RESUMO
Adipsin is a serine protease that is secreted by adipocytes into the bloodstream; it is deficient in several animal models of obesity, representing a striking example of defective gene expression in this disorder. Recombinant mouse adipsin was purified and its biochemical and enzymatic properties were studied in order to elucidate the function of this protein. Activated adipsin has little or no proteolytic activity toward most substrates but has the same activity as human complement factor D, cleaving complement factor B when it is complexed with activated complement component C3. Like authentic factor D, adipsin can activate the alternative pathway of complement, resulting in red blood cell lysis. Decreased (58 to 80 percent) complement factor D activity, relative to lean controls, was observed as a common feature of several experimental models of obesity, including the ob/ob, db/db, and monosodium glutamate (MSG)-injected mouse and the fa/fa rat. These results suggest that adipsin and the alternative pathway of complement may play an unexpected but important role in the regulation of systemic energy balance in vivo.
Assuntos
Enzimas Ativadoras do Complemento/metabolismo , Fator D do Complemento/metabolismo , Obesidade/imunologia , Serina Endopeptidases/metabolismo , Tecido Adiposo/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Via Alternativa do Complemento , Cricetinae , DNA/genética , Regulação da Expressão Gênica , Humanos , Immunoblotting , Camundongos , Dados de Sequência Molecular , Obesidade/genética , Obesidade/metabolismo , RNA Mensageiro/metabolismo , Proteínas Recombinantes , Serina Endopeptidases/genética , Serina Endopeptidases/isolamento & purificação , Especificidade por Substrato , TransfecçãoRESUMO
The release of adipsin, a serine proteinase with complement factor D activity, from 3T3-L1 adipocytes was measured by quantitative immunoblotting. This protein is secreted constitutively from 3T3-L1 adipocytes, and there is a 2-fold increase in the amount of adipsin released from cells treated with insulin for 1 to 10 min. Longer exposure to insulin had no further effect on the rate of adipsin release. Adipsin does not appear to be anchored by a glycosylphosphatidylinositol moiety, since adipsin which was been released with Triton X-114 from an intracellular membrane fraction partitions into the aqueous phase. Using a previously described procedure for the isolation of vesicles containing the insulin-responsive intracellular glucose transporters (GT vesicles), we show here that these GT vesicles contain an insulin-responsive pool of adipsin. Thus, insulin stimulates the secretion of a soluble protein, adipsin, as well as translocation to the plasma membrane of integral membrane proteins, including the glucose transporter, the transferrin receptors, and the insulin-like growth factor II receptor.
Assuntos
Tecido Adiposo/metabolismo , Insulina/farmacologia , Animais , Compartimento Celular/efeitos dos fármacos , Linhagem Celular , Fator D do Complemento , Membranas Intracelulares/metabolismo , Camundongos , Peso Molecular , Proteínas de Transporte de Monossacarídeos/metabolismo , Taxa Secretória/efeitos dos fármacos , Serina Endopeptidases , SolubilidadeRESUMO
The risks inherent in the use of homologous blood products have increased efforts toward identifying alternatives to transfusion. We have previously shown that the administration of recombinant human erythropoietin (rhEpo) enhances the erythropoietic response to acute blood loss. Recombinant human interleukin-3 (rh-IL-3) is a hematopoietic growth factor that has been shown to act synergistically with rhEpo in accelerating erythropoiesis in vitro. The purpose of this study in a primate model was to determine if the administration of rhIL-3 in combination with rhEpo could augment the erythropoietic response to acute blood loss more than rhEpo therapy alone. Twenty-four adult male baboons were randomized into four groups. The induction of acute normovolemic anemia to a hematocrit of 20% was accomplished via exchange-transfusion with 6% hetastarch. The groups were then treated for 7 consecutive days with the following growth factors: group I (n = 7), no growth factors; group II (n = 5), rhIL-3 alone (100 micrograms/kg/d); group III (n = 6), rhEpo alone (1000 U/kg/d); group IV (n = 6), rhEpo (1000 U/kg/d) plus rhIL-3 (100 micrograms/kg/d). All animals received folate, vitamin B12, and intravenous iron-dextran immediately following the exchange-transfusion. Response to therapy was monitored for 35 days. There were no adverse reactions following growth factor administration. The analysis of erythropoietic rates between study days 1 through 11, as determined via linear regression analysis, revealed that hematocrits increased significantly faster in the groups receiving rhEpo compared to controls. The administration of rhIL-3, however, did not increase the rate of erythropoiesis when compared to controls, nor did it augment response when added to the rhEpo regimen. The results of this study demonstrate that the administration of rhIL-3 alone had no significant effect on erythropoiesis in this setting of acute blood loss. Further, despite promising in vitro data, rhIL-3 provided no additional stimulation of erythropoiesis in animals receiving rhEpo. Nevertheless, the study confirms that the pharmacologic acceleration of erythropoiesis by rhEpo alone remains an attractive alternative to homologous transfusion.
