SARS-CoV GDH株N蛋白全长基因克隆及其可溶性表达

目的构建SARS冠状病毒N蛋白的原核表达质粒,诱导重组蛋白表达并纯化,鉴定其抗原性。方法以我国SARS冠状病毒GDH株总RNA为模板,采用RT-PCR技术扩增N蛋白的全长基因,TA克隆后测序。构建pET-23d的N基因表达载体,用IPTG诱导目的蛋白表达,利用硫酸铵沉淀、分子筛层析及离子交换层析纯化重组蛋白,免疫印迹鉴定重组蛋白。结果RT-PCR扩增出1269bpSARS冠状病毒N蛋白的基因片段,其序列分析结果与SARS-CoVGD01、BJ01株的同源性为99.92%;该基因在大肠杆菌表达系统中高效表达,占可溶性蛋白的33.57%,表达产物为非融合的可溶性蛋白,Westernblot结果显示重组N蛋白具有良好的抗原性;纯化后重组N蛋白纯度为92.9%。结论成功构建了SARS冠状病毒N蛋白的重组表达质粒,并在大肠杆菌中以非融合蛋白的形式得到高效的可溶性表达,为SRAS的诊断和疫苗的研制奠定了基础。     

Risk factors for death in patients with severe asthma

OBJECTIVE:

To identify risk factors for death among patients with severe asthma.

METHODS:

This was a nested case-control study. Among the patients with severe asthma treated between December of 2002 and December of 2010 at the Central Referral Outpatient Clinic of the Bahia State Asthma Control Program, in the city of Salvador, Brazil, we selected all those who died, as well as selecting other patients with severe asthma to be used as controls (at a ratio of 1:4). Data were collected from the medical charts of the patients, home visit reports, and death certificates.

RESULTS:

We selected 58 cases of deaths and 232 control cases. Most of the deaths were attributed to respiratory causes and occurred within a health care facility. Advanced age, unemployment, rhinitis, symptoms of gastroesophageal reflux disease, long-standing asthma, and persistent airflow obstruction were common features in both groups. Multivariate analysis showed that male gender, FEV₁ pre-bronchodilator < 60% of predicted, and the lack of control of asthma symptoms were significantly and independently associated with mortality in this sample of patients with severe asthma.

CONCLUSIONS:

In this cohort of outpatients with severe asthma, the deaths occurred predominantly due to respiratory causes and within a health care facility. Lack of asthma control and male gender were risk factors for mortality.     

Phonomechanocardiographic study of innocent murmurs in children

At the National Institute of Pediatrics DIF (formerly IMAN), a comparative study was performed in 157 healthy children. Ninety four (59.8%), had an innocent cardiac murmur, and 63 children (40.1%) had no heart murmur detectable. The presence of innocent murmurs was more frequent in pre-school and school age; murmurs of basal location were predominant. The murmurs were brief midsystolic and of the ejective type. All of them had the characteristics of a vibratory murmur of sinusoidal type, with diagonal radiation and low frequency. One more dynamic of pharmacological tests were performed in 60 children. In 88.5% of the cases, the murmur showed left behavior during the Valsalva monouver. Only in 15.7% during the Azoulay maneuver suggested right origin of the murmur. In 70% the murmur decreased with orthostatism and in the children who inhaled amyl nitrite, the murmur showed a behavior suggestive of aortic ejective origin. The comparison between the groups with and without murmurs showed that the heart rate was lower for those children with murmurs (P less than 0.05), the left ventricle ejection time was shorter in children with murmurs (P less than 0.01), but instead the preejection period was longer in children with murmurs (P less than 0.05). These differences let us point out that in children with murmurs the blood flow during the early systole is higher than in those without murmurs. This conditions probably a determinant in the origin of the innocent murmur.     

Measurement of functioning hepatocyte mass via [99mTc]galactosyl-neoglycoalbumin

Technetium-99m-galactosyl-neoglycoalbumin (TcNGA) is a synthetic radiolabeled ligand specific for hepatic binding protein (HBP), a receptor that resides exclusively on hepatocytes.In vivo measurement of receptor concentration was obtained via kinetic analysis of liver and blood time-activity data obtained during the hepatic clearance of intravenously administered TcNGA. The purpose of this study was to assess receptor concentration as a measure of the functioning hepatocyte mass. Therefore, TcNGA and dualinjection indocyanine green maximal removal rate (ICG R_max) studies were performed on nine patients with hepatic cirrhosis associated or not with hepatocellular carcinoma. Receptor concentration was compared with ICG R_max, which is a validated method for the estimation of the functioning hepatocyte mass. The correlation coefficient was 0.76 (P=0.017). It is concluded that HBP concentration ([HPB]_o) as measured by functional imaging is a measure of functioning hepatocyte mass. This implies that measurement of an individual's receptor concentration by using nuclear medicine techniques provides an objective index of hepatic functional mass and supports attempts to rigorously evaluate [HBP]_o for its clinical efficacy.     

