CpG-Induced Th1-Type Response in the Downmodulation of Early Development of Allergy and Inhibition of B7 Expression on T Cells of Newborn Mice

Several differences have been described between neonatal and adult immune responses. The predisposition in early life to Th2-type response or tolerance makes it a susceptible period for infections and allergic sensitization. The aim of this work was to evaluate the effects of CpG-containing oligodeoxynucleotides on neonatal and adult immunization with ovalbumin and Blomia tropicalis extract and compare the CpG effects on B and T cells of neonatal and adult mice. Mice that received CpG showed reduced immunoglobulin E (IgE) antibody production in both neonatal and adult periods, in parallel to increased IgG2a antibody levels. We observed that spleen cells of mice that received CpG in early life produced increased amounts of interferon-gamma upon anti-CD3 stimulation. Negative regulation of IgE response was more pronounced in adult than neonate mice; further, CpG decreased anaphylactic antiovalbumin IgG1 only in adults. Also, an upregulation of toll-like receptor 9 expression was detected in adult B cells, but not in neonatal, upon CpG stimuli. Neonatal B cells showed enhanced interleukin (IL)-10 expression and decreased IL-6 levels than adult B cells in response to CpG. When we analyzed in vitro activation of CD4+ T cells, an increased expression of B7 molecules on T cells in neonates was suppressed by CpG. Altogether, we verified qualitative and quantitative evidences regarding CpG effect on neonatal and adult allergens immunizations, which points to the importance of understanding neonatal immune system to establish immunomodulatory strategies for prevention of allergic diseases.

Coordinated expression of ER, PR and HER2 define different prognostic subtypes among poorly differentiated breast carcinomas

Aims: Histological grade is one of the most important prognostic factors in breast carcinomas, but poorly differentiated neoplasms still have quite heterogeneous biological behaviour, since they can be genetically classified as basal-like, HER2+ or even luminal. The aim was to analyse the frequency of oestrogen receptor (ER), progesterone receptor (PR) and HER2 expression profiles among breast carcinomas with < 10% tubular formation, and their correlation with classic prognostic factors. Methods and results: One hundred and thirty-four samples of paraffin-embedded tumours were studied retrospectively. The tumours were classified in to four groups by their ER/PR/HER2 profile: (i) ER+ and/or PR+ but HER2-; (ii) ER+ and/or PR+ and HER2+; (iii) ER- and/or PR- but HER2+; and (iv) ER-, PR- and HER2- (triple-negative). The histological features of triple-negative and HER2+ carcinomas overlap. The only difference was the expression of basal cytokeratins (basal CK), which was more frequent among triple-negative carcinomas. Basal-CK expression defined a more aggressive group of tumours, according to the pathological features, regardless of the immunohistochemical profile. Conclusions: Group 1 and 2 tumours (ER+ and/or PR+ tumours with or without HER2 expression) were not statistically different, suggesting that poorly differentiated carcinomas with hormone receptors correspond to the luminal B type of tumour. Among poorly differentiated breast carcinomas, the classic profile associated with basal-CK identifies distinct subtypes equivalent to those seen by genetic classification.

Acute hyperglycemia rapidly stimulates VEGF mRNA translation in the kidney. Role of angiotensin type 2 receptor (AT2)

