RESUMO
This study provides a safe and low-cost in-house protocol for RT-qPCR-based detection of SARS-CoV-2 using mouthwash-saliva self-collected specimens to achieve clinical and epidemiological surveillance in a real-time web environment applied to ambulatory populations. The in-house protocol comprises a mouthwash-saliva self-collected specimen, heat virus inactivation, and primers to target virus N-gene region and the human RPP30-gene. Aligning with 209 SARS-CoV-2 sequences confirmed specificity including the Alpha variant from the UK. Development, validation, and statistical comparison with official nasopharyngeal swabbing RT-qPCR test were conducted with 115 specimens of ambulatory volunteers. A web-mobile application platform was developed to integrate a real-time epidemiological and clinical core baseline database with mouthwash-saliva RT-qPCR testing. Nine built-in algorithms were generated for decision-making on testing, confining, monitoring, and self-reports to family, social, and work environments. Epidemiological and clinical follow-up and SARS-CoV-2 testing generated a database of 37,351 entries allowing individual decision-making for prevention. Mouthwash-saliva had higher sensitivity than nasopharyngeal swabbing in detecting asymptomatic and mild symptomatic cases with 720 viral copy number (VCN)/mL as the detection limit (Ct = 37.6). Cycling threshold and viral loading were marginally different (p = 0.057) between asymptomatic (35 Ct ± 2.8; 21,767.7 VCN/mL, range 720-77,278) and symptomatic (31.3 Ct ± 4.5; 747,294.3 VCN/mL, range 1433.6-3.08 × 106). We provided proof-of-concept evidence of effective surveillance to target asymptomatic and moderate symptomatic ambulatory individuals based on integrating a bio-safety level II laboratory, self-collected, low-risk, low-cost detection protocol, and a real-time digital monitoring system. Mouthwash-saliva was effective for SARS-CoV-2 sampling for the first time at the community level.
Assuntos
COVID-19 , Antissépticos Bucais , Teste para COVID-19 , Feminino , Humanos , SARS-CoV-2 , Saliva , Manejo de EspécimesRESUMO
BACKGROUND: The metabolic clearance of prolactin (PRL) is partially executed by the kidney. Here, we investigate the urine excretion of PRL in patients with Diabetes Mellitus and renal impairment. METHODS: Serum and urine samples were collected from male, mestizo patients in central Mexico employing a cross-sectional study design. Ninety-eight individuals had either no diabetes and normal renal function (control), diabetes and normal renal function, or diabetes with impaired renal function. PRL was determined by a chemiluminescent immunometric assay; protein, albumin, and creatinine were evaluated using quantitative colorimetric assays. The results were analyzed using ANOVA-testing. RESULTS: Patients with Diabetes Mellitus and renal impairment had significantly higher urine PRL levels than patients with Diabetes Mellitus and normal renal function and control patients. Higher urine PRL levels were associated with lower glomerular filtration rates, higher serum creatinine, and higher urinary albumin-to-creatinine ratios (UACR). Urine PRL levels correlated positively with UACR. Serum PRL levels were similar among groups. CONCLUSIONS: Patients with Diabetes Mellitus and impaired renal function demonstrate a high urinary PRL excretion. Urinary PRL excretion in the context of proteinuria could contribute to PRL dysregulation in renal impairment.
