RESUMO
Objectives: To examine the epidemiology of ß-lactam resistance in 'clonal group 258' (CG258), a successful KPC clonal group, over 14 years. Methods: Isolates were collected from 1999 to 2013 for a study of antibiotic resistance in Enterobacteriaceae in New York City; 515 bloodstream isolates had antibiotic susceptibility data available and 436 were available for a CG258 PCR assay. The 56 resulting CG258 isolates were characterized by MLST, capsular type and ESBL and KPC carriage. KPC-positive isolates were assessed for common KPC plasmid types, KPC subtype and Tn4401 isoform. Results: RT-PCR revealed 56 isolates were CG258. Seventeen of the 56 CG258 isolates were phenotypically susceptible to all carbapenems (all KPC negative). Five out of 17 susceptible isolates were of the cps-2 (wzi154) capsule type; none was cps-1 (wzi29). Nineteen out of 28 KPC-2 isolates were cps-1 (wzi29) and 8/10 KPC-3 isolates carried cps-2 (wzi154); however, cps-2 (wzi154) predominated among KPC-2-positive isolates in 2003 and 2004. KPC-2 was first detected in 2003 and KPC-3 was first detected in 2006. KPC-harbouring plasmids pKpQIL (all Tn4401a) and pBK30683 (all Tn4401d) were detected in 16/38 and 6/38 carbapenem-resistant isolates, respectively. Discussion: CG258-lineage Klebsiella pneumoniae isolates were completely absent in 1999, but common in 2003. Twenty-one percent of CG258 isolates were susceptible to carbapenems in addition to lacking both common ESBL and blaKPC-mediated resistance. The cps-2 (wzi154) capsule type was common in both these susceptible isolates and in early KPC-2-harbouring isolates, suggesting it was the initial capsule type in CG258. Carbapenem-resistant isolates carried common KPC-harbouring plasmids with the same KPC and Tn4401 isoforms, suggesting frequent clonal spread.
Assuntos
Enterobacteriáceas Resistentes a Carbapenêmicos/efeitos dos fármacos , Enterobacteriáceas Resistentes a Carbapenêmicos/isolamento & purificação , Epidemias , Infecções por Klebsiella/epidemiologia , Klebsiella pneumoniae/efeitos dos fármacos , Klebsiella pneumoniae/isolamento & purificação , Resistência beta-Lactâmica , Enterobacteriáceas Resistentes a Carbapenêmicos/classificação , Enterobacteriáceas Resistentes a Carbapenêmicos/genética , Humanos , Klebsiella pneumoniae/classificação , Klebsiella pneumoniae/genética , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Cidade de Nova Iorque/epidemiologia , Plasmídeos/análise , Reação em Cadeia da Polimerase , Estudos Retrospectivos , beta-Lactamases/genéticaRESUMO
Hereditary transthyretin amyloidosis with polyneuropathy (ATTR-PN) results from specific TTR gene mutations. In this study, we generated two induced pluripotent stem cell (iPSC) lines derived from ATTR-PN patients with heterozygous TTR gene mutations (Ala97Ser and Phe64Leu). These iPSC lines exhibited normal morphology, karyotype, high pluripotency marker expression, and differentiation into cells representing all germ layers. The generation of these iPSC lines serve as a valuable tool for investigating the mechanisms of ATTR-PN across various cell types and facilitating patient-specific in vitro amyloidosis modeling.
Assuntos
Neuropatias Amiloides Familiares , Células-Tronco Pluripotentes Induzidas , Polineuropatias , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Pré-Albumina/genética , Pré-Albumina/metabolismo , Neuropatias Amiloides Familiares/genética , Neuropatias Amiloides Familiares/metabolismo , Polineuropatias/genética , Polineuropatias/metabolismo , Mutação/genéticaRESUMO
We generated two induced pluripotent stem cell (iPSC) lines from peripheral blood mononuclear cells (PBMCs) of breast cancer patients carrying germline ATM mutations, a gene associated with a 7% prevalence in breast cancer. These iPSC lines displayed typical morphology, expressed pluripotency markers, maintained a stable karyotype, and retained the ability to differentiate into the three germ layers. These patient-specific iPSC lines hold great potential for mechanistic investigations and the development of drug screening strategies aimed at addressing ATM-related cancer.
