Investigation of the in vivo activity of CYP3A in Brazilian volunteers: comparison of midazolam and omeprazole as drug markers

Objective This study compares midazolam with omeprazole as marker drugs for the evaluation of CYP3A activity in nine healthy self-reported white Brazilian volunteers. Methods Omeprazole was also used to evaluate the CYP2C19 phenotype. The volunteers received p.o. 20 mg omeprazole, and blood samples were collected 3.5 h after drug administration. After a washout period of 10 days, the volunteers received p.o. 15 mg midazolam maleate, and serial blood samples were collected up to 6 h after administration of the drug. CYP2C19 was genotyped for the allelic variants CYP2C19*1, CYP2C19*2, CYP2C19*3, and CYP2C19*17. Analysis of omeprazole, hydroxyomeprazole, omeprazole sulfone, and midazolam in plasma was carried out by LC-MS/MS. Results The volunteers genotyped as CYP2C19*1*17, CYP2C19*17*17, CYP2C19*1*1 (n=8), or CYP2C19*17*2 (n=1) presented a median hydroxylation index (omeprazole/hydroxyomeprazole) of 1.35, indicating that all of them were extensive metabolizers of CYP2C19. The volunteers (n=9) presented a 0.12 log of the omeprazole/sulfone ratio and a median oral clearance of midazolam of 17.89 ml min(-1) kg(-1), suggesting normal CYP3A activity. Conclusions Orthogonal regression analysis between midazolam clearance and log of the plasma concentrations of the omeprazole/omeprazole sulfone ratio (R=-0.7544, P < 0.05) suggests that both midazolam and omeprazole can be used as markers of CYP3A activity in the population investigated.

Liver HLA-G expression is associated with multiple clinical and histopathological forms of chronic hepatitis B virus infection

As the mechanisms leading to the persistence of hepatitis B virus (HBV) infection are poorly understood and as the histocompatibility leucocyte antigen (HLA)-G is well described as a tolerogenic molecule, we evaluated HLA-G expression in 74 specimens of HBV liver biopsies and in 10 specimens obtained from previously healthy cadaver liver donors. HBV specimens were reviewed and classified by the METAVIR score, and HLA-G expression was assessed by immunohistochemistry. No HLA-G expression was observed in control hepatocytes. In contrast, 57 (77%) of 74 HBV specimens showed soluble and membrane-bound HLA-G expression in hepatocytes, biliary epithelial cells or both. No associations between the intensity of HLA-G expression and patient age or gender, HBeAg status, severity of liver fibrosis, and grade of histological findings were observed. Although significance was not reached (P = 0.180), patients exhibiting HLA-G expression presented a higher median HBV DNA viral load (105 copies/mL) than those who did not express HLA-G (103.7 copies/mL). These results indicate that HLA-G is expressed in most cases of chronic HBV infection in all stages and may play a role in the persistency of HBV infection.

Abnormal cell-cycle expression of the proteins p27, mdm2 and cathepsin B in oral squamous-cell carcinoma infected with human papillomavirus

Since the role of human papillomavirus (HPV) infection in oral carcinogenesis is still unclear, the purpose of this study was to verify the association between the expression of p27, mdm2 and cathepsin B and by HPV-related oral lesions. Fifty-five oral biopsies were studied and HPV detection and typing (6/11, 16, 18, 31 and 33) were performed using polymerase chain reaction techniques. The distribution p27, mdm2 and cathepsin B was determined by immunohistochemistry. Twenty-one (38%) out of the 55 oral lesions tested positive for HPV, of which 6(33%) were HPV 6/11, 1 (5%) was HPV 16,14 (72%) were HPV 18 and none was HPV 33/31. Among the 55 biopsies, immunopostivity for p27, mdm2 and cathepsin B was observed in 17 (30.9%), 37 (67.2%) and 37 (67.2%), respectively. Among 21 HPV-positive oral lesions, immunopostivity of mdm2, p27 and cathepsin B was found, respectively, in 6 (33%) out of 18 benign lesions (BL), 4(22%) out of 18 potential malignant epithelial lesions (PMEL) and 11(57.9%) out of 19 malignant lesions (ML). High-risk HPV types may be associated with oral carcinoma, by cell-cycle control dysregulation, contributing to oral carcinogenesis and the overexpression of mdm2, p27 and cathepsin B. (C) 2009 Elsevier GmbH. All rights reserved.