Assuntos
Nefropatias Diabéticas/diagnóstico , Rim/metabolismo , Prolactina/urina , Eliminação Renal , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Albuminúria/diagnóstico , Albuminúria/fisiopatologia , Albuminúria/urina , Biomarcadores/sangue , Biomarcadores/urina , Estudos de Casos e Controles , Creatinina/sangue , Creatinina/urina , Estudos Transversais , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/fisiopatologia , Nefropatias Diabéticas/urina , Retinopatia Diabética/etiologia , Retinopatia Diabética/fisiopatologia , Retinopatia Diabética/urina , Taxa de Filtração Glomerular , Humanos , Rim/fisiopatologia , Masculino , México , Pessoa de Meia-Idade , Regulação para Cima , Adulto JovemRESUMO
Prolactin (PRL), originally associated with milk secretion, is known to have a wide variety of biological actions and diverse sites of production beyond the pituitary gland. Recent studies have demonstrated that PRL is synthesized in retinal tissue. To gain insights into the functional role of PRL in the mammalian retina, we mapped the distribution of the PRL protein and the expression and localization of the PRL receptor (PRLR) in the retina of adult rats and green monkeys. PRL was examined in retinal sections by double immunolabeling combining anti-PRL antibodies with antibodies specific for glutamine synthetase (labeling Müller cells), glial fibrillary acidic protein (labeling astrocytes), or neuronal nuclei protein (labeling neurons). PRL was detected throughout the rat retina: in the photoreceptor outer segments, Müller cells, interneurons, ganglion cells, and astrocytes. The PRLR was examined by RT-PCR, in situ hybridization, immunohistochemistry, and Western blot. The long isoform of the PRLR was localized in the photoreceptor nuclear layer, inner nuclear layer, and ganglion cell layer of rat retina. The monkey retina showed a similar distribution of PRL and PRLR immunoreactivities. These findings suggest that PRL functions as a local regulator of various cell types in the mammalian retina.
Assuntos
Mamíferos/metabolismo , Prolactina/metabolismo , Receptores da Prolactina/metabolismo , Retina/metabolismo , Animais , Astrócitos/metabolismo , Chlorocebus aethiops/metabolismo , Hibridização In Situ/métodos , Interneurônios/metabolismo , Masculino , Células Fotorreceptoras de Vertebrados/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Wistar/metabolismo , Receptores da Prolactina/genética , Células Ganglionares da Retina/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Especificidade da EspécieRESUMO
Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17beta-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) alpha(1)A-, alpha(1)B-, alpha(1)D-, alpha(2)A-, alpha(2)C-, and beta(1)-AR, and showed for the first time that mRNAs for alpha(2)B-, beta(2)- and beta(3)-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17beta-estradiol, alpha(1)B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while alpha(1)B- and alpha(1)D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1-10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.
Assuntos
Estradiol/fisiologia , Regulação da Expressão Gênica/fisiologia , Hormônio Liberador de Gonadotropina/metabolismo , Neurônios/metabolismo , Receptores Adrenérgicos alfa/biossíntese , Receptores Acoplados a Proteínas G/biossíntese , Animais , Linhagem Celular Transformada , Hormônio Liberador de Gonadotropina/biossíntese , Hormônio Liberador de Gonadotropina/genética , Camundongos , Neurônios/fisiologia , Sistemas Neurossecretores/citologia , Sistemas Neurossecretores/metabolismo , Sistemas Neurossecretores/fisiologia , Receptores Adrenérgicos alfa/genética , Receptores Adrenérgicos alfa/fisiologia , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/fisiologia , Receptores de Kisspeptina-1RESUMO
Attachment of leukocytes to endothelial cells is an essential step for the extravasation and recruitment of cells at sites of inflammation. The pituitary hormone prolactin (PRL) is involved in the inflammatory process. Here, we show that treatment with PRL of human peripheral blood mononuclear cells (PBMC) stimulates their adhesion to human umbilical vein endothelial cells (HUVEC) activated by interleukin-1beta. Stimulation of adhesion by PRL is mediated via integrins leukocyte functional antigen-1 (LFA-1) and very late antigen-4 (VLA-4), because immunoneutralization of both integrins prevents PRL action. Also, PRL promotes the adhesion of PBMC to immobilized intercellular adhesion molecule-1 and fibronectin, ligands for LFA-1 and VLA-4, respectively. Stimulation of integrin-mediated cell adhesion by PRL may involve the activation of chemokine receptors, because PRL upregulates the expression of the G-protein-coupled chemokine receptor CXCR3 in PBMC, and pertussis toxin, a specific G-protein inhibitor, blocks PRL stimulation of PBMC adhesion to HUVEC. In addition, PRL stimulates tyrosine phosphorylation pathways leading to leukocyte adhesion. PRL triggered the tyrosine phosphorylation of Janus kinase-2, of signal transducer and activator of transcription-3 and 5, and of the focal adhesion protein paxillin. Furthermore, genistein, a tyrosine kinase inhibitor, blocked PRL-stimulated adhesion of PBMC and Jurkat T-cells to HUVEC. These results suggest that PRL promotes integrin-mediated leukocyte adhesion to endothelial cells via chemokine receptors and tyrosine phosphorylation signaling pathways.
