Modulation of the Endocannabinoids N-Arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) on Executive Functions in Humans.

Animal studies point to an implication of the endocannabinoid system on executive functions. In humans, several studies have suggested an association between acute or chronic use of exogenous cannabinoids (Δ9-tetrahydrocannabinol) and executive impairments. However, to date, no published reports establish the relationship between endocannabinoids, as biomarkers of the cannabinoid neurotransmission system, and executive functioning in humans. The aim of the present study was to explore the association between circulating levels of plasma endocannabinoids N-arachidonoylethanolamine (AEA) and 2-Arachidonoylglycerol (2-AG) and executive functions (decision making, response inhibition and cognitive flexibility) in healthy subjects. One hundred and fifty seven subjects were included and assessed with the Wisconsin Card Sorting Test; Stroop Color and Word Test; and Iowa Gambling Task. All participants were female, aged between 18 and 60 years and spoke Spanish as their first language. Results showed a negative correlation between 2-AG and cognitive flexibility performance (r = -.37; p<.05). A positive correlation was found between AEA concentrations and both cognitive flexibility (r = .59; p<.05) and decision making performance (r = .23; P<.05). There was no significant correlation between either 2-AG (r = -.17) or AEA (r = -.08) concentrations and inhibition response. These results show, in humans, a relevant modulation of the endocannabinoid system on prefrontal-dependent cognitive functioning. The present study might have significant implications for the underlying executive alterations described in some psychiatric disorders currently associated with endocannabinoids deregulation (namely drug abuse/dependence, depression, obesity and eating disorders). Understanding the neurobiology of their dysexecutive profile might certainly contribute to the development of new treatments and pharmacological approaches.

Human genetic selection on the MTHFR 677C>T polymorphism.

BACKGROUND The prevalence of genotypes of the 677C>T polymorphism for the MTHFR gene varies among humans. In previous studies, we found changes in the genotypic frequencies of this polymorphism in populations of different ages, suggesting that this could be caused by an increase in the intake of folate and multivitamins by women during the periconceptional period. The aim was to analyze changes in the allelic frequencies of this polymorphism in a Spanish population, including samples from spontaneous abortions (SA). METHODS A total of 1305 subjects born in the 20th century were genotyped for the 677C>T polymorphism using allele specific real-time PCR with Taqman probes. A section of our population (n = 276) born in 1980-1989 was compared with fetal samples (n = 344) from SA of unknown etiology from the same period. RESULTS An increase in the frequency of the T allele (0.38 vs 0.47; p < 0.001) and of the TT genotype (0.14 vs 0.24; p < 0.001) in subjects born in the last quarter of the century was observed. In the 1980-1989 period, the results show that the frequency of the wild type genotype (CC) is about tenfold lower in the SA samples than in the controls (0.03 vs 0.33; p < 0.001) and that the frequency of the TT genotype increases in the controls (0.19 to 0.27) and in the SA samples (0.20 to 0.33 (p < 0.01)); r = 0.98. CONCLUSION Selection in favor of the T allele has been detected. This selection could be due to the increased fetal viability in early stages of embryonic development, as is deduced by the increase of mutants in both living and SA populations.

R-ESHAP as salvage therapy for patients with relapsed or refractory diffuse large B-cell lymphoma: the influence of prior exposure to rituximab on outcome. A GEL/TAMO study.

BACKGROUND The role of re-treatment with rituximab in aggressive B-cell lymphomas still needs to be defined. This study evaluated the influence of prior exposure to rituximab on response rates and survival in patients with diffuse large B-cell lymphoma treated with rituximab plus etoposide, cytarabine, cisplatinum and methylprednisolone (R-ESHAP). DESIGN AND METHODS We retrospectively analyzed 163 patients with relapsed or refractory diffuse large B-cell lymphoma who received R-ESHAP as salvage therapy with a curative purpose. Patients were divided into two groups according to whether rituximab had been administered (n=94, "R+" group) or not (n=69, "R-" group) prior to R-ESHAP. RESULTS Response rates were significantly higher in the R- group in the univariate but not in the multivariate analysis. In the analysis restricted to the R+ group, we observed very low complete remission and overall response rates in patients with primary refractory disease (8% and 33%, respectively), as compared to those in patients who were in first partial remission (41% and 86%) or who had relapsed disease (50% and 75%) (p<0.01 in both cases). Overall, 60% and 65% of patients in the R+ and R- groups, respectively, underwent stem-cell transplantation after the salvage therapy. With a median follow-up of 29 months (range, 6-84), patients in the R+ group had significantly worse progression-free survival (17% vs. 57% at 3 years, p<0.0001) and overall survival (38% v 67% at 3 years, p=0.0005) than patients in the R- group. Prior exposure to rituximab was also an independent adverse prognostic factor for both progression-free survival (RR: 2.0; 95% CI: 1.2-3.3, p=0.008) and overall survival (RR: 2.2; 95% CI: 1.3-3.9, p=0.004). CONCLUSIONS R-ESHAP was associated with a high response rate in patients who were not refractory to upfront rituximab-based chemotherapy. However, the survival outcome was poor for patients previously exposed to rituximab, as compared to in those who had not previously been treated with rituximab.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Expression of the transcription factor NFAT2 in human neutrophils: IgE-dependent, Ca2+- and calcineurin-mediated NFAT2 activation.

