RESUMO
BACKGROUND: Racial and ethnic groups in the USA differ in the prevalence of posttraumatic stress disorder (PTSD). Recent research however has not observed consistent racial/ethnic differences in posttraumatic stress in the early aftermath of trauma, suggesting that such differences in chronic PTSD rates may be related to differences in recovery over time. METHODS: As part of the multisite, longitudinal AURORA study, we investigated racial/ethnic differences in PTSD and related outcomes within 3 months after trauma. Participants (n = 930) were recruited from emergency departments across the USA and provided periodic (2 weeks, 8 weeks, and 3 months after trauma) self-report assessments of PTSD, depression, dissociation, anxiety, and resilience. Linear models were completed to investigate racial/ethnic differences in posttraumatic dysfunction with subsequent follow-up models assessing potential effects of prior life stressors. RESULTS: Racial/ethnic groups did not differ in symptoms over time; however, Black participants showed reduced posttraumatic depression and anxiety symptoms overall compared to Hispanic participants and White participants. Racial/ethnic differences were not attenuated after accounting for differences in sociodemographic factors. However, racial/ethnic differences in depression and anxiety were no longer significant after accounting for greater prior trauma exposure and childhood emotional abuse in White participants. CONCLUSIONS: The present findings suggest prior differences in previous trauma exposure partially mediate the observed racial/ethnic differences in posttraumatic depression and anxiety symptoms following a recent trauma. Our findings further demonstrate that racial/ethnic groups show similar rates of symptom recovery over time. Future work utilizing longer time-scale data is needed to elucidate potential racial/ethnic differences in long-term symptom trajectories.
Assuntos
Depressão , Transtornos de Estresse Pós-Traumáticos , Humanos , Criança , Depressão/psicologia , Transtornos de Ansiedade , Ansiedade/epidemiologia , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Etnicidade/psicologiaRESUMO
AIM: It is well-known that enterococci are abundant in the environment; however, the role of surface water as a reservoir of antimicrobial-resistant enterococci remains largely undefined. In this study, surface water samples were collected over a 2-year period from the Upper Oconee watershed, Athens, GA to examine enterococci and their antimicrobial resistance. METHODS AND RESULTS: Approximately 97% (445/458) of the samples were positive for enterococci and a total of 637 enterococci were isolated. The predominant species were Enterococcus casseliflavus (33·6%) followed by Enterococcus faecalis (26·5%) and Enterococcus hirae (13·2%). Regardless of species, the highest levels of resistance were to lincomycin (88·5%) and tetracycline (13%); isolates also exhibited resistance to newer antimicrobials, daptomycin (8·9%) and tigecycline (6·4%). Multidrug resistance (resistance ≥3 antimicrobial classes) was observed to as many as five classes of antimicrobials. Resistant enterococci appeared to be randomly dispersed over the seasons rather than clustered by species or antimicrobial resistance. CONCLUSIONS: This study demonstrated that surface waters contain a large population of diverse species of antimicrobial-resistant enterococci, including resistance to new antimicrobials. SIGNIFICANCE AND IMPACT OF THE STUDY: These results may indicate the potential of human intestinal illness and/or colonization of the human gut with resistant enterococci as enterococci correlate with increased disease risk to humans during recreational exposure to water.
Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana , Enterococcus/isolamento & purificação , Água Doce/microbiologia , Microbiologia da Água , Farmacorresistência Bacteriana/efeitos dos fármacos , Enterococcus/classificação , Enterococcus/efeitos dos fármacos , Monitoramento Ambiental , Georgia , Humanos , Testes de Sensibilidade MicrobianaRESUMO
INTRODUCTION: Prior studies have demonstrated inconsistent development and utilisation of radiographers in the reporting of radiographs, and there is ongoing consideration of the level at which such radiographers should be educated to and operating at. This study aimed to explore and evaluate expectation and utilisation of radiographers currently, or training in, reporting in projection radiography across one integrated care system (ICS). METHODS: A multi-method approach was utilised, with document analysis of projection radiography reporting role job descriptions and person specifications and an online survey of managers and clinical leads. A single ICS in the north of England formed the setting for the study. RESULTS: This study demonstrated variation in implementation and utilisation of the role across trusts within the ICS. Inconsistencies in scope, expected underpinning education and role activity were identified. Radiographers autonomously reporting in projection radiography were titled advanced practitioners, however are not expected to achieve national educational standards for such roles and are not empowered to work at this level of practice by their employers. It was acknowledged that staffing pressures hinder appropriate role utilisation and reporting capacity. CONCLUSION: Inconsistent development and utilisation of radiographers in such roles may hamper collaboration and service delivery across a network. Identifying variation and working towards role standardisation could promote cross-organisational working and improve career progression opportunities. IMPLICATIONS FOR PRACTICE: Scoping the reporting radiographer workforce may assist and guide future imaging service and workforce planning.
