Group A streptococcal (GAS) infections and autoimmunity are associated with the onset of a spectrum of neuropsychiatric disorders in children, with the prototypical disorder being Sydenham chorea (SC). Our aim was to develop an animal model that resembled the behavioral, pharmacological, and immunological abnormalities of SC and other streptococcal-related neuropsychiatric disorders. Male Lewis rats exposed to GAS antigen exhibited motor symptoms (impaired food manipulation and beam walking) and compulsive behavior (increased induced-grooming). These symptoms were alleviated by the D2 blocker haloperidol and the selective serotonin reuptake inhibitor paroxetine, respectively, drugs that are used to treat motor symptoms and compulsions in streptococcal-related neuropsychiatric disorders. Streptococcal exposure resulted in antibody deposition in the striatum, thalamus, and frontal cortex, and concomitant alterations in dopamine and glutamate levels in cortex and basal ganglia, consistent with the known pathophysiology of SC and related neuropsychiatric disorders. Autoantibodies (IgG) of GAS rats reacted with tubulin and caused elevated calcium/calmodulindependent protein kinase II signaling in SK-N-SH neuronal cells, as previously found with sera from SC and related neuropsychiatric disorders. Our new animal model translates directly to human disease and led us to discover autoantibodies targeted against dopamine D1 and D2 receptors in the rat model as well as in SC and other streptococcal-related neuropsychiatric disorders.
Gprc5a functions as a tumor suppressor in mouse lung, and human GPRC5A may share this property. The Gprc5a-deficient mouse is a novel model to study lung carcinogenesis and chemoprevention.
Purpose: Retinoic acid receptor- 2 (RAR- 2 ) expression is suppressed in oral premalignant lesions and head and neck squamous cell carcinomas (HNSCCs). This study was conducted to determine whether RAR- 2 gene expression in such lesions can be silenced by promoter methylation.Experimental Design: RAR- 2 methylation was analyzed in DNA samples from 22 pairs of primary HNSCC and adjacent normal epithelium, 124 samples of oral leukoplakia, and 18 HNSCC cell lines using methylation-specific PCR. RAR- 2 promoter was methylated in 67, 56, and 53% of HNSCC tumors, HNSCC cell lines, and microdissected oral leukoplakia specimens, respectively. RAR- 2 hypermethylation was confirmed by sodium bisulfite-PCR combined with restriction enzyme digestion analysis and by random cloning and sequencing of bisulfite-treated DNA isolates.Results: Significantly higher RAR- 2 hypermethylation levels were found in tumor tissue compared with adjacent normal tissue (P ؍ 0.002). RAR- 2 methylation in the cell lines was correlated with loss of RAR- 2 expression (P ؍ 0.013) and inversely related to the presence of mutated p53 (P ؍ 0.025). The demethylating agent 5-aza-2-deoxycytidine (5-aza-CdR) restored RAR- 2 inducibility by all-trans-retinoic acid (ATRA) in some of the cell lines, which posses a methylated RAR- 2 promoter. In some cell lines, this effect was associated with increased growth inhibition after combined treatment with 5-aza-CdR and ATRA.Conclusions: RAR- 2 silencing by methylation is an early event in head and neck carcinogenesis; 5-Aza-CdR can restore RAR- 2 inducibility by ATRA in most cell lines, and the combination of 5-aza-CdR and ATRA is more effective in growth inhibition than single agents.
Mouse models can be useful for increasing the understanding of lung tumorigenesis and assessing the potential of chemopreventive agents. We explored the role of inflammation in lung tumor development in mice with knockout of the tumor suppressor Gprc5a. Examination of normal lung tissue and tumors from 51 Gprc5a+/+ (adenoma incidence 9.8%; adenocarcinoma 0%) and 38 Gprc5a−/− mice (adenoma 63%, adenocarcinoma 21%) revealed macrophages infiltration into lungs of 45% of the Gprc5a−/− mice and 8% of Gprc5a+/+ mice and the direct association of macrophages with 42% of adenomas and 88% of adenocarcinomas in the knockout mice. Gprc5a−/− mouse lungs contained higher constitutive levels of proinflammatory cytokines and chemokines and were more sensitive than lungs of Gprc5a+/+ mice to stimulation of NF-κB activation by lipopolysaccharide (LPS) in vivo. Studies with epithelial cells cultured from tracheas of Gprc5a−/− and Gprc5a+/+ mice revealed that Gprc5a loss is associated with increased cell proliferation, resistance to cell death in suspension, and increased basal, TNFα-induced, and LPS-induced NF-κB activation, which were reversed partially in Gprc5a−/− adenocarcinoma cells by re-expression of Gprc5a. Compared to Gprc5a+/+ cells, the Gprc5a−/− cells produced higher levels of chemokines and cytokines and their conditioned medium induced more extensive macrophage migration. Silencing Gprc5a and the p65 subunit of NF-κB in Gprc5a+/+ and Gprc5a−/− cells, respectively reversed these effects. Thus, Gprc5a−/− loss enhances NF-κB activation in lung epithelial cells leading to increased autocrine and paracrine interactions, cell autonomy and enhanced inflammation, which may synergize in the creation of a tumor promoting microenvironment.