Assuntos
Adesão Celular/efeitos dos fármacos , Endotélio Vascular/efeitos dos fármacos , Integrinas/metabolismo , Leucócitos Mononucleares/efeitos dos fármacos , Prolactina/farmacologia , Adulto , Relação Dose-Resposta a Droga , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Genisteína/farmacologia , Humanos , Integrinas/imunologia , Células Jurkat , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Fosforilação , RNA Mensageiro/metabolismo , Receptores CXCR3 , Receptores de Quimiocinas/genética , Receptores de Quimiocinas/metabolismo , Tirosina/metabolismo , Regulação para CimaRESUMO
PURPOSE: Disruption of the anti-angiogenic environment of the retina leads to neovascular eye diseases, including retinopathy of prematurity (ROP). Prolactin (PRL), the hormone originally associated with milk secretion, is proteolytically processed to 16K-PRL, a fragment with potent antiangiogenic, proapoptotic effects. Whether 16K-PRL is produced in eyes of patients with ROP and promotes the regression of intraocular blood vessels associated with this disease was investigated. METHODS: PRL was quantified in the aqueous humor, subretinal fluid, and serum from patients with stage 5 ROP and in patients with non-neovascular eye disorders. Intraocular expression of PRL was evaluated by RT-PCR, in situ hybridization, and Western blot analysis. AntiPRL antibodies were injected intravitreously in neonatal rats, and apoptosis of hyaloid vessels determined by TUNEL and ELISA. RESULTS: PRL was elevated in ocular fluids and serum from ROP patients. There was no correlation between PRL in ocular fluids and its level in serum, whereas PRL in aqueous humor and subretinal fluid were significantly correlated. PRL mRNA was expressed in blood vessels and leukocytes within retrolental fibrovascular membranes of ROP patients, and these membranes contained a 16 kDa immunoreactive PRL. The 16K-PRL isoform was more concentrated in subretinal fluid than in serum and was generated from PRL by subretinal fluid proteases. Intravitreous injection of neutralizing antiPRL antibodies inhibited the apoptosis of hyaloid vessels in neonatal rats. CONCLUSIONS: 16K-PRL derived from PRL internalized from the circulation or synthesized intraocularly can stimulate apoptosis-induced vascular regression and contribute to the development and progression of ROP.
Assuntos
Neovascularização Patológica/prevenção & controle , Prolactina/metabolismo , Retinopatia da Prematuridade/metabolismo , Animais , Anticorpos/administração & dosagem , Apoptose/efeitos dos fármacos , Western Blotting , Pré-Escolar , Ensaio de Imunoadsorção Enzimática , Líquido Extracelular/metabolismo , Feminino , Humanos , Hibridização In Situ , Marcação In Situ das Extremidades Cortadas , Lactente , Recém-Nascido , Injeções , Masculino , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Prolactina/genética , Prolactina/imunologia , Isoformas de Proteínas/genética , Isoformas de Proteínas/imunologia , Isoformas de Proteínas/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Retinopatia da Prematuridade/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Corpo Vítreo/irrigação sanguíneaRESUMO
Prolactin (PRL) has been implicated as a modulator of immune function, and some of its actions may be linked to NO synthesis. Because NO acts as a mediator of inflammation, we speculated that an inflammatory milieu could unmask pathways by which PRL could affect NO synthesis. Here, we show that pro-inflammatory cytokines induce the expression of PRL receptors in pulmonary fibroblasts, allowing PRL to inhibit cytokine-induced NO production and the expression of the inducible nitric oxide synthase (iNOS). Inhibition of iNOS expression by PRL correlates with the phosphorylation of STAT-5b (signal transducer and activator of transcription 5b) and the suppression of expression of IRF-1 (interferon regulatory factor 1), a transcription factor for iNOS. These results reveal previously unrecognized mechanisms by which PRL and PRL receptors may play significant modulatory roles during immune-inflammatory processes.