Identification of HAVCR1 gene haplotypes associated with mRNA expression levels and susceptibility to autoimmune diseases

Human HAVCR1 gene maps on 5q33.2, a region linked with susceptibility to allergic and autoimmune diseases. The aims of the present study were to define the haplotypes of HAVCR1 gene taking into account both HapMap Project SNP haplotypes and exon 4 variants, to investigate a possible relationship between these haplotypes and mRNA expression levels, and to assess whether HAVCR1 gene is involved in susceptibility to rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Genotyping of three ins/del variants in the exon 4 was performed by fragment length analysis. Five tag SNPs genotypes and mRNA levels were determined using TaqMan assays. We defined four major haplotypes in our population: the two major haplotypes (named haplotypes A and B) bear both the 5383_5397del variant and the two most common SNP sets found in the CEU population. Quantification analysis revealed that genotype B/B had the highest median of mRNA expression levels (vs. BX + XX, p < 0.0001). Additionally, frequency of the genotype BB was significantly higher in RA patients than in controls (12.3 vs. 5.9% in controls, p = 0.0046, p _c = 0.014, OR = 2.23, 95% CI 1.23–4.10). Our results support a relationship between HAVCR1 haplotypes and mRNA expression levels, and suggest an association of this gene with autoimmune diseases.     

1,4-Diazepane-2-ones as novel inhibitors of LFA-1

The design, synthesis, and biological evaluation of 1,4-diazepane-2-ones as novel LFA-1 antagonists from a scaffold-based combinatorial library are described. Initial optimization of the library lead has resulted in high-affinity antagonists of the LFA-1/ICAM-1 interaction, such as compounds 18d and 18e with IC(50) values of 110 and 70 nM, respectively.     

BIRB796 inhibits all p38 MAPK isoforms in vitro and in vivo

The compound BIRB796 inhibits the stress-activated protein kinases p38alpha and p38beta and is undergoing clinical trials for the treatment of inflammatory diseases. Here we report that BIRB796 also inhibits the activity and the activation of SAPK3/p38gamma. This occurs at higher concentrations of BIRB796 than those that inhibit p38alpha and p38beta and at lower concentrations than those that inhibit the activation of JNK isoforms. We also show that at these concentrations, BIRB796 blocks the stress-induced phosphorylation of the scaffold protein SAP97, further establishing that this is a physiological substrate of SAPK3/p38gamma. Our results demonstrate that BIRB796, in combination with SB203580, a compound that inhibits p38alpha and p38beta, but not the other p38 isoforms, can be used to identify physiological substrates of SAPK3/p38gamma as well as those of p38alpha and p38beta.     

Localization and survival of Azospirillum brasilense Az39 in soybean leaves

In recent years, foliar inoculation has gained acceptance among the available methods to deliver plant beneficial micro-organisms to crops under field conditions. Colonization efficiency by such micro-organisms largely depends on their ability to survive when applied on the leaves. In this work, we evaluated the survival and localization of Azospirillum brasilense Az39 (Az39) in excised soybean leaves. Scanning electron microscopy and confocal laser scanning microscopy of a red fluorescent-transformed variant of Az39 were used to determine bacterial localization, while the most probable number and plate count methods were applied for bacterial quantification. Microscopic observations indicated a decrease in the number of Az39 cells on the leaf surface at 24 h after treatment, whereas midribs and cell–cell junctions of the inner leaf epidermis became highly populated zones. The presence of Az39 inside xylem vessels was corroborated at 6 h after bacterization. Az39 population did not significantly decrease throughout 24 h. We could visualize Az39 cells on the surface and in internal tissues of soybean leaves and recover them through culture methodologies. These results evidence the survival capacity of Az39 on and inside leaves and suggest a previously unnoticed endophytic potential for this well-known plant growth-promoting rhizobacteria strain.     

Translational control of NKT cell cytokine production by p38 MAPK

NKT cells are known to rapidly produce a large amount of cytokines upon activation. Although a number of signaling pathways that regulate the development of NKT cells have been identified, the signaling pathways involved in the regulation of NKT cell cytokine production remain unclear. In this study, we show that the p38 MAPK pathway is dispensable for the development of NKT cells. However, NKT cell cytokine production and NKT-mediated liver damage are highly dependent on activation of this pathway. p38 MAPK does not substantially affect cytokine gene expression in NKT cells, but it regulates the synthesis of cytokines through the Mnk-eIF4E pathway. Thus, in addition to gene expression, translational regulation by p38 MAPK could be a novel mechanism that contributes to the overall production of cytokine by NKT cells.     