Angiotensin II (Ang II) and vascular endothelial growth factor (VEGF) are important mediators of kidney injury in diabetes. Acute hyperglycemia increased synthesis of intrarenal Ang I and Ang II and resulted in activation of both Ang II receptors, AT1 and AT2, in the kidney. Losartan (specific AT1 antagonist) or PD123319 (specific AT2 antagonist) did not affect hyperglycemia but prevented activation of renal AT1 and AT2, respectively. In murine renal cortex, acute hyperglycemia increased VEGF protein but not mRNA content after 24 h, which suggested translational regulation. Blockade of AT2, but not AT1, prevented increase in VEGF synthesis by inhibiting translation of VEGF mRNA in renal cortex. Acute hyperglycemia increased VEGF expression in wild type but not in AT2 knockout mice. Binding of heterogeneous nuclear ribonucleoprotein K to VEGF mRNA, which stimulates its translation, was prevented by blockade of AT2, but not AT1. The Akt-mTOR-p70(S6K) signaling pathway, involved in the activation of mRNA translation, was activated in hyperglycemic kidneys and was blocked by the AT2 antagonist. Elongation phase is an important step of mRNA translation that is controlled by elongation factor 1A (eEF1A) and 2 (eEF2). Expression of eEF1A and activity of eEF2 was higher in kidney cortex from hyperglycemic mice and only the AT2 antagonist prevented these changes. To assess selectivity of translational control of VEGF expression, we measured expression of fibronectin (FN) and laminin beta 1 (lam beta 1): acute hyperglycemia increased FN expression at both protein and mRNA levels, indicating transcriptional control, and did not affect the expression of lam beta 1. To confirm results obtained with PD123319, we induced hyperglycemia in AT2 knockout mice and found that in the absence of AT2, translational control of VEGF expression by hyperglycemia was abolished. Our data show that acute hyperglycemia stimulates Ang II synthesis in murine kidney cortex, this leads to AT2 activation and stimulation of VEGF mRNA translation, via the Akt-mTOR-p70(S6K) signaling pathway. Our data show that exclusive translational control of protein expression in the kidney by acute hyperglycemia is not a general phenomenon, but do not prove that it is restricted to VEGF. (C) 2010 Elsevier Inc. All rights reserved.

High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

Personality traits in patients with juvenile myoclonic epilepsy

There is evidence of personality disorders in patients with juvenile myoclonic epilepsy (JME). To date, there have been no published quantitative studies on personality traits in JME. The aim of the work described here was to study a group of patients with JME and quantitatively measure personality traits. We evaluated 42 patients (mean age: 26.57 years, SD: 8.38) and 42 controls (mean age: 26.96, SD: 8.48) using a validated personality inventory, the Temperament and Character Inventory (TCI). We applied two scores, one for the Beck Depression Inventory and one for the State-Trait-Anxiety Inventory, as depression and anxiety may impact the performance of these patients on the TCI. We compared both groups on TCI scales using analysis of covariance with Beck Depression Inventory and State-Trait-Anxiety Inventory scores as covariates. Patients with JME obtained significantly higher scores on Novelty Seeking (P=0.001) and Harm Avoidance (P=0.002) and significantly lower scores on Self-Directedness (P=0.001). Patients with JME have a higher expression of impulsive personality traits that demand early recognition to avoid further consequences and facilitate social insertion, consequently avoiding future stigma. (C) 2011 Elsevier Inc. All rights reserved.

Estrogen receptor-positive breast carcinomas in younger women are different from those of older women: A pathological and immunohistochemical study

The higher frequency of triple-negative and HER-2-positive tumors detected in younger patients has been suggested as an explanation for the more aggressive tumor types observed in this age group. However, estrogen receptor (ER)-positive tumors are the most frequent subtype of breast carcinomas identified, even in younger patients. In this retrospective study, the morphological and immunohistochemical profiles of ER-positive breast carcinomas from women 35 yrs and younger that were diagnosed between 1997 and 2007 were evaluated. From these cases, 213 were selected based on the availability of pathology reports and paraffin blocks. For comparison, 117 consecutive cases of breast carcinomas diagnosed in patients >60 yrs from 2006 were included. Paraffin-embedded tumors were stained for expression of ER, progesterone receptor (PR), human epidermal growth factor receptor 2 (HER-2). Ki-67 antigen, epidermal growth factor receptor (EGFR), cytokeratin 5/6, p53, vimentin, CD117, and p63 using tissue microarrays. ER-positive carcinomas were diagnosed in 120 (56.1%) samples of the younger patient group and in 92 (78.6%) samples of the older patient group. Of these ER-positive carcinomas, 48 (40%) from the younger patient group presented the subtype luminal A, compared with 53 (57.6%) from the older patient group (p=0.01). Tumors from the younger patient group were also associated with increased vascular involvement, co-expression of HER-2, and decreased expression of CD117. These results highlight differences in expression markers and the pathology of ER-positive tumors detected in younger women, with a notable characteristic being co-expression of HER-2. (C) 2010 Elsevier Ltd. All rights reserved.

