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Introduction. Mucoepidermoid carcinoma (MEC) of the lung is a rare form of lung cancer that is classified into low grade and high grade based on histological features. Surgical resection is the primary treatment for low-grade MEC with excellent outcomes, while high-grade MEC is a more aggressive form of malignancy. Clinical Case. We report a case of a 46-year-old woman who presented with dyspnea on exertion. Imaging studies revealed a mass involving the right upper lobe bronchus. Bronchoscopy, surgical resection, and pathological examination revealed a low-grade MEC with tumor-free margins. No adjuvant treatment was given. Discussion. Primary pulmonary MEC is a rare type of lung cancer with only few reported cases. This patient illustrates a typical presentation for low-grade MEC wherein surgical resection is considered curative. In contrast, high-grade MEC is a more aggressive malignancy with a poorer outcome. The role of targeted therapy directed against EGFR or a novel CRTC1-MAML2 fusion protein expressed in some high-grade tumors is yet to be determined.
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Multicenter trials have shown that hematopoietic cell transplantation (HCT) has an excellent outcome in children with sickle cell disease (SCD). As a single center, we performed a total of 11 transplants in 10 patients (6 males, 4 females) with SCD between 1997 and 2005. Eight patients had hemoglobin SS disease and two patients had HbSbeta(0) thalassemia. The median age of HCT was 10.1 (range 2.8-16.3) years. All donors were HLA-identical siblings; six patients received bone marrow (BM), two patients received mobilized peripheral blood, one patient received umbilical cord blood (UCB) and one patient received both UCB and BM from the same donor. Myeloablative conditioning regimen consisted of busulfan, horse antithymocyte globulin and cyclophosphamide. One patient had a gradual decrease in donor chimerism to 15% and subsequently received a second bone marrow transplant using a reduced intensity conditioning regimen consisting of alemtuzumab, fludarabine and melphalan leading to stable full engraftment. Currently, 9 out of 10 patients are alive with a median follow-up of 5.5 (range 2.9-11) years. As a single institution's experience with HCT in children with SCD, we report an excellent outcome, and a second HCT may be considered for patients with impending engraftment failure as a cure for SCD.
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Anemia Falciforme/terapia , Transplante de Células-Tronco Hematopoéticas , Adolescente , Criança , Pré-Escolar , Feminino , Seguimentos , Transplante de Células-Tronco Hematopoéticas/efeitos adversos , Humanos , Masculino , Doadores de Tecidos , Condicionamento Pré-Transplante , Resultado do TratamentoRESUMO
INTRODUCTION: Achalasia is a primary motility disorder of the esophagus that is treated most effectively with operative myotomy. Excellent outcomes with laparoscopic myotomy and fundoplication are well known. Heller myotomy utilizing a computer-enhanced (robotic) laparoscopic platform allows for a more precise dissection by utilizing the superior optics of a 3-dimensional camera and greater degrees of freedom provided by robotic instrumentation. How this affects outcome and quality of life is unknown. METHODS: We assessed patients' health perceptions using a standardized, validated, health-related, disease-specific quality-of-life metric. Sixty-one consecutive patients undergoing laparoscopic or robotic myotomy over a 6-year period were evaluated prospectively. All operations were performed using intraoperative manometric and endoscopic guidance and all except 5 patients had a fundoplication. The effects of the operation on health-related quality of life were evaluated with the Short Form (SF-36) Health Status Questionnaire and a disease-specific gastroesophageal reflux disease activity (GERD) activity index (GRACI) preoperatively and postoperatively. All patients completed the questionnaire at both time points. Patient scores were compared using 2-way repeated measures analyses of variance followed by the Tukey test. Operative time, estimated blood loss, duration of stay, intraoperative complication, and postoperative complications were analyzed. RESULTS: Thirty-seven patients had laparoscopic and 24 patients had robotic Heller myotomy. There was an increase in SF-36 overall evaluation of health postoperatively compared with preoperatively in both groups (P < .05). The robotic myotomy patients had better SF-36 Role Functioning (emotional) and General Health Perceptions (P < .05) compared with the laparoscopic group. The GRACI showed an equivalent improvement in severity of symptoms in both groups (P < .05). Operative time was 287 +/- 9 minutes for laparoscopic cases and 355 +/- 23 minutes for robotic cases. Estimated blood loss and duration of stay were not different between groups. There were 3 operative esophageal perforations (8%) during laparoscopic myotomy and all were repaired immediately. There were no perforations or operative complications in the robotic group. Neither group had any additional complications. CONCLUSIONS: Minimally invasive operative myotomy improves functional status and overall evaluation of health in patients with achalasia. Robotic myotomy had no intraoperative esophageal perforations compared with an 8% intraoperative rate during laparoscopic myotomy. Heller myotomy with partial fundoplication using a robotic platform appears to be a more precise and safer operation than laparoscopic myotomy with improved quality-of-life indices postoperatively compared with laparoscopic myotomy with fewer complications; this suggests that, in skilled hands, the robotic platform may be safer, with improved quality-of-life outcomes.