Assuntos
Competência Clínica , Motivação , Humanos , Radiografia , Inglaterra , Pessoal Técnico de SaúdeRESUMO
AIMS: Childhood adversities (CAs) predict heightened risks of posttraumatic stress disorder (PTSD) and major depressive episode (MDE) among people exposed to adult traumatic events. Identifying which CAs put individuals at greatest risk for these adverse posttraumatic neuropsychiatric sequelae (APNS) is important for targeting prevention interventions. METHODS: Data came from n = 999 patients ages 18-75 presenting to 29 U.S. emergency departments after a motor vehicle collision (MVC) and followed for 3 months, the amount of time traditionally used to define chronic PTSD, in the Advancing Understanding of Recovery After Trauma (AURORA) study. Six CA types were self-reported at baseline: physical abuse, sexual abuse, emotional abuse, physical neglect, emotional neglect and bullying. Both dichotomous measures of ever experiencing each CA type and numeric measures of exposure frequency were included in the analysis. Risk ratios (RRs) of these CA measures as well as complex interactions among these measures were examined as predictors of APNS 3 months post-MVC. APNS was defined as meeting self-reported criteria for either PTSD based on the PTSD Checklist for DSM-5 and/or MDE based on the PROMIS Depression Short-Form 8b. We controlled for pre-MVC lifetime histories of PTSD and MDE. We also examined mediating effects through peritraumatic symptoms assessed in the emergency department and PTSD and MDE assessed in 2-week and 8-week follow-up surveys. Analyses were carried out with robust Poisson regression models. RESULTS: Most participants (90.9%) reported at least rarely having experienced some CA. Ever experiencing each CA other than emotional neglect was univariably associated with 3-month APNS (RRs = 1.31-1.60). Each CA frequency was also univariably associated with 3-month APNS (RRs = 1.65-2.45). In multivariable models, joint associations of CAs with 3-month APNS were additive, with frequency of emotional abuse (RR = 2.03; 95% CI = 1.43-2.87) and bullying (RR = 1.44; 95% CI = 0.99-2.10) being the strongest predictors. Control variable analyses found that these associations were largely explained by pre-MVC histories of PTSD and MDE. CONCLUSIONS: Although individuals who experience frequent emotional abuse and bullying in childhood have a heightened risk of experiencing APNS after an adult MVC, these associations are largely mediated by prior histories of PTSD and MDE.
Assuntos
Transtorno Depressivo Maior , Transtornos de Estresse Pós-Traumáticos , Adulto , Humanos , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Idoso , Transtornos de Estresse Pós-Traumáticos/epidemiologia , Transtornos de Estresse Pós-Traumáticos/etiologia , Transtornos de Estresse Pós-Traumáticos/diagnóstico , Transtorno Depressivo Maior/psicologia , Depressão/psicologia , Inquéritos e Questionários , Veículos AutomotoresRESUMO
B lymphocyte-enriched cell populations cultured with mitogens in initial suspension cultures formed colonies in soft agar when the same mitogenic agent was present in the lower layer of a two-layer soft agar system. Colony formation depended upon the presence of T cells in the initial culture, and was optimal after an initial 72-h culture with phytohemagglutinin (PHA; 12.5 microliters/ml), pokeweed mitogen (PWM; 2.5 micrograms/ml), or protein A (10 micrograms/ml). The colonies could be picked from the agar and propagated by feeding every 3 d with medium supplemented with a growth factor-containing tissue culture supernate. The growth factor-containing supernate was prepared by stimulating pools of human peripheral blood mononuclear cells for 72 h with PHA or PWM. The lines propagated in this manner were membrane Ig+, lacked sheep erythrocyte rosette-forming ability, and did not ingest latex. They lacked the Epstein-Barr nuclear antigen (EBNA) and had 46 chromosomes. Such lines have been propagated for over 1 yr. One line (BL1) was subjected to limiting dilution cloning and a line, BL1.1, was prepared that contained 96% lambda-bearing cells and no kappa-bearing cells. This line was also EBNA negative. This procedure can thus be used to prepare and clone long-term lines of nontransformed human B lymphocytes.
