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Diagnosing and treating patients with acute or early HIV-1 infection (AEHI) is an important strategy to prevent HIV-1 transmission. We used qualitative methods to understand factors that facilitate adjustment to AEHI diagnosis, prompt linkage to care and initiation of antiretroviral treatment (ART). Twenty-three AEHI patients (12 women, 11 men) included 18 participants identified at health facilities, and 5 participants identified in a sex worker cohort. Of these, 17 participants (9 women, 8 men) participated in qualitative interviews about their AEHI status 2 weeks after diagnosis. Thirteen participants (7 women, 6 men) returned for a second interview 12 weeks after diagnosis. Interviews explored participants' experiences at the time of and following their diagnosis, and examined perceptions about ART initiation and behavior change recommendations, including disclosure and partner notification. A grounded theory framework was used for analysis, eliciting three important needs that should be addressed for AEHI patients: 1) the need to better understand AEHI and accept one's status; 2) the need to develop healthy strategies and adjust to the reality of AEHI status; and 3) the need to protect self and others through ART initiation, adherence, safer sex, and disclosure. A preliminary conceptual framework to guide further intervention and research with AEHI populations is proposed.
Assuntos
Ajustamento Emocional , Infecções por HIV/psicologia , Adulto , Antirretrovirais/uso terapêutico , Estudos de Coortes , Busca de Comunicante , Revelação , Diagnóstico Precoce , Feminino , Teoria Fundamentada , Infecções por HIV/diagnóstico , Infecções por HIV/tratamento farmacológico , HIV-1 , Humanos , Quênia , Masculino , Pesquisa Qualitativa , Profissionais do SexoRESUMO
OBJECTIVES: To demonstrate the value of routine, basic sexually transmitted infection (STI) screening at enrolment into an HIV-1 vaccine feasibility cohort study and to highlight the importance of soliciting a history of receptive anal intercourse (RAI) in adults identified as "high risk". METHODS: Routine STI screening was offered to adults at high risk of HIV-1 upon enrolment into a cohort study in preparation for HIV-1 vaccine trials. Risk behaviours and STI prevalence were summarised and the value of microscopy assessed. Associations between prevalent HIV-1 infection and RAI or prevalent STI were evaluated with multiple logistic regression. RESULTS: Participants had a high burden of untreated STI. Symptom-directed management would have missed 67% of urethritis cases in men and 59% of cervicitis cases in women. RAI was reported by 36% of male and 18% of female participants. RAI was strongly associated with HIV-1 in men (adjusted odds ratio (aOR) 3.8; 95% CI 2.0 to 6.9) and independently associated with syphilis in women (aOR 12.9; 95% CI 3.4 to 48.7). CONCLUSIONS: High-risk adults recruited for HIV-1 prevention trials carry a high STI burden. Symptom-directed treatment may miss many cases and simple laboratory-based screening can be done with little cost. Risk assessment should include questions about anal intercourse and whether condoms were used. STI screening, including specific assessment for anorectal disease, should be offered in African research settings recruiting participants at high risk of HIV-1 acquisition.
Assuntos
Vacinas contra a AIDS , HIV-1 , Doenças Retais/prevenção & controle , Infecções Sexualmente Transmissíveis/prevenção & controle , Cervicite Uterina/prevenção & controle , Doenças Vaginais/prevenção & controle , Adulto , Doenças do Ânus/prevenção & controle , Feminino , Infecções por HIV/prevenção & controle , Humanos , Quênia , Masculino , Programas de Rastreamento , Anamnese , Dor/etiologia , Pacientes , Doença Inflamatória Pélvica/diagnóstico , Medição de Risco , Fatores de Risco , Comportamento SexualRESUMO
BACKGROUND: HIV rapid tests (RT) are a quick and non-technically demanding means to perform HIV voluntary counselling and testing (VCT) but understanding their limitations is vital to delivering quality VCT. OBJECTIVE: To determine the sensitivity and specificity of HIV rapid tests used for research and voluntary counselling and testing at four sites in East Africa. DESIGN: Cross-sectional study. SETTING: Masaka District, Uganda; a sugar plantation in Kakira, Uganda; Coastal Villages in the Kilifi District of Kenya; and the Urban slum of Kangemi located West of Nairobi, Kenya. SUBJECTS: Six thousands two hundred and fifty five consenting volunteers were enrolled into the study, and 675 prevalent HIV infections were identified. RESULTS: The RT sensitivity tended to be high for all assays at all sites (97.63-100%) with the exception of the Uni-Gold assay (90.24% in Kangemi, 96.58% in Kilifi). Twenty four RT results were recorded as 'weak positives', 22 (92%) of which were negative by ELISA. There was a high rate of RT false positives in Uganda (positive predictive values ranging from 45.70% to 86.62%). CONCLUSIONS: The sensitivity and specificity of the RT varied significantly across sites. The rate of RT misclassification in Uganda suggests that a multiple test algorithm may be preferable to a single test as screener for HIV VCT.