Individual vulnerability to escalated aggressive behavior by a low dose of alcohol: decreased serotonin receptor mRNA in the prefrontal cortex of male mice

Low to moderate doses of alcohol consumption induce heightened aggressive behavior in some, but not all individuals. Individual vulnerability for this nonadaptive behavior may be determined by an interaction of genetic and environmental factors with the sensitivity of alcohol`s effects on brain and behavior. We used a previously established protocol for alcohol oral self-administration and characterized alcohol-heightened aggressive (AHA) mice as compared with alcohol non-heightened (ANA) counterparts. A week later, we quantified mRNA steady state levels of several candidate genes in the serotonin [5-hydroxytryptamine (5-HT)] system in different brain areas. We report a regionally selective and significant reduction of all 5-HT receptor subtype transcripts, except for 5-HT(3), in the prefrontal cortex of AHA mice. Comparable gene expression profile was previously observed in aggressive mice induced by social isolation or by an anabolic androgenic steroid. Additional change in the 5-HT(1B) receptor transcripts was seen in the amygdala and hypothalamus of AHA mice. In both these areas, 5-HT(1B) mRNA was elevated when compared with ANA mice. In the hypothalamus, AHA mice also showed increased transcripts for 5-HT(2A) receptor. In the midbrain, 5-HT synthetic enzyme, 5-HT transporter and 5-HT receptors mRNA levels were similar between groups. Our results emphasize a role for postsynaptic over presynaptic 5-HT receptors in mice which showed escalated aggression after the consumption of a moderate dose of alcohol. This gene expression profile of 5-HT neurotransmission components in the brain of mice may suggest a vulnerability trait for alcohol-heightened aggression.

Lower cytokine secretion ex vivo by natural killer T cells in HIV-infected individuals is associated with higher CD161 expression

Objective: Natural killer T (NKT) cells are efficiently targeted by HIV and severely reduced in numbers in the circulation of infected individuals. The functional capacity of the remaining NKT cells in HIV-infected individuals is poorly characterized. This study measured NKT cell cytokine production directly ex vivo and compared these responses with both the disease status and NKT subset distribution of individual patients. Methods: NKT cell frequencies, subsets, and ex-vivo effector functions were measured in the peripheral blood mononuclear cells of HIV-infected patients and healthy controls by flow cytometry. We measured cytokines from NKT cells after stimulation with either a-galactosyl ceramide-loaded CD1d dimers (DimerX-alpha GalCer) or phorbol myristate acetate and ionomycin. Results: The frequencies of NKT cells secreting interferon-gamma and tumor necrosis factor-alpha were significantly lower in HIV-infected patients than healthy controls after DimerX-alpha GalCer treatment, but responses were similar after treatment with phorbol myristate acetate and ionomycin. The magnitude of the interferon-gamma response to DimerX-alpha GalCer correlated inversely with the number of years of infection. Both interferon-gamma and tumor necrosis factor-alpha production in response to DimerX-alpha GalCer correlated inversely with CD161 expression. Conclusion: The ex-vivo Th1 responses of circulating NKT cells to CD1d-glycolipid complexes are impaired in HIV-infected patients. NKT cell functions may be progressively lost over time in HIV infection, and CD161 is implicated in the regulation of NKT cell responsiveness. (C) 2009 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins

Studies of RET gene expression and acetylcholinesterase activity in a series of sporadic Hirschsprung`s disease

