Method for efficient transfection of in vitro-transcribed mRNA into SK-N-AS and HEK293 cells: difference in the toxicity of nuclear EGFP compared to cytoplasmic EGFP

Here we report a method for efficient transfection of in vitro-transcribed mRNA into two different types of human adherent cells, the neuroblastoma cell line SK-N-AS, and the transformed kidney cell line HEK293. By using newly trypsinized adherent cells in suspension and Lipofectaminetrade mark 2000, we detected a transfection efficiency of 80-90% in both cell lines and a cell viability of 90% in SK-N-AS and 60% in HEK293, 24 h after transfection when using cytoplasmic enhanced green fluorescent protein (EGFP)-mRNA. We have evaluated the different effects of the generally used EGFP that mainly localizes to the cytoplasm and nuclear EGFP, where the nuclear EGFP are more toxic to the cells than the cytoplasmic EGFP. In order to develop a null experiment, we constructed a short non-functional mRNA including a nuclear localization signal and evaluated the concentrations at which mRNA encoding nuclear proteins can be added without a general toxicity, depending on the fact that the proteins are localized to the nucleus. For both SK-N-AS and HEK293 cells, a concentration of up to 100 ng mRNA in 10(5) cells, encoding a nuclear protein with no other function, did not affect the cells. For evaluation of the method, we screened four different human mRNAs, PDG, DFFA, CORT and PEX14, for their ability to affect cell proliferation in these cells. PEX14 was the only gene that significantly (p=0.03) reduced cell proliferation for both cell types, DFFA significantly (p=0.04) reduced cell proliferation in SK-N-AS but not in HEK293 cells. PGD and CORT did not have any effect on cell proliferation. We have developed an easy method for efficient delivery of in vitro-transcribed mRNA into the adherent cell lines, SK-N-AS and HEK293. This method is useful for a quick screening of how different genes affect cell proliferation.     

Continuity and change

This Themed Issue consists of three reviews and 11 original articles authored by internationally respected industrial and academic pharmacologists from across three continents. It derives from the highly successful symposium on ‘The H₃ and H₄ histamine receptors: the antihistamines for the 21^st century’, which took place at EPHAR 2008 in Manchester University, and encompasses new roles, new compounds and exciting new therapeutic areas for histamine.     

Neglected dorsal dislocation of the scaphoid

Isolated dislocation of the scaphoid is very rare. A 45-year old male, industrial worker reported two and half months after injury with wrist pain and swelling on the dorsum of left wrist. He was diagnosed as neglected dorsal dislocation of scaphoid. Proximal row carpectomy with capsular interposition was done stabilizing the distal carpus on the radius using Kirschner wires. At-12 months follow-up the patient had good wrist function and was satisfied with the outcome of the treatment. We hereby report this neglected dorsal dislocation of scaphoid in view of rarity and discuss the various options for management.     

Rare mutations in the CYP21A2 gene detected in congenital adrenal hyperplasia

ObjectivesThe purpose of this study was to identify and determine the frequencies of rare CYP21A2 gene mutations in patients with 21-hydroxylase deficiency (21-OHD) in the Cypriot population.Design and methodsDirect sequencing and MLPA analysis of the CYP21A2 gene.ResultsA group of families with 21-OHD were screened for the presence of rare CYP21A2 gene mutations. The rare V304M missense mutation was detected as compound heterozygous in two females with the nonclassical (NC) form of congenital adrenal hyperplasia (CAH). The rare F306insT was also detected in a female with severe salt wasting in the homozygous state and in cis in both alleles with the V281L mutation. Lastly, the rare A391T missense mutation was reported in a female patient with NC-CAH. A carrier rate of 2.1% for the V304M was also observed in a cohort of healthy controls.ConclusionsThe frequency of V304M mutation among Cypriots is high and the first reported so far and patients characterized as compound heterozygotes or heterozygotes are most readily identified by a mild phenotype of CAH. Thus, V304M should be included in the panel of mutations associated with the NC forms of 21-OHD.     

HPLC Determination of Mepiquat Chloride in Commercial Pesticide Formulations

A simple, fast and precise High Performance Liquid Chromatographic method with diode array detector was developed and single laboratory validated for the determination of mepiquat chloride in soluble concentrate pesticide formulations. From the results obtained, the repeatability of the method expressed as relative standard deviation (%RSD) was found to be 0.3%, and the limit of detection was 0.02 μg/mL. The accuracy of the whole procedure estimated by the comparison of the results obtained with the reference values, and was found to be acceptable as t _cal < t _crit. The precision of the method also considered acceptable as the experimental repeatability relative standard deviation (RSD_r) was lower than the interlaboratory relative standard deviation (RSD_R), calculated by the Horwitz equation.     

Therapeutic benefit of blocking interleukin‐6 activity with an anti–interleukin‐6 receptor monoclonal antibody in rheumatoid arthritis: A randomized,double‐blind,placebo‐controlled,dose‐escalation trial

Objective

To investigate the safety and efficacy of MRA, a recombinant human anti–interleukin‐6 (anti–IL‐6) receptor monoclonal antibody of the IgG1 subclass that inhibits the function of IL‐6, in patients with established rheumatoid arthritis (RA).

Methods

A randomized, double‐blind, placebo‐controlled, dose‐escalation trial was conducted in 45 patients with active RA, as defined by the American College of Rheumatology (ACR) revised criteria. Patients were sequentially allocated to receive a single intravenous dose of either 0.1, 1, 5, or 10 mg/kg of MRA or placebo. The primary efficacy end point was meeting the ACR 20% response criteria at week 2 after treatment.

Results

Demographic features were similar between treatment groups. At week 2, a significant treatment difference was observed between the 5 mg/kg of MRA and placebo, with 5 patients (55.6%) in the MRA cohort and none in the placebo cohort achieving ACR 20% improvement. There was no statistically significant difference in the ACR 20% response between the other 3 MRA cohorts and placebo at week 2. The mean disease activity score at week 2 in those who received 5 mg/kg and 10 mg/kg of MRA was 4.8 and 4.7 (P < 0.001 and P < 0.001 by analysis of variance), respectively. These mean scores were statistically significantly lower than those in the 0.1‐ and 1‐mg/kg MRA and the placebo cohorts (6.4, 6.2, and 7.0, respectively). The erythrocyte sedimentation rate and C‐reactive protein values fell significantly in the 5‐ and 10‐mg/kg MRA cohorts and normalized 2 weeks after treatment. Seventeen patients (5, 4, 6, 2, and 0 patients in the placebo, 0.1‐, 1‐, 5‐, and 10‐mg/kg MRA cohorts, respectively) required corticosteroid or disease‐modifying antirheumatic drug treatment because of active disease before study end. They were regarded as nonresponders from the time they received these treatments. Diarrhea was the most common adverse event, occurring in 8% of patients. Seven patients (15.6%) reported a severe adverse event (3, 1, 2, and 2 patients in the placebo, 0.1‐, 1‐, and 10‐mg/kg MRA cohorts). There were no serious adverse events that were thought to be related to the study drug.

Conclusion

This is the first randomized controlled trial showing that inhibition of IL‐6 significantly improved the signs and symptoms of RA and normalized the acute‐phase reactants. Further research with multiple dosing is necessary to define the most appropriate therapeutic regimen of MRA in RA.