RESUMO
Due to the current limited knowledge about the role of filamin A (FLNA) in pituitary tumour progression, we aimed to analyse FLNA expression levels and its impact on aggressive markers of pituitary neuroendocrine tumours (PitNETs), using an integrative approach of in vivo and in vitro models and human samples. An increase in the expression levels of FLNA was observed in the advanced tumoural stages of the hyperplastic adenomatous pituitary model, concomitant with a decrease in cell proliferation and with a modification in the subcellular localisation of this protein. Similarly, overexpression of FLNA in the somatolactotropic GH3 cell line induced a decrease in the cell proliferation, promoted a migratory phenotype, increased invasion activity, and decreased the prolactin secretion. Cyclin D1 (CCND1) and cyclin-dependent kinase 4 (CDK4) expression increased in both models in correlation with the increase observed in FLNA levels. When human tissues were analysed a significant increase of FLNA was observed in PitNETs compared to normal pituitary gland, with heterogeneous intracellular localisation. Higher levels of FLNA expression were observed in tumours with invasive characteristics. These results underline the crucial roles of FLNA as a modulator of pathological markers and as a potential prognostic marker in pituitary tumours.
Assuntos
Adenoma , Tumores Neuroendócrinos , Neoplasias Hipofisárias , Humanos , Neoplasias Hipofisárias/metabolismo , Filaminas/genética , Filaminas/metabolismo , Hipófise/metabolismoRESUMO
Hepatitis E virus (HEV) is a public health concern globally, causing acute viral hepatitis in humans. Genotype-3 HEV (HEV-3), the most frequently genotype detected in South America, is zoonotic and the main reservoirs are the domestic pig and wild boar. Circulation of HEV-3 in Argentina has been confirmed in humans as well as in pig herds, wild boar and environmental waters. However, data are scarce mainly due to the inaccessibility of serological assays in this country. In order to provide insights in the epidemiology of HEV in swine in Argentina, we developed an indirect ELISA based on the native recombinant protein ORF2 and conducted a serological survey to determine the prevalence of seropositive swine in small-scale pig farms in the central region of Argentina. The method was evaluated in a panel of 157 serum samples, resulting in relative sensitivity of 98.6 % (95 % CI 95 %-100 %) and relative specificity of 97.7 % (95 % CI 94 %-100 %) compared to a commercial test. An almost perfect agreement was obtained between the two tests (Kappa index of 0.961). A survey on 294 samples from 49 small-scale farms resulted in a seropositivity rate of 54 %. Seropositive animals were found in 34 out of 49 (69.4 %) farms. Most of the farms (70.6 %) had over 50 % of seropositive animals. The wide spreading of HEV in the swine population of Tandil, Argentina, underscore the need to better understand the epidemiology of HEV in the region, enabling the implementation of targeted interventions to mitigate the impact of this virus on public health.
Assuntos
Vírus da Hepatite E , Hepatite E , Doenças dos Suínos , Humanos , Suínos , Animais , Hepatite E/epidemiologia , Hepatite E/veterinária , Argentina/epidemiologia , Doenças dos Suínos/epidemiologia , Filogenia , Sus scrofa , Vírus da Hepatite E/genética , Ensaio de Imunoadsorção Enzimática/veterinária , RNA Viral/genéticaRESUMO
Background: Hepatitis E virus (HEV) infection is a common cause of acute clinical hepatitis worldwide and is emerging as a disease in Argentina. It is primarily transmitted through contaminated water and food, following the fecal-oral route. Furthermore, is a zoonotic disease with swine as the primary reservoir. Prevalence of HEV infection in humans in several regions of Argentina remains unknown. Objectives: (i) Determine the seroprevalence of HEV among the human population in Tandil, Buenos Aires, Argentina; (ii) Evaluate its association with demographic, socioeconomic and other risk exposures variables, and (iii) Describe and analyze spatial patterns related to HEV infection. Methods: From August 2020 to July 2021, serum samples were collected from 969 individuals aged 1-80 years. Seroprevalence and 95% Confidence Interval was determined. To assess the factors associated with the presence of anti-HEV antibodies, associations between the variables and seropositivity were evaluated through bivariate and multivariate analysis. Spatial scanning for clusters of positivity was carried out. Factors associated with these clusters were also assessed. Results: Anti-HEV antibodies were detected in 4.64% (IC 95% 3.27-6.02) of samples. Dark urine was associated with seropositivity (p = 0.02). Seropositivity was linked with the presence of natural water courses near their households (p = 0.02); the age (p = 0.04); and previous travel to Europe (p = 0.04). A spatial cluster of low rates of HEV seropositivity was detected, with greater distance of the households to water courses associated to the cluster, and male sex inversely associated to it. Discussion and conclusion: This study is the first study to investigate the prevalence of HEV in the population from Tandil, Buenos Aires, Argentina. Considering HEV infection in the differential diagnosis in individuals presenting acute hepatitis is highlighted. The incorporation of HEV testing into blood screening policies should be mandatory. Factors related to the infection and spatial patterns of high and low risk were determined, and should be considered when implementing specific preventive measures.