Assuntos
Sorodiagnóstico da AIDS/métodos , Aconselhamento Diretivo/estatística & dados numéricos , Infecções por HIV/diagnóstico , HIV-1/isolamento & purificação , Adolescente , Adulto , Algoritmos , Estudos Transversais , Ensaio de Imunoadsorção Enzimática , Estudos de Viabilidade , Feminino , Infecções por HIV/epidemiologia , Infecções por HIV/fisiopatologia , Humanos , Masculino , Pessoa de Meia-Idade , Valor Preditivo dos Testes , Kit de Reagentes para Diagnóstico , Sensibilidade e Especificidade , Adulto JovemRESUMO
A novel transcript of the GH gene has been identified in ocular tissues of chick embryos. It is, however, unknown whether this transcript (small chicken GH, scGH) is translated. This possibility was therefore assessed. The expression of scGH mRNA was confirmed by RT-PCR, using primers that amplified a 426-bp cDNA of its coding sequence. This cDNA was inserted into an expression plasmid to transfect HEK 293 cells, and its translation was shown by specific scGH immunoreactivity in extracts of these cells. This immunoreactivity was directed against the unique N terminus of scGH and was associated with a protein of 16 kDa, comparable with its predicted size. Most of the immunoreactivity detected was, however, associated with a 31-kDa moiety, suggesting scGH is normally dimerized. Neither protein was, however, present in media of the transfected HEK cells, consistent with scGH's lack of a signal sequence. Similar moieties of 16 and 31 kDa were also found in proteins extracted from ocular tissues (neural retina, pigmented epithelium, lens, cornea, choroid) of embryos, although they were not consistently present in vitreous humor. Specific scGH immunoreactivity was also detected in these tissues by immunocytochemistry but not in axons in the optic fiber layer or the optic nerve head, which were immunoreactive for full-length GH. In summary, we have established that scGH expression and translation occurs in ocular tissues of chick embryos, in which its localization in the neural retina and the optic nerve head is distinct from that of the full-length protein.
Assuntos
Regulação da Expressão Gênica no Desenvolvimento , Hormônio do Crescimento/genética , Biossíntese de Proteínas , Sequência de Aminoácidos , Animais , Sequência de Bases , Western Blotting , Células Cultivadas , Embrião de Galinha , Galinhas , Hormônio do Crescimento/metabolismo , Humanos , Imuno-Histoquímica , Rim/citologia , Dados de Sequência Molecular , Nervo Óptico/embriologia , Nervo Óptico/fisiologia , Retina/embriologia , Retina/fisiologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa , TransfecçãoRESUMO
Intravenous injection of cells and their tissue culture supernatants (CS) from human lymphoblastoid cell lines (LCL) induced the formation of lesions on the chorioallantoic membrane (CAM) of the chicken embryo. Injection of cells and CS from non-LCL and normal human lymphocytes induced few or no lesions. Irradiated chick embryos were more sensitive to lesion formation than were nonirradiated embryos. The log10 CAM lesions induced in irradiated (500 rads) embryos were a linear function of the log10 cells (from LCL) in the inoculum; the slope was 1.0, within experimental error. The formation of CAM lesions did not depend on the presence of Epstein-Barr virus (EBV) since lesions were also induced by cells and extracts derived from EBV genome-free LCL. Lesion-inducing activity associated with CS was filterable through 0.22-mu filters, sedimented at 78,000 x g, and sensitive to inactivation by heat (56 degrees C for 30 min), UV irradiation, chloroform, sera from chickens immunized against CS, and certain human sera. Lesion-inducing activity associated with cells and extracts was resistant to 5,000 rads of gamma-radiation. B2/B2 embryos (the B locus is the major histocompatibility locus of chickens) were more sensitive to lesion formation than were B15/B21 and outbred embryos; this suggested a genetic influence on lesion formation. Our data suggest that the irradiated chicken embryo may be a highly sensitive and useful means for the detection of an unidentified or unknown agent or agents that may play an important role in human oncogenic lymphocyte transformation or might interact with transforming viruses.
