Isolation of putative dengue virus receptor molecules by affinity chromatography using a recombinant E protein ligand

Nucleotide sequences coding for the full-length envelope (E) glycoprotein gene of dengue virus type 4 was amplified using an RT-PCR method from infected C6/36 cells and cloned into pPROEx-Hta expression vector. The expression of the recombinant E protein in Escherichia coli was confirmed by Western blot using a polyclonal anti-dengue polyclonal antibody. The His-tagged fusion protein was obtained from the bacterial cellular extracts in almost pure form by immobilized metal affinity chromatography and the recombinant protein retained its ability to bind to 40 and 45 kDa proteins, previously described as putative receptors for dengue virus in C6/36 cells. To purify the 40 and 45 kDa molecules, a total protein extract from C6/36 cells was passed through an affinity chromatography column using immobilized recombinant E protein. After washing with isotonic buffer, elution was accomplished using a high salt buffer. The two proteins obtained, with molecular weights of 40 and 45 kDa, were recognized by dengue 4 virus, in virus overlay protein binding assay. This procedure allows further characterization of molecules that could be involved in dengue binding and entry.     

Alterations in iron metabolism in chronic anti-HCV positive hepatitis. The relationship with response to interferon treatment

A prospective study in which the existence of iron metabolism alterations (increase in the serum levels of iron and ferritin and the presence of iron in liver tissue) in a group of 53 patients diagnosed with chronic anti-HCV positive hepatitis was performed. The aim of the study was to determine whether these parameters influence the response to interferon treatment. Elevations were observed in the serum levels of iron and/or ferritin in 17 (32%) of the patients. Higher than normal values of serum iron or ferritin in pretreatment analyses were associated with worse therapeutic response. The basal serum levels of iron and ferritin were significantly higher in non responding patients. No relationship was found between the presence of iron in the hepatic parenchyma and response to interferon treatment.     

Renal tubular acidosis in the Silver-Russell syndrome

Several patients with the Silver-Russell syndrome (SRS) attending our Genetics Clinic were diagnosed as having persistent metabolic acidosis. Since this abnormality has not been reported previously in the SRS, we reexamined 33 SRS patients to evaluate the frequency and type of metabolic acidosis, the clinical and laboratory findings, and the growth pattern in SRS patients with and without metabolic acidosis. Among them, 14 had a consistent decrease in HCO levels. Renal studies in acidotic patients showed urine pH of 5.8 and 24 h urine calcium of <2.4 mg/kg/24 h; serum creatinine, excretion of glucose, and aminoacids were normal, as were renal ultrasound and excretory urography findings. These data supported the diagnosis of renal tubular acidosis, probably type II; the patients were treated with oral bicarbonate and acidosis was corrected successfully. Clinical manifestations were similar in acidotic and non-acidotic patients. The nutritional indices at diagnosis and at last evaluation (at least 8 months after diagnosis) were abnormally low in all patients; however, acidotic patients, treated with bicarbonate, showed an improvement of nutritional status particularly in the weight/height index, although the difference between groups after follow-up did not reach statistical significance. We suggest that metabolic acidosis due to renal tubular acidosis, probably type II, may occur in children with the SRS and should be looked for and treated in all patients. © 1995 Wiley-Liss, Inc.     

Measurement of basal tear turnover using a standardized protocol

Background: The aim of this study was to compare basal tear turnover values of healthy volunteers in different countries. Methods: Healthy volunteers aged between 20 and 70 years were selected in three European cities. Basal tear turnover values were calculated according to a standardized protocol from the decay of the fluorescein concentration in tears after instillation of 1-l drop of fluorescein in the conjunctival sac. Fluorescein concentration was measured with identical commercial fluorophotometers. A monoexponential decay of fluorescein was assumed to represent basal tear flow. Results: The mean tear turnover values were 13.1%/ min ± 4.6 SD (n=4), 16.0%/ min ± 5.2 SD (n=24) and 17.5%/ min ± 3.4 SD (n = 20) in Clermont-Ferrand (France), Leiden (The Netherlands) and Madrid (Spain), respectively. The differences between the values were not significant (Mann-Whitney test P > 0.09). Conclusions: The tear turnover in the different cities was similar. The methods used were simple and the software easy to use.Concerted Action, supported in part by the European Commission, on Ocular Fluorometry: Standardization and Instrumentation Development of the 4th European Community Medical and Health Research Programme (No. MR 4*/0314/P).     