Assuntos
Linfócitos B/citologia , Ativação Linfocitária , Linfócitos B/imunologia , Linhagem Celular , Células Cultivadas , Células Clonais/citologia , Células Clonais/imunologia , Substâncias de Crescimento/farmacologia , Humanos , Fito-Hemaglutininas/farmacologia , Mitógenos de Phytolacca americana/farmacologia , Receptores de Antígenos de Linfócitos B , Proteína Estafilocócica A/farmacologia , Linfócitos T/citologia , Fatores de TempoRESUMO
Introduction of the CBA/N X-linked gene into C3H mice has resulted in the establishment of a new strain of mice that has profound immunologic defects. B cells from these mice show significantly impaired in vitro immune responses to the T cell-independent type 1 antigen trinitrophenyl-Brucella abortus (TNP-BA) as well as markedly reduced proliferative responses to a number of B cell mitogens when compared with the responses of the parental control mice. The in vivo response of such mice to TNP-BA is, however, comparable to that of CBA/N mice. Furthermore, B cells from C3.CBA/N mice are unresponsive to the plaque-forming cell enhancing effects induced by EL4-derived supernatant in the presence of TNP-BA, unlike B cells obtained from CBA/N or C3H/Hen mice whose responsiveness to TNP-BA can be significantly enhanced in the presence of EL4-derived supernatant. The model we have presented to best explain these results suggests that B cells from C3.CBA/N mice can be stimulated only under conditions in which they can interact with carrier-specific T cell help and not under conditions where factor-dependent responses are dominant.
Assuntos
Antígenos de Bactérias/imunologia , Linfócitos B/imunologia , Camundongos Endogâmicos/imunologia , Mitógenos/farmacologia , Animais , Citotoxicidade Celular Dependente de Anticorpos , Brucella abortus/imunologia , Divisão Celular , Feminino , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos CBA , Baço/citologia , Linfócitos T/imunologia , Trinitrobenzenos/imunologiaRESUMO
A monoclonal antibody, gamma-120, was raised against a highly purified gamma-glutamyltransferase (gamma GT) from human primary hepatoma. The antibody preferentially bound to the small subunit of gamma GT from human hepatoma and kidney as evidenced by immunoblot analysis. Weak binding to the normal liver enzyme could be detected by solid-phase enzyme-linked immunosorbent assay (ELISA). With the use of this antibody, an ELISA was developed for the quantitation of immunoreactive gamma GT in human serum. Sera of 188 normal control subjects displayed a low level (9.4 micrograms/ml) of immunoreactive gamma GT. Highly elevated levels of immunoreactive gamma GT were detected in the sera of patients with primary hepatoma, metastatic liver cancer, pancreatic cancer, and certain types of lung cancer. Slightly elevated levels of immunoreactive gamma GT were seen in the sera of patients with liver cirrhosis. The levels of gamma GT were within a normal range in the sera of patients with gastrointestinal cancers and patients with nonmalignant diseases such as hepatitis and gallstones. The antibody has been shown to be useful for the diagnosis of some of the neoplastic diseases.
Assuntos
Anticorpos Monoclonais/imunologia , Carcinoma Hepatocelular/enzimologia , Neoplasias Hepáticas/enzimologia , Neoplasias/enzimologia , gama-Glutamiltransferase/sangue , Especificidade de Anticorpos , Ensaio de Imunoadsorção Enzimática , Humanos , gama-Glutamiltransferase/imunologiaRESUMO
An enzyme-linked immunosorbent assay for human immunoreactive gamma-glutamyl transpeptidase (gamma-GTP) was developed. This assay was found to be simple, reproducible, and sensitive to 10 ng of the enzyme. Serum immunoreactive gamma-GTP content was significantly elevated in patients with various malignant tumors including liver cell cancer, lung cancer, gastric cancer, esophageal cancer, and colorectal cancer. On the other hand, in sera of patients with nonneoplastic diseases, the immunoreactive gamma-GTP content was not significantly elevated. No correlation was found between the serum levels of gamma-GTP determined by enzymatic assay and enzyme-linked immunosorbent assay, which indicates that, due to the presence of endogenous inhibitors and/or activators in sera, the enzyme activity may not reflect the true amount of enzyme protein. The measurement of immunoreactive gamma-GTP protein in sera appears to be useful for the detection and monitoring of certain malignant tumors.
Assuntos
Neoplasias/enzimologia , gama-Glutamiltransferase/sangue , Reações Cruzadas , Ensaio de Imunoadsorção Enzimática , Humanos , Soros Imunes/imunologia , gama-Glutamiltransferase/imunologiaRESUMO
The regulation of arginine vasopressin (AVP) gene transcription in the paraventricular nucleus (PVN) was studied in rat hypothalamic organotypic cultures using intronic in situ hybridization. While AVP heteronuclear (hn) RNA was not detected in the PVN under basal conditions, a marked induction of AVP hnRNA was observed after 2 and 3 h incubation of slices with forskolin. In contrast to the stimulatory effects of forskolin, phorbol 12-myristate 13-acetate (PMA) was completely ineffective in inducing AVP hnRNA in the PVN at any time examined (1-3 h). Forskolin-induced AVP hnRNA expression was unaffected by blockage of neurotransmission by the sodium channel inhibitor, tetrodotoxin, indicating that forskolin acts directly on AVP cells in the PVN. Dual staining in situ hybridization of forskolin-stimulated hypothalamic sections using both radio labeled AVP hnRNA and digoxigenin-labeled CRH mRNA probes revealed colocalization of both transcripts, indicating AVP hnRNA is expressed in the parvocellular neurons. The data demonstrate that cAMP directly activates AVP gene transcription in parvocellular neurons of the PVN.
