Phenotypic dissection of the mouse Ren1d knockout by complementation with human renin.

Normal renin synthesis and secretion is important for the maintenance of juxtaglomerular apparatus architecture. Mice lacking a functional Ren1d gene are devoid of renal juxtaglomerular cell granules and exhibit an altered macula densa morphology. Due to the species-specificity of renin activity, transgenic mice are ideal models for experimentally investigating and manipulating expression patterns of the human renin gene in a native cellular environment without confounding renin-angiotensin system interactions. A 55-kb transgene encompassing the human renin locus was crossed onto the mouse Ren1d-null background, restoring granulation in juxtaglomerular cells. Correct processing of human renin in dense core granules was confirmed by immunogold labeling. After stimulation of the renin-angiotensin system, juxtaglomerular cells contained rhomboid protogranules with paracrystalline contents, dilated rough endoplasmic reticulum, and electron-lucent granular structures. However, complementation of Ren1d-/- mice with human renin was unable to rescue the abnormality seen in macula densa structure. The juxtaglomerular apparatus was still able to respond to tubuloglomerular feedback in isolated perfused juxtaglomerular apparatus preparations, although minor differences in glomerular tuft contractility and macula densa cell calcium handling were observed. This study reveals that the human renin protein is able to complement the mouse Ren1d-/- non-granulated defect and suggests that granulopoiesis requires a structural motif that is conserved between the mouse Ren1d and human renin proteins. It also suggests that the altered macula densa phenotype is related to the activity of the renin-1d enzyme in a local juxtaglomerular renin-angiotensin system.

Evaluation of Multiple Immunoassay Technology Platforms to Select the Anti-Drug Antibody Assay Exhibiting the Most Appropriate Drug and Target Tolerance.

The aim of this study was, at the assay development stage and thus with an appropriate degree of rigor, to select the most appropriate technology platform and sample pretreatment procedure for a clinical ADA assay. Thus, ELISA, MSD, Gyrolab, and AlphaLISA immunoassay platforms were evaluated in association with target depletion and acid dissociation sample pretreatment steps. An acid dissociation step successfully improved the drug tolerance for all 4 technology platforms and the required drug tolerance was achieved with the Gyrolab and MSD platforms. The target tolerance was shown to be better for the ELISA format, where an acid dissociation treatment step alone was sufficient to achieve the desired target tolerance. However, inclusion of a target depletion step in conjunction with the acid treatment raised the target tolerance to the desired level for all of the technologies. A higher sensitivity was observed for the MSD and Gyrolab assays and the ELISA, MSD, and Gyrolab all displayed acceptable interdonor variability. This study highlights the usefulness of evaluating the performance of different assay platforms at an early stage in the assay development process to aid in the selection of the best fit-for-purpose technology platform and sample pretreatment steps.

Declining trends in the proportion of non-viral sexually transmissible infections reported by STD clinics in the US, 2000-10.

UNLABELLED: Background Recent budget shortfalls may have resulted in decreases in the number of sexually transmissible infections (STIs) reported from sexually transmitted disease clinics (STDCs) in the United States (US). The objective of this study was to examine the proportion of cases reported from STDCs for three non-viral STIs in the last decade. METHODS: Data from the national surveillance database on primary and secondary (P&S) syphilis, gonorrhoea and chlamydia cases for 2000-10 were extracted. The percentage of cases reported by STDCs for the nation and for each of the 48 contiguous states were then computed. Finally, the χ(2) trend test for proportions was used to determine the annual average decrease/increase in the percentage of cases reported by STDCs for the nation and for each state. RESULTS: RESULTS demonstrate that the average annual declines in the proportion of P&S syphilis, gonorrhoea, and chlamydia cases reported from STDCs were 1.43% (P<0.01), 1.31% (P<0.01), and 0.31% (P<0.01), respectively. Additionally, most of the states with statistically significant trends (P<0.05) in the proportion of cases reported by STDCs had negative slopes: 86% (25/29) for P&S syphilis, 89% (34/38) for gonorrhoea, and 63% (27/43) for chlamydia. CONCLUSION: These results document the declining role of STDCs in STI prevention and control efforts in the US. Further studies are needed to assess the direct or indirect impact of the decline in the proportion of cases from STDCs on the overall STI control and prevention efforts in the US and its implications for the future.

A pharmaceutical comparison of different commercially available imiquimod 5% cream products.

BACKGROUND: Alternatives to the innovator product for imiquimod 5% cream are currently marketed in South America and the People's Republic of China. METHODS: Seven alternative imiquimod 5% cream products were compared with the innovator product using physiochemical tests for cream appearance, pH, drug content and presence of crystals, as well as in vitro release testing of drug using Franz diffusion cells. RESULTS: In contrast to the innovator product, which had no crystalline imiquimod, significant amounts of suspended crystalline imiquimod were found in six of the seven alternative products. In vitro release rates of imiquimod were significantly slower in these six products compared with the innovator (p<0.001). In vitro release rates of imiquimod were significantly faster than the innovator (p<0.05) for the one alternative product without crystals. CONCLUSIONS: The clinical relevance of the differences observed is unknown; however, they raise concerns about whether these alternatives are therapeutically equivalent. While a generic topical imiquimod would almost certainly require clinical studies of therapeutic equivalence for approval in countries with more stringent regulatory environment, vigilance is warranted regarding importation of pharmaceutical products labeled as 'identical' in the absence of adequate evaluations.

Capillary electrophoresis of DNA.

Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single-base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.

Capillary electrophoresis of DNA.

Capillary electrophoresis (CE) is an alternative to conventional slab gel electrophoresis for the separation of DNA fragments. CE offers a number of advantages over slab gel separations in terms of speed, resolution, sensitivity, and data handling. Separation times are generally only a few minutes and the DNA is detected either by UV absorption or by fluorescent labeling. The quantity of DNA required for separation is in the nanogram range. Single-base resolution can be obtained on fragments up to several hundred base pairs. In the presence of appropriate standards, fragments can be accurately sized based on relative electrophoretic mobility. A protocol for the analysis of synthetic oligonucleotides in a flowable matrix is described in this unit.