A novel homozygous splice acceptor site mutation of KISS1R in two siblings with normosmic isolated hypogonadotropic hypogonadism

Context: Loss-of-function mutations of the kisspeptin-1 receptor gene, KISS1R, have been identified in patients with normosmic isolated hypogonadotropic hypogonadism (nIHH). Objective: To investigate KISS1R defects in patients with absent or delayed puberty. Patients: We investigated KISS1R gene defects in a cohort of 99 Brazilian patients with nIHH or constitutional delay of puberty (CDP). Methods: The entire coding region of KISS1R was amplified by PCR followed by automatic sequencing. In addition, screening for KISS1R exonic deletions was performed by multiplex ligation-dependent probe amplification. Results: One novel homozygous KISS1R mutation was identified in two siblings with nIHH. This variant was an insertion/deletion (indel) mutation characterized by the deletion of three nucleotides (GCA) at position -2 to -4, and by the insertion of seven nucleotides (ACCGGCT) at the same position, within the 30 splice acceptor site of intron 2 of KISS1R. The brothers who carried this KISS1R mutation had no clinical evidence of pubertal development at the ages of 14 and 20 years. Computational analysis of this indel mutation predicted the generation of an abnormal protein. In addition, a new heterozygous KISS1R variant (p.E252Q) was identified in a male patient with sporadic nIHH. However, in vitro studies of this variant did not demonstrate functional impairment. Only known polymorphisms were identified in patients with CDP. Conclusion: Loss-of-function mutations of KISS1R represents a rare cause of nIHH, and was absent in patients with CDP. We have described a novel KISS1R homozygous splice acceptor site mutation in the familial form of nIHH.

TAC3/TACR3 Mutations Reveal Preferential Activation of Gonadotropin-Releasing Hormone Release by Neurokinin B in Neonatal Life Followed by Reversal in Adulthood

Context: Mutations in TAC3 and TACR3 (encoding neurokinin B and its receptor) have been identified in Turkish patients with idiopathic hypogonadotropic hypogonadism (IHH), but broader populations have not yet been tested and genotype-phenotype correlations have not been established. Objective: A broad cohort of normosmic IHH probands was screened for mutations in TAC3/TACR3 to evaluate the prevalence of such mutations and define the genotype/phenotype relationships. Design and Setting: The study consisted of sequencing of TAC3/TACR3, in vitro functional assays, and neuroendocrine phenotyping conducted in tertiary care centers worldwide. Patients or Other Participants: 345 probands, 18 family members, and 292 controls were studied. Intervention: Reproductive phenotypes throughout reproductive life and before and after therapy were examined. Main Outcome Measure: Rare sequence variants in TAC3/TACR3 were detected. Results: In TACR3, 19 probands harbored 13 distinct coding sequence rare nucleotide variants [three nonsense mutations, six nonsynonymous, four synonymous (one predicted to affect splicing)]. In TAC3, one homozygous single base pair deletion was identified, resulting in complete loss of the neurokinin B decapeptide. Phenotypic information was available on 16 males and seven females with coding sequence variants in TACR3/TAC3. Of the 16 males, 15 had microphallus; none of the females had spontaneous thelarche. Seven of the 16 males and five of the seven females were assessed after discontinuation of therapy; six of the seven males and four of the five females demonstrated evidence for reversibility of their hypogonadotropism. Conclusions: Mutations in the neurokinin B pathway are relatively common as causes of hypogonadism. Although the neurokinin B pathway appears essential during early sexual development, its importance in sustaining the integrity of the hypothalamic-pituitary-gonadal axis appears attenuated over time. (J Clin Endocrinol Metab 95: 2857-2867, 2010)

Screening of autosomal gene deletions in patients with hypogonadotropic hypogonadism using multiplex ligation-dependent probe amplification: detection of a hemizygosis for the fibroblast growth factor receptor 1