Assuntos
Neoplasias da Mama , Células-Tronco Pluripotentes Induzidas , Humanos , Feminino , Células-Tronco Pluripotentes Induzidas/metabolismo , Neoplasias da Mama/genética , Leucócitos Mononucleares/metabolismo , Mutação/genética , Mutação em Linhagem Germinativa , Proteínas Mutadas de Ataxia Telangiectasia/genéticaRESUMO
Dilated cardiomyopathies (DCM) are one of the main causes of heart failure as one ages. BAG3 is a chaperone protein that is heavily implicated in the development of DCM and speed of progression toward heart failure. Here we generate two human iPSC lines from individuals with mutations in exon 3 of BAG3 and provide validation of their pluripotency and ability to differentiate toward the three primary germ layers. These two cell lines can help our understanding of BAG3 and its role in DCM by providing a good model for BAG3 inactivation and insufficiency.
Assuntos
Cardiomiopatia Dilatada , Insuficiência Cardíaca , Células-Tronco Pluripotentes Induzidas , Humanos , Cardiomiopatia Dilatada/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteínas Reguladoras de Apoptose/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Mutação , Éxons , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
Germline pathogenic variants in the BRCA2 gene are strongly correlated with an elevated risk of developing breast cancer. Two specific BRCA2 variants, c.8167G>C (p.Asp2723His) and c.1583del (p.Asn528fs), have been identified from individuals with a family history of breast cancer. Here we generated two iPSC lines from breast cancer patients who are heterozygous carriers of these two variants. These iPSCs exhibit pluripotency and demonstrate the capability to differentiate into three germ layers. These iPSC lines represent a valuable resource for personalized pre-clinical research, offering new opportunities to explore the underlying mechanisms of breast cancer and develop targeted therapeutic approaches.
Assuntos
Neoplasias da Mama , Células-Tronco Pluripotentes Induzidas , Humanos , Feminino , Neoplasias da Mama/genética , Genes BRCA2 , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação em Linhagem Germinativa , Mutação , Proteína BRCA2/genética , Proteína BRCA2/metabolismoRESUMO
The strength of the learned relation between two events, a model for causal perception, has been found to depend on their overall statistical relation, and might be expected to be related to both training trial frequency and trial duration. We report five experiments using a rapid-trial streaming procedure containing Event 1-Event 2 pairings (A trials), Event 1-alone (B trials), Event 2-alone (C trials), and neither event (D trials), in which trial frequencies and durations were independently varied. Judgements of association increased with increasing frequencies of A trials and decreased with increasing frequencies of both B and C trials but showed little effect of frequency of D trials. Across five experiments, a weak but often significant effect of trial duration was also detected, which was always in the same direction as trial frequency. Thus, both frequency and duration of trials influenced learning, but frequency had decidedly stronger effects. Importantly, the benefit of more trials greatly outweighed the observed reduction in effect size caused by a proportional decrease in trial duration. In experiment 5, more trials of proportionately shorter duration enhanced effects on contingency judgments despite a shortening of the training session. We consider the observed 'frequency advantage' with respect to both frequentist models of learning and models based on information. (PsycInfo Database Record (c) 2022 APA, all rights reserved).
Assuntos
Julgamento , Aprendizagem , HumanosRESUMO
Vasoactive Intestinal Peptide (VIP) is the major physiological agonist of the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) chloride channel activity. VIP functions as a neuromodulator and neurotransmitter secreted by neurons innervating all exocrine glands. VIP is also a potent vasodilator and bronchodilator that regulates exocrine gland secretions, contributing to local innate defense by stimulating the movement of water and chloride transport across intestinal and tracheobronchial epithelia. Previous human studies have shown that the rich intrinsic neuronal networks for VIP secretion around exocrine glands could be lost in tissues from patients with cystic fibrosis. Our research has since confirmed, in vitro and in vivo, the need for chronic VIP exposure to maintain functional CFTR chloride channels at the cell surface of airways and intestinal epithelium, as well as normal exocrine tissues morphology [1]. The goal of the present study was to examine changes in VIP in the lung, duodenum and sweat glands of 8- and 17-weeks old F508del/F508del mice and to investigate VIPergic innervation in the small intestine of CF mice, before important signs of the disease development. Our data show that a low amount of VIP is found in CF tissues prior to tissue damage. Moreover, we found a specific reduction in VIPergic and cholinergic innervation of the small intestine. The general innervation of the primary and secondary myenteric plexus was lost in CF tissues, with the presence of enlarged ganglionic cells in the tertiary layer. We propose that low amount of VIP in CF tissues is due to a reduction in VIPergic and cholinergic innervation and represents an early defect that constitutes an aggravating factor for CF disease progression.