Assuntos
Procedimentos Cirúrgicos do Sistema Digestório/métodos , Acalasia Esofágica/psicologia , Acalasia Esofágica/cirurgia , Fundoplicatura/métodos , Qualidade de Vida , Robótica , Adulto , Idoso , Idoso de 80 Anos ou mais , Procedimentos Cirúrgicos do Sistema Digestório/efeitos adversos , Perfuração Esofágica/etiologia , Feminino , Fundoplicatura/efeitos adversos , Nível de Saúde , Humanos , Complicações Intraoperatórias , Laparoscopia/efeitos adversos , Masculino , Pessoa de Meia-Idade , Satisfação do Paciente , Cirurgia Assistida por Computador , Inquéritos e QuestionáriosRESUMO
OBJECTIVE: Pulmonary arteriovenous malformations may cause progressive cyanosis after cavopulmonary anastomosis and may develop as a result of abnormal angiogenesis. We used immunohistochemistry to determine whether angiogenic proteins are increased in the lungs of children after cavopulmonary anastomosis. METHODS: Lung specimens were obtained from 13 children after cavopulmonary anastomosis and from 6 control subjects. Specimens were stained with antibodies against vascular endothelial growth factor and its receptor (flk-1/KDR), basic fibroblast growth factor, alpha-smooth muscle actin, CD31, collagen IV, fibronectin, and proliferating cell nuclear antigen. Staining was graded on a scale of 0 to 3. Vessels positive for proliferating cell nuclear antigen were counted in 10 fields per specimen, and the results were averaged. RESULTS: After cavopulmonary anastomosis, patients demonstrated increased staining for vascular endothelial growth factor (P =.03) and its receptor (P =.03) and decreased staining for CD31 (P =.004). Proliferating cell nuclear antigen staining in patients was equivalent to that for control subjects (P =.9). CONCLUSIONS: Lung biopsy specimens from children after cavopulmonary anastomosis demonstrate increased expression of vascular endothelial growth factor and its receptor. These data confirm earlier findings that blood vessels forming after cavopulmonary anastomosis may have reduced intercellular junctions (decreased CD31 staining). Despite the increased numbers of pulmonary vessels that are present in these patients, these vessels are not highly proliferative (proliferating cell nuclear antigen staining equivalent to that of control subjects). These results suggest that vascular endothelial growth factor may be a mediator of angiogenesis in the lungs of children after cavopulmonary anastomosis; however, other factors, such as vascular dilation and remodeling, may also be important.
