TGF-beta and CD23 are involved in nitric oxide production by pulmonary macrophages activated by beta-glucan from Paracoccidioides brasiliensis

Pulmonary macrophages (PM), which are CD11b/CD18(+) and CD23(+), may be involved in the onset of inflammatory events caused by Paracoccidioides brasiliensis in the lungs. In the present study, we measured the nitric oxide (NO) and interleukin in PM production after intratracheal (i.t.) inoculation of an enriched beta-glucan cell wall fraction from P. brasiliensis (Fraction F1). BALB/c and C57/BL6 (B6) mice were i.t. treated with Fraction F1, and their PM were restimulated in vitro with LPS and interferon-gamma up to 14 days after treatment. Macrophages BALB/c mice produced less NO than PM from B6 mice. The lower NO production was caused by higher production of TGF-beta by pulmonary macrophages of BALB/c and was abrogated by anti-TGF-beta MoAb in vitro and in vivo. Other interleukins such as IL-10, IL-4 and a combination of IL-1, TNF-alpha and IL-6 were not involved in NO production induced by Fraction F1. Expression of CD11b increases and expression of CD23 decreases on PM of BALB/c mice after in vivo treatment whereas PM of B6 mice do not show a variation of their phenotype. Moreover, the ability of pulmonary macrophages to induce lymphocyte proliferation was reduced in mixed cultures of CD11b(+) or CD23(+) macrophages but was restored when lymphocytes were cultivated in the presence of NO inhibitor (L-NMMA). Thus, the results presented herein indicate that in BALB/c but not in B6 mice TGF- is strongly induced by Fraction 1 in PM in vivo and suppresses NO production. Low NO production by PM is associated with a change in CD11b/CD23 expression and with a high lymphocyte proliferative response. Thus, CD11b(+)/CD23(+) PM modulate NO and TGF-beta production in the pulmonary microenvironment.

Mycobacterium tuberculosis Culture Filtrate Proteins plus CpG Oligodeoxynucleotides Confer Protection to Mycobacterium bovis BCG-Primed Mice by Inhibiting Interleukin-4 Secretion

Culture filtrate proteins (CFP) are potential targets for tuberculosis vaccine development. We previously showed that despite the high level of gamma interferon (IFN-gamma) production elicited by homologous immunization with CFP plus CpG oligodeoxynucleotides (CFP/CpG), we did not observe protection when these mice were challenged with Mycobacterium tuberculosis. In order to use the IFN-gamma-inducing ability of CFP antigens, in this study we evaluated a prime-boost heterologous immunization based on CFP/CpG to boost Mycobacterium bovis BCG vaccination in order to find an immunization schedule that could induce protection. Heterologous BCG-CFP/CpG immunization provided significant protection against experimental tuberculosis, and this protection was sustained during the late phase of infection and was even better than that conferred by a single BCG immunization. The protection was associated with high levels of antigen-specific IFN-gamma and interleukin-17 (IL-17) and low IL-4 production. The deleterious role of IL-4 was confirmed when IL-4 knockout mice vaccinated with CFP/CpG showed consistent protection similar to that elicited by BCG-CFP/CpG heterologous immunization. These findings show that a single dose of CFP/CpG can represent a new strategy to boost the protection conferred by BCG vaccination. Moreover, different immunological parameters, such as IFN-gamma and IL-17 and tightly regulated IL-4 secretion, seem to contribute to the efficacy of this tuberculosis vaccine.

Mouse Leydig cells express multiple P2X receptor subunits

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 mu M ivermectin induced an increase (131.2 +/- 5.9%) and 3 mu M ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X(2/4/6), is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X(2) subunit.