Assuntos
Fibrose Cística/metabolismo , Duodeno/inervação , Duodeno/metabolismo , Pulmão/inervação , Pulmão/metabolismo , Glândulas Sudoríparas/inervação , Glândulas Sudoríparas/metabolismo , Peptídeo Intestinal Vasoativo/biossíntese , Animais , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
The CFTR chloride channel is regulated by phosphorylation at PKA and PKC consensus sites within its regulatory region (R-region) through a mechanism, which is still not completely understood. We used a split-CFTR construct expressing the N-term-TMD1-NBD1 (Front Half; FH), TMD2-NBD2-C-Term (Back Half; BH), and the R-region as separate polypeptides (Split-R) in BHK cells, to investigate in situ how different phosphorylation conditions affect the R-region interactions with other parts of the protein. In proximity ligation assays, we studied the formation of complexes between the R-region and each half of the Split-CFTR. We found that at basal conditions, the density of complexes formed between the R-region and both halves of the split channel were equal. PKC stimulation alone had no effect, whereas PKA stimulation induced the formation of more complexes between the R-region and both halves compared to basal conditions. Moreover, PKC + PKA stimulation further enhanced the formation of FH-R complexes by 40% from PKA level. In cells expressing the Split-R with the two inhibitory PKC sites on the R-region inactivated (SR-S641A/T682A), density of FH-R complexes was much higher than in Split-R WT expressing cells after PKC or PKC + PKA stimulation. No differences were observed for BH-R complexes measured at all phosphorylation conditions. Since full-length CFTR channels display large functional responses to PKC + PKA in WT and S641A/T682A mutant, we conclude that FH-R interactions are important for CFTR function. Inactivation of consensus PKC site serine 686 (S686A) significantly reduced the basal BH-R interaction and prevented the PKC enhancing effect on CFTR function and FH-R interaction. The phospho-mimetic mutation (S686D) restored basal BH-R interaction and the PKC enhancing effect on CFTR function with enhanced FH-R interaction. As the channel function is mainly stimulated by PKA phosphorylation of the R-region, and this response is known to be enhanced by PKC phosphorylation, our data support a model in which the regulation of CFTR activation results from increased interactions of the R-region with the N-term-TMD1-NBD1. Also, serine S686 was found to be critical for the PKC enhancing effect which requires a permissive BH-R interaction at basal level and increased FH-R interaction after PKC + PKA phosphorylation.
RESUMO
Inhibition of cyclooxygenase (COX)-2 is reported to suppress growth and induce apoptosis in human esophageal adenocarcinoma (EADC) cells, although the precise biologic mechanism is unclear. In this study we tested the hypothesis that the antitumor activity of COX-2 inhibitors may involve modulation of basic fibroblast growth factor (FGF-2), which is overexpressed in EADC. We evaluated the effects of NS-398, a selective COX-2 inhibitor, on FGF-2 expression and proliferation of EADC cell lines that express COX-2 and those that do not. We also correlated COX-2 and FGF-2 expression with clinico-pathologic findings and outcome in a well-characterized series of surgically resected EADC tissues. Seg-1 cells robustly expressed COX-2 and FGF-2, whereas Bic-1 cells expressed neither transcript. FGF-2 was reduced to undetectable levels in Seg-1 cells following NS-398 treatment, but increased within 4 h of drug removal. NS-398 significantly inhibited the growth of Seg-1 cells, and this effect was ameliorated by addition of exogenous FGF-2. In contrast, NS-398 had no effect on Bic-1 cell proliferation and FGF-2 alone had no effect on proliferation of either cell line. NS-398, or a neutralizing anti-FGF-2 antibody, induced apoptosis in Seg-1 cells, and these effects were inhibited by addition of exogenous FGF-2. COX-2 protein was strongly expressed in 46% (10/22) of EADCs, and was associated with a trend towards reduced disease-free survival. These findings indicate that the antitumor effects of COX-2 inhibition in EADC cells may be mediated via suppression of FGF-2, and that COX-2 may be a clinically relevant molecular marker in the management of human EADC.