Assuntos
Arginina Vasopressina/genética , AMP Cíclico/fisiologia , Neurônios/fisiologia , Núcleo Hipotalâmico Paraventricular/fisiologia , Transcrição Gênica/fisiologia , Animais , Colforsina/farmacologia , Neurônios/efeitos dos fármacos , Técnicas de Cultura de Órgãos , Núcleo Hipotalâmico Paraventricular/citologia , RNA Nuclear Heterogêneo/metabolismo , Ratos , Ratos Sprague-Dawley , Tetrodotoxina/farmacologiaRESUMO
Interleukin-1-beta (IL-1beta) can promote inflammation by up-regulating vascular adhesion molecules and inhibit inflammation by activating the hypothalamic-pituitary-adrenal (HPA) axis to produce anti-inflammatory glucocorticoids. In this study, chronic morphine was shown to suppress IL-1beta-induction of corticotropin releasing factor (CRF) mRNA and plasma corticosterone levels. Leukocyte-endothelial adhesion (LEA) in rat mesenteric venules increased during IL-1beta- and FMLP-induced inflammation. Chronic morphine potentiated the LEA response to either IL-1beta or FMLP alone, and greatly enhanced LEA in response to combined IL-1beta and FMLP. Thus, it appears that chronic morphine exposure may promote a potentially damaging inflammatory reaction by disrupting the balance between IL-1beta-mediated local inflammation and the anti-inflammatory effects of the HPA axis.
Assuntos
Sistema Hipotálamo-Hipofisário/efeitos dos fármacos , Inflamação/metabolismo , Interleucina-1/farmacologia , Morfina/administração & dosagem , Sistema Hipófise-Suprarrenal/efeitos dos fármacos , Animais , Adesão Celular/efeitos dos fármacos , Corticosterona/sangue , Hormônio Liberador da Corticotropina/biossíntese , Hormônio Liberador da Corticotropina/genética , Esquema de Medicação , Sinergismo Farmacológico , Retroalimentação/efeitos dos fármacos , Retroalimentação/fisiologia , Sistema Hipotálamo-Hipofisário/metabolismo , Hipotálamo/metabolismo , Inflamação/induzido quimicamente , Injeções Intraventriculares , Leucócitos/citologia , Leucócitos/efeitos dos fármacos , Masculino , Mesentério/irrigação sanguínea , Mesentério/citologia , N-Formilmetionina Leucil-Fenilalanina/farmacologia , Sistema Hipófise-Suprarrenal/metabolismo , RNA Mensageiro/biossíntese , Ratos , Vênulas/citologia , Vênulas/efeitos dos fármacosRESUMO
In a previous study we described two distinct neuronal phenotypes in rat dorsal root ganglia based on immunocytochemical assays for the neuronal intermediate filament proteins, peripherin and low-molecular-weight neurofilaments [Goldstein M. E. et al. (1991) J. Neurosci. Res. 30, 92-104]. In this paper we have extended this classification by using in situ hybridization to localize and evaluate the levels of various cytoskeletal and neuropeptide messenger RNAs within the peripherin-immunoreactive and peripherin-immunoreactive-negative neurons found in embryonic day 15 and 20, postnatal day 2 and adult dorsal root ganglia. We found in postnatal and adult dorsal root ganglia in vivo that the large, peripherin-immunoreactive-negative neurons, which are intensely stained by low-molecular-weight neurofilament antibodies, also contain high levels of low, medium and high-molecular-weight neurofilament messenger RNAs, whereas the smaller peripherin-immunoreactive neurons do not. On the other hand, both cell types contained comparable levels of peripherin and alpha-tubulin messenger RNA. The presence of peripherin messenger RNA but no peripherin immunoreactivity in the large cells suggested either a translational or post-translational regulation of this polypeptide, or rapid clearance of this protein from the perikaryon into the axon. In adult dorsal root ganglia, more than 50% of the peripherin-immunoreactive neurons also contained high levels of substance P and/or calcitonin gene-related peptide messenger RNAs, while less than 20% of the large peripherin-immunoreactive-negative neurons did. The attainment of these phenotypic characteristics during development in vivo was studied by northern blot and in situ hybridization histochemistry. In early embryonic stages (embryonic days 15-16), virtually all neurons were peripherin-immunoreactive and were positive for peripherin, alpha-tubulin and low-molecular-weight neuro-filament messenger RNAs, suggesting a homogeneous population. By embryonic day 20, the two adult phenotypes became clearly evident, and were fully established by postnatal day 2. In cultures of embryonic day 15 dorsal root ganglion neurons grown in the presence of nerve growth factor, peripherin and low-molecular-weight neurofilament messenger RNAs were expressed in all neurons, even after nine days in vitro, similar to embryonic dorsal root ganglia in vivo. Nerve growth factor supplemented by skeletal and heart muscle extracts did up-regulate neurofilament gene expression, but not to the extent characteristic of the peripherin-immunoreactive-negative adult phenotype. These results suggest that development of the mature phenotype of dorsal root ganglion neurons occurs by postnatal day 2 in vivo and is dependent upon target contact and/or target-derived factors.