CCR2 receptor is essential to activate microbicidal mechanisms to control Toxoplasma gondii infection in the central nervous system

Chemokines comprise a structurally related family of cytokines that regulate leukocyte trafficking. Because infection with Toxoplasma gondii can induce an important inflammatory reaction that, if left uncontrolled, can lead to death, we investigated the role of the chemokine receptor CCR2 in T gondii infection. We orally infected CCR2(-/-) mice with five ME-49 T gondii cysts and monitored morbidity, survival, and immune response thereafter. The CCR2(-/-) mice displayed higher susceptibility to infection as all mice died on day 28 after infection. Despite similar Th1 responses, a more evident anti-inflammatory response was induced in the peripheral organs of CCR2(-/-) mice compared with wild-type C57BL/6 mice. Additionally, CCR2-/- mice presented greater parasitism and a milder inflammatory reaction in their peripheral organs with lesser CD4(+) and MAC-1(+) and greater CD8(+) cell migration. The parasite load decreased in these organs in CCR2(-/-) mice but remained uncontrolled in the central nervous system. Additionally, we observed down-regulated inducible nitric oxide synthase expression in peripheral organs from CCR2(-/-) mice that was associated with a small nitric oxide production by spleen macrophages. In conclusion, in the absence of CCR2, another mechanism is activated to control tissue parasitism in peripheral organs. Nevertheless, CCR2 is essential for the activation of microbicidal mediators that control T gondii replication in the central nervous system.

Role of Interleukin (IL)-18 in experimental paracoccidioidomycosis

Interleukin (IL)-18 has been regarded as a Th1 type cytokine involved in many fungal and parasitic infections. Since there have been no studies, as of yet, evaluating the role of this cytokine in paracoccidioidomycosis (PCM), we assessed the function of IL-18 by using an experimental PCM model. Our results showed that IL-18 knockout (IL-18-/-) BALB/c were more resistant to Paracoccidioides brasiliensis than their littermate controls (WT). In fact, mortality rate was higher in WT mice and in the first month of infection, the number of colony forming units of the etiologic agent recovered from the lungs was greater in WT mice. In histopathological analyses, well-formed granulomas were seen in both WT and IL-18-/- mice. However, substantial differences were observed at the second month of infection when epithelioid cells predominated in the lesions of IL-18-/- mice, which could infer that IL-18 postpones pulmonary healing. The levels of IL-10 were significantly higher in IL-18 sufficient mice at early stages of infection and therefore account for the delayed fungal clearance observed in WT mice. TNF- augmented later in the infection of WT mice, seemingly to compensate high levels of IL-10. Our results demonstrated that IL-18 has a critical role in protecting BALB/c mice against disseminated PCM.

Indirect immune detection of ecdysone receptor (EcR) during the formation of DNA puffs in Bradysia hygida (Diptera, Sciaridae)

Gene amplification occurs in Bradysia hygida salivary glands, at the end of the fourth larval instar. The hormone 20-hydroxyecdysone (20E) triggers this process, which results in DNA puff formation. Amplified genes are activated in two distinct groups. The activity of the first group is dependent on high levels of 20E, while the second group needs low hormone levels. Consequently, the salivary glands of B. hygida constitute an interesting biological model to study how 20E, and its receptors, affect gene amplification and activity. We produced polyclonal antibodies against B. hygida EcR (BhEcR). In western blots a polypeptide of about 66 kDa was detected in salivary gland extracts. The antibodies were also used for indirect immune-localization of BhEcR in polytene chromosomes. RNA-polymerase II was also immune-detected. We did not detect the receptor in chromosome C where the first and second groups of DNA puffs form during DNA puff anlage formation, but it was present during puff expansion. During the active phase of both groups of DNA puffs, RNA polymerase II co-localized with BhEcR. After puff regression, these antigens were not detected. Apparently, EcR plays a direct role in the transcription of amplified genes, but its role in gene amplification remains enigmatic.