Assuntos
Adenocarcinoma/enzimologia , Ciclo-Oxigenase 2/química , Inibidores de Ciclo-Oxigenase/farmacologia , Neoplasias Esofágicas/enzimologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Nitrobenzenos/farmacologia , Sulfonamidas/farmacologia , Adenocarcinoma/tratamento farmacológico , Adenocarcinoma/patologia , Idoso , Apoptose , Western Blotting , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ciclo-Oxigenase 2/biossíntese , Ciclo-Oxigenase 2/genética , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/patologia , Feminino , Fator 2 de Crescimento de Fibroblastos/genética , Imunofluorescência , Humanos , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
Fibroblast growth factor-2 (FGF-2) is a potent heparin-binding protein with growth-promoting and anti-apoptotic activity. Transcription of the GFG/NUDT6 gene on the opposite DNA strand generates an overlapping antisense RNA (FGF-AS) implicated in the post-transcriptional regulation of FGF-2. C6 glioma cells coordinately express FGF-2 and FGF-AS mRNA in a cell cycle-dependent manner. Cellular FGF-2 immunoreactivity was also cell cycle-dependent, with marked nuclear accumulation during S-phase. Stable transfection and overexpression of the FGF-AS RNA resulted in suppression of total cellular FGF-2, and a reduction in nuclear accumulation of FGF-2 isoforms. Serum stimulation of growth-arrested wild-type cells evoked a rapid nuclear translocation of FGF-2, and cell cycle re-entry. FGF-AS transfectants, in contrast, showed a significant delay in recovery of both nuclear FGF-2 staining and S-phase re-entry. Similar results were observed when cells were released from aphidicolin-induced G1 arrest or subjected to heat shock. These findings indicate that FGF-AS RNA inhibits expression and cell cycle-dependent nuclear accumulation of FGF-2, and this is associated with a marked delay in S-phase progression. The results suggest that the endogenous FGF antisense RNA may play a significant functional role in the regulation of FGF-2 dependent cell proliferation in FGF-2 expressing cells.
Assuntos
Ciclo Celular , Núcleo Celular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/genética , Glioma/patologia , RNA Antissenso/genética , Animais , Fator 2 de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/metabolismo , Expressão Gênica , Regulação da Expressão Gênica , Transporte Proteico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Soro , Frações SubcelularesRESUMO
PURPOSE: The basic fibroblast growth factor (FGF-2) gene is bidirectionally transcribed to generate overlapping sense and antisense (FGF-AS) mRNAs. FGF-AS has been implicated in the post-transcriptional regulation of FGF-2 expression. The aim of this study was to characterize FGF-2 and FGF-AS in esophageal cancer and to correlate their expression with clinicopathologic findings and outcome. EXPERIMENTAL DESIGN: Reverse transcription-PCR was used to study FGF-2 and FGF-AS mRNA expression (normalized to glyceraldehyde-3-phosphate dehydrogenase) in 48 esophageal cancers relative to matched histologically normal esophageal epithelia (internal control). We used Cox proportional hazards analysis to calculate hazard ratios for recurrence and survival of patients with underexpression relative to the overexpression of FGF-2 and/or FGF-AS. RESULTS: Overexpression of FGF-2 mRNA, by comparison with tumors underexpressing FGF-2, was associated with significantly increased risk for tumor recurrence (hazard ratio, 3.80; 95% confidence interval, 1.64-8.76) and reduced overall survival (hazard ratio, 2.11; 95% confidence interval, 1.0-4.58). When the effects of FGF-2 and FGF-AS were considered simultaneously, the association of FGF-2 mRNA overexpression with recurrence and mortality was even more pronounced, whereas FGF-AS mRNA overexpression was associated with reduced risk for recurrence and improved survival. CONCLUSIONS: Overexpression of FGF-2 mRNA is associated with tumor recurrence and reduced survival after surgical resection of esophageal cancer and that these risks are reduced in tumors coexpressing the FGF-AS mRNA. These data support the hypothesis that FGF-AS is a novel tumor suppressor that modulates the effect of FGF-2 expression and may have potential clinical application to the development of novel therapeutic strategies.
