Serum IL-17F does not predict poor response to IM IFNß-1a in relapsing-remitting MS.

OBJECTIVE: There is a significant unmet need for serum biomarkers in relapsing-remitting multiple sclerosis (RRMS) that are predictive of therapeutic response to disease-modifying therapies. Following a recent Stanford study which reported that pretreatment levels of serum interleukin (IL)-17F could predict poor response to interferon-ß (IFNß) therapy, we sought to validate the finding using samples from a large clinical trial. METHODS: The validation cohort included 54 good responders (GR) and 64 poor responders (PR) selected from 762 subjects with RRMS from the IM IFNß-1a dose comparison study (Avonex study C94-805). Subjects were classified as GR and PR based on the number of relapses, Expanded Disability Status Scale score, and new and enlarging T2 lesions on MRI. Serum samples were assayed for IL-17F using a multiplexed Luminex assay and for IL-17F/F using an ELISA. Replicate aliquots from the Stanford study were also assayed to assure reproducibility of methods. RESULTS: Median pretreatment and post-treatment serum IL-17F levels were not statistically significantly different between GR and PR, and serum IL-7/IL-17F ratios were also not predictive of response status. Replicate aliquots from the Stanford study showed good correlation to their original cohort (r = 0.77). CONCLUSIONS: We were unable to validate the finding that serum IL-17F is a predictor of PR in a large independent cohort of subjects with RRMS. Differences in patient populations and methodology might explain the failure to validate the results from the Stanford study.

Identification of genes regulated during osteoblastic differentiation by genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Although several independent studies of gene expression patterns during osteoblast differentiation in cultures from calvaria and other in vitro models have been reported, only a small portion of the mRNAs expressed in osteoblasts have been characterized. We have previously analyzed the behavior of several known markers in osteoblasts, using Affymetrix GeneChip murine probe arrays (27,000 genes). In the present study we report larger groups of transcripts displaying significant expression modulation during the culture of osteoblasts isolated from mice calvaria. The expression profiles of 601 such regulated genes, classified in distinct functional families, are presented and analyzed here. Although some of these genes have previously been shown to play important roles in bone biology, the large majority of them have never been demonstrated to be regulated during osteoblast differentiation. Despite the fact that the precise involvement of these genes in osteoblast differentiation and function needs to be evaluated, the data presented herein will aid in the identification of genes that play a significant role in osteoblasts. This will provide a better understanding of the regulation of osteoblast differentiation and maturation.

Behavior of osteoblast, adipocyte, and myoblast markers in genome-wide expression analysis of mouse calvaria primary osteoblasts in vitro.

Several genes, such as alkaline phosphatase, osteocalcin, and Cbfa1/Osf2, are known to be regulated during osteoblastic differentiation and are commonly used as "osteoblast markers" for in vitro or in vivo studies. The number of these genes is very limited, however, and it is of major interest to identify new genes that are activated or repressed during the process of osteoblast differentiation and bone formation as well as to extend the available information on gene families relevant to this particular differentiation pathway. To identify such genes, we have implemented a genome-wide analysis by determining changes in expression levels of 27,000 genes during in vitro differentiation of primary osteoblasts isolated from mouse calvaria. This study focuses on the description of the analytical and filtering process applied; on the transcriptional analysis of well-established "bone," "adipocyte," and "muscle" pathway markers; and on a description of the regulation profiles for genes recently described in the Skeletal Gene Database. We also demonstrate that new array technologies constitute reliable and powerful tools to monitor the transcription of genes involved in osteoblastic differentiation, allowing a more integrated vision of the biological pathways regulated during osteoblast commitment, differentiation, and function.

Bayesian estimation of fold-changes in the analysis of gene expression: the PFOLD algorithm.

A general and detailed noise model for the DNA microarray measurement of gene expression is presented and used to derive a Bayesian estimation scheme for expression ratios, implemented in a program called PFOLD, which provides not only an estimate of the fold-change in gene expression, but also confidence limits for the change and a P-value quantifying the significance of the change. Although the focus is on oligonucleotide microarray technologies, the scheme can also be applied to cDNA based technologies if parameters for the noise model are provided. The model unifies estimation for all signals in that it provides a seamless transition from very low to very high signal-to-noise ratios, an essential feature for current microarray technologies for which the median signal-to-noise ratios are always moderate. The dual use, as decision statistics in a two-dimensional space, of the P-value and the fold-change is shown to be effective in the ubiquitous problem of detecting changing genes against a background of unchanging genes, leading to markedly higher sensitivities, at equal selectivity, than detection and selection based on the fold-change alone, a current practice until now.