Consequences of lifetime isolated growth hormone (GH) deficiency and effects of short-term GH treatment on bone in adults with a mutation in the GHRH-receptor gene

Growth hormone (GH) influences bone mass maintenance. However, the consequences of lifetime isolated GH deficiency (IGHD) on bone are not well established. We assessed the bone status and the effect of 6 months of GH replacement in GH-naive adults with IGHD due to a homozygous mutation of the GH-releasing hormone (GHRH)-receptor gene (GHRHR). We studied 20 individuals (10 men) with IGHD at baseline, after 6 months of depot GH treatment, and 6 and 12 months after discontinuation of GH. Quantitative ultrasound (QUS) of the heel was performed and serum osteocalcin (OC) and C-terminal cross-linking telopeptide of type I collagen (ICTP) were measured. QUS was also performed at baseline and 12 months later in a group of 20 normal control individuals (CO), who did not receive GH treatment. At baseline, the IGHD group had a lower T-score on QUS than CO (-1.15 +/- 0.9 vs. -0.07 +/- 0.9, P < 0.001). GH treatment improved this parameter, with improvement persisting for 12 months post-treatment (T-score for IGHD = -0.59 +/- 0.9, P < 0.05). GH also caused an increase in serum OC (baseline vs. pGH, P < 0.001) and ICTP (baseline vs. pGH, P < 0.01). The increase in OC was more marked during treatment and its reduction was slower after GH discontinuation than in ICTP. These data suggest that lifetime severe IGHD is associated with significant reduction in QUS parameters, which are partially reversed by short-term depot GH treatment. The treatment induces a biochemical pattern of bone anabolism that persists for at least 6 months after treatment discontinuation.

Cultural Consonance, a 5HT2A Receptor Polymorphism, and Depressive Symptoms: A Longitudinal Study of Gene x Culture Interaction in Urban Brazil

In this study in urban Brazil we examine, as a predictor of depressive symptoms, the interaction between a single nucleotide polymorphism in the 2A receptor in the serotonin system (-1438G/A) and cultural consonance in family life, a measure of the degree to which an individual perceives her family as corresponding to a widely shared cultural model of the prototypical family. A community sample of 144 adults was followed over a 2-year-period. Cultural consonance in family life was assessed by linking individuals` perceptions of their own families with a shared cultural model of the family derived from cultural consensus analysis. The -1438G/A polymorphism in the 2A serotonin receptor was genotyped using a standard protocol for DNA extracted from leukocytes. Covariates included age, sex, socioeconomic status, and stressful life events. Cultural consonance in family life was prospectively associated with depressive symptoms. In addition, the interaction between genotype and cultural consonance in family life was significant. For individuals with the A/A variant of the -1438G/A polymorphism of the 2A receptor gene, the effect of cultural consonance in family life on depressive symptoms over a 2-year-period was larger (beta = -0.533, P < 0.01) than those effects for individuals with either the G/A (beta = -0.280, P < 0.10) or G/G (beta = -0.272, P < 0.05) variants. These results are consistent with a process in which genotype moderates the effects of culturally meaningful social experience on depressive symptoms. Am. J. Hum. Biol. 21:91-97, 2009. (C) 2008 Wiley-Liss, Inc.

Loss-of-function mutations in the genes encoding prokineticin-2 or prokineticin receptor-2 cause autosomal recessive Kallmann syndrome

Context: Physiological activation of the prokineticin pathway has a critical role in olfactory bulb morphogenesis and GnRH secretion in mice. Objective: To investigate PROK2 and PROKR2 mutations in patients with hypogonadotropic hypogonadism (HH) associated or not with olfactory abnormalities. Design: We studied 107 Brazilian patients with HH (63 with Kallmann syndrome and 44 with normosmic HH) and 100 control individuals. The coding regions of PROK2 and PROKR2 were amplified by PCR followed by direct automatic sequencing. Results: In PROK2, two known frameshift mutations were identified. Two brothers with Kallmann syndrome harbored the homozygous p. G100fsX121 mutation, whereas one male with normosmic HH harbored the heterozygous p. I55fsX56 mutation. In PROKR2, four distinct mutations (p. R80C, p. Y140X, p. L173R, and p. R268C) were identified in five patients with Kallmann syndrome and in one patient with normosmic HH. These mutations were not found in the control group. The p. R80C, p. L173R, and p. R268C missense mutations were identified in the heterozygous state in the HH patients and in their asymptomatic first-degree relatives. In addition, nomutations of FGFR1, KAL1, GnRHR, KiSS-1, or GPR54 were identified in these patients. Notably, the new nonsense mutation (p. Y140X) was identified in the homozygous state in an anosmic boy with micropenis, bilateral cryptorchidism, and high-arched palate. His asymptomatic parents were heterozygous for this severe defect. Conclusion: We expanded the repertoire of PROK2 and PROKR2 mutations in patients with HH. In addition, we show that PROKR2 haploinsufficiency is not sufficient to cause Kallmann syndrome or normosmic HH, whereas homozygous loss-of-function mutations either in PROKR2 or PROK2 are sufficient to cause disease phenotype, in accordance with the Prokr2 and Prok2 knockout mouse models.