Indications for liver transplantation in the cyclosporine era

One hundred seventy orthotopic liver transplants were performed under conventional immunosuppression with azathioprine and steroids with 1- and 5-year survivals of 32.9 per cent and 20.0 per cent, respectively. Since the introduction of cyclosporine-prednisone therapy in March 1980, 313 primary orthotopic liver transplants have been performed. Actuarial survivals at 1 and 5 years have improved to 69.7 per cent and 62.8 per cent, respectively. Biliary atresia is now the most common indication for liver replacement. In adults, primary biliary cirrhosis and sclerosing cholangitis have become more common indications for transplantation, and alcoholic cirrhosis and primary liver malignancy as indications have declined. Early enthusiasm for liver transplantation in patients with hepatic cancer has been tempered by the finding that recurrence is both common and rapid. An increasing number of patients with inborn errors of metabolism originating in the liver are receiving transplants, including patients with Wilson's disease, tyrosinemia, alpha-1-antitrypsin deficiency, glycogen storage disease, familial hypercholesterolemia, and hemochromatosis. Survival in this group of patients has been excellent (74.4 per cent at 1 and 5 years). A hemophiliac who received a transplant for postnecrotic cirrhosis has survived and may have been cured of his hemophilia. About 20 per cent of patients require retransplantation for rejection, technical failure, or primary graft failure. Only four of the patients receiving retransplants under conventional immunosuppression survived beyond 6 months, and all died within 14 months of retransplantation. Sixty-eight patients have received retransplants under cyclosporine-prednisone. Thirty-one patients are surviving, all for at least 1 year. Six of the twelve patients requiring a third transplant are alive 2 to 3 years after the primary operation. An aggressive approach to retransplantation in the patient with a failed graft is justified.     

Physical exercise and voluntary hyperventilation in childhood absence epilepsy.

The aim of this study was to compare the effects of a physical exercise test and of voluntary hyperventilation between controls and children with absence epilepsy. Eighteen children (6 controls and 12 epileptics) were studied during rest (R), a maximal physical exercise test (15 min; PE), recovery (REC) and voluntary hyperventilation (3 min; VHPV). EEG and ECG were recorded during the experiment; respiratory parameters were measured to quantify PE; plasma levels of pH, lactate, pyruvate, glucose and antiepileptic drugs were determined. A decrease in the number of absences was observed during PE whereas an increase was observed during VHPV. We found significant positive correlations between the number of children with absences, the total number of absences for each state, frequency of absences per minute and the corresponding mean plasma pH, which demonstrate that the lower the pH is, the fewer absences occur. On the other hand, there was no relationship between the number of absences and the values of other parameters. Relations between variations of the plasma value of the pH, and thus the probable cerebral value of pH, and neuronal excitability are discussed. Our results indicate that children who suffer absence epilepsy should not be discouraged from sport practice.     

Adenoviral infections in pediatric liver transplant recipients

Over a 5 1/2-year period, 22 of 262 children receiving liver transplants developed adenoviral infections. Five had adenoviral hepatitis in the allograft, caused by serotype 5. All five were treated for rejection, either just before or at the time of infection. Liver biopsy specimens had characteristic histological appearance, and diagnosis of adenoviral infection was confirmed with monoclonal antiadenoviral antibodies, electron microscopy, and by culture of liver tissue. In the remaining 17 patients, adenovirus was isolated from urine, stool, throat secretions, and/or blood samples, but none had any detectable visceral infection. Serotypes 1 and 2 predominated, similar to children not receiving transplants during the same time period. Three of the patients with hepatitis are alive and well; two died of liver failure. Adenoviral hepatitis did not recur in the second allograft of a patient who underwent retransplantation for combined rejection and adenoviral hepatitis, and appears, therefore, not to be a contraindication to retransplantation when liver failure ensues.     

Orthotopic liver transplantation for alpha-1-antitrypsin deficiency: an experience in 29 children and ten adults

Thirty-nine patients (29 children and ten adults) underwent OLT for liver disease associated with A1AD from March 1980 to March 1986. Thirty of thirty-six patients (83%) with available data were homozygous phenotype PiZZ. The other six were Pi heterozygotes, being either PiMZ or PiSZ. The mean A1A activity in homozygous and heterozygous patients was 38.8 mg/dL and 114.3 mg/dL respectively. Eight patients died during the first 3 months after OLT (20%). The 5-year actuarial survival is 83% and 60% in pediatric and adult recipients respectively. Today 30 (76%) of the recipients are alive, with follow-ups of 8 to 64 months (average 27 months). The quality of life in the surviving patients is excellent.     

Transplantation of multiple abdominal viscera

Two children with the short-gut syndrome and secondary liver failure were treated with evisceration and transplantation en bloc of the stomach, small intestine, colon, pancreas, and liver. The first patient died perioperatively, but the second lived for more than 6 months before dying of an Epstein-Barr virus-associated lymphoproliferative disorder that caused biliary obstruction and lethal sepsis. There was never evidence of graft rejection or of graft-vs-host disease in the long-surviving child. The constituent organs of the homograft functioned and maintained their morphological integrity throughout the 193 days of survival.     

Clinical considerations in orthotopic liver transplantation

Progress in immunosuppression, surgical techniques, and perioperative care has promoted orthotopic liver transplantation from an experimental procedure to an accepted clinical treatment. Orthotopic liver transplantation, in turn, has changed the treatment of terminal liver disease from care that is largely treatment of symptoms and support to cure, but at the price of major surgery and life-long immunosuppression. This article reviews the current status of liver transplantation as practiced at the University of Pittsburgh.