Development of a quantitative antigen-specific cell-based ELISA for the 7G7/B6 monoclonal antibody directed toward IL-2Ralpha

Interleukin-2 receptor alpha (IL-2Ralpha, CD25) has been identified as a valuable target for immunotherapy. The 7G7/B6 monoclonal antibody, a mouse IgG2a kappa, recognizes an epitope of the IL-2Ralpha peptide, other than that identified by anti-Tac. This antibody is currently being explored for potential therapeutic and diagnostic applications. Here, we show a cell-based enzyme-linked immunosorbent assay (CbELISA) method for quantitative measurement of the binding activity of the 7G7/B6 antibody to the Kit-225-iG3 cell line expressing IL-2Ralpha antigen on the cell surface. The cell- and antigen-specificity of the assay was established using specific cell lines and irrelevant control antibodies. Satisfactory binding curves were demonstrated with Kit-225-iG3 cells grown between 3 and 25 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The assay shows reproducible dose-response curves in the concentration range of 10-1000 ng/ml. The assay validation data presented here indicate that this CbELISA assay is quantitative, reproducible, robust, precise, and can be used to test the biological activity, lot to lot comparison, and stability of 7G7/B6 monoclonal antibody.     

Impact of vitamin A supplementation through different dosages of red palm oil and retinol palmitate on preschool children

Red palm oil (5 ml and 10 ml), ground nut oil fortified with 400 and 800 retinol equivalent retinol palmitate, and ground nut oil (5 and 10 ml), were administered to six groups of preschool children (four experimental and two control groups) in randomly assigned balwadis of Ramanathapuram District of Tamil Nadu for a period of 7 months, to monitor the difference in the efficacy of the mode of supplementation and the optimum dose for improving vitamin A status. Results show that red palm oil groups recorded more gain in retinol and beta-carotene levels compared to other dosage groups, and that administration of 10 ml did not offer any substantial improvement over the 5-ml daily dose.     

The independent association of renal dysfunction and arrhythmias in critically ill patients

STUDY OBJECTIVES: The purpose of this study was to quantify the impact of baseline renal dysfunction on incidence and occurrence of cardiac arrhythmias in the coronary ICU. BACKGROUND: Renal dysfunction is an established predictor of all-cause mortality in the ICU setting. We set out to evaluate the independent contributory effect of renal dysfunction to arrhythmias and mortality in this population. DESIGN AND SETTING: We analyzed a prospective coronary care unit registry of 12,648 admissions by 9,557 patients over 8 years at a single, tertiary center. An admission serum creatinine level was available for 9,544 patients. Those patients not receiving long-term dialysis were classified into quartiles of corrected creatinine clearance with cutpoints of 46.2 mL/min/72 kg (group 1), 63.1 mL/min/72 kg, and 81.5 mL/min/72 kg. Dialysis patients (n = 527) were considered as a fifth comparison group (group 5). MEASUREMENTS AND RESULTS: Baseline characteristics including older age, African-American race, diabetes, hypertension, history of previous coronary disease, and heart failure were incrementally more common with increasing renal dysfunction strata. There were graded, independent increased risks for accelerated idioventricular rhythm (relative risk [RR], 2.43; 95% confidence interval [CI], 1.40 to 4.20; p = 0.002), sustained ventricular tachycardia (RR, 2.07; 95% CI, 1.02 to 4.22; p = 0.04), ventricular fibrillation (RR, 2.42; 95% CI, 1.13 to 5.15; p = 0.02), and complete heart block (RR, 3.64; 95% CI, 1.77 to 7.48; p = 0.0004, group 5 vs group 1). CONCLUSIONS: We conclude that baseline renal function is a powerful, independent predictor of cardiac arrhythmias in the coronary ICU population.     

Familial exudative vitreoretinopathy (FEVR). Clinical profile and management

PURPOSE: To report our experience with the diagnosis and management of Familial Exudative Vitreoretinopathy (FEVR) in a predominantly older Indian population.. METHODS: This prospective interventional non-comparative case series included 38 patients of FEVR and their 23 family members. The diagnosis was established by clinical examination, fluorescein angiography and family screening. Prophylactic photocoagulation/cryotherapy or surgical treatment was done depending on the severity of the disease. RESULTS: The mean age of the patients was 23.6 years. The fundus/fluorescein angiographic findings in 116 eyes of our 61 patients (6 eyes phthisical) were as follows: forty eight (41.4%) eyes had only peripheral avascular zone, 8 (6.9%) eyes had peripheral new vessels, and 35 (30.1%) eyes had retinal detachments (RD)--10 (8.6%) exudative, 5 (4.3%) tractional and 20 (17.2%) rhegmatogenous. Prophylactic photocoagulation or cryotherapy was done in 34 eyes for retinal holes, local exudative detachments and bleeding new vessels. All the eyes retained stable vision over a mean follow-up of 16 months. Only 14 RDs were suitable for surgery: scleral buckling, vitrectomy or both. The reattachment rate was 85.7% (12 of 14) and the best-corrected visual acuity (BCVA) improved to 5/60 or better in 50% of these eyes over a 2-year follow-up. CONCLUSIONS: FEVR appears to be more common than reported. Timely diagnosis and intervention is essential in view of the lifelong progression of the disease, late exacerbations, frequent involvement of family members, and poor surgical results. A high index of suspicion, family screening and early prophylaxis are recommended to prevent avoidable blindness from this underdiagnosed disease.     

