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BACKGROUND: Yearly, almost six percent, which is more than 1,000.000 people, in the Netherlands receive mental health treatment, which usually improves their quality of life. Concurrently, mental healthcare professionals recognize clinically refractory cases in which improvement fails to occur, with severe ongoing burdens for patients. The Dutch Centre for Consultation and Expertise (CCE) is available to support such refractory cases. The Dutch government's (financial) facilitation of consultation through the CCE is unique in the world. CCE consultations provide therefore unique insight into and an overview of refractory cases in mental health services. The objective of this study was to gain insight into the commonalities underlying the reasons for CCE consultations and the solutions proposed that play roles in (the reduction of) refractory cases for which consultation has been requested. METHODS: This descriptive study was conducted with quantitative and qualitative data from 472 CCE consultations in the Netherlands. Using descriptive statistics and thematic content analysis, four exemplary situations were distilled from the qualitative data. RESULTS: Most (83%) cases in the sample could be explained with four exemplary situations involving self-harm (24.2%), aggression (21.8%), self-neglect (24.4%), and socially unacceptable behavior (12.5%), respectively. Each situation could be characterized by a specific interaction pattern that unintentionally maintained or aggravated the situation. At the time of closure of the consultation applicants' questions had been answered and their situations had improved in 60.4% of cases. CONCLUSIONS: This study offers an overview of approaches that provided new perspectives for patients and professionals in many refractory cases in the Dutch mental health services.
Assuntos
Serviços de Saúde Mental , Qualidade de Vida , Humanos , Encaminhamento e Consulta , Pessoal de Saúde , Países BaixosRESUMO
BACKGROUND: Self-harming behavior is a frequent problem seen in patients admitted to a closed ward in a psychiatric hospital. Little is known about prevalence and characteristics of this behavior as well as the preceding triggering factors. AIM: To gain insights in the self-harming behavior of patients admitted to a closed ward in a psychiatric hospital. METHOD: From September 2019 till January 2021 was gathered information on self-harming incidents and aggressive behavior towards others or objects, of 27 patients admitted to the closed department of the Centre Intensive Treatment (Centrum Intensieve Behandeling). RESULTS: 20 of 27 patients examined (74%) showed 470 incidents of self-harming behavior. Head banging (40.9%) and self-harming using straps/ropes (29.7%) occured most. Tension/stress as triggering factor was mentioned most (19.1%). Self-harming behavior occured more during evenings. Besides self-harm, a high degree of aggressive behavior towards others or objects was registered. CONCLUSION: This study delivers insights in self-harming behavior of patients admitted to closed psychiatric departments that can be used for prevention and treatment.
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Unidade Hospitalar de Psiquiatria , Comportamento Autodestrutivo , Humanos , Comportamento Autodestrutivo/epidemiologia , Agressão/psicologia , Pacientes Internados , HospitalizaçãoRESUMO
BACKGROUND: Yearly, over 1.000.000 people receive mental health care treatment in the Netherlands. Treatment usually results in improvement in quality of life. Concurrently, each professional recognizes clinically refractory cases in which improvement fails to occur with severe ongoing burden for the client. In the Netherlands, for these clinically refractory cases the Centre of Consultation and Expertise (CCE) is available. The CCE is an independent nation-wide organisation offering free consultations to care providers. Therefore, CCE-consultations provide a unique insight in and overview of refractory cases. AIM: Providing overview of and insight into backgrounds and themes that play a role in (the reduction of) refractory cases. METHOD: Descriptive study of quantitative and qualitative data from 472 consultations in mental health care. RESULTS: 83% of cases could be explained with 4 exemplary vignettes of refractoriness: self-harm, aggression, self-neglect and socially unacceptable behaviour. CONCLUSION: Refractory cases result from an interaction pattern that unintentionally maintains or aggravates the situation. This study offers an overview of approaches that proved to be helpful in providing new perspective for clients and professionals in many therapy refractory cases in Dutch mental health care.