Assuntos
Gânglios Espinais/citologia , Gânglios Espinais/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Glicoproteínas de Membrana , Proteínas do Tecido Nervoso , Neurônios/fisiologia , Animais , Northern Blotting , Proteínas do Olho/metabolismo , Coração/fisiologia , Imuno-Histoquímica , Hibridização In Situ , Proteínas de Filamentos Intermediários/metabolismo , Peso Molecular , Músculo Esquelético/fisiologia , Proteínas de Neurofilamentos/biossíntese , Proteínas de Neurofilamentos/genética , Neuropeptídeos/biossíntese , Neuropeptídeos/metabolismo , Sondas de Oligonucleotídeos , Periferinas , Fenótipo , Sondas RNA , RNA Mensageiro/biossíntese , RNA Mensageiro/genética , Ratos , Extratos de Tecidos/farmacologiaRESUMO
In vitro and ex vivo tissue models provide a useful level of biological organization for cytoprotection studies positioned between cultured cells and intact animals. We have used 2 such models, primary tissue cultures of winter flounder renal secretory epithelium and ex vivo preparations of rat intestinal tissues, the latter to access the microcirculation of exposed mesentery tissues. Herein we discuss studies indicating that differentiated functions are altered in thermotolerant or cytoprotected tissues. These functions include transepithelial transport in renal epithelium and attachment and transmigration of leukocytes across vascular endothelium in response to mediators of inflammation. Evidence pointing to inflammation as a major venue for the heat shock response in vertebrates continues to mount. One such venue is wound healing. Heat shock proteins are induced early in wound responses, and some are released into the extracellular wound fluid where they appear to function as proinflammatory cytokines. However, within responding cells in the wound, heat shock proteins contribute to the acquisition of a state of cytoprotection that protects cells from the hostile environment of the wound, an environment created to destroy pathogens and essentially sterilize the wound. We propose that the cytoprotected state is an anti-inflammatory state that contributes to limiting the inflammatory response; that is, it serves as a brake on inflammation.
Assuntos
Citoproteção/imunologia , Transtornos de Estresse por Calor/patologia , Resposta ao Choque Térmico/imunologia , Cicatrização/imunologia , Animais , Adesão Celular/imunologia , Cloretos/farmacocinética , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Células Epiteliais/metabolismo , Transtornos de Estresse por Calor/imunologia , Transtornos de Estresse por Calor/fisiopatologia , Inflamação/imunologia , Inflamação/patologia , Inflamação/fisiopatologia , Rim/metabolismo , Rim/patologia , Rim/fisiopatologia , Leucócitos/imunologia , Leucócitos/patologia , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Ratos , Compostos de Zinco/farmacocinéticaRESUMO
Heat and a variety of other stressors cause mammalian cells and tissues to acquire cytoprotection. This transient state of altered cellular physiology is nonproliferative and antiapoptotic. In this study, male Wistar rats were stress conditioned with either stannous chloride or gallium nitrate, which have immunosuppressive effects in vivo and in vitro, or heat shock, the most intensively studied inducer of cytoprotection. The early stages of inflammation in response to topical suffusion of mesentery tissue with formyl-methionyl-leucyl-phenylalanine (FMLP) were monitored using intravital microscopy. Microvascular hemodynamics (venular diameter, red blood cell velocity [Vrbc], white blood cell [WBC] flux, and leukocyte-endothelial adhesion [LEA]) were used as indicators of inflammation, and tissue levels of inducible Hsp70, determined using immunoblot assays, provided a marker of cytoprotection. None of the experimental treatments blocked decreases in WBC flux during FMLP suffusion, an indicator of increased low-affinity interactions between leukocytes and vascular endothelium known as rolling adhesion. During FMLP suffusion LEA, an indicator of firm attachment between leukocytes and vascular endothelial cells increased in placebo and gallium nitrate-treated animals but not in heat- and stannous chloride-treated animals, an anti-inflammatory effect. Hsp70 was not detected in aortic tissue from placebo and gallium nitrate-treated animals, indicating that Hsp70-dependent cytoprotection was not present. In contrast, Hsp70 was detected in aortic tissues from heat- and stannous chloride-treated animals, indicating that these tissues were in a cytoprotected state that was also an anti-inflammatory state.