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Malformações Arteriovenosas/etiologia , Malformações Arteriovenosas/patologia , Cianose/etiologia , Cianose/patologia , Fatores de Crescimento Endotelial/análise , Derivação Cardíaca Direita/efeitos adversos , Linfocinas/análise , Neovascularização Patológica/etiologia , Neovascularização Patológica/patologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/análise , Artéria Pulmonar/anormalidades , Receptores Proteína Tirosina Quinases/análise , Receptores de Fatores de Crescimento/análise , Adolescente , Malformações Arteriovenosas/cirurgia , Biópsia , Estudos de Casos e Controles , Criança , Pré-Escolar , Progressão da Doença , Técnica de Fontan , Humanos , Imuno-Histoquímica , Lactente , Neovascularização Patológica/cirurgia , Antígeno Nuclear de Célula em Proliferação/análise , Receptores de Fatores de Crescimento do Endotélio Vascular , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
The synthesis and characterization of new nanoscale container molecules 7 and 8 are described. They are covalent hybrids of deepened, self-folding cavitands and metalloporphyrins. In receptor 7, the Zn-porphyrin wall is directly built onto the cavitand skeleton. Host 8 features a large unimolecular cavity containing two cavitands attached with the Zn-porphyrin wall. Its dimensions, approximately 10 x 25 A, place it among the largest synthetic hosts prepared to date. A series of adamantyl- and pyridyl-containing guests 14-20 of various lengths were prepared and used to determine the hosts' binding abilities in solution using UV/vis and (1)H NMR spectroscopy. Intramolecular hydrogen bonds at the upper rims of the cavitands resist the unfolding of the inner cavities and thereby increase the energetic barrier to guest exchange. The exchange is slow on the NMR time scale (at < or =300 K), and kinetically stable complexes result. When the cavities and metalloporphyrins participate simultaneously in the binding event, very high affinities for guests are found (-DeltaG295 up to 10 kcal x mol(-1) in toluene), to which the porphyrin fragments contribute significantly (-DeltaG295 up to 6 kcal x mol(-1)). The pairwise selection of two different guests by molecular container 8 is reported, and the termolecular complex formed raises the possibility of metal-catalyzed bimolecular reactions in these containers.
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Porfirinas/síntese química , Amidas/química , Cristalografia por Raios X , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação Molecular , Porfirinas/química , Espectrofotometria Ultravioleta , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.
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Antígenos HLA/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Ferro/metabolismo , Proteínas de Membrana , Receptores da Transferrina/metabolismo , Linhagem Celular , Antígenos HLA/química , Células HeLa , Hemocromatose/genética , Proteína da Hemocromatose , Antígenos de Histocompatibilidade Classe I/química , Homeostase , Humanos , Receptores da Transferrina/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
OBJECTIVE: Pulmonary arteriovenous malformations cause progressive cyanosis in children after cavopulmonary anastomosis and may be due to abnormal angiogenesis. We determined the microvessel density, a marker of angiogenesis, in the lungs of children after cavopulmonary anastomosis. METHODS: Lung biopsy specimens were obtained from 8 children after cavopulmonary anastomosis and from 4 control patients. Three of the 8 children undergoing cavopulmonary anastomosis had clinical and angiographic evidence of pulmonary arteriovenous malformations, whereas the other 5 were free of symptoms. Routine histologic and immunohistologic stains were performed with a primary antibody to von Willebrand factor. Microvessel staining for von Willebrand factor was determined for 10 fields (200x) per patient. RESULTS: Patients with and without pulmonary arteriovenous malformations after cavopulmonary anastomosis demonstrated significantly increased microvessel density compared with control subjects (32.7 +/- 2.8 vs 9.3 +/- 4.6, P =.02, and 31.5 +/- 15.7 vs 9.3 +/- 4.6, P =.01, respectively). There was no difference in microvessel density in children with and without clinically apparent pulmonary arteriovenous malformations after cavopulmonary anastomosis (P =.9). The children with pulmonary arteriovenous malformations had numerous greatly dilated vessels that were absent in the asymptomatic children after cavopulmonary anastomosis. CONCLUSIONS: After cavopulmonary anastomosis, pulmonary microvessel density is increased even in the absence of clinically apparent pulmonary arteriovenous malformations, supporting the presence of a constant angiogenic stimulus. Children with clinically apparent pulmonary arteriovenous malformations possess large numbers of greatly dilated pulmonary microvessels, which are absent in asymptomatic children after cavopulmonary anastomosis. These results suggest that the transition to clinically apparent pulmonary arteriovenous malformations may be due to mechanisms that lead to vessel dilation and remodeling.
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Malformações Arteriovenosas/etiologia , Pulmão/irrigação sanguínea , Neovascularização Patológica , Artéria Pulmonar/cirurgia , Veia Cava Superior/cirurgia , Análise de Variância , Anastomose Cirúrgica/efeitos adversos , Anastomose Cirúrgica/métodos , Biópsia , Criança , Pré-Escolar , Feminino , Técnica de Fontan/efeitos adversos , Cardiopatias Congênitas/cirurgia , Humanos , Técnicas Imunoenzimáticas , Lactente , Pulmão/patologia , Masculino , Microcirculação , Complicações Pós-Operatórias , Resultado do TratamentoRESUMO
[structure: see text] Host-guest complexes of a new open-ended cavitand show unprecedented stabilities. Simultaneous binding in the cavity and at the metalloporphyrin affects both the kinetics and the thermodynamics of caviplex formation.