Assuntos
Saúde Mental , Qualidade de Vida , Encaminhamento e Consulta , Humanos , Países BaixosRESUMO
BACKGROUND: Since January 1, 2012, nurse practitioners (NP) working in mental health care are allowed to prescribe psychotropic medication. So far, there has been very little research on the results of this decision that now let NPS share with doctors prescribing psychotropic drugs. AIM: To provide insight into how patients and psychiatrists experience the prescribing behaviours of NPS and how NPS themselves regard their extended role. METHOD: We performed an explorative study in which we used the data given in prescriptions written by NPS, questionnaires exploring patients' experiences and semi-structured interviews with psychiatrists and NPS. RESULTS: Between May 2014 and May 2015, 13 NPS wrote 3542 prescriptions for 565 unique patients. On the whole, patients, psychiatrists and NPS expressed positive views on the prescribing of psychotropic medication by NPS. CONCLUSION: Our research project confirms that the various stakeholders are satisfied with the prescribing practices of NPS. A follow-up study is needed in order to ascertain whether there are qualitative differences between the prescriptions of NPS and those of doctors.
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Prescrições de Medicamentos/enfermagem , Profissionais de Enfermagem , Enfermagem Psiquiátrica/métodos , Psicotrópicos/uso terapêutico , Prescrições de Medicamentos/estatística & dados numéricos , Revisão de Uso de Medicamentos , HumanosRESUMO
BACKGROUND: Resistance to temozolomide (TMZ) greatly limits chemotherapeutic effectiveness in glioblastoma (GBM). Here we analysed the ability of the Inhibitor-of-apoptosis-protein (IAP) antagonist birinapant to enhance treatment responses to TMZ in both commercially available and patient-derived GBM cells. METHODS: Responses to TMZ and birinapant were analysed in a panel of commercial and patient-derived GBM cell lines using colorimetric viability assays, flow cytometry, morphological analysis and protein expression profiling of pro- and antiapoptotic proteins. Responses in vivo were analysed in an orthotopic xenograft GBM model. RESULTS: Single-agent treatment experiments categorised GBM cells into TMZ-sensitive cells, birinapant-sensitive cells, and cells that were insensitive to either treatment. Combination treatment allowed sensitisation to therapy in only a subset of resistant GBM cells. Cell death analysis identified three principal response patterns: Type A cells that readily activated caspase-8 and cell death in response to TMZ while addition of birinapant further sensitised the cells to TMZ-induced cell death; Type B cells that readily activated caspase-8 and cell death in response to birinapant but did not show further sensitisation with TMZ; and Type C cells that showed no significant cell death or moderately enhanced cell death in the combined treatment paradigm. Furthermore, in vivo, a Type C patient-derived cell line that was TMZ-insensitive in vitro and showed a strong sensitivity to TMZ and TMZ plus birinapant treatments. CONCLUSIONS: Our results demonstrate remarkable differences in responses of patient-derived GBM cells to birinapant single and combination treatments, and suggest that therapeutic responses in vivo may be greatly affected by the tumour microenvironment.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Apoptose/efeitos dos fármacos , Neoplasias Encefálicas/patologia , Dacarbazina/análogos & derivados , Dipeptídeos/farmacologia , Glioblastoma/patologia , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Animais , Western Blotting , Caspase 8/efeitos dos fármacos , Caspase 8/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Dacarbazina/farmacologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Citometria de Fluxo , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Microscopia de Contraste de Fase , Transplante de Neoplasias , Temozolomida , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The dismal outlook for patients with the most aggressive and common form of adult brain cancer, glioblastoma (GBM), motivates a search for new therapeutic strategies and targets for this aggressive disease. Here we review the findings to date on the role of Eph family receptor tyrosine kinases and their ephrin ligands in brain cancer. Expression of the Eph family of cell surface proteins is generally downregulated to very low levels in normal adult tissues making them particularly attractive for directed therapeutic targeting. Recent Eph targeting studies in pre-clinical models of GBM have been very encouraging and may provide an avenue to treat these highly refractory aggressive tumours.