Assuntos
Transformação Celular Neoplásica , Membranas Extraembrionárias/patologia , Linfócitos/microbiologia , Vírus Oncogênicos/isolamento & purificação , Virologia/métodos , Alantoide/patologia , Animais , Anticorpos Antivirais , Bromodesoxiuridina/farmacologia , Linfoma de Burkitt/microbiologia , Linhagem Celular , Embrião de Galinha/efeitos da radiação , Clorofórmio/farmacologia , Córion/patologia , Temperatura Alta , Humanos , Idoxuridina/farmacologia , Leucemia/microbiologia , Raios Ultravioleta , Replicação Viral/efeitos dos fármacosRESUMO
The goal of pharmacogenetic research is to assist clinicians in predicting patient response to medications when genetic variations are identified. The pharmacogenetic variation of antiepileptic drug response and side effects has yielded findings that have been included in drug labeling and guidelines. The goal of this review is to provide a brief overview of the pharmacogenetic research on antiepileptic drugs. It will focus on findings that have been included in drug labeling, guidelines, and candidate pharmacogenetic variation. Overall, several genes have been included in guidelines by national and international organizations; however, much work is needed to implement and evaluate their use in clinical settings.
RESUMO
We have examined the expression of TNF-alpha and its receptors, TNFR1 and TNFR2, during gastrulation in the chick embryo, and have investigated the possible role of this factor in the control of cell death at this early stage of development. TNF-alpha, immunoreactive at approximately 17 kD, was found both in vivo and in vitro, most intensely associated with the cell surface and cytoskeleton of endoderm cells. TNFR2 was especially immunoreactive in endoderm cells of the marginal zone. TNFR1 was found in nuclei throughout the embryo. Embryos also showed widespread expression of both the bcl-2 and Bax gene products, which are both associated with cell death pathways. Intact embryos in culture were sensitive to the addition of TNF-alpha (approx. 110 ng/ml), responding by significantly increasing the incidence of DNA fragmentation in cells from all tissues of the embryo. This effect was abrogated by immunological pre-absorption of the cytokine. Cultured cells from these embryos also responded to the addition of agonistic antibodies to TNF-alpha receptors by increasing DNA fragmentation. A similar response to TNF-alpha antiserum by cultured cells appeared to be related to a concomitant decrease in cell-substratum adhesion caused by the antibody. Decreased cell adhesion, induced non-specifically with anti-integrin antiserum, also resulted in increased DNA fragmentation. TNF-alpha, synthesized and secreted by the embryo itself, may be able to exert a paracrine effect on the incidence of cell death in tissues of the embryo, and the cell death process may be related to the expression of bcl-2 and Bax gene products. The influence of TNF-alpha may be exerted by the activation of cell death signalling pathways directly, or indirectly through perturbation of the cytoskeleton or of integrin-mediated cell adhesion.
RESUMO
Although cell death has long been recognized to be a significant element in the process of embryonic morphogenesis, its relationships to differentiation and its mechanisms are only now becoming apparent. This new appreciation has come about not only through advances in the understanding of cell death in parallel immunological and pathological situations, but also through progress in developmental genetics which has revealed the roles played by death in the cell lineages of invertebrate embryos. In this review, we discuss programmed cell death as it is understood in developmental situations, and its relationship to apoptosis. We describe the morphological and biochemical features of apoptosis, and some methods for its detection in tissues. The occurrence of programmed cell death during invertebrate development is reviewed, as well as selected examples in vertebrate development. In particular, we discuss cell death in the early vertebrate embryo, in limb development, and in the nervous system.