Assuntos
Gálio/farmacologia , Resposta ao Choque Térmico/imunologia , Imunossupressores/farmacologia , Inflamação/imunologia , Compostos de Estanho/farmacologia , Animais , Adesão Celular/imunologia , Citoproteção/imunologia , Endotélio Vascular/imunologia , Endotélio Vascular/patologia , Endotélio Vascular/fisiopatologia , Hemodinâmica/imunologia , Hemodinâmica/fisiologia , Temperatura Alta , Hipertermia Induzida , Immunoblotting , Inflamação/patologia , Inflamação/fisiopatologia , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Leucócitos/efeitos da radiação , Masculino , N-Formilmetionina Leucil-Fenilalanina/metabolismo , Ratos , Ratos WistarRESUMO
The magnocellular oxytocin (OT) and vasopressin (VP) neurons of the hypothalamo-neurohypophysial system are exceptional cell biological models to study mechanisms of cell-specific gene expression and neurosecretion of neuropeptides in the central nervous system. Single cell differential gene expression experiments have further defined these phenotypes by identifying novel and distinct regulatory molecules in these neurons. Transgenic mouse studies have led to the intergenic region (IGR) hypothesis, which states that the DNA sequences between the OT- and VP-genes contain critical enhancer sites for their cell-specific expression. The recent cloning and sequencing of the human IGR, and its comparison with the mouse IGR sequence has identified conserved sequences as putative, cell-specific enhancer sites which are now being evaluated by biolistic transfections of organotypic hypothalamic cultures. With these data, it is possible to target the gene expression of specific molecules to magnocellular neurons both in vivo and in vitro, in order to perturb and/or visualize neurosecretory and other processes.
Assuntos
Regulação da Expressão Gênica , Sistema Hipotálamo-Hipofisário/fisiologia , Neurônios/fisiologia , Animais , Arginina Vasopressina/genética , Modelos Neurológicos , Ocitocina/genéticaRESUMO
The use of hypothalamic organotypic cultures for the long-term study of mechanisms in magnocellular neurones (MCNs) of the hypothalamic-neurohypophysial system has been limited by the relatively poor maintenance of the vasopressin MCNs in vitro. Recent studies have shown that addition of ciliary neurotrophic factor (CNTF) to the media significantly reduced the apoptosis of both oxytocin and vasopressin MCNs. Here, we studied various temporal factors in the CNTF treatment that can influence the efficacy of MCN survival. Immunohistochemistry was used to identify and count surviving vasopressin and oxytocin MCNs in the supraoptic nucleus (SON) in hypothalamic slices cultured in the presence of CNTF (10 ng/ml media) for various time intervals, and in situ hybridization for vasopressin mRNA was used to evaluate the vasopressin mRNA gene expression in the SON under the same conditions. The presence of CNTF in the medium for 10 days produced a maximal increase in the survival of vasopressin MCNs (by 11-fold) and in the survival of oxytocin-MCNs (by approximately four-fold) over controls. These effects persisted for an additional 7-10 days even in the absence of CNTF. The ability of CNTF to increase survival of the MCNs or increase vasopressin mRNA levels in the SON required that the CNTF be present during the initial 7-10 days of culture. CNTF failed to rescue vasopressin or oxytocin MCNs when added to the media only for the last 7 days of a total of 14 days in vitro. Similar results were observed when SON vasopressin mRNA levels were measured. These results indicate that the presence of CNTF is required at the outset to rescue the vasopressin and oxytocin MCN from axotomy induced apoptosis, and that, after 10 days in CNTF, the MCNs no longer require the CNTF for survival.
