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The body of a deceased human and 4 deceased cats were found in a house while a fire was being extinguished. As a result of these findings, arson, homicide, and animal death investigations were opened. As part of the animal death investigation, all of the cats were submitted for veterinary forensic autopsies. All cats had soot on the fur and had soot deposits within the oral cavity, esophagus, and respiratory tract. Two cats had soot within the stomach. Cardiac blood was analyzed for carboxyhemoglobin using a CO-oximeter, and all cats had levels >65%. The cause of death was determined to be due to toxic smoke inhalation from the structure fire. Case findings support the potential use of CO-oximeter for determination of carboxyhemoglobin levels in cats and continued research in this area of forensic practice.
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Intoxicação por Monóxido de Carbono , Doenças do Gato , Incêndios , Piromania , Gatos , Humanos , Animais , Homicídio , Fuligem/análise , Carboxihemoglobina/análise , Oximetria/veterinária , Intoxicação por Monóxido de Carbono/veterináriaRESUMO
SmB6 is a strongly correlated mixed-valence Kondo insulator with a newly discovered surface state, proposed to be of non-trivial topological origin. However, the surface state dominates electrical conduction only below T∗ ≈ 4 K (ref. ), limiting its scientific investigation and device application. Here, we report the enhancement of T∗ in SmB6 under the application of tensile strain. With 0.7% tensile strain we report surface-dominated conduction at up to a temperature of 240 K, persisting even after the strain has been removed. This can be explained in the framework of strain-tuned temporal and spatial fluctuations of f-electron configurations, which might be generally applied to other mixed-valence materials. We note that this amount of strain can be induced in epitaxial SmB6 films via substrate in potential device applications.
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BACKGROUND: Certain glomerulopathies are associated with increased levels of CD80 (B7-1). We measured the urinary excretion of CD80, podocyturia and proteinuria in controls and in subjects with Fabry disease either untreated or on enzyme replacement therapy (ERT). METHODS: Cross-sectional study including 65 individuals: controls (n = 20) and Fabry patients (n = 45, 23 of them not on ERT and 22 on ERT). Variables included age, gender, urinary protein/creatinine ratio (UPCR), estimated glomerular filtration rate (eGFR), urinary uCD80/creatinine ratio (uCD80) and podocyturia. CD80 mRNA expression in response to lyso-Gb3, a bioactive glycolipid accumulated in Fabry disease, was studied in cultured human podocytes. RESULTS: Controls and Fabry patients did not differ in age, eGFR and gender. However, UPCR, uCD80 and podocyturia were significantly higher in Fabry patients than in controls. As expected, Fabry patients not on ERT were younger and a higher percentage were females. Non-ERT Fabry patients had less advanced kidney disease than ERT Fabry patients: UPCR was lower and eGFR higher, but uCD80 and podocyturia did not differ between non-ERT or ERT Fabry patients. There was a significant correlation between uCD80 and UPCR in the whole population (r 0.44, p 0.0005) and in Fabry patients (r 0.42, p 0.0046). Lyso-Gb3 at concentrations found in the circulation of Fabry patients increased uCD80 expression in cultured podocytes. CONCLUSIONS: Fabry disease is characterized by early occurrence of increased uCD80 excretion that appears to be a consequence of glycolipid accumulation. The potential for uCD80 excretion to reflect early, subclinical renal Fabry involvement should be further studied.
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Antígeno B7-1/urina , Doença de Fabry/patologia , Doença de Fabry/urina , Podócitos/metabolismo , Podócitos/patologia , Adolescente , Adulto , Idoso , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Estudos de Casos e Controles , Células Cultivadas , Criança , Doença de Fabry/metabolismo , Feminino , Glicolipídeos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Esfingolipídeos/metabolismo , Adulto JovemRESUMO
Animal sexual abuse (ASA) involves harm inflicted on animals for the purposes of human sexual gratification and includes such terms as bestiality, zoophilia, zoosadism, animal sexual assault, and others. The prevalence of ASA is not known, although it may be more common than is currently perceived. Veterinarians have the skills required to identify and document cases of ASA. This article reviews the terminology, legal definitions and forms of ASA, and its social and psychological context. An investigative approach is outlined, including an alternate light source examination; collection of swabs for DNA analysis; sampling vaginal washes, rectal washes, and toenails for trace evidence and biologic analyses; radiographic studies; and a complete forensic necropsy, including histopathology. Gross lesions identified in ASA victims include injuries to the anus, rectum, penis, scrotum, nipples, and vagina; the presence of foreign bodies; and abrasions, bruising, and other evidence of nonaccidental injury. Specialized procedures, including examination using alternate light sources and screening tests to identify human seminal fluid within samples from ASA victims, are of potential value but have not been validated for use in animals.