Assuntos
Neoplasias Encefálicas/metabolismo , Glioblastoma/metabolismo , Receptores da Família Eph/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias Encefálicas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Efrinas/fisiologia , Glioblastoma/tratamento farmacológico , Humanos , Terapia de Alvo Molecular , Inibidores de Proteínas Quinases/farmacologia , Inibidores de Proteínas Quinases/uso terapêutico , Receptores da Família Eph/genética , Receptores da Família Eph/metabolismo , Transdução de SinaisRESUMO
The role of astrocytes is becoming increasingly important to understanding how glioblastoma (GBM) tumor cells diffusely invade the brain. Yet, little is known of the contribution of extracellular vesicle (EV) signaling in GBM/astrocyte interactions. We modeled GBM-EV signaling to normal astrocytes in vitro to assess whether this mode of intercellular communication could support GBM progression. EVs were isolated and characterized from three patient-derived GBM stem cells (NES+/CD133+) and their differentiated (diff) progeny cells (NES-/CD133-). Uptake of GBM-EVs by normal primary astrocytes was confirmed by fluorescence microscopy, and changes in astrocyte podosome formation and gelatin degradation were measured. Quantitative mass spectrometry-based proteomics was performed on GBM-EV stimulated astrocytes. Interaction networks were generated from common, differentially abundant proteins using Ingenuity® (Qiagen Bioinformatics) and predicted upstream regulators were tested by qPCR assays. Podosome formation and Cy3-gelatin degradation were induced in astrocytes following 24-h exposure to GBM-stem and -diff EVs, with EVs released by GBM-stem cells eliciting a greater effect. More than 1700 proteins were quantified, and bioinformatics predicted activations of MYC, NFE2L2, FN1, and TGFß1 and inhibition of TP53 in GBM-EV stimulated astrocytes that were then confirmed by qPCR. Further qPCR studies identified significantly decreased Δ133p53 and increased p53ß in astrocytes exposed to GBM-EVs that might indicate the acquisition of a pro-inflammatory, tumor-promoting senescence-associated secretory phenotype (SASP). Inhibition of TP53 and activation of MYC signaling pathways in normal astrocytes exposed to GBM-EVs may be a mechanism by which GBM manipulates astrocytes to acquire a phenotype that promotes tumor progression.
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Astrócitos/metabolismo , Neoplasias Encefálicas/metabolismo , Vesículas Extracelulares/metabolismo , Glioblastoma/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Idoso , Diferenciação Celular , Linhagem Celular Tumoral , Senescência Celular , Vesículas Extracelulares/ultraestrutura , Gelatina/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Nanopartículas/ultraestrutura , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Tamanho da Partícula , Fenótipo , Podossomos/metabolismo , Isoformas de Proteínas/metabolismo , Proteólise , Proteoma/metabolismoRESUMO
High-grade glioma (HGG) is an incurable brain cancer. The transcriptomes of cells within HGG tumors are highly heterogeneous. This renders the tumors unresponsive or able to adapt to therapeutics targeted at single pathways, thereby causing treatment failure. To overcome this, we focused on cyclin-dependent kinase 7 (CDK7), a ubiquitously expressed molecule involved in two major drivers of HGG pathogenesis: cell cycle progression and RNA polymerase-II-based transcription. We tested the activity of THZ1, an irreversible CDK7 inhibitor, on patient-derived primary HGG cell lines and ex vivo HGG patient tissue slices, using proliferation assays, microarray analysis, high-resolution respirometry, cell cycle analysis and in vivo tumor orthografts. The cellular processes affected by CDK7 inhibition were analyzed by reverse transcriptase-quantitative PCR, western blot, flow cytometry and immunofluorescence. THZ1 perturbed the transcriptome and disabled CDK activation, leading to cell cycle arrest at G2 and DNA damage. THZ1 halted transcription of the nuclear-encoded mitochondrial ribosomal genes, reducing mitochondrial translation and oxidative respiration. It also inhibited the expression of receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor-α (PDGFR-α), reducing signaling flux through the AKT, extracellular-signal-regulated kinase 1/2 (ERK1/2) and signal transducer and activator of transcription 3 (STAT3) downstream pathways. Finally, THZ1 disrupted nucleolar, Cajal body and nuclear speckle formation, resulting in reduced cytosolic translation and malfunction of the spliceosome and thus leading to aberrant mRNA processing. These findings indicate that CDK7 is crucial for gliomagenesis, validate CDK7 as a therapeutic target and provide new insight into the cellular processes that are affected by THZ1 and induce antitumor activity.
RESUMO
The human EphA3 gene was discovered in a pre-B acute lymphoblastic leukemia (pre-B-ALL) using the EphA3-specific monoclonal antibody (mAb), IIIA4, which binds and activates both human and mouse EphA3. We use two models of human pre-B-ALL to examine EphA3 function, demonstrating effects on pre-B-cell receptor signaling. In therapeutic targeting studies, we demonstrated antitumor effects of the IIIA4 mAb in EphA3-expressing leukemic xenografts and no antitumor effect in the xenografts with no EphA3 expression providing evidence that EphA3 is a functional therapeutic target in pre-B-ALL. Here we show that the therapeutic effect of the anti-EphA3 antibody was greatly enhanced by adding an α-particle-emitting 213Bismuth payload.