Assuntos
Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Fator 2 de Crescimento de Fibroblastos/biossíntese , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica , Oligonucleotídeos Antissenso/farmacologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Diferenciação Celular , Linhagem Celular Tumoral , Intervalo Livre de Doença , Neoplasias Esofágicas/mortalidade , Feminino , Fator 2 de Crescimento de Fibroblastos/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Microscopia Confocal , Microscopia de Fluorescência , Pessoa de Meia-Idade , Análise Multivariada , Modelos de Riscos Proporcionais , Processamento Pós-Transcricional do RNA , RNA Mensageiro/metabolismo , Recidiva , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Fatores de Tempo , Resultado do TratamentoRESUMO
Fibroblast growth factor-2 (FGF-2) is a growth and survival factor whose expression is elevated in many hematopoietic malignancies. A natural antisense RNA (FGF-AS) has been implicated in the posttranscriptional regulation of FGF-2 mRNA expression. We demonstrate for the first time that FGF sense and antisense RNAs are coordinately expressed and translated in hematopoietic cells and tissues. Cytokine stimulation of growth-arrested K562 cells elicited a rapid transient increase in FGF-AS mRNA expression followed by a slower but sustained increase in FGF-2 mRNA. This was accompanied by a marked increase in the expression and nuclear translocation of FGF-2 and the FGF-AS encoded protein, GFG/NUDT6. These findings suggest a role for both FGF-2 and GFG proteins in the cell survival and proliferation of lymphoid and myeloid tumor cells.
Assuntos
Fator 2 de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/genética , Leucemia/genética , Processamento Alternativo , Animais , Linhagem Celular Tumoral , Sobrevivência Celular , Primers do DNA , Humanos , Leucemia/patologia , Prolactina/farmacologia , RNA Mensageiro/genética , Ratos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transcrição Gênica , Células Tumorais CultivadasRESUMO
In an attempt to develop an animal model for thyroid-associated ophthalmopathy (TAO) we have genetically immunized BALB/c and outbred (CD-1) mice with cDNAs encoding the thyroid and eye muscle shared protein G2s and full length human thyrotropin receptor (TSHr). Firstly, BALB/c mice were immunized with cDNAs for G2s and the TSHr, alone or in tandem with cDNAs for interleukin (IL)4 or IL12. Control mice were immunized with empty vehicle only. Sera from the great majority of experimental mice contained antibodies against a G2s fusion protein and the flavoprotein (Fp) subunit of mitochondrial succinate dehydrogenase, the "64 kDa protein", with the greatest levels being found at sacrifice (17 wk). Antibody levels in mice immunized with G2s + TSHr or G2s + IL12 were generally higher than those in mice immunized with G2s only. TSHr antibodies (TRAb), measured as TSH binding inhibition, were detected in only two mice. On histological examination of the orbits, mild edema, eye muscle fiber separation and mast cell infiltration in and around the eye muscles were found in the majority of experimental mice, but not in control mice. Splenocytes were transferred from selected G2s-immunized mice to normal syngeneic litter mates. None of the transfer mice had serum antibodies against G2s, Fp or TSHr but their orbital tissue showed the same degree of mast cell infiltration as primary mice. No major histological changes were observed in the thyroid or other skeletal muscle in either primary or transfer mice. Similar results were observed in CD-1 mice although, overall, the model was better expressed than in BALB/c mice. In these mice, serum anti-G2s antibody levels were not significantly different between the various experimental groups except at 16 wk, when they were slightly greater than in control animals. Anti-Fp antibodies were detected at 12, 14 and 16 wk, in all experimental groups, including those immunized with G2s only, and were greatest in mice immunized with TSHr alone. TRAb levels were greatest in mice immunized with both G2s and the TSHr in the presence of TL4, but not IL12. The finding of negative anti-G2s but positive anti-Fp antibodies in some CD-1 mice suggests that eye muscle damage and Fp release must have been mediated by T lymphocytes, rather than antibodies, targeting G2s or some other as yet unidentified cell membrane antigen. Histological changes in the orbit were similar to those observed in BALB/c mice although mast cell numbers were greater, in both primary and transfer mice. Overall, the greatest histological changes were observed in CD-1 mice immunized with both G2s + TSHr + IL4. None of the animals became overtly hyperthyroid or hypothyroid during the course of the study although several of the CD-1 mice had abnormal TSH or T4 levels. These results indicate that we have established a valid model for human ophthalmopathy using the novel thyroid and eye muscle expressed protein G2s, now recognized as a fragment of the winged-helix transcription factor Foxp1, and TSHr, and that G2s and the TSHr are both primary antigens in TAO. Reactivity against a TSHr-like protein may be the first event leading to ophthalmopathy in humans with TAO and experimental mice and eye muscle damage may result from autoimmunity against G2s and Fp as a result of "antigen spreading".