P>Objective Congenital hypogonadotropic hypogonadism with anosmia (Kallmann syndrome) or with normal sense of smell is a heterogeneous genetic disorder caused by defects in the synthesis, secretion and action of gonadotrophin-releasing hormone (GnRH). Mutations involving autosomal genes have been identified in approximately 30% of all cases of hypogonadotropic hypogonadism. However, most studies that screened patients with hypogonadotropic hypogonadism for gene mutations did not include gene dosage methodologies. Therefore, it remains to be determined whether patients without detected point mutation carried a heterozygous deletion of one or more exons. Measurements We used the multiplex ligation-dependent probe amplification (MLPA) assay to evaluate the potential contribution of heterozygous deletions of FGFR1, GnRH1, GnRHR, GPR54 and NELF genes in the aetiology of GnRH deficiency. Patients We studied a mutation-negative cohort of 135 patients, 80 with Kallmann syndrome and 55 with normosmic hypogonadotropic hypogonadism. Results One large heterozygous deletion involving all FGFR1 exons was identified in a female patient with sporadic normosmic hypogonadotropic hypogonadism and mild dimorphisms as ogival palate and cavus foot. FGFR1 hemizygosity was confirmed by gene dosage with comparative multiplex and real-time PCRs. Conclusions FGFR1 or other autosomal gene deletion is a possible but very rare event and does not account for a significant number of sporadic or inherited cases of isolated GnRH deficiency.

Targeting endothelin ET(A) and ET(B) receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ET(A) and ET(B) receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ET(B) receptor antagonism, but rather by endothelin ET(A) receptor antagonism. Furthermore, endothelin ET(A), but not ET(B), receptor antagonism inhibited antigen-induced PGE(2) production, whereas either selective or combined blockade of endothelin ET(A) and/or ET(B) receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Effects of nitric oxide on neutrophil influx depends on the tissue: role of leukotriene B(4) and adhesion molecules

Background and purpose: We investigated the effect of nitric oxide synthase (NOS) inhibition on polymorphonuclear cell (PMN) influx in zymosan or lipopolysaccharide (LPS)-induced arthritis and peritonitis. Experimental approach: Wistar rats received intra-articular (i.art.) zymosan (30-1000 mu g) or LPS (1-10 mu g). Swiss C57/Bl6 mice genetically deficient in intercellular adhesion molecule-1 (ICAM-1(-/-)) or in beta(2)-integrin (beta(2)-integrin(-/-)) received zymosan either i.art. or i.p. PMN counts, leukotriene B(4) (LTB(4)), tumour necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) levels were measured in joint and peritoneal exudates. Groups received the NOS inhibitors N(G)-nitro-L-arginine methyl ester (LN), nitro-L-arginine, N-[3-(aminomemethyl) benzyl] acetamide or aminoguanidine, prior to zymosan or LPS, given i.p. or s.c. in the arthritis and peritonitis experiments respectively. A group of rats received LN locally (i.art. or i.p.), 30 min prior to 1 mg zymosan i.art. Key results: Systemic or local NOS inhibition significantly prevented PMN migration in arthritis while increasing it in peritonitis, regardless of stimuli, concentration of NOS inhibitors and species. NOS inhibition did not alter TNF-alpha and IL-10 but decreased LTB(4) in zymosan-induced arthritis. LN administration significantly inhibited PMN influx into the joints of ICAM-1(-/-) and beta(2)-integrin(-/-) mice with zymosan-arthritis, while not altering PMN influx into the peritoneum of mice with zymosan-peritonitis. Conclusions and implications: Nitric oxide has a dual modulatory role on PMN influx into joint and peritoneal cavities that is stimulus-and species-independent. Differences in local release of LTB(4) and in expression of ICAM-1 and beta(2)-integrin account for this dual role of NO on PMN migration.

Leukotriene B(4) is essential for selective eosinophil recruitment following allergen challenge of CD4(+) cells in a model of chronic eosinophilic inflammation