Assuntos
Anemia/sangue , Eritropoese/efeitos dos fármacos , Eritropoetina/farmacologia , Interleucina-3/farmacologia , Doença Aguda , Análise de Variância , Anemia/tratamento farmacológico , Animais , Contagem de Células Sanguíneas/efeitos dos fármacos , Modelos Animais de Doenças , Quimioterapia Combinada , Eritropoetina/uso terapêutico , Interleucina-3/uso terapêutico , Masculino , Papio , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêuticoRESUMO
PURPOSE: To evaluate a prototype densitometer traceable to primary optical standards and compare its performance to an EPSON Expression(®) 10000XL flatbed scanner (the Epson) for quantitative radiochromic film (RCF) dosimetry. METHODS: A prototype traceable laser densitometry system (LDS) was developed to mitigate common film scanning artifacts, such as positional scan dependence and high noise in low-dose regions, by performing point-based measurements of RCF suspended in free-space using coherent light. The LDS and the Epson optical absorbance scales were calibrated up to 3 AU, using reference materials calibrated at a primary standards laboratory and a scanner calibration factor (SCF). Calibrated optical density (OD) was determined for 96 Gafchromic(®) EBT3 film segments before and after irradiation to one of 16 dose levels between 0 and 10 Gy, exposed to (60)Co in a polymethyl-methacrylate (PMMA) phantom. The sensitivity was determined at each dose level and at two rotationally orthogonal readout orientations to obtain the sensitometric response of each RCF dosimetry system. LDS rotational scanning dependence was measured at nine angles between 0°and 180°, due to the expected interference between coherent light and polarizing EBT3 material. The response curves were fit to the analytic functions predicted by two physical response models: the two-parameter single-hit model and the four-parameter percolation model. RESULTS: The LDS and the Epson absorbance measurements were linear to primary optical standards to within 0.2% and 0.3% up to 2 and 1 AU, respectively. At higher densities, the LDS had an over-response (2.5% at 3 AU) and the Epson an under-response (3.1% and 9.8% at 2 and 3 AU, respectively). The LDS and the Epson SCF over the applicable range were 0.968% ± 0.2% and 1.561% ± 0.3%, respectively. The positional scan dependence was evaluated on each digitizer and shown to be mitigated on the LDS, as compared to the Epson. Maximum EBT3 rotational dependence was found to have a strong dependence on dose (0.1% and 34% at 30 mGy and 5 Gy, respectively). The preferred EBT3 polymerization axis angle was constant within experimental uncertainties. In its most sensitive orientation, the LDS-measured EBT3 sensitivity was 7.13 × 10(-4) ± 9.2 × 10(-6) AU/mGy, which represented a 4.5 fold increase over the Epson of 1.58 × 10(-4) ± 9.8 × 10(-6) AU/mGy. To first order approximations, EBT3 response was linear up to 500 mGy to within 0.80% and to within 7.5% for the most sensitive LDS and the Epson orientations, respectively. The corresponding single-hit and percolation model relative residual norms were 0.082 and 0.074 for LDS as compared to 0.29 and 0.18 for the Epson, which represented a significant increase in LDS-measured agreement with the simple physical model. Less sensitive LDS and the Epson orientations showed a marked decrease in the physical model agreement, which suggested that suboptimal readout device characteristics may be the origin of the complex sensitometric functional forms currently required for accurate RCF dosimetry. CONCLUSIONS: The prototype densitometer was shown to be superior to a conventional scanner for quantitative RCF dosimetry based on physical models of film response. The Epson was shown to be a reliable tool for routine RCF dosimetry in a clinical setting, yet calibration to primary optical standards did not mitigate the necessity for complex, empirical functional form fitting.