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Porfirinas/química , Indicadores e Reagentes , Espectroscopia de Ressonância Magnética , Piridinas/química , Espectrofotometria UltravioletaRESUMO
OBJECTIVE: Vascular endothelial growth factor and basic fibroblast growth factor are potent stimulators of angiogenesis. Children with cyanotic congenital heart disease often experience the development of widespread formation of collateral blood vessels, which may represent a form of abnormal angiogenesis. We undertook the present study to determine whether children with cyanotic congenital heart disease have elevated serum levels of vascular endothelial growth factor and basic fibroblast growth factor. METHODS: Serum was obtained from 22 children with cyanotic congenital heart disease and 19 children with acyanotic heart disease during cardiac catheterization. Samples were taken from the superior vena cava, inferior vena cava, and a systemic artery. Vascular endothelial growth factor and basic fibroblast growth factor levels were measured in the serum from each of these sites by enzyme-linked immunosorbent assay. RESULTS: Vascular endothelial growth factor was significantly elevated in the superior vena cava (P =.04) and systemic artery (P =.02) but not in the inferior vena cava (P =.2) of children with cyanotic congenital heart disease compared to children with acyanotic heart disease. The mean vascular endothelial growth factor level, determined by averaging the means of all 3 sites, was also significantly elevated (P =.03). Basic fibroblast growth factor was only significantly elevated in the systemic artery (P =.02). CONCLUSION: Children with cyanotic congenital heart disease have elevated systemic levels of vascular endothelial growth factor. These findings suggest that the widespread formation of collateral vessels in these children may be mediated by vascular endothelial growth factor.
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Fatores de Crescimento Endotelial/sangue , Fator 2 de Crescimento de Fibroblastos/sangue , Cardiopatias Congênitas/sangue , Linfocinas/sangue , Criança , Cianose/sangue , Feminino , Humanos , Lactente , Masculino , Isoformas de Proteínas/sangue , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
INTRODUCTION: Pulmonary arteriovenous malformations are a common cause of progressive cyanosis in children after cavopulmonary anastomoses. We analyzed the pulmonary histologic characteristics from children in whom pulmonary arteriovenous malformations developed after procedures that resulted in pulmonary arterial blood flow devoid of hepatic venous effluent. METHODS: We performed routine histologic studies, immunohistochemical staining, and electron microscopic analysis of peripheral lung biopsy specimens from 2 children with angiographically proven pulmonary arteriovenous malformations. Microvessel density was determined with a computer-assisted, morphometric analysis system. RESULTS: Histologic examination demonstrated large, dilated blood vessels ("lakes") and clustered, smaller vessels ("chains") in the pulmonary parenchyma. Microvessel density was significantly greater in these patients than in age-matched controls (P =.01). Immunohistochemistry demonstrated uniform staining for type IV collagen and alpha-smooth muscle actin, weak staining for the endothelial marker CD31 (cluster of differentiation, PECAM-1), and negative staining for proliferating cell nuclear antigen. Electron microscopy revealed endothelial irregularity, a disorganized basement membrane, and increased numbers of collagen and actin filaments beneath the endothelium. CONCLUSIONS: This study represents an attempt to characterize the histologic features of pulmonary arteriovenous malformations in children with congenital heart disease who have pulmonary arterial blood flow devoid of hepatic venous effluent. The histologic correlate of this condition appears to be greatly increased numbers of thin-walled vessels. Immunohistochemistry suggests that the rate of cellular proliferation is not increased in these lesions. The development of these techniques may provide a standardized histologic approach for this condition and aid in understanding its etiology.