The purpose is to present the studies of RET gene expression and acetylcholinesterase activity in 23 patients operated for Hirschsprung`s disease (HD). The patients underwent either transanal endorectal pull-through or Duhamell`s procedure. Full-thickness intestinal samples from the three different segments (ganglionic, intermediate and aganglionic) were collected. Each tissue sample was divided in two portions, one for AChE histochemical staining and the other for examination of RET mRNA expression level. All patients had an uneventful postoperative recovery. In all patients, the AChE stainings demonstrated the absence of activity in the ganglionic area, the marked increase of positive fibers in the aganglionic area, and little increase of positive fibers in the intermediate area. In the ganglionic and intermediate areas, all patients (100%) showed significant RET gene expression. In the aganglionic area, 18 patients (78.3%) did not present gene expression and the other five patients (21.7%) presented gene expression that was similar to the ganglionic and intermediate areas. The results reinforce the conclusion that the method of AChE staining is effective for the diagnosis of intestinal aganglionosis and confirm the knowledge that genes beyond RET may be implicated in the genesis of sporadic cases of HD.

Topical interleukin-1 receptor antagonist inhibits inflammatory cell infiltration into the cornea

Interleukin (IL)-1 alpha and beta are important modulators of many functions of corneal epithelial and stromal cells that occur following injury to the cornea, including the influx of bone marrow-derived inflammatory cells into the stroma attracted by chemokines released from the stroma and epithelium. In this study, we examined the effect of topical soluble IL-1 receptor antagonist on bone marrow-derived cell influx following corneal epithelial scrape injury in a mouse model. C57BL/6 mice underwent corneal epithelial scrape followed by application of IL-1 receptor antagonist (Amgen, Thousand Oaks, CA) at a concentration of 20 mg/ml or vehicle for 24 h prior to immunocytochemical detection of marker CD11b-positive cells into the stroma. In two experiments, topical IL-1 receptor antagonist had a marked effect in blocking cell influx. For example, in experiment 1, topical IL-1 receptor antagonist markedly reduced detectible CD11b-positive cells into the corneal stroma at 24 It after epithelial injury compared with the vehicle control (3.5 +/- 0.5 (standard error of the mean) cells/400x field and 13.9 +/- 1.2 cells/400x field, respectively, p < 0.01). A second experiment with a different observer performing cell counting had the same result. Thus, the data demonstrate conclusively that topical IL-1 receptor antagonist markedly down-regulates CD-11b-positive monocytic cell appearance in the corneal stroma. Topical IL-1 receptor antagonist could be an effective adjuvant for clinical treatment of corneal conditions in which unwanted inflammation has a role in the pathophysiology of the disorder. (c) 2008 Elsevier Ltd. All rights reserved.

Novel inactivating mutations in the GH secretagogue receptor gene in patients with constitutional delay of growth and puberty

Background: A limited number of mutations in the GH secretagogue receptor gene (GHSR) have been described in patients with short stature. Objective: To analyze GHSR in idiopathic short stature (ISS) children including a subgroup of constitutional delay of growth and puberty (CDGP) patients. Subjects and methods: The GHSR coding region was directly sequenced in 96 independent patients with ISS, 31 of them with CDGP, in 150 adults, and in 197 children with normal stature. The pharmacological consequences of GHSR non-synonymous variations were established using in vitro cell-based assays. Results: Five different heterozygous point variations in GHSR were identified (c.-6 G>C, c.251G>T (p.Ser84Ile), c.505G>A (p.Ala169Thr), c.545 T>C (p.Val182Ala), and c.1072G>A (p.Ala358Thr)), all in patients with CDGP. Neither these allelic variants nor any other mutations were found in 694 alleles from controls. Functional studies revealed that two of these variations (p.Ser84Ile and p. Val182Ala) result in a decrease in basal activity that was in part explained by a reduction in cell surface expression. The p.Ser84Ile mutation was also associated with a defect in ghrelin potency. These mutations were identified in two female patients with CDGP (at the age of 13 years, their height SDS were -2.4 and -2.3). Both patients had normal progression of puberty and reached normal adult height (height SDS of -0.7 and -1.4) without treatment. Conclusion: This is the first report of GHSR mutations in patients with CDGP. Our data raise the intriguing possibility that abnormalities in ghrelin receptor function may influence the phenotype of individuals with CDGP.