Assuntos
Vírus da Hepatite E , Hepatite E , Humanos , Masculino , Suínos , Animais , Argentina/epidemiologia , Estudos Soroepidemiológicos , Hepatite E/epidemiologia , Hepatite E/diagnóstico , Anticorpos Anti-Hepatite , Fatores de Risco , ÁguaRESUMO
The hepatitis E virus (HEV) is an emergent zoonotic virus causing viral hepatitis worldwide. Clinically, hepatitis E is not easily distinguished from other types of acute viral hepatitis. There is a need for HEV diagnostic assays to detect and prevent interspecies transmission among susceptible populations. Nanobodies (Nbs) are expressed recombinantly in different systems, produced with high yields, and have superior physicochemical properties compared with conventional antibodies (Ab). Several Nbs against ORF2, the capsid protein and main antigen, were selected and produced in E. coli. Nb39 and Nb74 specifically recognized HEV ORF2 (genotypes 3 and 4). A competitive ELISA (cELISA) was developed and validated using a reference panel of human (n = 86) and swine sera (n = 116) tested in comparison with a commercial kit. The optimal cutoff values determined by ROC analysis were 69.16% (human) and 58.76% (swine); the sensitivity and specificity were high: 97.4% (95% CI 86.5-99.5%) and 95.8% (95% CI 86.0-98.8%) for human vs. 100% (95% CI 93.5-100%) and 98.3% (95% CI 91.0-99.7%) for swine. Further, the cELISA detected total anti-HEV antibodies in wild boar, deer, and mice. To our knowledge, this is the first report of production of Nbs against HEV-3 ORF2 for diagnostic purposes.
Assuntos
Cervos , Vírus da Hepatite E , Anticorpos de Domínio Único , Humanos , Animais , Camundongos , Suínos , Escherichia coli , Anticorpos , Ensaio de Imunoadsorção EnzimáticaRESUMO
Introduction: Intracellular communication is essential for the maintenance of the anterior pituitary gland plasticity. The aim of this study was to evaluate whether GPCR-Gαi modulates basic fibroblast growth factor (FGF2)-induced proliferative activity in normal pituitary cell populations. Methods: Anterior pituitary primary cell cultures from Wistar female rats were treated with FGF2 (10ng/mL) or somatostatin analog (SSTa, 100nM) alone or co-incubated with or without the inhibitors of GPCR-Gαi, pertussis toxin (PTX, 500nM), MEK inhibitor (U0126, 100µM) or PI3K inhibitor (LY 294002, 10 µM). Results: FGF2 increased and SSTa decreased the lactotroph and somatotroph BrdU uptak2e (p<0.05) whereas the FGF2-induced S-phase entry was prevented by SSTa co-incubation in both cell types, with these effects being reverted by PTX, U0126 or LY294002 pre-incubation. The inhibition of lactotroph and somatotroph mitosis was associated with a downregulation of c-Jun expression, a decrease of phosphorylated (p) ERK and pAKT. Furthermore, SSTa was observed to inhibit the S-phase entry induced by FGF2, resulting in a further increase in the number of cells in the G1 phase and a concomitant reduction in the number of cells in the S phases (p< 0.05), effects related to a decrease of cyclin D1 expression and an increase in the expression of the cell cycle inhibitors p27 and p21. Discussion: In summary, the GPCR-Gαi activated by SSTa blocked the pro-proliferative effect of FGF2 in normal pituitary cells via a MEK-dependent mechanism, which acts as a mediator of both anti and pro-mitogenic signals, that may regulate the principal effectors of the G1 to S-phase transition.
Assuntos
Fator 2 de Crescimento de Fibroblastos , Hipófise , Animais , Feminino , Ratos , Proliferação de Células , Fator 2 de Crescimento de Fibroblastos/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Fosfatidilinositol 3-Quinases/metabolismo , Ratos Wistar , Hipófise/citologia , Hipófise/efeitos dos fármacosRESUMO
Toxicological studies have revealed that DEHP exposure during pregnancy may induce developmental disorders, especially in male offspring, leading to morphological and functional alterations in the reproductive system by mechanisms that should be investigated. Thus, the aim of this work was to analyze the testicular toxicity induced by an environmentally relevant DEHP dose during development and its impact on FLNA, a protein that participates in the blood-testis barrier assembly. We used male Wistar rats exposed to DEHP during pregnancy and lactation. The results showed that DEHP exposure during development and lactation increased body weight, decreased gonadal weight and shortened anogenital distance. This phthalate induced morphological changes in the testis, suggestive of hypospermatogenesis. DEHP exposure decreased the number of FLNA positive cells and the expression of FLNA and claudin-1 in prepubertal testes. Furthermore, DEHP inhibited FLNA and claudin-1 protein expression in adult male rats. These results indicated that exposure to DEHP during gestation and lactation perturbed testis development and suggested that FLNA is a target protein of DEHP, possibly contributing to the phthalate-induced damage on BTB.