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Malformações Arteriovenosas/patologia , Cianose/complicações , Cardiopatias Congênitas/complicações , Artéria Pulmonar/anormalidades , Veias Pulmonares/anormalidades , Anastomose Cirúrgica/efeitos adversos , Angiografia , Malformações Arteriovenosas/diagnóstico por imagem , Malformações Arteriovenosas/etiologia , Biópsia , Capilares/diagnóstico por imagem , Capilares/ultraestrutura , Criança , Pré-Escolar , Cianose/cirurgia , Feminino , Seguimentos , Átrios do Coração/cirurgia , Cardiopatias Congênitas/cirurgia , Veias Hepáticas/cirurgia , Humanos , Pulmão/irrigação sanguínea , Pulmão/ultraestrutura , Masculino , Artéria Pulmonar/patologia , Veias Pulmonares/patologia , Veia Cava Superior/cirurgiaRESUMO
A candidate gene, HFE, was recently described in patients with hereditary hemochromatosis (HH) and found to contain a missense mutation leading to a cysteine to tyrosine substitution (C282Y). A second mutation, H63D, was also found in the gene. This study was undertaken to determine the HFE genotype in liver transplant recipients clinically diagnosed with HH and those incidentally found to have increased iron deposition in their explanted livers and to evaluate whether biochemical or histological hepatic iron indices (HIIs) correlated with homozygosity for the C282Y mutation. We identified 15 patients clinically diagnosed with various liver disorders other than HH who had increased liver iron deposits among 918 adult patients who underwent liver transplantation from 1988 to 1995. Four additional patients were clinically diagnosed as having HH. Archival explant liver tissue was evaluated for the histological HII according to the method of Deugnier et al, in which an index greater than 0.15 suggests homozygosity for HH. The HII was computed according to established methods, with a value greater than 1.9 suggesting homozygosity for HH. A portion of liver tissue was subjected to DNA genotyping using polymerase chain reaction-amplified products. Two of 4 patients with clinically suspected HH were homozygous for C282Y, and 2 patients had neither mutation. One of the 15 patients not suspected to have HH was a C282Y homozygote, 1 was a C282Y heterozygote, 6 were H63D heterozygotes, and 7 had neither mutation. The histological HII was consistent with HH in 13 patients, whereas the HII was consistent with HH in 6 patients. Thus, in patients with end-stage liver disease, despite fulfilling the established clinical criteria for HH using biochemical and histological parameters, only a minority of patients were homozygous for the C282Y mutation. Hepatic iron overload may result from other causes, and in end-stage liver disease, an elevated HII may not accurately predict HH. Other factors that either control or lead to iron absorption may explain iron overload in these patients.
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Antígenos HLA/genética , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Transplante de Fígado , Mutação , Adulto , Idoso , Feminino , Genótipo , Homozigoto , Humanos , Ferro/metabolismo , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Hepatopatias/metabolismo , Masculino , Pessoa de Meia-IdadeRESUMO
OBJECTIVE: Radial artery harvesting for coronary artery bypass may lead to digit ischemia if collateral hand circulation is inadequate. The modified Allen's test is the most common preoperative screening test used. Unfortunately, this test has high false-positive and false-negative rates. The purpose of this study was to compare the results of a modified Allen's test with digit pressure change during radial artery compression for assessing collateral circulation before radial artery harvest. METHODS: One hundred twenty-nine consecutive patients were studied before coronary artery bypass operations. A modified Allen's test was performed with Doppler ultrasound to assess blood flow in the superficial palmar arch before and during radial artery compression. A decreased audible Doppler signal after radial artery compression was considered a positive modified Allen's test. First and second digit pressures were measured before and during radial artery compression. A decrease in digit pressure of 40 mm Hg or more (digit DeltaP) with radial artery compression was considered positive. RESULTS: Seven of 14 dominant extremities (50%) and 8 of the 16 nondominant extremities (50%) with a positive modified Allen's test had a digit DeltaP of less than 40 mm Hg (false positive). Sixteen of 115 dominant extremities (14%) and 5 of 112 nondominant extremities (4%) with a negative Allen's test had a digit DeltaP of 40 mm Hg or more with radial artery compression (false negative). CONCLUSION: Use of the modified Allen's test for screening before radial artery harvest may unnecessarily exclude some patients from use of this conduit and may also place a number of patients at risk for digit ischemia from such harvest. Direct digit pressure measurement is a simple, objective method that may more precisely select patients for radial artery harvest. Additional studies are needed to define objective digital pressure criteria that will accurately predict patients at risk for hand ischemia after radial harvest.