Assuntos
Apoptose , Invertebrados/crescimento & desenvolvimento , Vertebrados/crescimento & desenvolvimento , Animais , Extremidades/crescimento & desenvolvimento , Sistema Nervoso/citologia , Vertebrados/embriologiaRESUMO
Leptospirosis is difficult to distinguish from dengue fever without laboratory confirmation. Sporadic cases/clusters of leptospirosis occur in Puerto Rico, but surveillance is passive and laboratory confirmation is rare. We tested for leptospirosis using an IgM ELISA on sera testing negative for dengue virus IgM antibody and conducted a case-control study assessing risk factors for leptospirosis, comparing clinical/laboratory findings between leptospirosis (case-patients) and dengue patients (controls). Among 730 dengue-negative sera, 36 (5%) were positive for leptospirosis. We performed post mortem testing for leptospirosis on 12 available specimens from suspected dengue-related fatalities; 10 (83%) tested positive. Among these 10 fatal cases, pulmonary hemorrhage and renal failure were the most common causes of death. We enrolled 42 case-patients and 84 controls. Jaundice, elevated BUN, hyperbilirubinemia, anemia, and leukocytosis were associated with leptospirosis (p < .01 for all). Male sex, walking in puddles, rural habitation, and owning horses were independently associated with leptospirosis. Epidemiological, clinical, and laboratory criteria may help distinguish leptospirosis from dengue and identify patients who would benefit from early antibiotic treatment.
Assuntos
Dengue/diagnóstico , Leptospirose/diagnóstico , Vigilância da População/métodos , Adolescente , Adulto , Estudos de Casos e Controles , Criança , Pré-Escolar , Dengue/etiologia , Diagnóstico Diferencial , Feminino , Humanos , Incidência , Lactente , Leptospirose/etiologia , Leptospirose/mortalidade , Masculino , Prontuários Médicos , Pessoa de Meia-Idade , Porto Rico/epidemiologia , Fatores de RiscoRESUMO
We have used double-label confocal microscopy to examine the relationships between the orientation of filopodial extension in mesoderm cells and the orientation of fibronectin-rich extracellular matrix fibrils during chick embryo gastrulation. We fluorescently labeled mesoderm tissue dissected from donor embryos by immersion in carboxyfluorescein and then grafted it into unlabeled host embryos at the same stage of gastrulation. After further incubation, the host embryos were fixed, the endoderm removed, and the extracellular matrix was immunostained with antibodies to fibronectin conjugated to Texas Red. We found that both the general shape of the mesoderm cells and the orientation of filopodial extension were influenced by the surrounding matrix fibrils. Elongated shape was associated with individual fibrils which impinge on only one side of the cell. Similarly, filopodial extension followed a single fibronectin-rich fibril, although filopodia were also observed to be channeled between pairs of parallel fibrils. Cells attached to non-aligned regions of substratum showed no polarity. The mesoderm cells themselves apparently synthesize their own fibronectin, and deposit this on the cell surface not attached to the substratum. We conclude that individual fibronectin-rich substratum fibrils, in the size range 0.7-2.8 microns, are able to exert contact guidance on the mesoderm cells, despite the production of endogenous fibronectin by the cells themselves. These results support the contention that contact guidance is a physiological mechanism influencing the orientation and directionality of cells during the morphogenetic movements of embryogenesis.
Assuntos
Matriz Extracelular/fisiologia , Fibronectinas/análise , Gástrula/fisiologia , Mesoderma/ultraestrutura , Microscopia Confocal , Animais , Embrião de Galinha , Matriz Extracelular/química , Fluoresceínas , Corantes Fluorescentes , Microscopia de Fluorescência , XantenosRESUMO
Early chick embryos have previously been shown to express tumor necrosis factor-alpha-cross-reactive proteins (TNF alpha-CRPs) in a developmentally regulated manner, thus implicating these proteins in programmed cell death and in tissue remodeling. In this study, cells undergoing DNA fragmentation have been identified, using terminal deoxynucleotide transferase (TdT) mediated dUTP-biotin nick-end-labeling (TUNEL), during the embryonic development of the chick, between stages 18 and 29. DNA fragmentation is indicative of cells undergoing programmed cell death. TUNEL-positive cells were identified in several well documented areas of programmed cell death, including the limb buds, the heart, spinal motoneurons, dorsal root ganglia, and the ventral horn of the neural tube. In addition, other areas of cell death were identified including the floor plate and the mesonephros. In several locations, a close correlation was noted between the presence of TUNEL-positive cells and regions of TNF alpha-immunoreactivity. These regions included the ventral horn and marginal zone of the neural tube, spinal motoneurons, paravertebral ganglia, parts of the myotome, mesenchyme of the body wall, and the mesonephros. In addition, using the TNF alpha-sensitive L929-8 bioassay it was shown that homogenate of stage 18 chick embryos is cytotoxic to L929-8 cells and that this toxicity can be reduced using neutralizing antibodies to mouse TNF alpha. This bioassay allowed us to estimate the mean concentration of TNF alpha-like activity in embryo homogenate, which is within the range of physiological (pg/ml) levels of TNF alpha found in other systems. These results suggest that proteins with TNF alpha-like activity may have a role in programmed cell death in some tissues during early chick embryo development.