NFAT (nuclear factors of activated T cells) proteins constitute a family of transcription factors involved in mediating signal transduction. The presence of NFAT isoforms has been described in all cell types of the immune system, with the exception of neutrophils. In the present work we report for the first time the expression in human neutrophils of NFAT2 mRNA and protein. We also report that specific antigens were able to promote NFAT2 protein translocation to the nucleus, an effect that was mimicked by the treatment of neutrophils with anti-immunoglobulin E (anti-IgE) or anti-Fcepsilon-receptor antibodies. Antigens, anti-IgE and anti-FcepsilonRs also increased Ca2+ release and the intracellular activity of calcineurin, which was able to interact physically with NFAT2, in parallel to eliciting an enhanced NFAT2 DNA-binding activity. In addition, specific chemical inhibitors of the NFAT pathway, such as cyclosporin A and VIVIT peptide, abolished antigen and anti-IgE-induced cyclooxygenase-2 (COX2) gene upregulation and prostaglandin (PGE(2)) release, suggesting that this process is through NFAT. Our results provide evidence that NFAT2 is constitutively expressed in human neutrophils, and after IgE-dependent activation operates as a transcription factor in the modulation of genes, such as COX2, during allergic inflammation.

Prospective phase II trial of extended treatment with rituximab in patients with B-cell post-transplant lymphoproliferative disease.

BACKGROUND AND OBJECTIVES The elective treatment of patients with post-transplant lymphoproliferative disorders is controversial. The purpose of this trial was to evaluate the efficacy of treatment with extended doses of rituximab adapted to the response in patients with post-transplant lymphoproliferative disorders after solid organ transplantation. DESIGN AND METHODS This was a prospective, multicenter, phase II trial. Patients were treated with reduction of immunosuppression and four weekly infusions of rituximab. Those patients who did not achieve complete remission (CR) received a second course of four rituximab infusions. The primary end-point of the study was the CR rate. RESULTS Thirty-eight patients were assesable. One episode of grade 4 neutropenia was the only severe adverse event observed. After the first course of rituximab, 13 (34.2%) patients achieved CR, 8 patients did not respond, and 17 patients achieved partial remission. Among those 17 patients, 12 could be treated with a second course of rituximab, and 10 (83.3%) achieved CR, yielding an intention-to-treat CR rate of 60.5%. Eight patients excluded from the trial because of absence of CR were treated with rituximab combined with chemotherapy, and six (75%) achieved CR. Event-free survival was 42% and overall survival was 47% at 27.5 months. Fourteen patients died, ten of progression of their post-transplant lymphoproliferative disorder. INTERPRETATION AND CONCLUSIONS These results confirm that extended treatment with rituximab can obtain a high rate of CR in patients with post-transplant lymphoproliferative disorders after solid organ transplantation without increasing toxicity, and should be recommended as initial therapy for these patients.

Mutational spectrum of the SPG4 (SPAST) and SPG3A (ATL1) genes in Spanish patients with hereditary spastic paraplegia.

BACKGROUND Hereditary Spastic Paraplegias (HSP) are characterized by progressive spasticity and weakness of the lower limbs. At least 45 loci have been identified in families with autosomal dominant (AD), autosomal recessive (AR), or X-linked hereditary patterns. Mutations in the SPAST (SPG4) and ATL1 (SPG3A) genes would account for about 50% of the ADHSP cases. METHODS We defined the SPAST and ATL1 mutational spectrum in a total of 370 unrelated HSP index cases from Spain (83% with a pure phenotype). RESULTS We found 50 SPAST mutations (including two large deletions) in 54 patients and 7 ATL1 mutations in 11 patients. A total of 33 of the SPAST and 3 of the ATL1 were new mutations. A total of 141 (31%) were familial cases, and we found a higher frequency of mutation carriers among these compared to apparently sporadic cases (38% vs. 5%). Five of the SPAST mutations were predicted to affect the pre-mRNA splicing, and in 4 of them we demonstrated this effect at the cDNA level. In addition to large deletions, splicing, frameshifting, and missense mutations, we also found a nucleotide change in the stop codon that would result in a larger ORF. CONCLUSIONS In a large cohort of Spanish patients with spastic paraplegia, SPAST and ATL1 mutations were found in 15% of the cases. These mutations were more frequent in familial cases (compared to sporadic), and were associated with heterogeneous clinical manifestations.