Association of HLA-DR11 with the anaphylactoid reaction caused by nonsteroidal anti-inflammatory drugs

BACKGROUND: Several HLA alleles have been associated with asthma induced by nonsteroidal anti-inflammatory drugs (NSAIDs). The existence of HLA markers linked to other NSAID-induced reactions, such as cutaneous and anaphylactoid reactions, has not been established. OBJECTIVE: The purpose of our work was to study the HLA-DRB1 and HLA-DQB1 alleles in patients with cutaneous and anaphylactoid reactions caused by NSAIDs. METHODS: We have analyzed 114 HLA DRB1 and 26 HLA-DQB1 alleles in 21 patients with anaphylactoid reactions caused by NSAIDs, 47 patients who had exclusively cutaneous reactions during single-blind, placebo-controlled oral challenges with NSAIDs, and 167 tolerant control subjects (29 of whom had also had an IgE-mediated anaphylaxis to different agents). HLA-DRB1 and HLA-DQB1 alleles were typed by the polymerase chain reaction sequence-specific primers method with genomic DNA. RESULTS: The frequency of HLA-DR11 alleles was 58.8% in the anaphylactoid reaction group, compared with 15.9% in the NSAID-tolerant healthy control subjects (OR, 7:3; 95% confidence interval, 2.8-19.0; P <.02) and 6.3% in the group of the patients with a tolerance for NSAIDs and with IgE-mediated anaphylaxis (OR, 18.75; 95% confidence interval, 4.3-81.1; P <.004). No differences were observed among HLA-DR11 alleles analyzed. There were no significant HLA-DQB1 associations with NSAID-induced anaphylactoid reactions. Patients with cutaneous reactions had HLA frequencies that did not differ significantly from the tolerant control subjects. CONCLUSION: The HLA-DRB1*11 alleles showed a positive association with NSAID-induced anaphylactoid reactions.     

Left ventricular assist device improves survival in children with left ventricular dysfunction after repair of anomalous origin of the left coronary artery from the pulmonary artery

BACKGROUND: Repair of anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) in infants carries a high operative risk, particularly in infants with myocardial infarction and poor left ventricular function. The marked recovery of left ventricular function reported late after repair, however, suggests that an aggressive approach to repair should be undertaken. METHODS: Of 31 children undergoing primary repair of ALCAPA at our institution from 1987 to 1996, 26 were infants (6 weeks to 9 months old). All but 2 had severe left ventricular dysfunction, and 8 had moderate to severe mitral regurgitation. Seven children were unable to be weaned from cardiopulmonary bypass because of poor left ventricular function and elevated left atrial pressure. These 7 children were placed on mechanical left ventricular support using a centrifugal pump, with support ranging from 2.2 to 70.6 hours. RESULTS: One child died shortly after the start of left ventricular assist (2.2 hours), and another died of arrhythmia within 24 hours after successful decannulation. All 5 survivors had significant improvement in left ventricular function, with 2 requiring late mitral valve repair. CONCLUSIONS: Infants with ALCAPA who have severe left ventricular dysfunction represent a higher risk group for repair. However, with use of mechanical circulatory support in those unable to be weaned from cardiopulmonary bypass, a high survival rate can be achieved with good long-term recovery. We conclude that an aggressive approach to early repair in all children with ALCAPA is warranted, regardless of the degree of left ventricular dysfunction.