Assuntos
Apoptose , Fator de Necrose Tumoral alfa/análise , Animais , Sistema Nervoso Central/química , Sistema Nervoso Central/embriologia , Embrião de Galinha , Nervos Cranianos/química , Nervos Cranianos/embriologia , DNA/metabolismo , Extremidades/embriologia , Gânglios/química , Gânglios/embriologia , Coração/embriologia , Mesonefro/química , Miocárdio/química , Fatores de TempoRESUMO
Early chick embryos contain two beta-galactoside-binding lectins of 16 kDa and 14 kDa. using several antisera to these proteins, we have studied lectin expression at embryonic stages when the segregation and early differentiation of organ primordia are taking place. With antisera to the 16 kDa lectin that display similar immunoreactivity in immunoblot analysis, we show that these antisera exhibit varying immunoreactivity in embryo sections. One antiserum reacts preferentially with a matrix form of lectin while another detects mainly a cellular form of this protein. During early development, galactoside-binding lectins of the matrix type are expressed in the vitelline membrane, the outer and inner limiting membranes of the neural tube, the surface of the notochord and the coelomic surface of the cardiac rudiments. The cellular form of the lectin occurs in the intracellular yolk of early embryos, in the primordial germ cells, the myocardium, in the early myotome, and in a cohort of cells which are presumed to belong to the neural crest. Our results indicate that, although all of the antisera recognize the intracellular lectin of the extraembryonic endoderm, some antisera to the 16 kDa lectin exhibit preferential reactivity with different lectin isoforms. The extracellular matrix form of lectin is transiently expressed during early development at the stages when the segregation of organ primordia is occurring. It's expression could be related to the acquisition of polarity in developing epithelia. Results also suggest that various versions of the same protein may perform distinct developmental roles in the embryo.
Assuntos
Embrião de Galinha/fisiologia , Hemaglutininas/biossíntese , Animais , Blastoderma/citologia , Blastoderma/fisiologia , Embrião de Galinha/citologia , Embrião de Galinha/ultraestrutura , Desenvolvimento Embrionário e Fetal , Galactosídeos/metabolismo , Galectinas , Hemaglutininas/análise , Hemaglutininas/isolamento & purificação , Soros Imunes , Immunoblotting , Imuno-Histoquímica/métodos , Mesoderma/citologia , Mesoderma/fisiologia , Microscopia Imunoeletrônica/métodos , Peso MolecularRESUMO
GH receptor immunoreactivity is widely distributed within the rat pituitary gland, although apart from somatotrophs the cell types with GH receptor immunoreactivity have yet to be identified. It is also unknown whether this immunoreactivity reflects the presence of GH binding proteins (GHBPs) or authentic receptors. The possible colocalization of GHBPs and pituitary hormones in somatotrophs, lactotrophs, gonadotrophs, thyrotrophs, and corticotrophs was therefore examined using immunogold electron microscopy. Pituitary sections were indirectly immunostained with antibodies for GH, PRL, LH, FSH, TSH, or ACTH using gold-labeled immunoglobulin G. The same grids were also immunostained with a polyclonal antibody raised against rat GHBP using protein A conjugated to gold particles of a different size. Some sections were gold-labeled using a monoclonal antibody (MAb 4.3) raised against the unique hydrophobic tail of the GHBP, using gold-labeled immunoglobulin G. The cellular and ultrastructural distribution of immunoreactivity within the pituitary gland was similar after labeling with either GHBP antiserum. In all immunoreactive cells the labeling was most intense in secretory granules. Specific staining was not, however, demonstrated in the nucleus, in contrast with earlier findings using MAb 263. Moreover, while staining with MAb 263 appeared to be ubiquitous, some pituitary cells were not labeled by the polyclonal GHBP antiserum or by MAb 4.3. GHBP immunoreactivity was, however, colocalized with hormones in all pituitary cell-types, although in some cells GHBP immunoreactivity was not present in all secretory granules. These results clearly demonstrate the presence of GHBPs in adenohypophyseal cells, in which they appear to be stored or secreted with GH, PRL, LH, FSH, TSH, and ACTH. The function of GHBPs in the pituitary gland is, however, uncertain.
