Expression and purification of functional recombinant epitopes for the platelet antigens, PlA1 and PlA2

The platelet antigens, PlA1 and PlA2, are responsible for most cases of posttransfusion purpura (PTP) and neonatal alloimmune thrombocytopenia (NAIT) in the caucasian population and are determined by two allelic forms of the platelet glycoprotein GPIIIa gene. To study the interaction between these antigens and their respective antibodies, we inserted the sequence that encodes the signal peptide and the N- terminal 66 amino acids of the PlA1 form of GPIIIa into the expression vector pGEX1. To express the PlA2 antigen, nucleotide 196 of the PlA1 coding sequence was mutated to the PlA2 allelic form. When transformed and induced in Escherichia coli, the two constructs produce glutathione S-transferase (GST)/N-terminal GPIIIa fusion proteins, one containing leucine at position 33 (PlA1), the other proline (PlA2). These proteins are easily purified in milligram quantities using glutathione-Sepharose and react specifically with their respective antibodies by immunoblot and enzyme-linked immunosorbent assay. Antigenicity of the PlA1 fusion protein in reduced glutathione increases with time; moreover, the addition of oxidized glutathione accelerates this process, presumably because of formation of the native disulfide bonds. Neutralization assays indicate that the PlA1 fusion protein competes for all of the anti-PlA1 antibody in the serum of patients with PTP and NAIT that is capable of interacting with the surface of intact platelets. This study shows that the GST/N-terminal GPIIIa fusion proteins contain conformational epitopes that mimic those involved in alloimmunization, and that regions other than the amino terminal 66 amino acids of GPIIIa are not likely to contain or be required for the development of functional PlA1 epitopes. Furthermore, these recombinant proteins can be used for the affinity-purification of clinical anti-PlA1 antibodies and specific antibody identification by western blotting, making them useful in the diagnosis of patients alloimmunized to PlA1 alloantigens.     

The vestibular aqueduct in congenital deafness: evaluation by the axial projection

Thirty-two patients with congenital neurosensory hearing loss demonstrated dilatation of the peripheral limb of the vestibular aqueduct as visualized on pluridirectional tomograms. Multiple additional anomalies involving most frequently the cochlea (15 patients) but also the ossicles (three patients) or vestibule (one patient) were associated. The axial-pyramidal (Poschl) projection was of great value in evaluation of the vestibular aqueduct and cochlea. The technique, clinical correlation, and surgical implications are discussed.     

Prevalence of early stages of chronic kidney disease in healthy army personnel

Introduction: Chronic kidney disease (CKD) is associated with significant morbidity and mortality. Screening and detection of early stages of CKD can help institute interventions that may delay the progression of the disease. One aim was to study the prevalence of early stages of CKD in the Army.Methods: A cross-sectional study ofArmy Personnel in an Army cantt in Central India was carried out. All participants filled a structured questionnaire and anthropometric data was collected. Investigative profile included routine urine exam, semi-quantitative microalbuminuria (MAU), serum creatinine, lipid profile and fasting blood glucose. Glomerular Filteration rate (eGFR) was calculated using the Modification of Diet in Renal Diseases (MDRD) study equation.Result: A total of 1920 subjects were examined with 731 (38.07%) from Arms and 1189 (6I.93%) from Services. 348 were excluded and of the remaining 1572 subjects, 141 (8.97%) had MAU and 157 (9.99%) had deranged Albumin Creatinine Ratio (ACR). Mean eGFR by MDRD equation was 102 ± 25.84 ml/min/1.73m². Early CKD was seen in 150 (9.54%) with 84 (5.34%) in stage I CKD, 55 (3.5%) in stage II and 11 (0.7%) in stage III. Multiple logistic regression showed BMI > 23, the presence of DM and HTN were independent risk factors for CKD.Conclusion: 9.54 % of healthy army personnel were found to have early stages of CKD. Institution of screening programs can result in early detection of CKD.     

NEONATAL OUTCOME IN MECONIUM STAINED DELIVERIES — A PROSPECTIVE STUDY

This prospective study analyzes the neonatal outcome in deliveries complicated by meconium stained amniotic fluid. In a study of 1000 live born deliveries, meconium staining of amniotic fluid was seen in 50 (5%) deliveries. Out of these, 20 newborns (40%) developed classical signs of meconium aspiration syndrome and were managed according to a predetermined protocol. Multiparity, term deliveries, use of sedatives in mother, intrauterine growth retardation and prolonged labour were some of the risk factors for development of meconium aspiration syndrome in newborns. This study highlights the need for review of management protocol in newborns after meconium staining of the amniotic fluid, including the use of prophylactic antibiotics.KEY WORDS: Amniotic fluid, Delivery, Meconium aspiration, Respiratory distress syndrome     

PLASMID MEDIATED DRUG RESISTANCE IN VIBRIO CHOLERAE 0139 BENGAL

Sixteen strains of Vibrio cholerae were isolated from cases of diarrhoea. Out of these, 12 (75%) were identified as Vibrio cholerae 0139 synonym Bengal and 4 (25%) as Vibrio cholerae El Tor by standard biochemical and serological tests. Modified CAMP reaction in sheep blood agar showed that 0139 produced moderate hemolysis, El Tor produced wider zone of hemolysis whereas Classical Vibrio cholerae produced no zone of hemolysis (CAMP negative). Break point minimum inhibitory concentration (MIC) by agar dilution method showed that all 0139 strains were resistant to ampicillin 8 mg/L, streptomycin 1 mg/L, chloramphenicol 8 mg/L, sulphamethoxazole 32 mg/L and trimethoprim 0.3-128 mg/L, 58.3% were sensitive to gentamicin 1 mg/L, and all were sensitive to norfloxacin 1 mg/L and cefotaxime 1 mg/L. Resistance to trimethoprim, sulphamethoxazole, ampicillin and gentamicin in 5 strains could be transferred to E coli K-12 by conjugation experiment at a rate of 5×10⁻⁶ to 4×10⁻³. Distinct plasmid bands of 35.8 mega daltons could be seen in agarose gel electrophoresis.KEY WORDS: CAMP test, Drug resistance, Plasmid, Vibrio cholerae 0139.