Subcutaneous heat-coagulated egg white implants (EWI) induce chronic, intense local eosinophilia in mice, followed by asthma-like responses to airway ovalbumin challenge. Our goal was to define the mechanisms of selective eosinophil accumulation in the EWI model. EWI carriers were challenged i.p. with ovalbumin and the contributions of cellular immunity and inflammatory mediators to the resulting leukocyte accumulation were defined through cell transfer and pharmacological inhibition protocols. Eosinophil recruitment required Major Histocompatibility Complex Class It expression, and was abolished by the leukotriene B4 (LTB4) receptor antagonist CP 105.696, the 5-lipoxygenase inhibitor BWA4C and the 5-lipoxygenase activating protein inhibitor MK886. Eosinophil recruitment in EWI carriers followed transfer of: a) CD4(+) (but not CD4(-)) cells, harvested from EWI donors and restimulated ex vivo; b) their cell-free supernatants, containing LTB4. Restimulation in the presence of MK886 was ineffective. CC chemokine receptor ligand (CCL)5 and CCL2 were induced by ovalbumin challenge in vivo. mRNA for CCL17 and CCL11 was induced in ovalbumin-restimulated CD4(+) cells ex vivo. MK886 blocked induction of CCL17 Pretreatment of EWI carriers with MK886 eliminated the effectiveness of exogenously administered CCL11, CCL2 and CCL5. In conclusion, chemokine-producing, ovalburnin-restimulated CD4(+) cells initiate eosinophil recruitment which is strictly dependent on LTB4 production. (C) 2008 Elsevier Inc. All rights reserved.

CXCR2-specific chemokines mediate leukotriene B-4-dependent recruitment of neutrophils to inflamed joints in mice with antigen-induced arthritis

Objective. To investigate the mechanism underlying neutrophil migration into the articular cavity in experimental arthritis and, by extension, human-inflammatory synovitis. Methods. Antigen-induced arthritis (AIA) was generated in mice with methylated bovine serum albumin (mBSA). Migration assays and histologic analysis were used to evaluate neutrophil recruitment to knee joints. Levels of inflammatory mediators were measured by enzyme-linked immunosorbent assay. Antibodies and pharmacologic inhibitors were used in vivo to determine the role of specific disease mediators. Samples of synovial tissue and synovial fluid from rheumatoid arthritis (RA) or osteoarthritis patients were evaluated for CXCL1 and CXCL5 expression. Results. High levels of CXCL1, CXCL5, and leukotriene B-4 (LTB4) were expressed in the joints of arthritic mice. Confirming their respective functional roles, repertaxin (a CXCR1/CXCR2 receptor antagonist), anti-CXCL1 antibody, anti-CXCL5 antibody, and MK886 (a leukotriene synthesis inhibitor) reduced mBSA-induced neutrophil migration to knee joints. Repertaxin reduced LTB4 production in joint tissue, and neutrophil recruitment induced by CXCL1 or CXCL5 was inhibited by MK886, suggesting a sequential mechanism. Levels of both CXCL1 and CXCL5 were elevated in synovial fluid and were released in vitro by RA synovial tissues. Moreover, RA synovial fluid neutrophils stimulated with CXCL1 or CXCL5 released significant amounts of LTB4. Conclusion. Our data implicate CXCL1, CXCL5, and LTB4, acting sequentially, in neutrophil migration in AIA. Elevated levels of CXCL1 and CXCL5 in the synovial compartment of RA patients provide robust comparative data indicating that this mechanism plays a role in inflammatory joint disease. Together, these results suggest that inhibition of. CXCL1, CXCL5, or LTB4 may represent a potential therapeutic strategy in RA.

DIFFERENT ROLE OF THE VENTRAL MEDIAL PREFRONTAL CORTEX ON MODULATION OF INNATE AND ASSOCIATIVE LEARNED FEAR

Reversible inactivation of the ventral portion of medial prefrontal cortex (vMPFC) of the rat brain has been shown to induce anxiolytic-like effects in animal models based on associative learning. The role of this brain region in situations involving innate fear, however, is still poorly understood, with several contradictory results in the literature. The objective of the present work was to verify in male Wistar rats the effects of vMPFC administration of cobalt chloride (CoCl(2)), a selective inhibitor of synaptic activity, in rats submitted to two models based on innate fear, the elevated plus-maze (EPM) and light-dark box (LOB), comparing the results with those obtained in two models involving associative learning, the contextual fear conditioning (CFC) and Vogel conflict (VCT) tests. The results showed that, whereas CoCl(2) induced anxiolytic-like effects in the CFC and VCT tests, it enhanced anxiety in rats submitted to the EPM and LOB. Together these results indicate that the vMPFC plays an important but complex role in the modulation of defensive-related behaviors, which seems to depend on the nature of the anxiety/fear inducing stimuli. (C) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

Involvement of the lateral septal area in the expression of fear conditioning to context

Considering the evidence that the lateral septal area (LSA) modulates defensive responses, the aim of the present study is to verify if this structure is also involved in contextual fear conditioning responses. Neurotransmission in the LSA was reversibly inhibited by bilateral microinjections of cobalt chloride (CoCl(2), 1 mM) 10 min before or after conditioning or 10 min before re-exposure to the aversively conditioned chamber. Only those animals that received CoCl(2) before re-exposure showed a decrease in both cardiovascular and behavioral conditioned responses. These results suggest that the LSA participates in the expression, but not acquisition or consolidation, of contextual fear conditioning.