Assuntos
Anticorpos Monoclonais/uso terapêutico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Receptor EphA3/imunologia , Animais , Bismuto , Linhagem Celular Tumoral , Humanos , Imunoterapia , Camundongos , Receptor EphA3/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The acquisition of genetic alterations in tumor cells is a hallmark of cancer progression. Genetic alterations, including chromosomal sequence alterations and abnormal gene expression, increase the malignant potential of tumors by affecting pathways that regulate cell growth, cell death, tumor angiogenesis, and invasion/metastasis. We used an expression profiling technique, representational difference analysis, to identify genes the expressions of which are aberrantly increased in invasive breast carcinomas as compared with adjacent normal breast tissue from the same individual. Among the genes we identified was GIRK1, which encodes a 501 amino acid, G-protein inwardly rectifying potassium channel protein. We then measured GIRK1 mRNA expression in benign breast tissues, primary invasive breast carcinomas, and metastatic breast carcinomas from axillary lymph nodes using quantitative TaqMan reverse transcription-PCR and correlated the results with clinical parameters. We found that GIRK1 overexpression correlated with lymph node metastasis (P < 0.0029), and overexpression was greatest in tumors with more than one positive lymph node. These results indicate that GIRK1 may be useful as a biomarker for lymph node metastasis and possibly a pharmaceutical target.
Assuntos
Neoplasias da Mama/patologia , Canais de Potássio Corretores do Fluxo de Internalização , Canais de Potássio/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Mama/metabolismo , Mama/patologia , Neoplasias da Mama/genética , DNA Complementar/química , DNA Complementar/genética , Etiquetas de Sequências Expressas , Feminino , Canais de Potássio Corretores do Fluxo de Internalização Acoplados a Proteínas G , Regulação Neoplásica da Expressão Gênica , Humanos , Linfonodos/metabolismo , Linfonodos/patologia , Metástase Linfática , Pessoa de Meia-Idade , Dados de Sequência Molecular , Invasividade Neoplásica/genética , Invasividade Neoplásica/patologia , Reação em Cadeia da Polimerase/métodos , RNA/genética , RNA/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Análise de Sequência de DNARESUMO
SETTING: The joint Médecins Sans Frontières/Ministry of Health Multidrug-Resistant Tuberculosis (MDR-TB) Programme, Karakalpakstan, Uzbekistan. OBJECTIVE: Uzbekistan has high rates of MDR-TB. We aimed to understand patients' and prescribers' attitudes to anti-tuberculosis drug prescription, regulation and drug-taking behaviour. METHODS: Participants (12 patients, 12 practitioners) were recruited purposively. Data were gathered qualitatively using field notes and in-depth interviews and analysed thematically. FINDINGS: Our analysis highlighted two main themes. First, shame and stigma were reported to increase the likelihood of self-treatment and incorrect use of anti-tuberculosis drugs, most commonly at the initial stages of illness. A health system failure to promote health information was perceived, leading to wrong diagnoses and inappropriate therapies. Motivated by shame, patients hid their condition by resorting to drug treatment options outside the programme, compounding the risk of chaotic management and dissemination of erroneous information through lay networks. Second, positive influences on treatment were reported through patients, practitioners and peers working effectively together to deliver the correct information and support, which acted to normalise TB, reduce stigma and prevent misuse of anti-tuberculosis drugs. CONCLUSION: Effective case finding, patient support and community education strategies are essential. Patients, practitioners and peers working together can help reduce stigma and prevent misuse of anti-tuberculosis drugs.