Assuntos
Proteínas de Transporte/análise , Adeno-Hipófise/ultraestrutura , Hormônios Hipofisários/análise , Hormônio Adrenocorticotrópico/análise , Animais , Anticorpos Monoclonais , Grânulos Citoplasmáticos/química , Hormônio Foliculoestimulante/análise , Hormônio do Crescimento/análise , Imuno-Histoquímica , Hormônio Luteinizante/análise , Microscopia Imunoeletrônica , Adeno-Hipófise/química , Prolactina/análise , Ratos , Tireotropina/análiseRESUMO
We have previously shown that human platelets express matrix metalloproteinase-2 (MMP-2) and that the release of this enzyme during platelet activation mediates the ADP- and thromboxane-independent part of aggregation. We have now used immunogold electron microscopy, flow cytometry. Western blot analysis and zymography methods to study the ultrastructural localization of MMP-2 in human washed platelets. Platelet aggregation was stimulated by collagen and the MMP-2 immunoreactivity of platelets was followed during various stages of aggregation. In resting platelets, MMP-2 was randomly distributed in the platelet cytosol without detectable association with platelet granules. Platelet aggregation caused the translocation of MMP-2 from the cytosol to the extracellular space. During the early stages of aggregation, MMP-2 remained in close association with the platelet plasma membrane. We conclude that the interactions of MMP-2 with platelet surface membranes mediate the aggregatory response induced by this enzyme.
Assuntos
Plaquetas/enzimologia , Gelatinases/sangue , Metaloendopeptidases/sangue , Agregação Plaquetária , Difosfato de Adenosina/farmacologia , Transporte Biológico , Plaquetas/ultraestrutura , Membrana Celular/enzimologia , Colágeno/farmacologia , Citosol/enzimologia , Espaço Extracelular/enzimologia , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Metaloproteinase 2 da Matriz , Agregação Plaquetária/efeitos dos fármacosRESUMO
Growth hormone (GH) gene expression predominantly occurs in the pituitary gland, although it also occurs in many extrapituitary sites, including the brain. The cellular location and ontogeny of neural GH production is, however, largely unknown. This has therefore been determined during chick embryogenesis. In chicks, the brain develops from the neural tube at embryonic day (ED) 3. At this age, the divisions of the brain (the telencephalon, diencephalon, mesencephalon, metencephalon and myelencephalon) have intense GH immunoreactivity (GH-IR) (detected by two polyclonal antibodies and a monoclonal antibody for chicken GH). The otic and optic vesicles were also strongly GH immunoreactive, as were the Vth (semi-lunar), VIIth (facial), VIIIth (acoustic) and IXth (glossopharyngeal) nerve ganglia. This GH-IR was specific for GH and was lost when the antibodies were preabsorbed with recombinant chicken GH. The widespread distribution of GH-IR in the neural tissues of ED 3 embryos was mirrored by the distribution of GH receptor (GHR) immunoreactivity, detected by an antibody raised against the chicken GHR. In ED 6/ED 7 embryos, the neural retina of the eye and the epithelial and lens fiber cells were intensely stained for GH-IR, as was Rathke's pouch and the wall of the diencephalon. In contrast, only a few scattered cells were immunoreactive in the surrounding mesoderm. At ED 14, the GH-IR in the brain was restricted to specific tissues and cells. For instance, immunoreactive cells were present in the molecular and pyramidal layers of the cerebral cortex, in the gray matter of the cerebellum, in the choroid plexus, and in the walls of the ventricles. In summary, GH- and GHR-like proteins are abundant in neural tissues of the chick during the first third of incubation, becoming discretely localized to specific tissues and cells during later incubation. The localization of GH and GHR in these tissues, prior to the ontogeny of plasma GH, suggests autocrine or paracrine roles for GH during early embryogenesis.