Anxiolytic-like effects induced by blockade of transient receptor potential vanilloid type 1 (TRPV1) channels in the medial prefrontal cortex of rats

The endocannabinoid anandamide, in addition to activating cannabinoid type 1 receptors (CB1), may act as an agonist at transient receptor potential vanilloid type 1 (TRPV1) channels. In the periaqueductal gray, CB1 activation inhibits, whereas TRPV1 increases, anxiety-like behavior. In the medial prefrontal cortex (mPFC), another brain region related to defensive responses, CB1 activation induces anxiolytic-like effects. However, a possible involvement of TRPV1 is still unclear. In the present study, we tested the hypothesis that TRPV1 channel contributes to the modulation of anxiety-like behavior in the mPFC. Male Wistar rats (n = 5-7 per group) received microinjections of the TRPV1 antagonist capsazepine (1-60 nmol) in the ventral portion of the mPFC and were exposed to the elevated plus maze (EPM) or to the Vogel conflict test. Capsazepine increased exploration of open arms in the EPM as well as the number of punished licks in the Vogel conflict test, suggesting anxiolytic-like effects. No changes in the number of entries into the enclosed arms were observed in the EPM, indicating that there were no changes in motor activity. Moreover, capsazepine did not interfere with water consumption or nociceptive threshold, discarding potential confounding factors for the Vogel conflict test. These data suggest that TRPV1 in the ventral mPFC tonically inhibits anxiety-like behavior. TRPV1 could facilitate defensive responses opposing, therefore, the anxiolytic-like effects reported after local activation of CB1 receptors.

Identification of genetic alterations by multiple ligation-dependent probe amplification (MLPA) analysis in oligodendrogliomas

Concurrent deletion at 1p/19q is a common signature of oligodendrogliomas, and it may, be identified in low-grade tumours (grade II) suggesting it represents an early event in the development of these brain neoplasms. Additional non-random changes primarily involve CDKN2A, PTEN and EGFR. Identification of all of these genetic changes has become an additional parameter in the evaluation of the clinical patients` prognosis, including good response to conventional chemotherapy. Multiple ligation-dependent probe amplification (MLPA) analysis is a new methodology that allows an easy identification of the oligodendrogliomas` abnormalities in a single step. No need of the respective constitutional DNA from each patient is another advantage of this method. We used MLPA kits P088 and P105 to determine the molecular characteristics of a series of 40 oligodendrogliomas. Deletions at I p and 19q were identified in 45% and 65% of cases, respectively. Alterations of EGFR, CDKN2A, ERBB2, PTEN and TP53 were also identified in variable frequencies among 7% to 35% of tumours. These findings demonstrate that MLPA is a reliable technique to the detection of molecular genetic changes in oligodendrogliomas.

Spindle and chromosome configurations of in vitro-matured oocytes from polycystic ovary syndrome and ovulatory infertile women: a pilot study

To evaluate the meiotic spindle and chromosomal distribution of in vitro-matured oocytes from infertile nonobese women with PCOS and male or tubal causes of infertility (controls), and to compare in vitro maturation (IVM) rates between groups. Seventy four patients (26 with PCOS and 48 controls) undergoing stimulated cycles of oocyte retrieval for ICSI were selected prospectively. Thirteen PCOS patients and 27 controls had immature oocytes retrieved submitted to IVM. After IVM, oocytes showing extrusion of the first polar body were fixed and processed for evaluation of the meiotic spindle and chromosome distribution by immunofluorescence microscopy. There were no differences between PCOS and control groups with respect to IVM rates (50.0% and 42.9%, respectively) nor the percentage of meiotic abnormalities in metaphase II oocytes (35.3% and 25%, respectively). In vitro-matured oocytes obtained from stimulated cycles of nonobese PCOS did not have an increased ratio of meiotic abnormalities.