Monoclonal antibody production to human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase): high-specificity recognition in whole brain acetone powders and conservation of sequence between CNP1 and CNP2

Monoclonal antibodies against human and bovine 2′:3′-cyclic nucleotide 3′-phosphodiesterase (CNPase) were generated by fusing FOX-NY myeloma cells with spleen cells from RBF/Dn mice previously immunized with the purified brain antigens. The enzyme isolated from bovine brain was quite basic, with an isoelectric point of 9.71 and both the bovine and human enzymes consisted of a closely spaced doublet at approximately 44 and 46 kDa on SDS-PAGE. Six monoclonals were identified as strongly recognizing the enzyme on both ELISA plates and on immunoblots of whole brain protein. Four monoclonals very weakly cross-reacted with guinea pig myelin basic protein. In contrast with two previous reports, some of our monoclonal antibodies did immunostain 2 or 3 protein bands in peripheral nerve, two bands closely corresponding to those immunostained in central nervous system (CNS) myelin, the Wolfgram protein fraction and in acetone powders of whole brain. Each of the 6 monoclonals reacting strongly on immunoblots recognized the enzyme in from 2 to 5 of the species examined (human, bovine, rat, mouse and rabbit). In addition, all 6 monoclonals that immunostained the enzyme in whole brain, myelin and Wolfgram protein immunoblots recognized both CNP1 (44 kDa) and CNP2 (46 kDa). The two closely spaced protein bands observed on SDS-PAGE and previously stained on immunoblots of CNS CNPase using polyvalent rabbit anti-bovine CNPase antisera, and now different monoclonal antibodies, appear to be immunologically related and to contain highly conserved sequences.     

Clinical symptoms and signs in sore throat patients with large colony variant β-haemolytic streptococci groups C or G versus group A

BACKGROUND: The role of large colony streptococci groups C or G as pathogen agents in sore throat has been questioned. AIM: To analyse clinical features of patients with large colony streptococci groups C or G compared with patients with group A streptococci (GAS) and with negative cultures. DESIGN OF STUDY: Prospective study of patients with sore throat. SETTING: Two Norwegian general practices in Stokke and Kongsberg communities with 6500 patients.METHOD: Frequency of clinical features in the three patient categories including the four Centor criteria (fever, anterior cervical lymphadenopathy, tonsillar exudates, and lack of cough), degree of pain on swallowing, pharyngeal rubor, C-reactive protein (CRP) values, patient age between 3 and 14 years, and duration of symptoms before seeing the doctor. A logistic regression analysis to find independent predictors was performed. RESULTS: Out of 306 patients with a sore throat, 244 were adults and 62 were children under 10 years old; 40% were men. One hundred and twenty-seven had GAS, 33 had streptococci groups C or G, and 146 had negative throat cultures. Forty-eight per cent of the GAS patients and 45% of the C or G patients met three or four of the Centor criteria. The logistic regression revealed that in patients with GAS considerable pain on swallowing, an age of 3-14 years and a duration of symptoms of < or =3 days or less were significantly associated with GAS infection in addition to the Centor criteria. The same results were found when all streptococci were analysed together, in addition elevated CRP was significant. In patients with streptococci group C or G an elevated CRP-value was significantly associated. CONCLUSION: Patients with tonsillitis caused by streptococcus groups C or G have, to a large extent, the same clinical picture as patients with GAS. Large colony streptococci groups C and G should be considered as throat pathogens in line with GAS.     

Behavior of cryopreserved endothelial cells in different phases: Their application in the seeding of vascular prostheses

Various methods of cryopreservation of human endothelial cells (EC) were studied to determine their viability and behavior when seeded onto vascular prostheses made of polytetrafluoroethylene (PTFE). Three different protocols were used: (1) cyropreservation of whole umbilical vein, (2) cyropreservation of freshly extracted umbilical EC in suspension, and (3) cryopreservation of EC derived from a first subculture. Fresh EC and EC from a first subculture were used as controls. The viability and growth of these cells in culture media were studied, and basal prostacyclin levels were determined. The cells were assessed morphologically after they were seeded onto PTFE discs. Our results showed that the cryopreservation method that maintained the greatest viability was that in which previously cultured EC were used. Basal prostacyclin levels were significantly different following cyropreservation. However, when these cells were seeded onto PTFE discs their behavior was similar to that of fresh EC.We thank W. L. Gore & Associates for supplying the prostheses used in this study.Supported by a grant from the Comision Interministerial de Cieucia y Tecnologia SAF 92/0875.     

Intragastric blood flow in the etiology of duodenal ulcer--new aspects of surgical treatment

In the experiment on dogs and in patients with duodenal ulcer disease, it was established that the regulation of acid production besides the vagal nerves was mediated by the intragastric gastrin transport with the blood flow. A method of preoperative diagnosis of the intensity of intragastric blood flow permitting to define more accurately the indications for the choice of a method for surgical treatment has been developed. Supplementation of the selective proximal vagotomy with circular mucosectomy at the boundary between the antrum and body of a stomach enhances the effectiveness of operation due to reduction of the antral gastrin influence on the acid-producing zones of the stomach.     