Assuntos
Embrião de Galinha/metabolismo , Hormônio do Crescimento/metabolismo , Sistema Nervoso/embriologia , Sistema Nervoso/metabolismo , Animais , Encéfalo/embriologia , Encéfalo/metabolismo , Olho/embriologia , Olho/metabolismo , Técnicas Imunoenzimáticas , Receptores da Somatotropina/metabolismoRESUMO
Early embryonic growth is independent of pituitary growth hormone (GH), since it occurs prior to the differentiation of pituitary somatotrophs. Embryogenesis is therefore thought to be regulated by local growth factors. As GH is now known to be produced in many extrapituitary sites, in which it acts in an autocrine or paracrine manner, the possibility that extra-pituitary GH may participate in embryogenesis and organogenesis was assessed by determining the immunocytochemical presence and location of GH- and GH-receptor (GHR)-like proteins in the peripheral tissues of chick embryos during their 21-day incubation period. Immunoreactive (IR)-GH, detectable by a monoclonal and two polyclonal antibodies for chicken GH, was specifically and ubiquitously present in tissues of 3-day-old embryos. At embryonic day (ED) 5, IR-GH was widespread in ectodermal, mesodermal and endodermal tissues, but it was not present in every cell of each tissue. IR-GH was particularly abundant i! n the neural tube, notochord, limb bud, somites, heart, stomach, liver, kidney, Wolffian duct and the amnion. By ED8, IR-GH was still widespread and was now present in limb bud cartilage, although the heart and liver were no longer GH immunoreactive. GH receptor immunoreactivity was also present in most tissues and cells of ED3-ED8 embryos. These results demonstrate that extrapituitary GH is abundantly present during early embryogenesis, prior to the differentiation of pituitary somatotrophs (at ED12). Since GH- and GHR-like proteins are present in most tissues of the chick embryo, it is proposed that extrapituitary GH may act as a local growth factor during embryonic development.
Assuntos
Embrião de Galinha/metabolismo , Hormônio do Crescimento/análise , Receptores da Somatotropina/análise , Âmnio/química , Animais , Química Encefálica , Sistema Nervoso Central/química , Sistema Nervoso Central/embriologia , Coração Fetal/química , Idade Gestacional , Hormônio do Crescimento/genética , Imuno-Histoquímica , Rim/química , Botões de Extremidades/química , Fígado/química , Morfogênese , Notocorda/química , Adeno-Hipófise/química , RNA Mensageiro/análise , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Estômago/química , Distribuição Tecidual , Ductos Mesonéfricos/químicaRESUMO
In this review, methods currently available for the detection of cell death in tissues are surveyed, with special reference to techniques that allow the recognition of apoptotic cells in situ, either in whole mount specimens or in tissue sections. The techniques considered include: several variations on in situ DNA nick-end labelling methods, vital dyes, lysosomal enzyme histochemistry, transglutaminase expression, and immunocytochemical detection of several death-associated antigens. These methods are discussed in relation to their utility in detecting different stages of cell death, and also to their ability to distinguish between apoptotic and necrotic cell death.
Assuntos
Apoptose/fisiologia , Fenômenos Fisiológicos Celulares , Animais , DNA/química , DNA/metabolismo , Histocitoquímica , HumanosRESUMO
Upon receptor-mediated endocytosis of very-low-density lipoprotein (VLDL) and vitellogenin into growing chicken oocytes, the protein moieties of these lipoproteins are proteolytically cleaved. Unlike the complete lysosomal degradation in somatic cells, enzymatic ligand breakdown in oocytes generates a characteristic set of polypeptides, which enter yolk storage compartments for subsequent utilization by the embryo. Here, we demonstrate directly that the catalyst for the intraoocytic processing of both apolipoprotein B and vitellogenin is cathepsin D. The enzyme was purified from oocytic yolk, preovulatory follicle homogenates, and liver by affinity chromatography. When plasma VLDL and vitellogenin were incubated with the purified enzyme, fragments indistinguishable from those found in yolk were generated from both precursors under identical, mildly acidic conditions. Amino-terminal sequencing of the pure enzyme demonstrated 88% identity with mammalian cathepsin Ds over 34 residues. On the basis of this information, a full-length clone specifying chicken preprocathepsin D was isolated from a chicken follicle cDNA library by screening with a human cathepsin D probe. Whereas previous studies have demonstrated that the receptors for lipoproteins in somatic cells and oocytes, respectively, of the chicken are the products of different genes, Southern and Northern blot hybridization experiments showed that the enzymes expressed in oocytes and liver are the product of a single gene, giving rise to a 3.3-kb transcript. The primary structure of the 335-residue mature protein suggests a high degree of conservation of known crucial features of aspartyl proteases; however, the absence of the so-called processing region and of an aromatic residue in a region thought to partake in catalysis raise questions with possible evolutionary implications.