Effect of a thrombin receptor (protease-activated receptor 1, PAR-1) gene polymorphism in chronic hepatitis C liver fibrosis

Background and Aim: Tissue injury leads to activation of coagulation and generation of thrombin. Inhibition of thrombin receptor protease-activated receptor 1 (PAR-1) has been shown to reduce liver fibrosis in animals. This study aimed to evaluate the effect of PAR-1 gene polymorphism on rate of liver fibrosis (RF) in chronic hepatitis C. Methods: Polymorphisms studied: C > T transition 1426 bp upstream of translation start site (-1426C/T), 13 bp repeat of preceding -506 5`-CGGCCGCGGGAAG-3` sequence (-506I/D), and A > T transversion in intervening sequence (IVS) 14 bp upstream of exon-2 start site (IVS-14A/T). A total of 287 European and 90 Brazilian patients were studied. Results: 1426C/T polymorphism: There was a trend to higher RF in patients with the TT genotype (P = 0.06) and an association between genotype CC and slow fibrosis (P = 0.03) in Europeans. In males, RF was significantly higher in those with the TT genotype compared to CT (P = 0.003) and CC (P = 0.007). There was a significant association between TT and fast fibrosis (P = 0.04). This was confirmed in an independent cohort of Brazilians where RF was higher in TT than in CC (P = 0.03). Analysis of -506I/D showed no difference in RF and distribution of slow/fast fibrosis among different genotypes in both populations. Analysis of IVS-14A/T showed no difference between genotypes. Conclusion: In conclusion, these findings suggest that PAR-1 receptor polymorphisms influence the progression of liver fibrosis.

Diagnostic value of interleukin-6 and C-reactive protein on early onset bacterial infection in preterm neonates with respiratory distress

Aims: To evaluate the C-reactive protein (CRP) and interleukin-6 (IL-6) as diagnostic tools for early onset infection in preterm infants with early respiratory distress (RD). Methods: CRP and IL-6 were quantified at identification of RD and 24 h after in 186 newborns. Effects of maternal hypertension, mode of delivery, Apgar score, birth weight, gestational age, mechanical ventilation, being small for gestational age (SGA), and the presence of infection were analyzed. Results: Forty-four infants were classified as infected, 42 as possibly infected, and 100 as uninfected. Serum levels of IL-6 (0 h), CRP (0 h), and CRP (24 h), but not IL-6 (24 h) were significantly higher in infected infants compared to the remaining groups. The best test for identification of infection was the combination of IL-6 (0 h) 36 pg/dL and/or CRP (24 h) 0.6 mg/dL, which yielded 93% sensitivity and 37% specificity. The presence of infection and vaginal delivery independently increased IL-6 (0 h), CRP (0 h) and CRP (24 h) levels. Being SGA also increased the CRP (24 h) levels. IL-6 (24 h) was independently increased by mechanical ventilation. Conclusions: The combination of IL-6 (0 h) and/or CRP (24 h) is helpful for excluding early onset infection in preterm infants with RD but the poor specificity limits its potential benefit as a diagnostic tool.