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Ponte de Artéria Coronária/métodos , Mãos/irrigação sanguínea , Artéria Radial/fisiologia , Artéria Radial/transplante , Adulto , Idoso , Idoso de 80 Anos ou mais , Determinação da Pressão Arterial/instrumentação , Determinação da Pressão Arterial/métodos , Determinação da Pressão Arterial/estatística & dados numéricos , Distribuição de Qui-Quadrado , Doença das Coronárias/fisiopatologia , Doença das Coronárias/cirurgia , Feminino , Humanos , Fluxometria por Laser-Doppler/instrumentação , Fluxometria por Laser-Doppler/métodos , Fluxometria por Laser-Doppler/estatística & dados numéricos , Masculino , Pessoa de Meia-Idade , Curva ROC , Fluxo Sanguíneo Regional , Reprodutibilidade dos Testes , Temperatura CutâneaRESUMO
Although nonoperative management of blunt splenic injury (NMBSI) has an established role in the overall management of adult patients with blunt splenic injury, the criteria by which patients are selected continue to be debated. The purpose of this study is to establish the effectiveness of a defined set of criteria that includes CT grading for the selection of patients for NMBSI by examining the outcomes of patients managed in this manner 1 year before with those 1 year after the implementation of this specific set of selection criteria. All patients hospitalized at St. Joseph Mercy Hospital over the time period April 1994 through July 1996 with blunt splenic injury were included. Patients who died in the Emergency Department were excluded. Patients admitted from April 1994 through April 1995 composed Group I, those treated before the specific selection criteria, whereas those admitted from July 1995 through July 1996 composed Group II, those treated after the implementation of selection criteria. The two groups were compared with respect to demographic parameters, Injury Severity Score, mechanism of injury and length of stay. Outcomes were compared between these two groups. Those patients successfully managed without operation were further compared with those for whom NMBSI was unsuccessful. A total of 57 patients met the criteria for study entry, 28 from Group I and 29 from Group II. There were no significant differences between these two groups with respect to age, sex, mechanism of injury, Injury Severity Score, or length of stay. Nine of 27 in Group I required immediate operation; 19 were initially managed nonoperatively. Four of 19 required delayed laparotomy for bleeding, and all required splenectomy. Between patients successfully managed nonoperatively and those requiring delayed operation, the only significant difference was CT grade (1.47 vs 3.5; P = 0.0001). In Group II, after the implementation of selection criteria that included CT grade, no patient required delayed operation. Eleven underwent immediate operation, whereas 18 were successfully managed nonoperatively. We conclude that, in the hemodynamically stable patient without clinical indication for laparotomy, CT grading of the splenic injury is a reliable criterion by which patients may be selected for nonoperative management.
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Baço/lesões , Tomografia Computadorizada por Raios X , Ferimentos não Penetrantes/diagnóstico por imagem , Ferimentos não Penetrantes/terapia , Adolescente , Adulto , Idoso , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Seleção de Pacientes , Baço/diagnóstico por imagem , Esplenectomia , Ferimentos não Penetrantes/cirurgiaRESUMO
YAC-based and bacterial-clone based STS-content maps were constructed that served as the framework physical maps for the positional cloning of a candidate gene for hereditary hemochromatosis. The YAC-based map comprises 43 YACs and 86 STS and spans approximately 8 Mb of DNA between the class I region of the major histocompatibility complex on human chromosome 6p21.3 and D6S276 in 6p22. Comparison with published maps revealed a hole in the MIT/Whitehead and CEPH YAC maps that includes the immediate region around the hemochromatosis gene itself. Approximately 3 Mb of DNA was covered by a bacterial clone contig that consists of 38 BACs, 45 PACs, 26 PI clones and one lambda phage. The bacterial clone-based STS map comprises 153 STSs. A contiguous block of 8 STSs could be amplified from both human chromosome 6 and 5. Further characterization of selected STSs and bacterial clones by radiation hybrid mapping and fluorescence in situ hybridization, respectively, revealed the presence of a multicopy DNA segment, more than one bacterial clone length in size, which is duplicated near the chromosome-6 centromere and part of which is present in multiple copies on chromosome 5. Possible implications of the incomplete public YAC-contig map and of the multicopy segment for physical mapping and linkage disequilibrium studies of the hemochromatosis candidate region are discussed.