Constitutive expression and enzymatic activity of Tan protein in brain and epidermis of Ceratitis capitata and of Drosophila melanogaster wild-type and tan mutants

The present report shows a partial biochemical characterization and life cycle expression of N-??-alanyldopamine hydrolase (Tan protein) in Ceratitis capitata and Drosophila melanogaster. This enzyme catalyzes the hydrolysis of N-??-alanyldopamine (NBAD), the main tanning precursor of insect brown cuticles. It also plays an important role in the metabolism of brain neurotransmitters, recycling dopamine and histamine. In contrast to NBAD-synthase, Tan is expressed constitutively in epidermis and does not respond directly to microbial challenge. Immunodetection experiments showed the novel localization of NBAD-hydrolase in the embryo central neural system and in different regions of the adult brain, in addition to optic lobes. We sequenced and characterized Drosophila mutants tan¹ and tan³. The latter appears to be a mutant with normal expression in neural tissue but weak one in epidermis.     

Specific calcineurin targeting in macrophages confers resistance to inflammation via MKP‐1 and p38

Macrophages contribute to tissue homeostasis and influence inflammatory responses by modulating their phenotype in response to the local environment. Understanding the molecular mechanisms governing this plasticity would open new avenues for the treatment for inflammatory disorders. We show that deletion of calcineurin (CN) or its inhibition with LxVP peptide in macrophages induces an anti‐inflammatory population that confers resistance to arthritis and contact hypersensitivity. Transfer of CN‐targeted macrophages or direct injection of LxVP‐encoding lentivirus has anti‐inflammatory effects in these models. Specific CN targeting in macrophages induces p38 MAPK activity by downregulating MKP‐1 expression. However, pharmacological CN inhibition with cyclosporin A (CsA) or FK506 did not reproduce these effects and failed to induce p38 activity. The CN‐inhibitory peptide VIVIT also failed to reproduce the effects of LxVP. p38 inhibition prevented the anti‐inflammatory phenotype of CN‐targeted macrophages, and mice with defective p38‐activation were resistant to the anti‐inflammatory effect of LxVP. Our results identify a key role for CN and p38 in the modulation of macrophage phenotype and suggest an alternative treatment for inflammation based on redirecting macrophages toward an anti‐inflammatory status.     

Expression of Babesia bovis rhoptry-associated protein 1 (RAP1) in Brucella abortus S19

Brucella abortus strain 19 (live vaccine) induces a strong humoral and cellular immune response and therefore, it is an attractive vector for the delivery of heterologous antigens. The objective of the present study was to express the rhoptry-associated protein (RAP1) of Babesia bovis in B. abortus S19, as a model for heterologous expression of immunostimulatory antigens from veterinary pathogens. A plasmid for the expression of recombinant proteins fused to the aminoterminal of the outer membrane lipoprotein OMP19 was created, pursuing the objective of increasing the immunogenicity of the recombinant antigen being expressed by its association to a lipid moiety. Recombinant strains of B. abortus S19 expressing RAP1 as a fusion protein either with the first amino acids of beta-galactosidase (S19pBB-RAP1) or B. abortus OMP19 (S19pBB19-RAP1) were generated. Plasmid stability and the immunogenicity of the heterologous proteins were analyzed. Mice immunized with S19pBB-RAP1 or S19pBB19-RAP1 developed specific humoral immune response to RAP1, IgG2a being the predominant antibody isotype. Furthermore, a specific cellular immune response to recombinant RAP1 was elicited in vitro by lymphocytes from mice immunized with both strains. Therefore, we concluded that B. abortus S19 expressing RAP1 is immunostimulatory and may provide the basis for combined heterologous vaccines for babesiosis and brucellosis.     