Assuntos
Modelos Animais de Doenças , Proteínas do Olho/imunologia , Doença de Graves/imunologia , Proteínas de Membrana/imunologia , Receptores da Tireotropina/imunologia , Animais , Anticorpos/sangue , Proteínas do Olho/genética , Feminino , Humanos , Proteínas de Membrana/genética , Camundongos , Camundongos Endogâmicos BALB C , Órbita/citologia , Órbita/patologia , Receptores da Tireotropina/genética , Tiroxina/sangue , Vacinas de DNA/imunologiaRESUMO
While many studies have shown a connection between stress and autoimmune disease, most of the evidence for stress contributing to the onset and course of autoimmune disease is circumstantial and the mechanisms by which stress affects autoimmune disease are not fully understood. The best circumstantial evidence for an effect of stress on autoimmune thyroid disease is the well-known relationship between the onset of Graves' hyperthyroidism and major stress but even this is debated. However, most of the recent case-control studies have supported stress as a factor that affects the onset and clinical course of Graves' disease. On the other hand, there have been few reports concerning the possible relationship between stress and Hashimoto's thyroiditis. Because the onset and course of Hashimoto's thyroiditis is generally insidious, the effect of stress on Hashimoto's thyroiditis might be overlooked. Numerous human and animal studies have demonstrated that psychological and physiologic stressors induce various immunologic changes. Stress affects the immune system either directly or indirectly through the nervous and endocrine systems. These immune modulations may contribute to the development of autoimmunity as well as the susceptibility to autoimmune disease in genetically predisposed individuals. Stress can be one of the environmental factors for thyroid autoimmunity.
Assuntos
Estresse Fisiológico/complicações , Estresse Fisiológico/imunologia , Tireoidite Autoimune/etiologia , Tireoidite Autoimune/imunologia , Animais , Progressão da Doença , Doença de Graves/etiologia , Doença de Graves/imunologia , Humanos , Sistemas Neurossecretores/patologiaRESUMO
The eye changes associated with Graves' hyperthyroidism can be classified into two subtypes, congestive ophthalmopathy (CO), in which inflammatory changes in the periorbital tissues predominate, and ocular myopathy (OM), in which eye muscle damage is the main feature. Antibodies against the flavoprotein (Fp) subunit of succinate dehydrogenase (SDH), the 64-kd protein, and G2s, a thyroid and eye muscle shared protein of unknown function, are good markers of eye muscle cell damage in patients with OM. Another antigen associated with ophthalmopathy is the flavine adenine nucleotide (FAD) cofactor of several mitochondrial enzymes, including SDH. We tested for serum antibodies against purified human recombinant Fp, FAD, and a G2s fusion protein, in patients with thyroid-associated ophthalmopathy (TAO) and control patients and subjects, in enzyme-linked immunosorbent assay. Antibodies against Fp were detected in 32% of patients with TAO, 30% with Graves' hyperthyroidism (GH), 16% with Hashimoto's thyroiditis (HT), in 14% of patients with multi-nodular goiter (MNG), and in 6% of normal subjects. Antibodies against FAD were found in 24%, 30%, 24%, and 14%, respectively, of these patients and in 12% of the normals, while antibodies against G2s were detected in 50% of patients with TAO, 40% with GH, 40% with HT, in 29% of patients with MNG, and in 7% of normals. We also tested for antibodies against SDH, FAD, and G2s in 12 patients with GH who developed CO (6 patients) or OM (6 patients) after treatment with antithyroid drugs. Of the 6 patients who developed OM, antibodies against SDH preceded the onset of eye disease in 4 and coincided with it in 2, antibodies against G2s preceded eye muscle disease in 5 and coincided with it in 1 patient while antibodies against FAD preceded the development of OM in 5 patients. Of the 6 patients who developed CO, antibodies against SDH were detected in only one patient and borderline levels were demonstrated in 1, while anti-FAD and anti-G2s each preceded the onset of eye signs in 6 patients. Positive sera from another group of patients with TAO, and a second group of normal subjects, were tested at increasing serum dilutions. Sera from the two groups showed similar dilution patterns, except for a few patients with TAO in whom increasing dilutions was associated