Interleukin-18 and interferon-gamma polymorphisms in Brazilian human immunodeficiency virus-1-infected patients presenting with lipodystrophy syndrome

Cytokines play important roles in the pathogenesis of lipodystrophy syndrome (LS). Single nucleotide polymorphisms (SNPs) at positions -607(C/A) and -137(C/G) in the promoter region of the interleukin-18 (IL-18) gene and at position +874(T/A) of the interferon-gamma (IFN-gamma) gene are related to the expression of these cytokines. To examine whether IL-18 and IFN-gamma polymorphisms are associated with LS, these SNPs were genotyped in 88 human immunodeficiency virus (HIV)-infected patients presenting LS, 79 HIV-infected without LS, and 133 healthy controls. The -607A allele, -607AA genotype, and -137G/-607A and -137C/-607A haplotypes in the IL-18 gene were over-represented in HIV patients presenting LS. The -137G/-607C haplotype was associated with protection against LS. These results indicate that the -607(C/A) SNP is associated with LS development in HIV-infected patients.

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.

Regulation of chemokine receptor by Toll-like receptor 2 is critical to neutrophil migration and resistance to polymicrobial sepsis

Patients with sepsis have a marked defect in neutrophil migration. Here we identify a key role of Toll-like receptor 2 (TLR2) in the regulation of neutrophil migration and resistance during polymicrobial sepsis. We found that the expression of the chemokine receptor CXCR2 was dramatically down-regulated in circulating neutrophils from WT mice with severe sepsis, which correlates with reduced chemotaxis to CXCL2 in vitro and impaired migration into an infectious focus in vivo. TLR2 deficiency prevented the down-regulation of CXCR2 and failure of neutrophil migration. Moreover, TLR2(-/-) mice exhibited higher bacterial clearance, lower serum inflammatory cytokines, and improved survival rate during severe sepsis compared with WT mice. In vitro, the TLR2 agonist lipoteichoic acid (LTA) down-regulated CXCR2 expression and markedly inhibited the neutrophil chemotaxis and actin polymerization induced by CXCL2. Moreover, neutrophils activated ex vivo by LTA and adoptively transferred into naive WT recipient mice displayed a significantly reduced competence to migrate toward thioglycolate-induced peritonitis. Finally, LTA enhanced the expression of G protein-coupled receptor kinases 2 (GRK2) in neutrophils; increased expression of GRK2 was seen in blood neutrophils from WT mice, but not TLR2(-/-) mice, with severe sepsis. Our findings identify an unexpected detrimental role of TLR2 in polymicrobial sepsis and suggest that inhibition of TLR2 signaling may improve survival from sepsis.

Modulation of anxiety-like behaviour by Transient Receptor Potential Vanilloid Type 1 (TRPV1) channels located in the dorsolateral periaqueductal gray

The endocannabinoid anandamide is a possible agonist at the Transient Receptor Potential Vanilloid Type 1 (TRPV1) channel, in addition to its agonist activity at cannabinoid type 1 (CB1) receptor. In the midbrain dorsolateral periaqueductal gray (dlPAC) our previous data showed that CB1 activation induces anxiolytic-like effects. However, the rote of TRPV1 has remained unclear. Thus, in the present study we tested the hypothesis that this channel would contribute to the modulation of anxiety-like behaviour in the dlPAG. Mate Wistar rats received local injections of the TRPV1 antagonist capsazepine (10-60 nmol) and were submitted to the elevated plus-maze (EPM) and to the Vogel test. In addition, animals received local injections of capsaicin (0.01-1nmol), a TRPV1 agonist, and were tested in the same models. In accordance with our hypothesis, capsazepine produced anxiolytic-like effects both in the EPM and in the Vogel test. Capsaicin mimicked these results, which might be attributed to its ability to quickly desensitize the channel. Altogether, our data suggest that, while CB1 receptors seem to inhibit aversive responses in the dlPAG, TRPV1 could facilitate them. Thus, CB1 and TRPV1 may have opposite functions in modulating anxiety-like behaviour in this region. (C) 2008 Elsevier B.V. and ECNP. All rights reserved.