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.

Angiotensin-converting enzyme inhibition augments the expression of rat elastase-2, an angiotensin II-forming enzyme

Becari C, Teixeira FR, Oliveira EB, Salgado MC. Angiotensin-converting enzyme inhibition augments the expression of rat elastase- 2, an angiotensin II-forming enzyme. Am J Physiol Heart Circ Physiol 301: H565-H570, 2011. First published May 20, 2011; doi:10.1152/ajpheart.00534.2010.-Mounting evidence suggest that tissue levels of angiotensin (ANG) II are maintained in animals submitted to chronic angiotensin-converting enzyme (ACE) inhibitor treatment. We examined the expression levels of transcripts for elastase-2, a chymostatin-sensitive serine protease identified as the alternative pathway for ANG II generation from ANG I in the rat vascular tissue and the relative role of ACE-dependent and -independent pathways in generating ANG II in the rat isolated carotid artery rings of spontaneously hypertensive rats (SHR) and Wistar normotensive rats (WNR) treated with enalapril for 7 days. Enalapril treatment decreased blood pressure of SHR only and resulted in significantly more elastase-2 mRNA expression in carotid artery of both enalapril-treated WNR and SHR. Captopril induced a comparable rightward shift of concentration-response curves to ANG I in vehicle and enalapril-treated rats, although this effect was of lesser magnitude in SHR group. Chymostatin induced a rightward shift of the dose response to ANG I in vehicle-treated and a decrease in maximal effect of 22% in enalapril-treated WNR group. Maximal response induced by ANG I was remarkably reduced by chymostatin in enalapril-treated SHR carotid artery (by 80%) compared with controls (by 23%). Our data show that chronic ACE inhibition was associated with augmented functional role of non-ACE pathway in generating ANG II and increased elastase-2 gene expression, suggesting that this protease may contribute as an alternative pathway for ANG II generation when ACE is inhibited in the rat vascular tissue.

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.

Apoptosis induced by Oropouche virus infection in HeLa cells is dependent on virus protein expression

Oropouche (OROV) is a single-stranded RNA arbovirus of the family Bunyaviridae, genus Orthobunyavirus, which has caused over half a million cases of febrile illness in Brazil in the past 30 years. OROV fever has been registered almost exclusively in the Amazon region, but global warming, deforestation and redistribution of vectors and animal reservoirs increases the risk of Oropouche virus emergence in other areas. OROV causes a cytolytical infection in cultured cells with characteristic cytopathic effect 48 h post-infection. We have studied the mechanisms of apoptosis induced by OROV in HeLa cells and found that OROV causes DNA fragmentation detectable by gel electrophoresis and by flow cytometric analysis of the Sub-G1 population at 36 h post-infection. Mitochondrial release of cytochrome C and activation of caspases 9 and 3 were also detected by western blot analysis. Lack of apoptosis induced by UV-inactivated OROV reveals that virus-receptor binding is not sufficient to induce cell death. Results obtained in cells treated with chloroquine and cycloheximide indicated that viral uncoating and replication are required for apoptosis induction by OROV. Furthermore, treatment of the cells with pan-caspase inhibitor prevented OROV-induced apoptosis without affecting virus progeny production. The results show that OROV infection in vitro causes apoptosis by an intracellular pathway involving mitochondria, and activated by a mechanism dependent on viral replication and protein synthesis. (C) 2010 Elsevier B.V. All rights reserved.