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Bem-Estar do Animal , Patologia Legal , Transtornos Parafílicos , Patologia Veterinária , Bem-Estar do Animal/legislação & jurisprudência , Animais , Autopsia/veterinária , Feminino , Patologia Legal/legislação & jurisprudência , Patologia Legal/métodos , Genitália/lesões , Humanos , Masculino , Patologia Veterinária/legislação & jurisprudência , Patologia Veterinária/métodos , Análise do Sêmen/veterináriaRESUMO
Semenogelins are proteins originating in the seminal vesicle and are useful markers for the presumptive identification of human semen. Detection of semenogelin can be done with a commercially available membrane test. In this study, a commercially available membrane test for human semenogelin proteins was used to assess for cross-reactivity in dog bodily fluids to allow for the potential utilization for detection of human semen in dog bodily fluids. The authors analyzed canine semen and other bodily fluids, including urine, saliva, vaginal secretions, fecal material, and blood. They also examined the distribution of human semenogelin I transcripts in the canine testis, prostate, and several bodily fluids by reverse transcription polymerase chain reaction. No cross-reactivity was observed in the canine bodily fluids tested except for a single rectal swab, which was negative on a second test. Further testing should be done to validate the use of this kit for screening samples from dogs suspected to have been victims of sexual abuse.
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Bem-Estar do Animal , Fitas Reagentes , Sêmen/imunologia , Proteínas Secretadas pela Vesícula Seminal/imunologia , Animais , Sangue/imunologia , Secreções Corporais/imunologia , Reações Cruzadas/imunologia , Cães , Fezes , Feminino , Humanos , Masculino , Transtornos Parafílicos/diagnóstico , Saliva/imunologia , Urina , Vagina/metabolismoRESUMO
BACKGROUND: Formal, structured annual appraisals are now a regulatory requirement for all doctors and must meet the standards stipulated by the General Medical Council (GMC), including connection with a designated body. AIMS: To investigate occupational physicians' opinions and experience of the first year of appraisals for revalidation. METHODS: Members of the Society of Occupational Medicine (SOM) were surveyed electronically regarding the appraisal process and its software. A quiz tested knowledge of the requirements and underlying principles and regulations of appraisal for revalidation. RESULTS: In total, 163 responses were received, with 57 free-text comments. Respondents spent a mean of 31h on the appraisal process (range 0-200h). Less than half of respondents said they had a clear understanding of the appraisal process. Most respondents (88 and 92%) reported difficulties with the appraisal process and the appraisal software. Only 22% felt the time taken for the appraisal process was proportionate to the value gained. Overall, there was support for appraisal and in particular for SOM appraisers, but only half of the respondents found it useful. The average quiz score was 11 out of 15 (74%) with only one respondent scoring full marks. About 25% of respondents were not aware that the responsible officer is obliged to act on any concerns raised. The free-text comments suggested that some physicians found the process costly and stressful. CONCLUSIONS: The findings suggest that the time requirements and process of appraisal for occupational physicians could be improved, and clearer, comprehensive and more accessible guidance should be developed.
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OBJECTIVE: Colocation of mental health screening, assessment, and treatment in primary care reduces stigma, improves access, and increases coordination of care between mental health and primary care providers. However, little information exists regarding older adults' attitudes about screening for mental health problems in primary care. The objective of this study was to evaluate older primary care patients' acceptance of and satisfaction with screening for depression and anxiety. METHODS: The study was conducted at an urban, academically affiliated primary care practice serving older adults. Study patients (N = 107) were screened for depression/anxiety and underwent a post-screening survey/interview to assess their reactions to the screening experience. RESULTS: Most patients (88.6%) found the length of the screening to be "just right." A majority found the screening questions somewhat or very acceptable (73.4%) and not at all difficult (81.9%). Most participants did not find the questions stressful (84.9%) or intrusive (91.5%); and a majority were not at all embarrassed (93.4%), upset (93.4%), or uncomfortable (88.8%) during the screening process. When asked about frequency of screening, most patients (72.4%) desired screening for depression/anxiety yearly or more. Of the 79 patients who had spoken with their physicians about mental health during the visit, 89.8% reported that it was easy or very easy to talk with their physicians about depression/anxiety. Multivariate results showed that patients with higher anxiety had a lower positive reaction to the screen when controlling for gender, age, and patient-physician communication. CONCLUSIONS: These results demonstrate strong patient support for depression and anxiety screening in primary care.
