Development of a quantitative cell-based ELISA, for a humanized anti-IL-2/IL-15 receptor beta antibody (HuMikbeta(1)), and correlation with functional activity using an antigen-transfected murine cell line

The HuMikbeta(1), a humanized IgG1 monoclonal antibody directed toward the IL-2/IL-15 receptor beta-chain (CD122), inhibits the actions of the inflammatory cytokine IL-15, and may be useful for immunotherapy of an array of autoimmune disorders as well as diseases associated with the retrovirus human T-cell lymphotrophic virus 1 (HTLV-1). In order to facilitate the production of material for clinical investigation, we developed a cell-based ELISA (CbELISA) for measuring the binding activity, as a potential biological activity marker, of the HuMikbeta(1) monoclonal antibody to a transfected 32D mouse cell line (32Dbeta) expressing IL-2Rbeta antigen on the cell surface. There is specific binding of HuMikbeta(1) to the transfected cell line, titrating out in the concentration range of 1-1,000 ng/ml. Under identical conditions, there was no binding of HuMikbeta(1) to the parent cell line 32D. Satisfactory binding curves with HuMikbeta(1) were obtained with 32Dbeta cells grown between 3 and 19 passages in culture and at seed densities of 2 x 10(5)-4 x 10(6) cells/well. The binding was specific for Mikbeta antibodies recognizing the IL-2/IL-15 receptor beta subunit as demonstrated by binding of HuMikbeta(1), Mikbeta(2) and Mikbeta(3) antibodies, and lack of binding of irrelevant humanized and chimeric antibodies and isotype-matched human IgG1 to the 32Dbeta cell. Also, the human IgG1 and irrelevant humanized and chimeric antibodies did not interfere with the HuMikbeta(1) binding. The assay could detect changes in binding activity of HuMikbeta(1) antibody under stressful conditions (heat and low pH) and the results paralleled the effect of stress on the physicochemical characteristics. More importantly, the binding activity shows an apparent correlation to inhibition of IL-15-induced proliferation of 32Dbeta cells with HuMikbeta(1). In conclusion, the cell-based ELISA method represents a simple, reproducible accurate quantitative assay for monitoring HuMikbeta(1) activity and could be used as a potency marker assay for monitoring the lot-lot consistency and functional stability of HuMikbeta(1) product.     

Repeatability of left ventricular dyssynchrony and function parameters in serial gated myocardial perfusion SPECT studies

Background  

The purpose of this study was to establish the repeatability of left-ventricular (LV) dyssynchrony and function parameters measured from serial gated myocardial perfusion SPECT (GMPS) studies.     

Paraffin-embedded tissue as a source of RNA for gene expression analysis in oral malignancy

OBJECTIVE: To assess the feasibility of using archival oral mucosal tissue to examine gene expression at the ribonucleic acid (RNA) level.
MATERIALS AND METHODS: We describe the isolation of RNA from 8 nm sections of formalin-fixed paraffin-embedded oral mucosal tissue. RNA was reverse transcribed and three candidate genes amplified by polymerase chain reaction (PCR). The ribosomal protein S14 gene is a housekeeping gene which has been used as an internal standard in several quantitative PCR protocols. The thymidine kinase (TK) gene is expressed at low levels in most tissues and, with a well-documented genomic organisation, is a useful tool for discrimination between genomic DNA and cDNA. The RIa gene is reported to be overexpressed in many cancer cell lines, in malignant tissue and in vitro transformed cellS. RESULTS: The S14 gene, the TK gene and the RIα gene of the cAMP-dependent protein kinase (PKA) were amplified successfully from formalin-fixed paraffin-embedded tissue sections. The TK primer pair is a useful additional tool in the unambiguous identification of RNA-derived species.
CONCLUSION: RNA suitable for reverse transcribed (RT)-PCR was extracted from archival oral mucosal tissue. This should permit rapid sequence analysis of transcribed tumor suppressor genes and oncogenes in this material. Furthermore, the RT-PCR approach described may allow quantification of gene expression in oral mucosal archival material processed in a standard fashion.     

Surfactant therapy for acute respiratory distress in severe pediatric burn injury: a case series

After severe burn injury, pediatric patients often succumb to complications of respiratory failure. Surfactant has been used to improve pulmonary gas exchange for severe respiratory distress in other pediatric populations but has not been studied in pediatric burn-injured patients. Here, the authors report a case series of seven severely burned pediatric patients who received surfactant for acute respiratory distress and severe hypoxemia. Seven cases were reviewed of pediatric patients who received surfactant for severe acute respiratory distress. Data analyzed included age, TBSA burned, height, weight, mechanism of injury, total intensive care unit days, hospital days, and ventilator days. Modes of ventilation, peak inspiratory pressure, oxygen requirement, arterial blood gas analysis, blood pressure, and heart rate were analyzed before and the day following surfactant therapy. Four patients had reduced oxygen requirements following surfactant administration (FiO(2): 0.66 ± 0.23-0.48 ± 0.025). Three patients showed no reduction in oxygen requirements (FiO(2): 0.95 ± 0.09-0.90 ± 0.0). The remaining four patients who had reduced oxygen requirements received surfactant earlier following their injury (4.8 ± 0.9 days postinjury vs 17.7 ± 8 days postinjury) and had less derangement in oxygenation before surfactant dosing (PaO(2):FiO(2) ratio: 105.2 ± 26.4 vs 64.5 ± 5.2). Surfactant therapy may offer a therapeutic option during acute respiratory distress for severely burned pediatric patients. Surfactant may be useful early in the course of severe hypoxemia and acute respiratory distress but may not be effective as a salvage modality.