Novel (60%) and recurrent (40%) androgen receptor gene mutations in a series of 59 patients with a 46,XY disorder of sex development.

BACKGROUND Androgen receptor (AR) gene mutations are the most frequent cause of 46,XY disorders of sex development (DSD) and are associated with a variety of phenotypes, ranging from phenotypic women [complete androgen insensitivity syndrome (CAIS)] to milder degrees of undervirilization (partial form or PAIS) or men with only infertility (mild form or MAIS). OBJECTIVE The aim of the study was to characterize the contribution of the AR gene to the molecular cause of 46,XY DSD in a series of Spanish patients. SETTING We studied a series of 133 index patients with 46,XY DSD in whom gonads were differentiated as testes, with phenotypes including varying degrees of undervirilization, and in whom the AR gene was the first candidate for a molecular analysis. METHODS The AR gene was sequenced (exons 1 to 8 with intronic flanking regions) in all patients and in family members of 61% of AR-mutated gene patients. RESULTS AR gene mutations were found in 59 individuals (44.4% of index patients), of whom 46 (78%) were CAIS and 13 (22%) PAIS. Fifty-seven different mutations were found: 21.0% located in exon 1, 15.8% in exons 2 and 3, 57.9% in exons 4-8, and 5.3% intronic. Twenty-three mutations (40.4%) had been previously described and 34 (59.6%) were novel. CONCLUSIONS AR gene mutation is the most frequent cause of 46,XY DSD, with a clearly higher frequency in the complete phenotype. Mutations spread along the whole coding sequence, including exon 1. This series shows that 60% of mutations detected during the period 2002-2009 were novel.

The diamine oxidase gene is associated with hypersensitivity response to non-steroidal anti-inflammatory drugs.

Non-steroidal anti-inflammatory drugs (NSAIDs) are the drugs most frequently involved in hypersensitivity drug reactions. Histamine is released in the allergic response to NSAIDs and is responsible for some of the clinical symptoms. The aim of this study is to analyze clinical association of functional polymorphisms in the genes coding for enzymes involved in histamine homeostasis with hypersensitivity response to NSAIDs. We studied a cohort of 442 unrelated Caucasian patients with hypersensitivity to NSAIDs. Patients who experienced three or more episodes with two or more different NSAIDs were included. If this requirement was not met diagnosis was established by challenge. A total of 414 healthy unrelated controls ethnically matched with patients and from the same geographic area were recruited. Analyses of the SNPs rs17740607, rs2073440, rs1801105, rs2052129, rs10156191, rs1049742 and rs1049793 in the HDC, HNMT and DAO genes were carried out by means of TaqMan assays. The detrimental DAO 16 Met allele (rs10156191), which causes decreased metabolic capacity, is overrepresented among patients with crossed-hypersensitivity to NSAIDs with an OR  = 1.7 (95% CI  = 1.3-2.1; Pc  = 0.0003) with a gene-dose effect (P = 0.0001). The association was replicated in two populations from different geographic areas (Pc  = 0.008 and Pc  = 0.004, respectively). CONCLUSIONS AND IMPLICATIONS: The DAO polymorphism rs10156191 which causes impaired metabolism of circulating histamine is associated with the clinical response in crossed-hypersensitivity to NSAIDs and could be used as a biomarker of response.

Gender-Specific Effects of Genetic Variants within Th1 and Th17 Cell-Mediated Immune Response Genes on the Risk of Developing Rheumatoid Arthritis.