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Transtornos de Ansiedade/diagnóstico , Prestação Integrada de Cuidados de Saúde/normas , Transtorno Depressivo/diagnóstico , Serviços de Saúde para Idosos/organização & administração , Programas de Rastreamento/organização & administração , Serviços de Saúde Mental/organização & administração , Satisfação do Paciente , Atenção Primária à Saúde/organização & administração , Adulto , Idoso , Feminino , Avaliação Geriátrica/métodos , Serviços de Saúde para Idosos/normas , Humanos , Masculino , Programas de Rastreamento/normasRESUMO
BACKGROUND: Despite its widespread use, the beneficial effect of low-dose fentanyl administered at induction of anesthesia on perioperative outcomes has not been studied in the ambulatory setting. Therefore, this study was designed to test the hypothesis that administration of small-dose fentanyl vs. saline during induction reduces coughing and movements without adversely affecting recovery after day-surgery. METHODS: One hundred consenting outpatients scheduled to undergo superficial surgical procedures under general anesthesia with a laryngeal mask airway (LMA) device for airway management were randomly assigned to one of two treatment groups: control (n = 50) or fentanyl (n = 50). After administration of 2 ml of the unlabelled study medication containing either fentanyl (100 µg) or saline, anesthesia was induced with lidocaine 30-50 mg and propofol 2 mg/kg IV followed by the insertion of an LMA device. General anesthesia was maintained using a propofol infusion, 75 µg/kg/min, and desflurane (2-5% end-tidal) in 100% oxygen. RESULTS: Coughing was observed in six (12%) and ten (20%) in the fentanyl and control group, respectively (P = 0.41). The incidence of movements during surgery was lower in the fentanyl group (18% vs. 31%, P < 0001). There were no significant differences in early and late recovery times or pain scores between the two groups. CONCLUSION: Administration of a small-dose of fentanyl at induction of anesthesia significantly reduced purposeful movements during day-surgery under propofol-desflurane anesthesia. No significant difference was found in coughing or recovery times.
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Procedimentos Cirúrgicos Ambulatórios , Analgésicos Opioides/administração & dosagem , Fentanila/administração & dosagem , Adulto , Tosse/induzido quimicamente , Método Duplo-Cego , Feminino , Fentanila/efeitos adversos , Humanos , Masculino , Pessoa de Meia-Idade , MovimentoRESUMO
Fifteen dogs were found dead in a house that was on fire. Several of these dogs were partially burned. Four dogs were submitted for postmortem examination, 2 of which were determined to have died prior to the fire. Of the 2 submitted fire fatalities, only 1 dog had burns on its body (dorsum and right side of body). Internally, both dogs had soot deposits mixed with mucus in the larynx, trachea, and primary bronchi. Microscopically, soot was identified within both airways and alveolar spaces. There were no macroscopic or microscopic indications of vital heat exposure. High levels of carboxyhemoglobin were detected in the 2 dogs tested. The findings in this case support the use of postmortem examination and toxicology testing to allow for determination of vital reaction to heat and fire fumes.