Impact of beta-carotene supplementation through red palm

A 10-month long feeding trial was conducted to assess the impact of beta-carotene supplementation through red palm oil (RPO) with the focus on vitamin A status, morbidity status and acceptability of an RPO-incorporated noon-meal as a dietary supplement among two cohorts of 409 (experimental) and 346 (control) preschool children in two southern districts of rural Tamil Nadu, selected by stratified random sampling. Information was gathered on ocular symptoms of vitamin A deficiency and anthropometry, and blood samples were drawn at baseline and final rounds for estimation of serum beta-carotene, retinol and tocopherol. Data about Socioeconomic Status (SES) were collected once during the study period, while information on attendance, consumption and morbidity was recorded by preschool teachers. The results showed the following. 1. Significant improvement in the vitamin A status of children in terms of disappearance of Bitot's spots (50.0 per cent) in the experimental group vs. 28.0 per cent in the control group. 2. After feeding of RPO, incidence rate of new Bitot's spots cases was low at 2.13 in the experimental children vs. 4.78 in control children. 3. Marked improvement in the serum beta-carotene levels after 10 months of feeding. 4. RPO is acceptable to children as an edible grade oil as there is no perceptible difference in the consumption pattern between experimental and control children.     

Sporovexins A-C and a new preussomerin analog: antibacterial and antifungal metabolites from the coprophilous fungus Sporormiella vexans

Sporovexins A-C (1-3) and 3'-O-desmethyl-1-epipreussomerin C (4) have been isolated from liquid cultures of the coprophilous fungus Sporormiella vexans (JS 306). The structures of these new metabolites were elucidated on the basis of MS and NMR analysis. Compounds 1 and 4 show antifungal activity against competitor fungi, as well as antibacterial effects.     

Expression of Fas and Fas ligand in Hodgkin's disease.

Fas and Fas ligand expression were investigated in twenty two cases of classical Hodgkin's disease (HD) by immunohistochemistry. While Reed-Sternberg (RS) cells in 7/22 (32%) cases expressed Fas ligand, reactive lymphoid cells expressed Fas ligand in only 2 (9%) cases. In 20/22 (91%) cases, the RS cells expressed Fas. A higher proportion of RS cells in the nodular sclerosis subtype expressed Fas as compared to the mixed cellularity subtype. In 18/22 (82%) cases, Fas expression was also noted in the reactive lymphoid cells. In eight cases, the reactive lymphoid cells were also analyzed by flow cytometry and a majority of them were CD4+CD45RO+. Most of these activated T-cells expressed Fas but were negative for Fas Ligand. To investigate the co-expression of Fas and Fas Ligand in the RS cells, six cases were subjected to Fas and Fas ligand immunostaining on consecutive sections. The co-expression was documented in the RS cells in four of six cases. These six cases with expression of both Fas and Fas ligand were investigated for the incidence of apoptosis. There was no statistically significant relationship between expression of Fas on reactive cells, expression of FasL on RS cells and the proportion of apoptotic reactive cells. In all these cases apoptosis was not observed in the RS cells. Thus Fas - FasL interactions may not lead to apoptosis of the RS cells.     

Burn injury and pulmonary sepsis: development of a clinically relevant model

BACKGROUND: Despite improvements in the early resuscitation of the critically injured, mortality from multiple organ failure has remained stable, with the lung often the first organ to fail. Early intubation and mechanical ventilation predispose patients to the development of pneumonia and respiratory failure. Our objective was to establish a murine model of combined injury, consisting of burn/trauma and pulmonary sepsis with reproducible end-organ responses and mortality. METHODS: Male B6D2F1 mice were divided into four groups: burn/infection (BI), burn (B), infection (I), and sham (S). Burned animals had a full-thickness 15% dorsal scald burn. BI and I groups were inoculated intratracheally with Pseudomonas aeruginosa (3-5 x 103 colony-forming units). S and B animals received saline intratracheally. All animals were resuscitated with 2 mL of intraperitoneal saline. Mortality was recorded at 24, 48, and 72 hours. Bacterial sepsis was confirmed by tissue Gram's stain of the lungs and positive organ and blood cultures for Pseudomonas aeruginosa. Femoral bone marrow cells were collected at 72 hours from surviving animals. Clonogenic potential was assessed by response to macrophage (M) colony-stimulating factor (CSF) and granulocyte-macrophage (GM) CSF in a soft agar assay and the data were represented as colonies per femur. Isolated alveolar macrophages and whole lung tissue were assayed for levels of the inflammatory cytokines tumor necrosis factor-alpha and interleukin-6. RESULTS: Mortality at 72 hours was 30% in BI, 12% in I, and <10% in B and S groups. Pneumonia was documented in all infected animals at 24 hours by Gram's stain and positive tissue cultures for Pseudomonas aeruginosa. Systemic sepsis as confirmed by blood, and remote organ cultures was seen in BI animals only. Significantly increased responsiveness to M-CSF stimulations was noted in all groups (BI, 8,291 +/- 1,402 colonies/femur; B, 6,357 +/- 806 colonies/femur; and I, 8,054 +/- 1,112 colonies/femur; p < 0.05) relative to sham (3,369 +/- 883 colonies/femur, p < 0.05). Maximal responsiveness to GM-CSF stimulation was noted in the BI group (11,932 +/- 982 colonies/femur, p < 0.05), and similar GM responsiveness was noted in all other groups (B, 7,135 +/- 548 colonies/femur; I, 7,023 +/- 810 colonies/femur; and S, 6,829 +/- 1,439 colonies/femur). Alveolar macrophage release of the proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 increased in all animals, but the magnitude of increase was not proportional to the strength of the inciting stimulus. CONCLUSION: Although minimal perturbations were seen after burn or pulmonary infection alone, the combined insult of burn and pulmonary sepsis resulted in statistically significant hematopoietic changes with increased monocytopoiesis. Only the combined injury resulted in systemic sepsis and significantly increased mortality. We have developed a clinically relevant model of trauma and pulmonary sepsis that will allow further clarification of the inflammatory response after injury and infection.