Assuntos
Dosimetria Fotográfica/instrumentação , Artefatos , Calibragem , Desenho de Equipamento , Dosimetria Fotográfica/métodos , Lasers , Modelos Teóricos , Imagens de Fantasmas , Polimetil Metacrilato , Doses de Radiação , Análise EspectralRESUMO
Adipsin gene expression is greatly diminished in certain forms of genetic and acquired obesity. In the present study we evaluate the time course for the development of adipsin deficiency in obesity and its regulation by the sympathomimetic-thermogenic drug mixture ephedrine and caffeine. Previously, it was unknown whether adipsin deficiency occurred before or after the development of massive obesity. In the first series of experiments in which mice were treated with monosodium glutamate (MSG) for the first week of life, we demonstrate that adipsin deficiency occurs early in the development of MSG-induced obesity as evidenced by decreased circulating adipsin concentrations by 1 week of age and deficient adipsin mRNA levels in white adipose tissue (WAT) by 2 weeks. In db/db mice, diminished circulating adipsin was noted at 2 weeks of age. In both models, decreased adipsin gene expression precedes the development of marked obesity. Little is known about the factors which regulate adipsin gene expression in obesity. Common to the ob/ob, db/db and MSG models is diminished thermogenesis and sympathetic nervous system activity. In a second series of experiments we sought to determine whether adipsin deficiency in obesity could be corrected by treatment with ephedrine and caffeine (E+C), a sympathomimetic-thermogenic mixture previously shown to increase thermogenesis and reverse obesity in some models. In the present study, E+C treatment of MSG obese mice reversed obesity and markedly increased serum adipsin and adipsin mRNA levels in WAT and brown adipose tissue (BAT). In ob/ob mice, however, E+C treatment produced a negligible increase in adipsin mRNA levels in WAT and BAT as well as serum adipsin concentrations and this correlated with only a very small decrease in obesity. Thus, the ability of E+C to increase adipsin gene expression correlated with its ability to reverse obesity in these two models. Finally, the effect of E+C on adipsin gene expression may not be exerted directly on the fat cell since treatment of cultured 3T3-F442A adipocytes and isolated rat adipocytes in primary culture produced no effect on adipsin mRNA or secreted protein despite a lipolytic effect as measured by increased glycerol release. In summary, decreased adipsin gene expression occurs early in the development of MSG and db/db obesity and is markedly increased in the MSG model by the sympathomimetic-thermogenic drug mixture, E+C, which also reverses obesity. Elucidation of the factors responsible for these effects may enhance our understanding of fat cell gene regulation and obesity.