Assuntos
Antituberculosos/uso terapêutico , Atitude do Pessoal de Saúde , Conhecimentos, Atitudes e Prática em Saúde , Adesão à Medicação , Padrões de Prática Médica , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológico , Adulto , Feminino , Humanos , Entrevistas como Assunto , Masculino , Pessoa de Meia-Idade , Educação de Pacientes como Assunto , Preconceito , Pesquisa Qualitativa , Autocuidado , Vergonha , Estigma Social , Tuberculose Resistente a Múltiplos Medicamentos/diagnóstico , Tuberculose Resistente a Múltiplos Medicamentos/epidemiologia , Tuberculose Resistente a Múltiplos Medicamentos/psicologia , Uzbequistão/epidemiologia , Adulto JovemRESUMO
Using polymerase chain reaction amplification and oligonucleotide probing, the activation of ras oncogenes in 24 benign and 20 malignant human thyroid neoplasms was examined. The frequency of ras oncogene activation was similar at all stages of tumorigenesis in this system, being found in 33% of adenomas overall (50% of microfollicular tumours), 53% of differentiated follicular carcinomas and 60% of undifferentiated carcinomas. This supports the contention that mutation of these oncogenes occurs at an early step in tumorigenesis. The predominant amino acid substitution in the differentiated tumours was glutamine to arginine at position 61 of Ha-ras or N-ras, but this mutation was not found in any of the undifferentiated tumours. It was noted that while transition mutations predominated in differentiated tumours (both benign and malignant), transversions were more common in the undifferentiated tumours.
Assuntos
Genes ras , Neoplasias da Glândula Tireoide/genética , Adenoma/genética , Adolescente , Adulto , Idoso , Carcinoma/genética , Feminino , Amplificação de Genes , Humanos , Masculino , Pessoa de Meia-Idade , MutaçãoRESUMO
One of the earliest cellular responses to growth factors is the rapid induction of primary response genes. One group of such genes was originally isolated as tetradecanoyl phorbol acetate (TPA) inducible sequences (TIS genes) from mouse 3T3 cells. Proteins encoded by the TIS genes include two transcription factors: TIS8 (also known as egr1/NGFIA/zif268) and TIS1 (also known as NGFIB/nur77/N10). We have examined the inducibility of these two genes in a skeletal muscle cell line in response to agents that have been reported to block muscle differentiation. We report here that basic fibroblast growth factor (bFGF) induced the expression of both TIS1 and TIS8 in mouse C2C12cells. Both genes were also inducible by TPA while forskolin which activates the cAMP-dependent pathway induced TIS1 but not TIS8. Down-regulation of protein kinase C (PKC) activity by TPA pretreatment repressed the bFGF induction of TIS1 but had little effect on the bFGF-stimulated expression of TIS8. Moreover, while both TPA and bFGF stimulated the hyperphosphorylation of c-RAF and the activity of MAP kinase, TPA pretreatment failed to block RAF phosphorylation or the stimulation of MAP kinase activity by bFGF. Induction of the two TIS genes in skeletal myoblasts therefore appeared to be dependent to different extents on the activation of protein kinase A (PKA), PKC and MAP kinase.
Assuntos
Proteínas de Ligação a DNA/genética , Músculos/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Colforsina/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteína Quinase C/metabolismo , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Chondrocytes isolated from normal adult human articular cartilage were infected with a retroviral vector encoding a temperature-sensitive mutant of the simian virus 40 large tumor antigen and a linked geneticin (G418)-resistance marker. G418-resistant colonies were then isolated, ring-cloned, and expanded in serum-containing media. Several immortalized chondrocyte cell lines were established from the clones that survived, some of which have been maintained in continuous culture for over 2 years. Despite serial subcultures and maintenance as monolayers, these cells retain expression of markers specific for cells of the lineage, namely type II collagen and aggrecan, detected immunocytochemically. We also examined the phenotype of three of these immortalized cell lines (designated HAC [human articular chondrocyte]) using a pellet culture system, and in this report, we present evidence that a prototype of these lines (HAC-F cells) expresses markers normally associated with hypertrophic chondrocytes. When HAC-F cells were cultivated in centrifuge tubes, for periods of up to 63 days, at 39 degrees C with mild and intermittent centrifugation they continued to express both lineage markers; total type II collagen/pellet remained stable, whereas there was a temporal decrease in cartilage-specific glycosaminoglycans content. In addition, in the presence of ascorbate but in the absence of a phosphate donor or inorganic phosphate supplement, the cells also begin to express a hypertrophic phenotype characterized by type X collagen synthesis and extensive mineralization of the extracellular matrix in late stage cultures. The mRNA encoding type X collagen was detected in the cell pellets by reverse transcriptase polymerase chain reaction as early as day 2, and anti-type X collagen immunoreactivity was subsequently localized in the matrix. The mineral was characterized by energy-dispersive X-ray microanalysis as containing calcium (Ca) and phosphorus (P) with a Ca:P peak height ratio close to that of mineralized bone tissue. The unexpected phenotype of this human chondrocyte cell line provides an interesting opportunity for studying chondrocyte maturation in vitro.