The present study was conducted to explore whether single nucleotide polymorphisms (SNPs) in Th1 and Th17 cell-mediated immune response genes differentially influence the risk of rheumatoid arthritis (RA) in women and men. In phase one, 27 functional/tagging polymorphisms in C-type lectins and MCP-1/CCR2 axis were genotyped in 458 RA patients and 512 controls. Carriers of Dectin-2 rs4264222T allele had an increased risk of RA (OR = 1.47, 95%CI 1.10-1.96) whereas patients harboring the DC-SIGN rs4804803G, MCP-1 rs1024611G, MCP-1 rs13900T and MCP-1 rs4586C alleles had a decreased risk of developing the disease (OR = 0.66, 95%CI 0.49-0.88; OR = 0.66, 95%CI 0.50-0.89; OR = 0.73, 95%CI 0.55-0.97 and OR = 0.68, 95%CI 0.51-0.91). Interestingly, significant gender-specific differences were observed for Dectin-2 rs4264222 and Dectin-2 rs7134303: women carrying the Dectin-2 rs4264222T and Dectin-2 rs7134303G alleles had an increased risk of RA (OR = 1.93, 95%CI 1.34-2.79 and OR = 1.90, 95%CI 1.29-2.80). Also five other SNPs showed significant associations only with one gender: women carrying the MCP-1 rs1024611G, MCP-1 rs13900T and MCP-1 rs4586C alleles had a decreased risk of RA (OR = 0.61, 95%CI 0.43-0.87; OR = 0.67, 95%CI 0.47-0.95 and OR = 0.60, 95%CI 0.42-0.86). In men, carriers of the DC-SIGN rs2287886A allele had an increased risk of RA (OR = 1.70, 95%CI 1.03-2.78), whereas carriers of the DC-SIGN rs4804803G had a decreased risk of developing the disease (OR = 0.53, 95%CI 0.32-0.89). In phase 2, we genotyped these SNPs in 754 RA patients and 519 controls, leading to consistent gender-specific associations for Dectin-2 rs4264222, MCP-1 rs1024611, MCP-1 rs13900 and DC-SIGN rs4804803 polymorphisms in the pooled sample (OR = 1.38, 95%CI 1.08-1.77; OR = 0.74, 95%CI 0.58-0.94; OR = 0.76, 95%CI 0.59-0.97 and OR = 0.56, 95%CI 0.34-0.93). SNP-SNP interaction analysis of significant SNPs also showed a significant two-locus interaction model in women that was not seen in men. This model consisted of Dectin-2 rs4264222 and Dectin-2 rs7134303 SNPs and suggested a synergistic effect between the variants. These findings suggest that Dectin-2, MCP-1 and DC-SIGN polymorphisms may, at least in part, account for gender-associated differences in susceptibility to RA.

Anaphylaxis caused by imported red fire ant stings in Málaga, Spain.

A 27-year-old woman suffered from anaphylaxis after being stung by Solenopsis invicta ants while she was handling wood from South America. The patient reported no previous adverse reactions to stings by other hymenopteran species. Intradermal skin tests with hymenoptera venom (Vespula vulgaris, Polistes species, Apis melifera) were negative. Serum specific immunoglobulin (Ig) E yielded positive results for S invicta (5.28 kU/L) and negative results for A melifera, Ves v 5 and Pol a 5. Immunodetection assays showed the presence of serum IgE against the Sol i 2 allergen. The patient had probably been stung previously although inadvertently by red fire ants while she handled infested wood from South America, and precautionary measures are thus advisable when this material is to be handled. To our knowledge this is the first case of anaphylaxis from red fire ant stings reported in Europe.

Role of Kras Status in Patients with Metastatic Colorectal Cancer Receiving First-Line Chemotherapy plus Bevacizumab: A TTD Group Cooperative Study

BACKGROUND In the MACRO study, patients with metastatic colorectal cancer (mCRC) were randomised to first-line treatment with 6 cycles of capecitabine and oxaliplatin (XELOX) plus bevacizumab followed by either single-agent bevacizumab or XELOX plus bevacizumab until disease progression. An additional retrospective analysis was performed to define the prognostic value of tumour KRAS status on progression-free survival (PFS), overall survival (OS) and response rates. METHODOLOGY/PRINCIPAL FINDINGS KRAS data (tumour KRAS status and type of mutation) were collected by questionnaire from participating centres that performed KRAS analyses. These data were then cross-referenced with efficacy data for relevant patients in the MACRO study database. KRAS status was analysed in 394 of the 480 patients (82.1%) in the MACRO study. Wild-type (WT) KRAS tumours were found in 219 patients (56%) and mutant (MT) KRAS in 175 patients (44%). Median PFS was 10.9 months for patients with WT KRAS and 9.4 months for patients with MT KRAS tumours (p=0.0038; HR: 1.40; 95% CI:1.12-1.77). The difference in OS was also significant: 26.7 months versus 18.0 months for WT versus MT KRAS, respectively (p=0.0002; HR: 1.55; 95% CI: 1.23-1.96). Univariate and multivariate analyses showed that KRAS was an independent variable for both PFS and OS. Responses were observed in 126 patients (57.5%) with WT KRAS tumours and 76 patients (43.4%) with MT KRAS tumours (p=0.0054; OR: 1.77; 95% CI: 1.18-2.64). CONCLUSIONS/SIGNIFICANCE This analysis of the MACRO study suggests a prognostic role for tumour KRAS status in patients with mCRC treated with XELOX plus bevacizumab. For both PFS and OS, KRAS status was an independent factor in univariate and multivariate analyses.