Assuntos
Envelhecimento/metabolismo , Cafeína/farmacologia , Efedrina/farmacologia , Obesidade/metabolismo , Serina Endopeptidases/metabolismo , Tecido Adiposo Marrom/fisiopatologia , Animais , Temperatura Corporal/efeitos dos fármacos , Fator D do Complemento , Combinação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Obesidade/induzido quimicamente , Obesidade/genética , RNA Mensageiro/metabolismo , Serina Endopeptidases/genética , Glutamato de Sódio , Simpatomiméticos/farmacologia , Fatores de TempoRESUMO
Ischemic neuropathy from sources other than diabetes is less common, but can be encountered in clinical practice. Diagnosis can be challenging, and many patients may be referred to the electrodiagnostic laboratory. Overlapping mononeuritis multiplex is a common presentation, but distal symmetric polyneuropathy and monomelic neuropathy patterns can be seen. Depending on the disease associated with ischemic neuropathy, a mononeuropathy or a sensory-motor, axonal-demyelinating peripheral neuropathy may be seen as well. The treatment of ischemic neuropathy varies depending on the associated disease. Prognosis can be poor in the case of amyloidosis and the primary vasculitides. The literature is limited to cross-sectional case series and rare longitudinal studies likely related to the incidence of the diseases. Further study is needed to fully define the extent of the neurologic consequences of peripheral ischemia and its significance clinically.
Assuntos
Arteriopatias Oclusivas/tratamento farmacológico , Arteriopatias Oclusivas/reabilitação , Isquemia/diagnóstico por imagem , Isquemia/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/reabilitação , Sistema Nervoso Periférico/irrigação sanguínea , Doença Aguda , Arteriopatias Oclusivas/diagnóstico , Doença Crônica , Terapia Combinada , Eletrodiagnóstico , Humanos , Isquemia/diagnóstico , Doenças do Sistema Nervoso Periférico/diagnóstico , Prognóstico , CintilografiaRESUMO
Marijuana has been proposed as treatment for a widening spectrum of medical conditions. Marijuana is a substance with many properties that may be applicable to the management of amyotrophic lateral sclerosis (ALS). These include analgesia, muscle relaxation, bronchodilation, saliva reduction, appetite stimulation, and sleep induction. In addition, marijuana has now been shown to have strong antioxidative and neuroprotective effects, which may prolong neuronal cell survival. In areas where it is legal to do so, marijuana should be considered in the pharmacological management of ALS. Further investigation into the usefulness of marijuana in this setting is warranted.
Assuntos
Esclerose Lateral Amiotrófica/tratamento farmacológico , Cannabis/uso terapêutico , Fitoterapia , Esclerose Lateral Amiotrófica/complicações , Esclerose Lateral Amiotrófica/fisiopatologia , Esclerose Lateral Amiotrófica/psicologia , Controle de Medicamentos e Entorpecentes/legislação & jurisprudência , Medicina Baseada em Evidências , Humanos , Seleção de Pacientes , Resultado do TratamentoRESUMO
The alternative complement pathway is best known for its role in humoral suppression of infectious agents. We have previously shown that adipose cells synthesize adipsin, the mouse homolog of human complement factor D, and that the synthesis of this protein is reduced in several rodent models of obesity. We show here that adipose cells and adipose tissue also synthesize two other essential components of the alternative pathway of complement, factors C3 and B, and activate the proximal portion of this pathway. This activation occurs in the absence of infectious agents and without triggering the terminal, lytic part of this pathway. We demonstrate the production in vitro of several polypeptides characteristic of complement activation that are known to have potent biological activities, including the anaphylatoxin C3a. Cultured adipocytes require stimulation with cytokines to activate complement, while explanted adipose tissue has no such requirement. The adipose tissue from obese mice is deficient in this localized activation of the alternative pathway. These results indicate that complement activation occurs in a localized site, adipose tissue, in normal mice and is impaired in a state of metabolic dysfunction. This suggests a novel function for the proximal portion of this complement pathway related to adipose cell biology or energy balance.