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Carboxihemoglobina/análise , Doenças do Cão/patologia , Incêndios , Lesão por Inalação de Fumaça/veterinária , Animais , Autopsia/veterinária , Brônquios/patologia , Queimaduras/veterinária , Doenças do Cão/etiologia , Cães , Laringe/patologia , Sistema Respiratório/patologia , Lesão por Inalação de Fumaça/patologia , Fuligem/toxicidade , Traqueia/patologiaRESUMO
Nitric acid in air is formed by atmospheric reactions of oxides of nitrogen and is removed primarily through deposition to surfaces, either as the gas or after conversion to particulate nitrate. Many of the surfaces and particles have organic coatings, but relatively little is known about the interaction of nitric acid with organic films. We report here studies of the interaction of gaseous HNO(3) with a self-assembled monolayer (SAM) formed by reacting 7-octenyltrichlorosilane [H(2)C=CH(CH(2))(6)SiCl(3)] with the surface of a germanium infrared-transmitting attenuated total reflectance (ATR) crystal that was coated with a thin layer of silicon oxide (SiO(x)). The SAM was exposed at 298 ± 2 K to dry HNO(3) in a flow of N(2), followed by HNO(3) in humid N(2) at a controlled relative humidity (RH) between 20-90%. For comparison, similar studies were carried out using a similar crystal without the SAM coating. Changes in the surface were followed using Fourier transform infared spectroscopy (FTIR). In the case of the SAM-coated crystal, molecular HNO(3) and smaller amounts of NO(3)(-) ions were observed on the surface upon exposure to dry HNO(3). Addition of water vapor led to less molecular HNO(3) and more H(3)O(+) and NO(3)(-) complexed to water, but surprisingly, molecular HNO(3) was still evident in the spectra up to 70% RH. This suggests that part of the HNO(3) observed was initially trapped in pockets within the SAM and shielded from water vapor. After increasing the RH to 90% and then exposing the film to a flow of dry N(2), molecular nitric acid was regenerated, as expected from recombination of protons and nitrate ions as water evaporated. The nitric acid ultimately evaporated from the film. On the other hand, exposure of the SAM to HNO(3) and H(2)O simultaneously gave only hydronium and nitrate ions. Molecular dynamics simulations of defective SAMs in the presence of HNO(3) and water predict that nitric acid intercalates in defects as a complex with a single water molecule that is protected by alkyl chains from interacting with additional water molecules. These studies are consistent with the recently proposed hydrophobic nature of HNO(3). Under atmospheric conditions, if HNO(3) is formed in organic layers on surfaces in the boundary layer, e.g. through NO(3) or N(2)O(5) reactions, it may exist to a significant extent in its molecular form rather than fully dissociated to nitrate ions.
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BACKGROUND: In 2009, the General Medical Council (GMC) updated its guidance on consent, introducing a new statement that employees should be offered the opportunity to view reports, before the reports are sent to the employer. AIMS: To investigate the effects of this change on the perceptions and practice of occupational physicians. METHODS: A cross-sectional survey of UK occupational physicians via the Society of Occupational Medicine e-newsletter, seeking their opinions of the anticipated and actual effects of the guidance on employers, employees, occupational physicians and occupational health services. RESULTS: Two hundred and ninety-five completed questionnaires were returned (estimated response rate 30%). Respondents included 25% of accredited UK specialists. Some reported improved standards and greater transparency, however the change was generally perceived as unfavourable, with employee and employer losses: 50% reported delay providing timely advice to employers and 35% reported delays in employees returning to work. Significant variation in practice and increased costs were reported, with variable effects on different services. Difficulties in areas such as pensions and health surveillance were reported. Some occupational physicians had moved to instant reporting; others had moved away from this to allow more care with wording of reports. CONCLUSIONS: We found significant variations in practice between occupational physicians and concerns of employee and employer losses as a result of changes to the GMC consent guidance. Clearer guidance on practical implementation was desired. The background ethical reasoning should be stated so that the parameters of the guidance are delineated and its reach should be clarified.
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Atitude do Pessoal de Saúde , Medicina do Trabalho/normas , Médicos , Análise de Variância , Estudos Transversais , Inglaterra/epidemiologia , Feminino , Guias como Assunto , Humanos , Masculino , Medicina do Trabalho/ética , Percepção Social , Inquéritos e QuestionáriosRESUMO
Live-cell imaging is a common data acquisition technique used by biologists to analyze cell behavior. Since manually tracking cells in a video sequence is extremely time-consuming, many automatic algorithms have been developed in the last twenty years to accomplish the task. However, none of these algorithms can yet claim robust tracking performance at the varying of acquisition conditions (e.g., cell type, acquisition device, cell treatments). While many visualization tools exist to help with cell behavior analysis, there are no tools to help with the algorithm's validation. This paper proposes CellTrackVis, a new visualization tool for evaluating cell tracking algorithms. CellTrackVis allows comparing automatically generated cell tracks with ground truth data to help biologists select the best-suited algorithm for their experimented pipeline. Moreover, CellTackVis can be used as a debugging tool while developing a new cell tracking algorithm to investigate where, when, and why each tracking error occurred.