Assuntos
Calcificação Fisiológica , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Colágeno/biossíntese , Adulto , Antibacterianos , Cálcio/análise , Linhagem Celular , Sobrevivência Celular , Condrócitos/virologia , Colágeno/genética , Resistência Microbiana a Medicamentos , Gentamicinas , Glicosaminoglicanos/análise , Humanos , Fenótipo , Fósforo/análise , RNA Mensageiro/análise , Retroviridae/genética , Vírus 40 dos Símios/genética , TemperaturaRESUMO
The purpose of this study was to further investigate the mechanism that limits thyroid growth in the presence of a sustained elevation of serum TSH. An in vitro thyroid follicle culture was used, thyroid function and growth being assessed by 131I- organification and [3H]thymidine incorporation into DNA, respectively. Normal thyroid follicles incorporated [3H]thymidine in response to added TSH and also organified 131I-. Follicles taken from rats previously given goitrogen for 80 days, however, organified 131I- in response to added TSH but did not incorporate [3H]thymidine. These in vitro results parallel those obtained in in vivo studies despite the disruption of thyroid architecture. We conclude that the growth desensitization seen in vivo during sustained serum TSH elevation is mediated by an intracellular change in the follicular cell (either at the receptor or postreceptor level) rather than by a locally acting chalone.
Assuntos
Glândula Tireoide/citologia , Tireotropina/farmacologia , Amitrol (Herbicida)/farmacologia , Animais , Divisão Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Iodo/metabolismo , Masculino , Ratos , Ratos Endogâmicos , Timidina/metabolismo , Glândula Tireoide/efeitos dos fármacosRESUMO
We have investigated the proliferative responses of rat thyroid follicular cells in serum-free culture to a range of growth factors including TSH, epidermal growth factor, and insulin, added singly or in combination. Follicles released from normal thyroids by collagenase/dispase digestion were cultured in suspension in agarose-coated microtiter plates to prevent monolayer formation. Growth responses were measured by [3H] thymidine incorporation and by autoradiography over successive 24- or 36-h periods. Insulin, even in the absence of other growth factors, stimulated [3H]thymidine incorporation in a concentration-dependent manner, rising from basal levels of 486 +/- (SE) 18 cpm to 4222 +/- 367 cpm/5 X 10(4) cells at 8 micrograms/ml. In contrast, TSH alone had no effect. In the presence of threshold levels (0.08 micrograms/ml) of insulin, however, there was a highly significant (P less than 0.001) response to TSH, [3H]thymidine incorporation rising from 1089 +/- 163 cpm in the absence of TSH to a maximum of 7548 +/- 585 with 1 mU/ml TSH. There was a synergistic interaction between insulin and TSH over the concentrations tested. Epidermal growth factor either alone or in combination with insulin failed to produce a significant response. Parallel autoradiographic studies were concordant with the [3H]thymidine incorporation data. We conclude that whereas in the absence of other growth factors TSH is unable to stimulate DNA synthesis in isolated rat thyroid follicles, the inclusion of just a single growth factor, insulin, permits a marked response. These observations emphasize the need for inclusion of appropriate permissive growth factor(s) when assessing the in vitro effect of a suspected tissue-specific mitogen.
Assuntos
Substâncias de Crescimento/farmacologia , Glândula Tireoide/citologia , Animais , Divisão Celular/efeitos dos fármacos , Células Cultivadas , DNA/biossíntese , Sinergismo Farmacológico , Fator de Crescimento Epidérmico/farmacologia , Células Epiteliais , Insulina/farmacologia , Masculino , Microscopia Eletrônica , Ratos , Ratos Endogâmicos , Glândula Tireoide/efeitos dos fármacos , Tireotropina/farmacologiaRESUMO
The effects of hyperthyroidism on experimental autoimmune thyroiditis were investigated in the rat. Rats were given T4 twice daily by sc injection in amounts sufficient to raise circulating hormone levels 10-fold 4 h after administration. Thyroiditis was induced by immunization with rat thyroglobulin (Tg) in complete Freund's adjuvant, and the severity of the disease was assessed by comparison with saline-treated controls. Thymic and splenic hypertrophy were found in T4-treated animals, whereas lymph node wt decreased. The levels of Tg antibodies did not differ between animals given saline and those given T4, but the expected sustained rise in control animals was not seen in those treated with T4; in addition, there was a significant decrease in the amount of Tg antibody produced by in vitro culture of lymph node lymphocytes from T4-treated rats. Continuous T4 administration lowered the number of T cells in the circulation, but the number of phenotypically identified helper cells remained the same. The most striking effects of T4 were to ameliorate the intensity of histologically defined thyroiditis and lower the response of lymph node T cells to the nonspecific mitogen, phytohemagglutinin. These results show that excessive T4 does not, as previously suggested, enhance the immune response in autoimmune thyroid disease: on the contrary, suppression is found with the dose and model we have used. In view of the magnitude of this effect, it is now important to identify the site of T4 action and investigate how this effect contributes to the autoimmune response in Graves' disease.