Lactoside-binding lectins (galectins) with molecular weights of about 14.5 kDa (galectin-1) and 29-35 kDa (galectin-3) bind preferentially to polylactosaminoglycan-containing glycoconjugates and have been found on the surface of tumour cells and implicated in cell-cell and cell-extracellular matrix adhesion and metastasis. We have demonstrated by immunoblotting that both galectin-1 and galectin-3 are present in extracts of endothelial cells cultured from bovine aorta, rat lung, mouse lung and mouse brain microvessels, whereas mouse hepatic sinusoidal endothelial cells expressed primarily galectin-1. These galectins were also localized by indirect immunofluorescent labelling on the surface of the different endothelial cells in culture and by immunohistochemical staining in human tissues in vivo. Anti-galectin-1 antibodies inhibited the adhesion of liver-preferring murine RAW117-H10 large-cell lymphoma cells to hepatic sinusoidal endothelial cells or lung microvessel endothelial cells in vitro. The data indicate that galectin-1 is expressed on the extracellular surface of endothelial cells and can mediate in part the adhesion of RAW117-H10 cells to liver microvessel endothelial cells.
Group A β-hemolytic streptococcal (GAS) infection is associated with a spectrum of neuropsychiatric disorders. The leading hypothesis regarding this association proposes that a GAS infection induces the production of auto-antibodies, which cross-react with neuronal determinants in the brain through the process of molecular mimicry. We have recently shown that exposure of rats to GAS antigen leads to the production of anti-neuronal antibodies concomitant with the development of behavioral alterations. The present study tested the causal role of the antibodies by assessing the behavior of naïve rats following passive transfer of purified antibodies from GAS-exposed rats. Immunoglobulin G (IgG) purified from the sera of GAS-exposed rats was infused directly into the striatum of naïve rats over a 21-day period. Their behavior in the induced-grooming, marble burying, food manipulation and beam walking assays was compared to that of naïve rats infused with IgG purified from adjuvant-exposed rats as well as of naïve rats. The pattern of in vivo antibody deposition in rat brain was evaluated using immunofluorescence and colocalization. Infusion of IgG from GAS-exposed rats to naïve rats led to behavioral and motor alterations partially mimicking those seen in GAS-exposed rats. IgG from GAS-exposed rats reacted with D1 and D2 dopamine receptors and 5HT-2A and 5HT-2C serotonin receptors in vitro. In vivo, IgG deposits in the striatum of infused rats colocalized with specific brain proteins such as dopamine receptors, the serotonin transporter and other neuronal proteins. Our results demonstrate the potential pathogenic role of autoantibodies produced following exposure to GAS in the induction of behavioral and motor alterations, and support a causal role for autoantibodies in GAS-related neuropsychiatric disorders.
The expression of lactoside-binding lectin L-31 was analyzed in normal mucosa and in primary and metastatic gastric carcinomas. Immunoblotting revealed L-31 lectin in extracts of normal and malignant gastric tissues from 26 patients. The L-31 level was higher in tumor than in normal tissue in 9/26 cases, similar in 14/26 cases, and lower in 3/26 cases. Anti-L-31 monoclonal antibodies (MAbs) were used in immunohistochemical analyses to compare lectin expression in specimens of primary gastric carcinomas and adjacent normal mucosa from 39 patients and in specimens of metastases and the corresponding primary gastric carcinomas from 74 patients. The lectin was detected in normal gastric epithelial cells and in all gastric carcinoma specimens, albeit in varying amounts. The L-31 level was significantly higher in the primary tumor than in adjacent normal tissue in 55% of the well-differentiated tubular carcinoma cases and in 50% of stage-III and -IV tumors. L-31 expression in liver metastases from well-differentiated tubular primary gastric carcinomas was higher in 31% of the cases relative to the corresponding primary cancers. Likewise, L-31 expression in metastases from poorly differentiated gastric carcinomas in lymph nodes was higher in 38% of the cases compared to the primary cancers. The higher expression of L-31 in primary cancers and metastases of certain types implicates this lectin in the metastatic phenotype, but the presence of L-31 in a primary cancer is not sufficient to allow the metastatic propensity of the tumor to be predicted.
Lung cancer continues to be a major deadly malignancy. The mortality of this disease could be reduced by improving the ability to predict cancer patients' survival. We hypothesized that genes differentially expressed among cells constituting an in vitro human lung carcinogenesis model consisting of normal, immortalized, transformed, and tumorigenic bronchial epithelial cells are relevant to the clinical outcome of non–small cell lung cancer (NSCLC). Multidimensional scaling, microarray, and functional pathways analyses of the transcriptomes of the above cells were done and combined with integrative genomics to incorporate the microarray data with published NSCLC data sets. Up-regulated (n = 301) and down-regulated genes (n = 358) displayed expression level variation across the in vitro model with progressive changes in cancer-related molecular functions. A subset of these genes (n = 584) separated lung adenocarcinoma clinical samples (n = 361) into two clusters with significant survival differences. Six genes, UBE2C, TPX2, MCM2, MCM6, FEN1, and SFN, selected by functional array analysis, were also effective in prognosis. The mRNA and protein levels of one these genes—UBE2C—were significantly up-regulated in NSCLC tissue relative to normal lung and increased progressively in lung lesions. Moreover, stage I NSCLC patients with positive UBE2C expression exhibited significantly poorer overall and progression-free survival than patients with negative expression. Our studies with this in vitro model have lead to the identification of a robust six-gene signature, which may be valuable for predicting the survival of lung adenocarcinoma patients. Moreover, one of those genes, UBE2C, seems to be a powerful biomarker for NSCLC survival prediction.
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