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Lassa virus (LASV), a Risk Group-4 zoonotic haemorrhagic fever virus, affects sub-Saharan African countries. Lassa fever, caused by LASV, results in thousands of annual deaths. Although decades have elapsed since the identification of the Natal multimammate mouse (Mastomys natalensis) as a natural reservoir of LASV, little effort has been made to characterize LASV infection in its reservoir. The natural route of infection and transmission of LASV within M. natalensis remains unknown, and the clinical impact of LASV in M. natalensis is mostly undescribed. Herein, using an outbred colony of M. natalensis, we investigate the replication and dissemination dynamics of LASV in this reservoir following various inoculation routes. Inoculation with LASV, regardless of route, resulted in a systemic infection and accumulation of abundant LASV-RNA in many tissues. LASV infection in the Natal multimammate mice was subclinical, however, clinical chemistry values were transiently altered and immune infiltrates were observed histologically in lungs, spleens and livers, indicating a minor disease with coordinated immune responses are elicited, controlling infection. Intranasal infection resulted in unique virus tissue dissemination dynamics and heightened LASV shedding, compared to subcutaneous inoculation. Our study provides important insights into LASV infection in its natural reservoir using a contemporary infection system, demonstrating that specific inoculation routes result in disparate dissemination outcomes, suggesting intranasal inoculation is important in the maintenance of LASV in the natural reservoir, and emphasizes that selection of the appropriate inoculation route is necessary to examine aspects of viral replication, transmission and responses to zoonotic viruses in their natural reservoirs.
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Reservatórios de Doenças/veterinária , Febre Lassa/veterinária , Vírus Lassa/fisiologia , Murinae/virologia , Doenças dos Roedores/virologia , Zoonoses Virais/virologia , Eliminação de Partículas Virais , Animais , Reservatórios de Doenças/virologia , Feminino , Humanos , Febre Lassa/transmissão , Febre Lassa/virologia , Vírus Lassa/genética , Masculino , Murinae/fisiologia , Doenças dos Roedores/transmissão , Zoonoses Virais/transmissãoRESUMO
A protein that may be an enkephalin precursor has been identified in extracts of bovine adrenal medulla. This protein (about 50,000 daltons) appears to contain seven copies of [Met]enkephalin and one copy of [Leu]enkephalin. Digestion with trypsin and carboxypeptidase B yields [Met]enkephalin and [Leu]enkephalin in a ratio of almost 7 to 1. The enkephalins were identified by chromatography and by their binding to opiate receptors. Some characteristics of several other adrenal peptides that may serve as intermediates in the biosynthesis of the enkephalins are presented.
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Medula Suprarrenal/análise , Grânulos Cromafim/análise , Sistema Cromafim/análise , Endorfinas/biossíntese , Encefalinas/biossíntese , Precursores de Proteínas/análise , Proteínas/análise , Animais , Bovinos , Encefalina Leucina , Encefalina Metionina , Peso Molecular , Oligopeptídeos/análise , Fragmentos de Peptídeos/análise , TripsinaRESUMO
The cyanobacterial pentapeptide nodularin (NOD), mainly produced by genus Nodularia, is a potent inhibitor of protein phosphatases PP1 and PP2A, and causes animal mortality. The few studies available indicate that NOD is a potential non-genotoxic carcinogen. In the present study we evaluated NOD (0.01, 0.1 and 1⯵g/ml) genotoxic activity in human hepatoma (HepG2) cells with the comet, γH2AX and cytokinesis block micronucleus cytome assays. In addition, induction of oxidative stress was studied. Moreover changes in the expression of selected genes from the P53 pathway, involved in the response to DNA damage (P53, GADD45α, CDKN1A, MDM2), apoptosis (BAX, BCL2) and oxidative stress (GPX1, GSR, GCLC, CAT, SOD1) were determined using qPCR. Non-cytotoxic concentrations induced time and dose dependant increase in reactive oxygen species (ROS) production and substantially increased the formation of oxidative DNA damage. In addition, elevated formation of micronuclei was detected. For the first time it has been shown that NOD deregulated the mRNA level of DNA damage (CDKN1A, GADD45α) and oxidative stress (GPX1, GSR, GCLC, CAT and SOD1) responsive genes and anti-apoptotic gene BCL2. Our results provide new evidence that NOD genotoxic effects are mediated through ROS production, already at low environmentally relevant concentrations.