Assuntos
Dietilexilftalato , Efeitos Tardios da Exposição Pré-Natal , Gravidez , Feminino , Ratos , Masculino , Animais , Humanos , Testículo/metabolismo , Dietilexilftalato/toxicidade , Dietilexilftalato/metabolismo , Filaminas/metabolismo , Claudina-1/metabolismo , Ratos Wistar , Lactação , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Efeitos Tardios da Exposição Pré-Natal/metabolismoRESUMO
INTRODUCTION: Macroautophagy is a lysosome-mediated degradation process that controls the quality of cytoplasmic components and organelles, with its regulation depending on autophagy-related proteins (Atg) and with Beclin1/Atg6 and microtubule-associated protein light chain 3 (LC3/Atg8) being key players in the mammalian autophagy. As reports on this mechanism in the field of pituitary neuropathology and neuroendocrinology are scarce, our study analyzed the ultrastructural signs of macroautophagy and the expression of Beclin1 and LC3 proteins in human functioning PitNETs and in experimental pituitary tumors. METHODS: A group of humans functioning PitNETs and an experimental lactotroph model in rats of the F344 strain stimulated with estradiol benzoate (BE) were used. Ultrastructural and molecular evidence of the macroautophagic process was evaluated using different techniques. RESULTS: In functioning PitNETs cohort, 60% exhibited evidence of macroautophagy, with a significant difference found for Beclin1 and LC3 between macro- and micro-PitNETs (p < 0.05). In the experimental model, the expression of both Beclin1 and LC3 proteins was immunopositive in normal and tumoral glands when analyzed by immunofluorescence, Western blot, and immunohistochemistry. In the experimental model, protein expression was associated with increased glandular size and weight. CONCLUSIONS: Our study revealed evidence of macroautophagy at the pituitary level and the important role of Beclin1 and LC3 in the progression of functioning PitNETs, implying that this mechanism participate in regulating pituitary cell growth.
Assuntos
Macroautofagia , Neoplasias Hipofisárias , Humanos , Ratos , Animais , Proteína Beclina-1 , Ratos Endogâmicos F344 , Autofagia , Proteínas Associadas aos Microtúbulos/metabolismo , Mamíferos/metabolismoRESUMO
Vancomycin (VAN) is unable to penetrate the outer membrane of Gram-negative bacteria and reach the target site. One approach to overcome this limitation is to associate it with compounds with permeabilizing or antimicrobial properties. Eudragit E100® (Eu) is a cationic polymer insufficiently characterized for its potential antimicrobial action. Eu-VAN combinations were characterized, the antimicrobial efficacy against Pseudomonas aeruginosa was evaluated and previous studies on the effects of Eu on bacterial envelopes were extended. Time-kill assays showed eradication of P. aeruginosa within 3-6 h exposure to Eu-VAN, whilst VAN was ineffective. Eu showed regrowth in 24 h and delayed colony pigmentation. Although permeabilization of bacterial envelopes or morphological alterations observed by TEM and flow cytometry after exposure to Eu were insufficient to cause bacterial death, they allowed access of VAN to the target site, since Eu-VAN/Van-FL-treated cultures showed fluorescent staining in all bacterial cells, indicating Van-FL internalization. Consequently, Eu potentiated the activity of an otherwise inactive antibiotic against P. aeruginosa. Moreover, Eu-VAN combinations exhibited improved physicochemical properties and could be used in the development of therapeutic alternatives in the treatment of bacterial keratitis.
Assuntos
Pseudomonas aeruginosa , Vancomicina , Antibacterianos/química , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Polímeros/farmacologia , Vancomicina/farmacologiaRESUMO
Among pituitary adenomas, prolactinomas are the most frequently diagnosed (about 50%). Dopamine agonists are generally effective in the treatment of prolactinomas. However, a subset of about 25% of patients does not respond to these agents. The management of drug-resistant prolactinomas remains a challenge for endocrinologists and new inhibitory treatments are needed. Pituitary activins inhibit lactotroph function. Its expression and action were found reduced in animal models of lactotroph hyperplasia (female mice overexpressing the B subunit of the human chorionic gonadotrophin and female mice knockout for dopamine receptor type 2). In these models, an oophorectomy avoids prolactinoma development. Hormonal replacement with oestradiol and/or progesterone is not enough to reach the tumor size observed in transgenic females. We postulated that the loss of gonadal inhibins after an oophorectomy contributes to prevent hyperplasia development. Here, we demonstrated that an oophorectomy at 2 months age recovers the following in adulthood: (i) pituitary activin expression, (ii) activin receptor expression specifically in lactotroph population, (iii) activin biological activity in lactotrophs with a concomitant reduction of Pit-1 expression. To summarize, when an oophorectomy is performed, inhibins are lost and the inhibitory action of pituitary activins on lactotroph population is recovered, helping to prevent lactotroph hyperplasia development. These results emphasize the importance of the inhibitory action of activins on lactotroph function, positioning activins as a good therapeutic target for the treatment of resistant prolactinomas.