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Mapeamento Cromossômico/métodos , Cromossomos Humanos Par 6 , Antígenos HLA/genética , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Sitios de Sequências Rotuladas , Bactérias/genética , Cromossomos Artificiais de Levedura , Clonagem Molecular , Elementos de DNA Transponíveis , Genoma Humano , Proteína da Hemocromatose , Humanos , Células Híbridas/efeitos da radiação , Hibridização in Situ Fluorescente , Repetições de Microssatélites/genética , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido NucleicoRESUMO
We recently reported the positional cloning of a candidate gene for hereditary hemochromatosis (HH), called HLA-H, which is a novel member of the major histocompatibility complex class I family. A mutation in this gene, cysteine 282 --> tyrosine (C282Y), was found to be present in 83% of HH patient DNAs, while a second variant, histidine 63 --> aspartate (H63D), was enriched in patients heterozygous for C282Y. The functional relevance of either mutation has not been described. Co-immunoprecipitation studies of cell lysates from human embryonic kidney cells transfected with wild-type or mutant HLA-H cDNA demonstrate that wild-type HLA-H binds beta2-microglobulin and that the C282Y mutation, but not the H63D mutation, completely abrogates this interaction. Immunofluorescence labeling and subcellular fractionations demonstrate that while the wild-type and H63D HLA-H proteins are expressed on the cell surface, the C282Y mutant protein is localized exclusively intracellularly. This report describes the first functional significance of the C282Y mutation by suggesting that an abnormality in protein trafficking and/or cell-surface expression of HLA-H leads to HH disease.
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Antígenos HLA/genética , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Microglobulina beta-2/metabolismo , Sequência de Bases , Membrana Celular/metabolismo , Regulação da Expressão Gênica , Hemocromatose/metabolismo , Proteína da Hemocromatose , Humanos , Dados de Sequência Molecular , Mutação , Microglobulina beta-2/genéticaRESUMO
Hereditary haemochromatosis (HH), which affects some 1 in 400 and has an estimated carrier frequency of 1 in 10 individuals of Northern European descent, results in multi-organ dysfunction caused by increased iron deposition, and is treatable if detected early. Using linkage-disequilibrium and full haplotype analysis, we have identified a 250-kilobase region more than 3 megabases telomeric of the major histocompatibility complex (MHC) that is identical-by-descent in 85% of patient chromosomes. Within this region, we have identified a gene related to the MHC class I family, termed HLA-H, containing two missense alterations. One of these is predicted to inactivate this class of proteins and was found homozygous in 83% of 178 patients. A role of this gene in haemochromatosis is supported by the frequency and nature of the major mutation and prior studies implicating MHC class I-like proteins in iron metabolism.
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Antígenos HLA/genética , Hemocromatose/genética , Antígenos de Histocompatibilidade Classe I/genética , Proteínas de Membrana , Alelos , Sequência de Aminoácidos , Sequência de Bases , Evolução Biológica , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 6 , Clonagem Molecular/métodos , Cisteína , Primers do DNA/química , Expressão Gênica , Genes MHC Classe I , Marcadores Genéticos , Haplótipos , Proteína da Hemocromatose , Humanos , Desequilíbrio de Ligação , Complexo Principal de Histocompatibilidade , Dados de Sequência Molecular , RNA Mensageiro/genética , Alinhamento de Sequência , Homologia de Sequência de AminoácidosRESUMO
Murine monoclonal antibody (MAb) G3-519 has been shown to recognize a conserved neutralizing epitope in the fourth constant (C4) region of the external glycoprotein gp120 of HIV-1. Inasmuch as this antibody effectively neutralized the infectivity of diverse HIV-1 isolates, it has been selected to be developed for passive immunization against HIV-1 infection in humans. In order to minimize the problem of immunogenicity of murine antibodies and to confer additional accessory immune functions, we have constructed mouse/human chimeric and humanized forms of the antibody. The chimeric antibody was constructed by cloning the murine variable regions and replacing the mouse constant regions with those from human Ig gamma 1,kappa. The humanized antibody was constructed using the human KAS variable region framework sequences as template. Engineering was guided by a three dimensional model of the murine variable region. The murine, chimeric and humanized forms of the antibody exhibited similar reactivity with the peptidic antigen in ELISA, and comparably neutralized the infectivity of HIV-1 in vitro. Taken together, our results show that the chimeric and humanized forms of G3-519 essentially retain the binding activity of the mouse parental antibody. Clinical development is planned to assess the prophylactic and therapeutic usefulness of these reshaped antibodies in humans.