with increased, then decreased, antibody levels. In this experiment the prevalences of the two antibodies were much greater in patients with TAO namely, 67% for anti-Fp and 89% for anti-G2s, while the prevalences in the normals were 11% and 22%, respectively. The reason for this apparent discrepancy is not clear but may reflect subject and assay differences. Because Fp is found within the mitochondrial membrane it is likely that the corresponding antibodies are produced after eye muscle necrosis, and do not play a role in its pathogenesis. The primary reaction in the eye muscle may be T-cell autoimmunity against G2s, although this has not been proven. The mechanism for the production of antibodies against G2s, FAD, and Fp in subjects who do not have ophthalmopathy is unclear. The significance of such antibodies in control subjects is presently being addressed in our laboratory.
Assuntos
Autoanticorpos/sangue , Proteínas do Olho , Doença de Graves/imunologia , Músculos Oculomotores/imunologia , Adulto , Idoso , Biomarcadores , Feminino , Bócio Nodular/imunologia , Humanos , Masculino , Proteínas de Membrana/imunologia , Pessoa de Meia-Idade , Succinato Desidrogenase/imunologia , Tireoidite Autoimune/imunologiaRESUMO
We assembled a new set of mRNA sequences from the leg hypodermis transcriptome of the wandering spider, Cupiennius salei. Each sequence was assembled to exhaustion in the 5' direction to detect all upstream open reading frames (uORFs) both in-frame and out-of-frame with the main open reading frame (mORF). We also counted nucleotide probabilities before and after the START codon of the mORF to establish the optimum Kozak consensus sequence. More than 80% of 5' sequences had uORFs before the mORF with a range of 1-16 uORFs. Kozak consensus strengths of uORFs were significantly weaker than mORFs. Random scrambling of 5' nucleotide positions did not give significantly different numbers, sizes, or Kozak consensus strengths of uORFs. Random simulations of 5' sequences using either equal or experimental distributions of nucleotides gave similar numbers of uORFs, with similar sizes and Kozak consensus strengths to experimental data. Abundance of mRNA for each gene was estimated by counting matching Illumina reads to assembled genes. Abundance was negatively correlated with numbers of uORFs, but not with 5' length. Our data are compatible with a random model of 5' mRNA sequence structure.
Assuntos
Regiões 5' não Traduzidas/genética , Códon de Iniciação/genética , Sequência Conservada/genética , Fases de Leitura Aberta/genética , Seleção Genética , Aranhas/genética , Transcriptoma/genética , Animais , Simulação por Computador , Evolução MolecularRESUMO
In clinical studies, skeletal myoblast (SKMB) transplantation late after myocardial infarction (MI) has minimal impact on left ventricular (LV) function. This may be related to our previous observation that the extent of SKMB engraftment is minimal in chronic MI when compared to acute MI, which correlates with decreased hepatocyte growth factor (HGF) expression, an important regulator of SKMB function. Here, we investigated delivery of exogenous HGF as a strategy for augmenting SKMB engraftment late after MI. Rats underwent SKMB transplantation 4 weeks after coronary ligation. HGF or vehicle control was delivered intravenously during the subsequent 2 weeks. LV function was assessed by MRI before and 2 weeks after SKMB transplantation. We evaluated HGF delivery, SKMB engraftment, and expression of genes associated with post-MI remodeling. Serum HGF was 6.2 ± 2.4 ng/mL after 2 weeks of HGF infusion (n = 7), but undetectable in controls (n = 7). LV end-diastolic volume and ejection fraction did not improve with HGF treatment (321 ± 27 mm(3), 42% ± 2% vs. 285 ± 33 mm(3), 43% ± 2%, HGF vs. control). MIs were larger in HGF-treated animals (50 ± 7 vs. 30 ± 6 mm(3), P = 0.046), but the volume of engrafted SKMBs or percentage of MIs occupied by SKMBs did not increase with HGF (1.7 ± 0.3 mm(3), 4.7% ± 1.9% vs. 1.4 ± 0.4 mm(3), 5.3% ± 1.6%, HGF vs. control). Expression of genes associated with post-infarction remodeling was not altered by HGF. Delivery of exogenous HGF failed to augment SKMB engraftment and functional recovery in chronic MI. Expression of genes associated with LV remodeling was not altered by HGF. Alternative strategies to enhance engraftment of SKMB must be explored to optimize the clinical efficacy of SKMB transplantation.