Assuntos
Fator Neurotrófico Ciliar/farmacologia , Neurônios/efeitos dos fármacos , Núcleo Supraóptico/citologia , Vasopressinas/fisiologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Técnica Indireta de Fluorescência para Anticorpo , Hipotálamo/citologia , Hipotálamo/efeitos dos fármacos , Hibridização In Situ , Técnicas de Cultura de Órgãos , Sondas RNA , Ratos , Ratos Sprague-Dawley , Núcleo Supraóptico/efeitos dos fármacosRESUMO
Neurons from hypothalamic paraventricular nuclei (PVN) and supraoptic nuclei (SON) from postnatal day 6-8 rats were enzymatically dissociated and separately maintained in monolayer cultures for 14 days. The osmotic pressure of the culture medium, based on Neurobasal medium (Life Technologies), was varied (255, 300 and 330 mOsm/l) by adjustment using mannitol. The survival of oxytocin (OT), vasopressin (VP) and oxytocin-vasopressin (OT/VP) coexpressing neurons were studied under these varied conditions, and the identification of the cell phenotypes in the cultures was carried out by using double-label immunofluorescence. Under control osmolar conditions (300 mOsm/l) equivalent numbers of OT and VP neurons were found in the SON (P = 0.8398) and PVN (P = 0.4721) cultures. The OT neurons' survival did not change in 255 or 330 mOsm media in the SON cultures, but the VP neurons in the SON cultures were significantly increased in 255 mOsm/l medium as compared to control (300 mOsm/l) medium (P = 0.0088). No significant changes were found in VP neuron survival in SON cultures between the 300-330 mOsm/l media (P = 0.2372). Similar data were obtained for the VP neurons in PVN-derived cultures, but the OT neurons in these cultures survived significantly better at 300 mOs/l than at 255 mOsm/l (P<0.0001), but were not significantly different at 330 mOsm/l (P = 0.1208). In general, the VP neurons were more vulnerable than OT neurons to increases of culture medium osmolarity with respect to their survival. The number of OT/VP coexpressing neurons was greater in SON-derived cell cultures as compared to PVN-derived cell cultures, and their numbers were higher in the lower osmolarity media. The effects of adding brain-derived neurotrophic factor (BDNF) to the culture medium on survival were determined. BDNF significantly increased the numbers of all three types of neurons in both PVN and SON cell cultures (P = 0.0001-0.0060). The phenotypically identified cells, cultured in the 300 mOsm/l medium, responded by depolarization or hyperpolarization when transferred to hypertonic or hypotonic perfusion salines, respectively.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/farmacologia , Hipotálamo/citologia , Neurônios/citologia , Ocitocina/fisiologia , Vasopressinas/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Anticorpos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Eletrofisiologia , Feminino , Imunofluorescência , Soluções Hipotônicas/farmacologia , Imunofenotipagem , Masculino , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Pressão Osmótica , Ocitocina/análise , Ocitocina/imunologia , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio/farmacologia , Vasopressinas/análise , Vasopressinas/imunologia , Equilíbrio Hidroeletrolítico/fisiologiaRESUMO
Rat and mouse hypothalami from postnatal animals containing highly differentiated neurones survive very well in long-term (>15 days in vitro, DIV) stationary organotypic cultures. Magnocellular oxytocin (OT) and vasopressin (VP) neurones are present in identifiable paraventricular (PVN), supraoptic (SON) and accessory (ACC) nuclei in these cultures. After 15 DIV in standard medium immunocytochemistry revealed 427 +/- 63 OT cells and 217 +/- 27 VP cells per cultured rat hypothalamus, and 380 +/- 72 OT cells and 622 +/- 91 VP cells per cultured mouse hypothalamus. Following a 7-day adaptation period in standard culture medium containing serum, the rat slice-explants survived very well after subsequent transfer to defined, serum- free media (SFM) for an additional 8 days. The number of OT cells surviving in SFM was 612 +/- 147 OT cells per cultured rat hypothalamus. Only 0.5% of the magnocellular OT and VP neurones in the cultures appeared to express both peptides. Experiments on c-fos gene expression in these cultures showed that while only 12% of the magnocellular OT and VP neurones contained barely detectable Fos protein in their nuclei under control conditions, potassium depolarization of these cultures for 3 h produced intense c-fos expression in 87-91% of these cells. Thus, magnocellular neurones in these cultures are sufficiently stable and responsive to permit long-term physiological and gene expression studies to be done under defined media conditions.