Predictive testing for Huntington's disease: II. Qualitative findings from a study of uptake in South Wales

Semi-structured interviews were conducted with a cohort of 22 test applicants who requested Huntington's disease (HD) predictive testing in South Wales, and a random sample of 32 non-requesters, drawn from the South Wales HD register. Apart from identifying differences between the groups, the study afforded the opportunity to listen, at length, to at-risk individuals' accounts of living at risk and their thoughts about predictive testing and genetic services. Emergent themes included difficulties in family communication and the uncertainties inherent in being at risk and undergoing testing. Important factors in decision making about testing were: moral imperatives to clarify one's genetic status; views about the controllability of the future; family attitudes and norms; and the impact of a test result on family members. At-risk individuals' perceptions of the genetics service were that contact with the service would result in pressure to be tested and a need for test applicants to present a favourable view of coping capacities to secure testing. In addition, there was an expectation of ongoing contact with HD families at the initiative of the service providers. Implications of the findings for the way in which predictive testing services are structured and introduced to the at-risk population are discussed.     

Sarcoglycanopathies: a multiplex molecular analysis for the most common mutations.

Sarcoglycanopathies (SGpathies) are highly frequent among severely affected limb-girdle muscular dystrophy patients. On the basis of the findings of 5 common mutations in the 4 sarcoglycan (SG) genes in the Brazilian population, we standardized a multiplex polymerase chain reaction-single-strand conformation polymorphism methodology for their concomitant analysis in DNA samples. The test was able to confirm the diagnosis in about 63% of new patients with a suspected SGpathy and was particularly important in patients in advanced stages of the disease, when obtaining a muscle biopsy for analysis may be very difficult. As common mutations have been described in several countries, this multiplex analysis could be useful for the diagnosis of SGpathies if established according to the most prevalent mutations in each population. Besides, even though the disorder studied is rare, the technique could be broadly applicable to other genes and disorders.     

Actin-free Gc globulin: a rapidly assessed biomarker of organ dysfunction in acute liver failure and cirrhosis.

Reductions in serum levels of Gc globulin, a hepatically synthesized component of the extracellular actin scavenger system responsible for complexing circulating actin and attenuating intravascular microthrombus formation, are associated with poor outcome in acute liver failure. Clinically applicable assays of the important actin-free fraction (Af-Gc) have not been available until now. We measured actin-free Gc globulin levels with a novel, rapid assay in 61 cases of acute liver failure (ALF) and in 91 patients with cirrhosis (40 of whom were clinically unstable with extrahepatic organ dysfunction), and studied associations with liver dysfunction, extrahepatic organ dysfunction, indices of disseminated coagulation, and outcome. Reductions in Af-Gc levels mirrored hepatic dysfunction and organ dysfunction in both groups, and discriminated patients with poor prognosis from those with good prognosis in the ALF cohort. Levels were lowest in patients with ALF (10% of control values), but levels were also markedly reduced in both unstable (28%) and stable (44%) patients with cirrhosis. Associations with markers of disseminated intravascular coagulation were seen in both groups, most notably in the cirrhosis cohort, supporting a pathophysiological role for reduced Af-Gc in the evolution of organ dysfunction. In acetaminophen-induced ALF, Af-Gc identified patients with poor prognosis as well as did the Acute Physiology and Chronic Health Evaluation (APACHE II) score (area under the receiver operating characteristic curve, 0.7), and in cirrhosis, Af-Gc was an independent predictor of mortality by multifactorial analysis. In conclusion, the importance of Af-Gc reductions in the development of multiple organ dysfunction in ALF and cirrhosis is highlighted, probably resulting from reduced hepatic production and peripheral exhaustion of this arm of the extracellular actin scavenger system.     

Relationship between oxidative stress and essential hypertension.

This study investigated the association of blood pressure with blood oxidative stress-related parameters in normotensive and hypertensive subjects. A cross-sectional design was applied to 31 hypertensive patients and 35 healthy normotensive subjects. All subjects were men between the ages of 35 and 60 years. Exclusion criteria were obesity, dyslipidemia, diabetes mellitus, smoking and current use of any medication. All patients underwent 24-h ambulatory blood pressure monitoring and sampling of blood and urine. Antioxidant enzymes activity, reduced/oxidized glutathione ratio (GSH/GSSG), and lipid peroxidation (malondialdehyde) were determined in erythrocytes. Parameters measured in the plasma of test subjects were plasma antioxidant status, lipid peroxidation (8-isoprostane), plasma vitamin C and E, and the blood pressure modulators renin, aldosterone, endothelin-1 and homocysteine. Daytime systolic and diastolic blood pressures of hypertensives were negatively correlated with plasma antioxidant capacity (r=-0.46, p<0.009 and r=-0.48, p<0.007), plasma vitamin C levels (r=-0.53, p<0.003 and r=-0.44, p<0.02), erythrocyte activity of antioxidant enzymes, and erythrocyte GSH/GSSG ratio, with hypertensives showing higher levels of oxidative stress. Blood pressures showed a positive correlation with both plasma and urine 8-isoprostane. Neither plasma vitamin E nor the assessed blood pressure modulator levels showed significant differences between the groups or correlation with blood pressures. These findings demonstrate a strong association between blood pressure and some oxidative stress-related parameters and suggest a possible role of oxidative stress in the pathophysiology of essential hypertension.