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Mutagênicos/toxicidade , Peptídeos Cíclicos/toxicidade , Apoptose/efeitos dos fármacos , DNA/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Simples/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Humanos , Estresse Oxidativo/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismoRESUMO
The ability of intact human red cells to scavenge extracellularly generated H2O2 and O2-, and to prevent formation of hydroxyl radicals and hypochlorous acid has been examined. Red cells inhibited oxidation of ferrocytochrome c by H2O2. Cells treated with aminotriazole no longer inhibited, indicating that protection was almost entirely due to intracellular catalase. Contribution by the GSH system was slight, and apparent only with low H2O2 concentrations when catalase was inhibited by aminotriazole. The cells were about a quarter as efficient at inhibiting cytochrome c oxidation as an equivalent concentration of purified catalase. No inhibition of O2(-)-dependent reduction of ferricytochrome c or nitroblue tetrazolium was observed, although extracted red cell superoxide dismutase inhibited nitroblue tetrazolium reduction at one fortieth the concentration of that in the cells. Red cells efficiently inhibited deoxyribose oxidation by hydroxyl radicals generated from H2O2, O2- and Fe(EDTA), and myeloperoxidase-dependent oxidation of methionine to methionine sulfoxide by stimulated neutrophils. Most of the red cell inhibition of hydroxyl radical production, and all the inhibition of methionine oxidation, was prevented by blocking intracellular catalase with aminotriazole. Thus red cells are able to efficiently scavenge H2O2, but not O2-, produced in their environment, and to inhibit formation of hydroxyl radicals and hypochlorous acid. They may therefore have an important role in extracellular antioxidant defense.
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Eritrócitos/metabolismo , Peróxido de Hidrogênio/sangue , Hidróxidos/sangue , Ácido Hipocloroso/sangue , Amitrol (Herbicida)/farmacologia , Catalase/antagonistas & inibidores , Catalase/sangue , Grupo dos Citocromos c/sangue , Eritrócitos/efeitos dos fármacos , Glutationa/sangue , Humanos , Radical Hidroxila , Metionina/análogos & derivados , Metionina/sangue , Neutrófilos/metabolismo , Nitroazul de Tetrazólio/sangue , Oxirredução , Espectrofotometria , Superóxido Dismutase/sangue , Superóxidos/sangueRESUMO
It is now recognized that IL-12 plays a predominant role in protective immunity against intracellular pathogens by promoting the development of T helper type 1 (Th1) responses. We here report the unexpected observations that IL-12 exerts differential effects on the maturation of "native" human CD4 T cells isolated from umbilical cord blood or from the blood of healthy adults. After priming in the presence of IL-12, naive cells of adult donors, defined as CD45R0- CD4+ T cells, acquire a Th1 phenotype whereas neonatal cells develop into effector cells producing high levels of IL-4 in addition to IFN-gamma. This effect of IL-12 on neonatal T cells is direct inasmuch as it is observed on highly purified CD4 T cells, however, it is not inhibited by CD8 T cells and natural killer cells. Unstimulated neonatal T cells which have been preincubated with IL-12 before the priming behave like adult T cells and acquire a Th1 phenotype after stimulation in the presence of IL-12. Given that IL-4 is a potent antagonist of Th1 responses, the finding that IL-12 promotes the maturation of neonatal T cells into IL-4 producers may explain the increased susceptibility of neonates to intracellular pathogens and should be taken into account for the development of vaccines to be used in the perinatal period.