Assuntos
Hipotálamo/metabolismo , Neurônios/metabolismo , Ocitocina/metabolismo , Vasopressinas/metabolismo , Animais , Polaridade Celular/fisiologia , Sobrevivência Celular/fisiologia , Imunofluorescência , Hipotálamo/citologia , Imuno-Histoquímica , Camundongos , Neurônios/fisiologia , Técnicas de Cultura de Órgãos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
The accuracy of oculoplethysmography (OPG) and carotid phonoangiography (CPA) singly and in combination, the Doppler velocity detector, and photoplethysmography (PPG) was checked by measurement of the degree of stenosis as shown on arteriograms in 308 internal carotid arteries. In a second study using arteriographic measurement in 210 internal carotid arteries, the comparative accuracy of the fluid-filled (Kartchner) and the air-filled (Zira) OPG, each with and without CPA, was assessed. In the first study the specificity in arteries with less than 40% diameter reduction varied from 88% for the PPG to 97% for the Doppler examination. The sensitivity in arteries with more than 40% diameter reduction varied from 17% for the Doppler examination to 80% for the combination of OPG plus CPA. For arteries with a reduction in diameter greater than 70%, the sensitivity varied from 67% for the CPA to 87% for the OPG plus CPA. The sensitivity of the OPG plus CPA for total occlusions was 93%. For bilateral carotid artery stenosis over 40%, the sensitivity varied from 50% for the CPA to 82% for the combined OPG plus CPA. In the second study, for arteries with less than 40% stenosis the specificity varied from 86% for the Zira computed readout to 93% for the OPG(K). In the second study, when retrospectively analyzed, the sensitivity for arteries with more than 40% stenosis varied from 74% for the Zira computed readout to 88% for the combined OPG(K) plus CPA. For arteries with greater than 70% diameter reduction the sensitivity varied from 79% for the Zira readout to 100% for OPG plus CPA. For bilateral carotid artery disease with greater than 40% diameter reduction, the sensitivity ranged from 50% for OPG(Z) to 77% for OPG(Z) plus CPA.
Assuntos
Doenças das Artérias Carótidas/diagnóstico , Artéria Carótida Interna/fisiopatologia , Angiografia/métodos , Velocidade do Fluxo Sanguíneo , Artérias Carótidas/diagnóstico por imagem , Doenças das Artérias Carótidas/fisiopatologia , Efeito Doppler , Estudos de Avaliação como Assunto , Reações Falso-Negativas , Reações Falso-Positivas , Humanos , Pletismografia/métodosRESUMO
Antisera against partially processed, unamidated forms of AVP and OT were raised and characterized by radioimmunoassay and immunocytochemistry. These antibodies, and antibodies that recognize fully processed, amidated forms of AVP and OT, were used together with various fractionation methods to study the content of prohormones, partially processed and fully processed forms of AVP and OT in the hypothalamo-neurohypophysial system of adult and fetal (E21) rats. The levels of cleaved AVP and OT in the fetus were lower than those of the adult (1 to 3 orders of magnitude for brain and pituitary, respectively), and the detection of cleaved OT in brain and pituitary was delayed compared to that of AVP. Pro-AVP cleavage efficiency in the adult and the fetus was high (99 and 95% cleavage, respectively) resulting in formation of fully processed amidated forms of AVP, with no detectable partially processed peptides. Pro-OT processing in the adult was very similar (over 99% cleavage) resulting in formation of fully processed amidated OT. However, Pro-OT processing efficiency in the fetus was very low and incomplete, resulting in 40% unprocessed precursor and the accumulation of C-terminally extended unamidated intermediate forms (OT-Gly, OT-Gly-Lys, and OT-Gly-Lys-Arg).
Assuntos
Arginina Vasopressina , Neurofisinas , Ocitocina/análogos & derivados , Precursores de Proteínas/metabolismo , Processamento de Proteína Pós-Traducional , Vasopressinas/metabolismo , Animais , Anticorpos/imunologia , Cromatografia em Gel , Reações Cruzadas , Eletroforese , Sistema Hipotálamo-Hipofisário/imunologia , Sistema Hipotálamo-Hipofisário/metabolismo , Imuno-Histoquímica , Masculino , Ocitocina/imunologia , Ocitocina/metabolismo , Precursores de Proteínas/imunologia , Radioimunoensaio , Ratos , Ratos Endogâmicos , Núcleo Supraóptico/imunologia , Núcleo Supraóptico/metabolismo , Núcleo Supraóptico/ultraestrutura , Vasopressinas/imunologiaRESUMO
The present study investigated the effect of a surgically induced varicocele on the dynamics of testicular blood flow. The surface vasculature of the normal and the varicocele-affected testis was examined utilizing intravital epi-illumination microscopy. Application of this technique to the study of the varicocele is new. Blood flow characteristics in surface veins were studied as the surface temperature of the testis was varied. Periodic, reproducible stoppages in blood flow, determined by direct observation of the red blood cells, were seen in seven of eight sham animals at the lower temperatures. These stoppages were abolished and blood flow increased at higher temperatures; stoppages reappeared at lower temperatures. The periodic stoppages were present in only one of eight rats with a proven varicocele (P less than 0.025) at any temperature studied. This loss of blood flow regulation may be the result of a loss of testicular arteriolar tone and may explain the increase in testicular blood flow and temperature elevation observed in association with a varicocele. These findings may provide new insights into the pathophysiology of the varicocele and highlight the need to study the microvascular sequelae of this vascular abnormality.