Assuntos
Tecido Adiposo/enzimologia , Proteínas do Sistema Complemento/metabolismo , Serina Endopeptidases/metabolismo , Células 3T3 , Animais , Northern Blotting , Fator D do Complemento , Via Alternativa do Complemento , Proteínas do Sistema Complemento/biossíntese , Proteínas do Sistema Complemento/genética , Humanos , Cinética , Camundongos , Testes de Precipitina , RNA Mensageiro/metabolismoRESUMO
Mural trophectoderm cells of the mouse embryo possess a phagocytic potential as early as 3.5 days post coitum (d.p.c.). This first differentiated function shows a graded variation along the embryonic-abembryonic axis, from a maximal activity in the non-dividing cells of the abembryonic pole to a complete lack of activity in the replicating polar trophectoderm overlying the inner cell mass (ICM). This pattern can be explained by a negative control exerted by the ICM. Addition of FGF4, a factor secreted by ICM cells, strongly inhibited phagocytosis while inducing resumption of DNA synthesis in mural trophectoderm cells, revealing a reversible, FGF4-dependent differentiation state. Under conditions in which a small cluster of mural trophectoderm cells (<10) had internalized large particles, these otherwise morphologically normal embryos could not implant in the uterus, indicating that cells at the abembryonic pole have a critical role in initiating the implantation process. At post-implantation stages (6.5-8.5 d.p.c.), the ectoplacental cone and secondary giant cells derived from the polar trophectoderm also contained active phagocytes, but at that stage, differentiation was not reversed by FGF4.
Assuntos
Desenvolvimento Embrionário e Fetal/imunologia , Fagocitose , Animais , Sangue , Diferenciação Celular , Técnicas de Cultura , Ectoderma , Implantação do Embrião , Feminino , Fator 4 de Crescimento de Fibroblastos , Fatores de Crescimento de Fibroblastos/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Fagocitose/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Fase S , Células-Tronco/citologiaRESUMO
BACKGROUND: Bartonella henselae has been identified as the causative agent of the neuroretinitis associated with cat scratch disease (CSD). Immunofluorescent antibody tests with good sensitivity and specificity are available to aid in diagnosis. Despite diagnostic advances, optimal management remains controversial. We present a case of documented B. henselae macular neuroretinitis managed without antibiotics and discuss antibiotic use in this condition. METHODS: We examined a young woman with macular neuroretinitis and established a diagnosis of CSD. Management consisted of a review of the literature, followed by educating her about the condition and close observation. We documented the course of her disease. RESULTS: We diagnosed neuroretinitis associated with B. henselae infection based on immunofluorescent antibody titres and clinical presentation. Our patient's neuroretinitis resolved promptly without antibiotic therapy. CONCLUSIONS: Macular neuroretinitis in CSD can be satisfactorily diagnosed with the use of fluorescent antibodies in the appropriate clinical setting. Optimal treatment for the disease has not been established and observation combined with patient education remains an appropriate option. The self-limited nature of the disease implies that treatment studies not using controls must be interpreted with great caution. Adverse drug reactions and other iatrogenic complications can be reduced by limiting antibiotic use in settings where a meaningful treatment benefit has not been established.
Assuntos
Bartonella henselae/isolamento & purificação , Doença da Arranhadura de Gato/complicações , Doença da Arranhadura de Gato/microbiologia , Neurite Óptica/etiologia , Retinite/etiologia , Adulto , Animais , Doença da Arranhadura de Gato/diagnóstico , Gatos , Feminino , Imunofluorescência , Humanos , Neurite Óptica/complicações , Neurite Óptica/microbiologia , Neurite Óptica/patologia , Papiledema/etiologia , Retinite/complicações , Retinite/microbiologia , Retinite/patologia , Escotoma/etiologiaRESUMO
Adipocyte differentiation is accompanied by the transcriptional activation of many new genes, including a putative lipid-binding protein termed adipocyte P2 (aP2). The aP2 gene contains a regulatory element (FSE2) 124 bases 5' to its start of transcription. This element binds nuclear factors in sequence-specific and differentiation-dependent fashion as determined by altered mobility in gel retardation assays. Deletion analysis of promoter-linked transfection assays and competition of these constructions in cells with a synthetic FSE2 element suggest that trans-acting factors bind to this region and act as negative regulators of aP2 gene activity in preadipocytes. c-fos appears to participate directly in this nucleoprotein complex, as demonstrated by the ability of antibodies to c-fos to disrupt specific binding of factors to the FSE2 sequence but not to factor-binding sequences from several other genes. Antibodies to c-fos specifically immunoprecipitate protein complexes covalently bound to FSE2 DNA via UV cross-linking.