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Linfócitos T CD4-Positivos/imunologia , Interferon gama/biossíntese , Interleucina-12/farmacologia , Interleucina-4/biossíntese , Adulto , Linfócitos T CD4-Positivos/efeitos dos fármacos , Células Cultivadas , Humanos , Recém-Nascido , Interleucina-1/farmacologia , Ativação Linfocitária , Proteínas Recombinantes/farmacologia , Fator de Necrose Tumoral alfa/farmacologia , Cordão Umbilical/citologiaRESUMO
To characterize genes that become upregulated with malignant transformation of human hepatocytes, a library of monoclonal antibodies was produced against the FOCUS hepatocellular carcinoma cell line. Antibody FB-50 reacted with an antigen that was highly expressed in 4 of 10 primary hepatocellular carcinomas, in all 20 cholangiocarcinomas we studied, and in a variety of transformed cell lines. This antigen was also highly expressed in neoplastic epithelial cells of breast and colon carcinomas in contrast to its low level of expression in normal hepatocytes and in non-neoplastic epithelial cells. Among the normal adult tissues studied, high levels were observed only in proliferating trophoblastic cells of the placenta and in adrenal glands. A 636-bp partial cDNA, isolated from a gamma GT11 expression library generated with HepG2 human hepatoblastoma cells, and a complete cDNA, generated by reverse transcriptase-PCR, identified the antigen as the human form of aspartyl(asparaginyl)beta-hydroxylase. This enzyme catalyzes posttranslational hydroxylation of beta carbons of specific aspartyl and asparaginyl residues in EGF-like domains of certain proteins. Analyses of extracts prepared from several human tumor cell lines compared to their normal tissue counterparts indicate that the increase in hydroxylase, approximately 10-fold, is controlled at the level of transcription and the protein is expressed in an enzymatically active form. In similar analyses, comparing hepatocellular carcinomas to adjacent uninvolved liver from five patients, enzymatic activity was much higher in the tumor tissue from the four patients whose immunoblots revealed increased hydroxylase protein in the malignant tissue. EGF repeats in the extracellular domain of Notch or its homologs contain the consensus sequence for hydroxylation. Deletion mutants lacking this domain are gain-of-function mutants, suggesting that the domain modulates signal transduction by the cytoplasmic domain. While the function imparted by beta hydroxylation is unknown, our studies raise the possibility that beta hydroxylation is regulated in proteins like the mammalian Notch homologs, whose cytoplasmic domains have been shown to be oncogenic.
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Antígenos de Superfície/genética , Carcinoma Hepatocelular/enzimologia , Colangiocarcinoma/enzimologia , Oxigenases de Função Mista/genética , Adulto , Sequência de Aminoácidos , Anticorpos Monoclonais , Sequência de Bases , Western Blotting , Neoplasias da Mama/imunologia , Carcinoma Hepatocelular/genética , Linhagem Celular Transformada , Colangiocarcinoma/genética , Clonagem Molecular , Neoplasias do Colo/imunologia , Biblioteca Gênica , Humanos , Imuno-Histoquímica , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Células Tumorais CultivadasRESUMO
Erythrina trypsin/tPA inhibitor (ETI) from the seeds of Erythrina caffra retains its native structure and inhibitory function after reducing its two disulfide bonds. In order to elucidate the specific role of these crosslinks, alanine residues were substituted for cysteines after cloning the gene in Escherichia coli. Expression of the recombinant inhibitor and the substitution mutants, C83A, CC39, 83AA, and CC132, 139AA, led to inclusion bodies. After solubilization in guanidinium-chloride (GdmCl)/dithiothreitol and oxidation in glutathione buffer, activity could be recovered at yields up to 80%. The mutant proteins exhibit full inhibitory function without detectable alterations of their native structure. However, their stability is reduced: at acid pH, where the oxidized natural inhibitor retains its native structure, the reduced wildtype protein and the mutants undergo at least partial denaturation, reflected by decreased pH ranges of stability: pH 5-7 for the reduced inhibitor, pH 2.5-8.5 for CC132, 139AA, and pH 3.5-8.5 for C83A and CC39, 83AA. Urea and GdmCl denaturation at pH 7 show hysteresis for both the oxidized inhibitor and the double mutant CC132, 139AA. In contrast, the reduced protein and the other mutants exhibit true equilibrium transitions at pH 7, with urea half-concentrations of 0.9 M and 1.9 M and GdmCl half-concentrations of 0.5 M and 1.0 M, respectively. The stability of Erythrina trypsin/tPA inhibitor follows the sequence: oxidized ETI > CC132, 139AA > CC39, 83AA and C83A > reduced ETI.