Assuntos
Anticorpos Monoclonais/biossíntese , Anticorpos Anti-HIV/biossíntese , HIV-1/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/genética , Clonagem Molecular , DNA/genética , Genes de Imunoglobulinas , Vetores Genéticos , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/terapia , Humanos , Hibridomas/imunologia , Imunização Passiva , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Testes de Neutralização , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/imunologiaRESUMO
Antibodies specific for membrane-bound Ig (mIg) but not for their secreted forms would be useful not only for studying the function of mIg but also for modulating B cell activities in vivo. We have proposed that the extracellular portions of the membrane anchor peptides of mIg can be used as antigenic sites for isotype-specific targeting of B cells. Clones containing the genes of human Ig alpha 1 or alpha 2 subclasses were isolated from a genomic DNA library. The gene segments encoding the membrane peptides and their flanking regions were amplified by polymerase chain reaction, subcloned into plasmid pUC19, and the DNA sequences were determined. Human alpha 1 and alpha 2 genes, like murine alpha gene, each has only one membrane exon. The sequences of the human alpha 1 and alpha 2 genes are almost identical in the membrane peptide-coding region. The mRNA from a human mIgA-expressing B cell line, DAKIKI, was isolated, its cDNA prepared, and the segments spanning the membrane peptide-coding region and a part of the constant domain 3 amplified by polymerase chain reaction. DNA sequences revealed that there are two isoforms of alpha 1-chain, resulting from the alternative splicing of the third constant domain of H chain to two acceptor sites in the membrane exon. One isoform has a segment of 32 and the other 26 amino acid residues in the extracellular portion of the membrane peptide. These segments may serve as isotype-specific antigenic epitopes for antibody targeting of mIgA-bearing B cells.
Assuntos
Genes de Imunoglobulinas , Isotipos de Imunoglobulinas/genética , Splicing de RNA , RNA Mensageiro/metabolismo , Receptores de Antígenos de Linfócitos B/genética , Sequência de Bases , DNA/análise , Humanos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
We have isolated and characterized mouse cDNA clones representing the entire coding region of vimentin. RNA blot analysis of different stages during development has revealed differential control in the expression of vimentin mRNA in the different tissues studied. The nucleotide sequence extends 1800 base pairs and contains the 466 amino acid mouse vimentin polypeptide chain, flanked by 90 base pairs 5' and 312 base pairs 3' untranslated region. Conformational analysis of the deduced amino acid sequence was used to localize the three known structural domains: a non-alpha-helical N-terminal head of 81 residues, a rod-like domain of 330 residues arising from three alpha-helices, and a non-alpha-helical C-terminal domain of 55 residues. Amino acid sequence comparisons with other species revealed high sequence conservation of mouse vimentin to hamster (98.7%), human (96%), and chicken (88%) protein. Computer sequence analysis also revealed domains of significant homology between different alpha helical regions of vimentin and the DNA binding-leucine zipper domain of several proto-oncogenes and transcription regulators. Specifically, 50-70% structural similarity was observed between the basic domain of the DNA binding region of the nuclear proto-oncogene products c-fos and its related antigen fra-1, c-jun and the cAMP-responsive DNA binding protein CREB, with part of the N-terminal half region of helix 1b of vimentin. When the leucine zipper domains of all these proteins were compared to vimentin, at least two different regions of similarity in the vimentin molecule were found reaching up to 53% for jun, 60% for fos, and 76% for CREB. Further analysis revealed several domains of significant similarity (50%) between all alpha-helices of the rod domain of vimentin and the N-terminal (approximately 210 residues) activation domain tpr of the oncogenic raf.