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Doenças Autoimunes/imunologia , Hipertireoidismo/imunologia , Tireoidite/imunologia , Animais , Anticorpos Anti-Idiotípicos/análise , Formação de Anticorpos , Peso Corporal , Ensaio de Imunoadsorção Enzimática , Feminino , Ativação Linfocitária , Ratos , Ratos Endogâmicos , Linfócitos T/classificação , Tireoglobulina/imunologia , Tireoidite/fisiopatologia , Tiroxina/sangue , Fatores de TempoRESUMO
The genetic manipulation of neural cells has advantage in both basic biology and medicine. Its utility has provided a clearer understanding of how the survival, connectivity, and chemical phenotype of neurones is regulated during, and after, embryogenesis. Much of this achievement has come from the recent generation by genetic means of reproducible and representative supplies of precursor cells which can then be analyzed in a variety of paradigms. Furthermore, advances made in the clinical use of transplantation for neurodegenerative disease have created a demand for an abundant, efficacious and safe supply of neural cells for grafting. This review describes how genetic methods, in juxtaposition to epigenetic means, have been used advantageously to achieve this goal. In particular, we detail how gene transfer techniques have been developed to enable cell immortalization, manipulation of cell differentiation and commitment, and the controlled selection of cells for purification or safety purposes. In addition, it is now also possible to genetically modify antigen presentation on cell surfaces. Finally, there is detailed the transfer of therapeutic products to discrete parts of the central nervous system (CNS), using neural cells as elegant and sophisticated delivery vehicles. In conclusion, once the epigenetic and genetic controls over neural cell production, differentiation and death have been more fully determined, providing a mixture of hard-wired elements and more flexibly expressed characteristics becomes feasible. Optimization of the contributions and interactions of these two controlling systems should lead to improved cell supplies for neurotransplantation.
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Genes Reguladores , Neurônios/citologia , Células-Tronco/citologia , Animais , Apoptose , Diferenciação Celular/genética , Divisão Celular/genética , Linhagem Celular , Separação Celular , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Vetores Genéticos/imunologia , Sobrevivência de Enxerto , Humanos , Camundongos , Fenótipo , Ratos , Transplante de Células-Tronco , TransfecçãoRESUMO
In order to investigate the molecular basis for the regulated expression of plasminogen activator inhibitor type 2 (PAI-2), we sought to identify monocyte-derived nuclear factors which interact with the PAI-2 gene. We have explored the application of Southwestern blot mapping as an approach for identifying specific DNA-protein interactions and targeting potential regulatory DNA elements. The procedure involves an initial global screening of a crude preparation of nuclear proteins with radiolabelled DNA fragments (200-300 bp) derived from a large region (8.8 kb) of PAI-2. The bound DNA fragments are eluted and their location within PAI-2 mapped by Southern blot hybridization analysis. We have used this procedure to examine the differential binding of nuclear factors from the U937 monocytic cell in the absence and in the presence of the differentiating agent, 12-phorbol 13-myristate acetate (PMA), in order to identify proteins that bind specifically to the 5' flanking promoter region and first intron of PAI-2. Eleven DNA-binding proteins ranging in molecular mass from 27 to 92 kDa were identified, and the results define three regions of the gene which contain DNA-binding sites which may be involved in the transcriptional regulation of PAI-2. Deletion analysis using a series of 5' deletion mutants spanning PAI-2 fused to a chloramphenicol acetyltransferase-encoding reporter gene (cat) demonstrates that two of the regions identified by Southwestern blot mapping contain elements which can function to modulate PAI-2 expression in transient transfections of U937 cells.