Role of Muscle c-Jun NH2-Terminal Kinase 1 in Obesity-Induced Insulin Resistance

Obesity caused by feeding of a high-fat diet (HFD) is associated with an increased activation of c-Jun NH₂-terminal kinase 1 (JNK1). Activated JNK1 is implicated in the mechanism of obesity-induced insulin resistance and the development of metabolic syndrome and type 2 diabetes. Significantly, Jnk1^−/− mice are protected against HFD-induced obesity and insulin resistance. Here we show that an ablation of the Jnk1 gene in skeletal muscle does not influence HFD-induced obesity. However, muscle-specific JNK1-deficient (M^KO) mice exhibit improved insulin sensitivity compared with control wild-type (M^WT) mice. Thus, insulin-stimulated AKT activation is suppressed in muscle, liver, and adipose tissue of HFD-fed M^WT mice but is suppressed only in the liver and adipose tissue of M^KO mice. These data demonstrate that JNK1 in muscle contributes to peripheral insulin resistance in response to diet-induced obesity.Obesity is a major risk factor for the development of insulin resistance, hyperglycemia, and metabolic syndrome that can lead to β-cell dysfunction and type 2 diabetes (8). The prevalence of human obesity represents a serious health problem in the United States. It is therefore important that we obtain a detailed understanding of the molecular mechanism that accounts for obesity-induced insulin resistance. Recent progress has led to the identification of signal transduction pathways that may mediate the effects of obesity on insulin resistance (14, 23).c-Jun NH₂-terminal kinase 1 (JNK1) represents one signaling pathway that has been implicated in the pathogenesis of metabolic syndrome and type 2 diabetes (21). JNK1 is activated when mice are fed a high-fat diet (HFD) (7). Moreover, Jnk1^−/− mice are protected against HFD-induced insulin resistance (7). The mechanism of protection is mediated, in part, by the failure of Jnk1^−/− mice to develop HFD-induced obesity (7). However, JNK1 can regulate insulin resistance independently of obesity. Thus, mice with an adipose tissue-specific JNK1 deficiency develop normal diet-induced obesity but exhibit selective protection against HFD-induced insulin resistance in both the liver and adipose tissue (16). These data indicate that adipose tissue JNK1 plays a critical role during the development of HFD-induced insulin resistance.The liver plays a key role in the insulin-stimulated disposal of blood glucose during the postprandial state because of reduced gluconeogenesis and increased glycogen synthesis (17). However, glucose uptake by skeletal muscle also makes a major contribution to insulin-stimulated glucose disposal (17). Muscle may therefore be an important target of obesity-induced JNK1 signaling and the regulation of glucose homeostasis.The purpose of this study was to test the role of JNK1 in muscle. Our approach was to examine the effect of a muscle-specific ablation of the Jnk1 gene in mice. We found that HFD-fed control wild-type (M^WT) mice and muscle-specific JNK1-deficient (M^KO) mice became similarly obese. However, M^KO mice were selectively protected against HFD-induced insulin resistance. This analysis demonstrates that muscle JNK1 contributes to the effects of obesity on insulin resistance.     

Evidence of New Risk Genetic Factor to Systemic Lupus Erythematosus: The UBASH3A Gene

The ubiquitin associated and Src-homology 3 (SH3) domain containing A (UBASH3a) is a suppressor of T-cell receptor signaling, underscoring antigen presentation to T-cells as a critical shared mechanism of diseases pathogenesis. The aim of the present study was to determine whether the UBASH3a gene influence the susceptibility to systemic lupus erythematosus (SLE) in Caucasian populations. We evaluated five UBASH3a polymorphisms (rs2277798, rs2277800, rs9976767, rs13048049 and rs17114930), using TaqMan® allelic discrimination assays, in a discovery cohort that included 906 SLE patients and 1165 healthy controls from Spain. The SNPs that exhibit statistical significance difference were evaluated in a German replication cohort of 360 SLE patients and 379 healthy controls. The case-control analysis in the Spanish population showed a significant association between the rs9976767 and SLE (Pc = 9.9E-03 OR = 1.21 95%CI = 1.07–1.37) and a trend of association for the rs2277798 analysis (P = 0.09 OR = 0.9 95%CI = 0.79–1.02). The replication in a German cohort and the meta-analysis confirmed that the rs9976767 (Pc = 0.02; Pc = 2.4E-04, for German cohort and meta-analysis, respectively) and rs2277798 (Pc = 0.013; Pc = 4.7E-03, for German cohort and meta-analysis, respectively) UBASH3a variants are susceptibility factors for SLE. Finally, a conditional regression analysis suggested that the most likely genetic variation responsible for the association was the rs9976767 polymorphism. Our results suggest that UBASH3a gene plays a role in the susceptibility to SLE. Moreover, our study indicates that UBASH3a can be considered as a common genetic factor in autoimmune diseases.