RESUMO
BACKGROUND: In a recent clinical trial, skeletal myoblast (SKMB) transplantation performed late after myocardial infarction (MI) did not improve left ventricular function. We hypothesized that (1) delaying SKMB transplantation until a chronic infarct scar has developed reduces engraftment, and (2) hepatocyte growth factor (HGF), a main regulator of SKMBs, is present in acute but not chronic MI, potentially influencing engraftment. METHODS: Rats underwent coronary artery ligation followed by SKMB transplantation immediately (n = 12) or delayed by 5 weeks (n = 11). The volume of engrafted SKMBs was quantified 6 weeks later. Hepatocyte growth factor was evaluated by computerized analysis of immunohistochemical labeling of rat heart sections 48 hours, 1 week, 2 weeks, and 5 weeks after coronary artery ligation. The impact of HGF on SKMB proliferation and its ability to protect against oxidative stress and hypoxia was evaluated in vitro. RESULTS: Skeletal myoblast transplantation immediately after MI resulted in an engraftment volume of 29.1 +/- 2.9 mm(3). However, delaying SKMB transplantation 5 weeks caused a 95% drop in engraftment (1.4 +/- 0.3 mm(3); p < 0.001). Hepatocyte growth factor labeling in MIs 48 hours after coronary artery ligation was similar to control myocardium (18.0 +/- 2.0 versus 16.8 +/- 1.3 units). However, HGF declined progressively at 1, 2, and 5 weeks after MI (9.1 +/- 1.4, 4.2 +/- 0.4, and 3.1 +/- 0.6 units, respectively; p < 0.05 versus 48 hours). Hepatocyte growth factor caused a dose-dependent increase in SKMB proliferation in vitro and protected against oxidative stress and hypoxia. CONCLUSIONS: These results demonstrate that engraftment of SKMBs is impaired when transplantation is delayed until a chronic infarct has developed. Hepatocyte growth factor in MI declines with time and may enhance engraftment of SKMBs transplanted early after MI. Delivery of exogenous HGF to enhance SKMB engraftment in chronic infarcts warrants further investigation.
Assuntos
Sobrevivência de Enxerto , Coração , Fator de Crescimento de Hepatócito/farmacologia , Mioblastos Esqueléticos/transplante , Infarto do Miocárdio/terapia , Animais , Hipóxia Celular , Proliferação de Células/efeitos dos fármacos , Fator de Crescimento de Hepatócito/fisiologia , Infarto do Miocárdio/patologia , Infarto do Miocárdio/fisiopatologia , Estresse Oxidativo , Ratos , Ratos Endogâmicos Lew , Função Ventricular EsquerdaRESUMO
Although there is a great deal of interest in women's health, research on the health and well being of women with disabilities has not increased. In this article we present internal and structural barriers to wellness activities experienced by women with disabilities. We also discuss women's actual and recommended strategies to address these barriers. Data were collected in six focus groups in urban and rural Ontario, Canada. The participants represented a diversity of disability, age, and ethnoracial backgrounds. Our findings suggest that individual and structural barriers exist for the women, with structural barriers (physical, informational, and systemic access) being predominant. Barriers prevented women from engaging in desired wellness activities. Women discussed actual strategies to address these barriers, such as collective efforts to buy nutritious foods and recommendations to create greater access (e.g., increase health professionals' training in disability issues).