate of degradation is indicative of an enhanced stoichiometry of coupling of this form of the receptor to G9 and G11.

Fab antibodies capable of blocking T cells by competitive binding have the identical specificity but a higher affinity to the MHC-peptide-complex than the T cell receptor.

In transplant rejection, graft versus host or autoimmune diseases T cells are mediating the pathophysiological processes. Compared to unspecific pharmacological immune suppression specific inhibition of those T cells, that are involved in the disease, would be an alternative and attractive approach. T cells are activated after their T cell receptor (TCR) recognizes an antigenic peptide displayed by the Major Histocompatibility Complex (MHC). Molecules that interact with MHC-peptide-complexes in a specific fashion should block T cells with identical specificity. Using the model of the SSX2 (103-111)/HLA-A*0201 complex we investigated a panel of MHC-peptide-specific Fab antibodies for their capacity blocking specific T cell clones. Like TCRs all Fab antibodies reacted with the MHC complex only when the SSX2 (103-111) peptide was displayed. By introducing single amino acid mutations in the HLA-A*0201 heavy chain we identified the K66 residue as the most critical binding similar to that of TCRs. However, some Fab antibodies did not inhibit the reactivity of a specific T cell clone against peptide pulsed, artificial targets, nor cells displaying the peptide after endogenous processing. Measurements of binding kinetics revealed that only those Fab antibodies were capable of blocking T cells that interacted with an affinity in the nanomolar range. Fab antibodies binding like TCRs with affinities on the lower micromolar range did not inhibit T cell reactivity. These results indicate that molecules that block T cells by competitive binding with the TCR must have the same specificity but higher affinity for the MHC-peptide-complex than the TCR.

Inverse agonism and neutral antagonism at alpha(1a)- and alpha(1b)-adrenergic receptor subtypes.

We have characterized the pharmacological antagonism, i.e., neutral antagonism or inverse agonism, displayed by a number of alpha-blockers at two alpha1-adrenergic receptor (AR) subtypes, alpha(1a)- and alpha(1b)-AR. Constitutively activating mutations were introduced into the alpha(1a)-AR at the position homologous to A293 of the alpha(1b)-AR where activating mutations were previously described. Twenty-four alpha-blockers differing in their chemical structures were initially tested for their effect on the agonist-independent inositol phosphate response mediated by the constitutively active A271E and A293E mutants expressed in COS-7 cells. A selected number of drugs also were tested for their effect on the small, but measurable spontaneous activity of the wild-type alpha(1a)- and alpha(1b)-AR expressed in COS-7 cells. The results of our study demonstrate that a large number of structurally different alpha-blockers display profound negative efficacy at both the alpha(1a)- and alpha(1b)-AR subtypes. For other drugs, the negative efficacy varied at the different constitutively active mutants. The most striking difference concerns a group of N-arylpiperazines, including 8-[2-[4-(5-chloro-2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro [4, 5] decane-7,9-dione (REC 15/3039), REC 15/2739, and REC 15/3011, which are inverse agonists with profound negative efficacy at the wild-type alpha(1b)-AR, but not at the alpha(1a)-AR.