Assuntos
Catepsina D/genética , Gema de Ovo/enzimologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Catepsina D/isolamento & purificação , Catepsina D/metabolismo , Galinhas , Clonagem Molecular , DNA , Humanos , Lipoproteínas VLDL/metabolismo , Dados de Sequência Molecular , Oócitos/metabolismo , Precursores de Proteínas/isolamento & purificação , Precursores de Proteínas/metabolismo , Homologia de Sequência de Aminoácidos , Vitelogeninas/metabolismoRESUMO
Leptospirosis has rarely been reported in Puerto Rico, although in the period from 1948 to 1952, 208 cases of leptospirosis and an island-wide seroprevalence of antibody to Leptospira of 14% were documented. In Puerto Rico in October 1996, following rainfall and a period of flooding generated by Hurricane Hortense, serum specimens of 4 patients with suspected dengue fever that were negative for dengue tested positive for Leptospira-specific IgM antibodies in a dipstick assay. Subsequently, we used an island-wide dengue laboratory-based surveillance system to determine the increase in leptospirosis after hurricane-generated floods. All anti-dengue IgM-negative patients (n = 142) with disease onset from August 8 to October 6, 1996 from prehurricane and posthurricane groups were investigated for leptospirosis. Laboratory-confirmed leptospirosis cases were defined as microscopic agglutination test titers > or = 1 :400 to 1 or more serovars, or positive immunohistochemistry in autopsy tissues. Four (6%) of 72 prehurricane and 17 (24%) of 70 posthurricane patients had laboratory-confirmed cases of leptospirosis (relative risk [RR] = 4.4, 95% confidence interval [CI] = 1.6-12.4). The mean age of case-patients was 34 years (range = 13-64). Eighteen (86%) of 21 confirmed case-patients were males, including one patient who died (31 years old). Patients were located in 18 (38%) of 48 municipalities that submitted serum samples. Clinical features significantly associated with leptospirosis were eye pain (RR = 1.5, 95% CI = 1.3-1.9), joint pain (RR = 1.4, 95% CI = 1.1-1.6), diarrhea (RR = 1.7, 95% CI = 1.2-2.5), and jaundice (RR = 3.3, 95% CI = 1.5-7.2). This study demonstrates the utility of a dengue laboratory-based surveillance system for the detection of an increase of leptospirosis, which most likely would have gone unrecognized. Leptospirosis is treatable with antibacterial agents; knowledge of this diagnosis may significantly reduce morbidity and mortality.
Assuntos
Dengue/epidemiologia , Desastres , Leptospirose/epidemiologia , Adolescente , Adulto , Anticorpos Antibacterianos/sangue , Anticorpos Antivirais/sangue , Dengue/virologia , Vírus da Dengue/imunologia , Feminino , Humanos , Imunoglobulina M/sangue , Leptospira interrogans/classificação , Leptospira interrogans/imunologia , Leptospira interrogans/isolamento & purificação , Leptospirose/microbiologia , Masculino , Pessoa de Meia-Idade , Vigilância da População , Porto Rico/epidemiologiaRESUMO
Outbreaks of yellow fever (YF) have never been recorded in Kenya. However, in September 1992, cases of hemorrhagic fever (HF) were reported in the Kerio Valley to the Kenya Ministry of Health. Early in 1993, the disease was confirmed as YF and a mass vaccination campaign was initiated. Cases of suspected YF were identified through medical record review and hospital-based disease surveillance by using a clinical case definition. Case-patients were confirmed serologically and virologically. We documented 55 persons with HF from three districts of the Rift Valley Province in the period of September 10, 1992 through March 11, 1993 (attack rate = 27.4/100,000 population). Twenty-six (47%) of the 55 persons had serologic evidence of recent YF infection, and three of these persons were also confirmed by YF virus isolation. No serum was available from the other 29 HF cases. In addition, YF virus was isolated from a person from the epidemic area who had a nonspecific febrile illness but did not meet the case definition. Five patients with confirmed cases of YF died, a case-fatality rate of 19%. Women with confirmed cases of YF were 10.9 times more likely to die than men (P = 0.010, by Fisher's exact test). Of the 26 patients with serologic or virologic evidence of YF, and for whom definite age was known, 21 (81%) were between 10 and 39 years of age, and 19 (73%) were males. All patients with confirmed YF infection lived in rural areas. There was only one instance of multiple cases within a single family, and this was associated with bush-clearing activity. This was the first documented outbreak of YF in Kenya, a classic example of a sylvatic transmission cycle. Surveillance in rural and urban areas outside the vaccination area should be intensified.