Assuntos
Tecido Adiposo/fisiologia , Diferenciação Celular , Regulação da Expressão Gênica , Proteínas de Neoplasias , Proteínas do Tecido Nervoso , Nucleoproteínas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proto-Oncogenes , Serina Endopeptidases , Fatores de Transcrição/genética , Fatores de Transcrição/fisiologia , Animais , Sequência de Bases , Proteínas de Transporte/genética , Mapeamento Cromossômico , Fator D do Complemento , Proteínas de Ligação a DNA/fisiologia , Endopeptidases/genética , Proteína 7 de Ligação a Ácidos Graxos , Proteínas de Ligação a Ácido Graxo , Glicerolfosfato Desidrogenase/genética , Camundongos , Regiões Promotoras GenéticasRESUMO
BACKGROUND: Children with certain neurologic diseases (hydrocephalus, meningomyelocele, or cerebral palsy) have been reported to manifest a high frequency of A-pattern strabismus and superior oblique overaction. However, it is not generally recognized whether children with strabismus who have superior oblique overaction are more likely to have concurrent neurologic diseases than those without superior oblique overaction. In this study, the authors examine this issue. METHODS: The authors retrospectively reviewed the medical records of all patients (n = 168) with overdepression of the downturned eye in adduction, who were examined between October 1989 and March 1992. A randomly selected population of children with strabismus who did not have overdepression of the eye on infraduction and adduction served as controls (n = 98). Patients with simulating or confounding conditions such as pseudo-superior oblique overaction, inferior rectus skew deviation (alternating skew on lateral gaze), and restrictive or paralytic strabismus, and who were older than 20 years of age, were excluded. RESULTS: One hundred twelve patients with true superior oblique overaction were analyzed. Of these 112 patients, 45 (40.2%) had concurrent neurologic abnormalities, compared with less than one fifth (17.3%) of control subjects (17 of 98) (P < or = 0.001). CONCLUSIONS: Children with strabismus who have superior oblique overaction were found to have higher prevalence of concurrent neurologic diseases than control subjects. Superior oblique overaction may represent a clinical marker for an associated neurologic dysfunction, possibly representing a form of skew deviation in some cases.
Assuntos
Doenças do Sistema Nervoso/complicações , Estrabismo/complicações , Adolescente , Adulto , Criança , Pré-Escolar , Feminino , Florida/epidemiologia , Humanos , Lactente , Masculino , Doenças do Sistema Nervoso/epidemiologia , Transtornos da Motilidade Ocular/complicações , Transtornos da Motilidade Ocular/epidemiologia , Prevalência , Distribuição Aleatória , Estudos Retrospectivos , Estrabismo/epidemiologiaRESUMO
A cDNA for human adipsin was isolated and shown to encode a protein sharing 98% amino acid sequence similarity with the protein sequence previously determined for purified natural human complement factor D. Like mouse adipsin, recombinant human adipsin displays the enzymatic activity of human complement factor D, cleaving complement factor B only when B is complexed with activated complement component C3. We conclude that human adipsin is equivalent to complement factor D and that adipsin is the homologue of factor D in rodents. Adipose tissue is a major site of synthesis of human adipsin/complement factor D mRNA, but unlike the case in rodents, human adipsin mRNA is also expressed in monocytes/macrophages. The data presented here, demonstrating the equivalence of human adipsin to complement factor D and its high level of expression in fat, suggest a previously unsuspected role for adipose tissue in immune system biology.