ProbeDesigner: for the design of probesets for branched DNA (bDNA) signal amplification assays.

MOTIVATION: The sensitivity and specificity of branched DNA (bDNA) assays are derived in part through the judicious design of the capture and label extender probes. To minimize non-specific hybridization (NSH) events, which elevate assay background, candidate probes must be computer screened for complementarity with generic sequences present in the assay. RESULTS: We present a software application which allows for rapid and flexible design of bDNA probesets for novel targets. It includes an algorithm for estimating the magnitude of NSH contribution to background, a mechanism for removing probes with elevated contributions, a methodology for the simultaneous design of probesets for multiple targets, and a graphical user interface which guides the user through the design steps. AVAILABILITY: The program is available as a commercial package through the Pharmaceutical Drug Discovery program at Chiron Diagnostics.

A branched DNA signal amplification assay for quantification of nucleic acid targets below 100 molecules/ml.

The branched DNA hybridization assay has been improved by the inclusion of the novel nucleotides, isoC and isoG, in the amplification sequences to prevent non-specific hybridization. The novel isoC, isoG-containing amplification sequences have no detectable interaction with any natural DNA sequence. The control of non-specific hybridization in turn permits increased signal amplification. Addition of a 14 site preamplifier was found to increase the signal/noise ratio 8-fold. A set of 74 oligonucleotide probes was designed to the consensus HIV POL sequence. The detection limit of this new HIV branched DNA amplifier assay was approximately 50 molecules/ml. The assay was used to measure viral load in 87 plasma samples of HIV- infected patients on triple drug therapy whose RNA titers were <500 molecules/ml. In all 11 patients viral load eventually declined to below the detection limit with the new assay.

Breast reconstruction after mastectomy.

This chapter includes brief descriptions of the major methods of reconstructive surgery, along with guidelines as to the optimal approach in a number of clinical situations. Considerations of the candidates for breast reconstruction and the psychologic benefits of reconstruction are also discussed.

The development and standardization of an enzyme-linked immunosorbent assay for the detection of antibodies to bovine herpesvirus 2.

A single dilution enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies to bovine herpesvirus 2 has been developed, standardized and compared with the virus neutralization test. The results of the two tests correlated well. A positive/negative threshold was established for the ELISA. The ELISA was reproducible, sensitive, rapid and specific.

Identifying health care stakeholders: a key to strategic implementation.

As the number and complexity of stakeholders for health care organizations has increased, health care managers have become more aware of the ability of these groups to thwart or facilitate the implementation of strategic plans. Most stakeholder models have focused on identification of groups within the usual, global definition of affecting or being affected by an organization's actions. The authors argue that stakeholders management is critical to the implementation of strategic plans. They provide a narrower, more operationally useful stakeholder definition and present a framework for assessing the relative importance of each stakeholder for a given situation. The situational evaluation of stakeholders is critical to successful implementation of strategy.

Establishment of a statistical base for use of ELISA in diagnostic serology for infectious bovine rhinotracheitis.

The critical statistical parameters of an enzyme-linked immunosorbent assay were determined to enable quantitation of antibody responses in cattle affected with infectious bovine rhinotracheitis. A system of controlling well-to-well variations in optical density reading across a microtitre plate was evolved and dose--response assays were carried out to determine the dilution of serum which gave the greatest discrimination between acute and convalescent sera from an infected animal. Use of a standard serum was studied in further assays. An increase in optical density value of 0.15 was set as a diagnostic criterion for a significantly rising antibody response. This compared well with the conventional criterion of a fourfold rise in virus neutralizing antibody titre.

Detection of bovine leukosis virus in bronchoalveolar lung washings and nasal secretions.

Cattle and sheep persistently infected with bovine leukosis virus (BLV) were studied for the presence of the virus in bronchoalveolar lung washings and nasal secretions. The virus was demonstrated in the cellular fraction of the lung washings in six out of nine cattle and in one out of six sheep. In no instance was bovine leukosis isolated from the cell-free bronchoalveolar lung washings. The virus was isolated from the nasal secretion of only one of six naturally infected milking cows despite frequent sampling; the virus-infected nasal secretion was from a sick 10-year-old cow. Bovine leukosis virus was not isolated from cellular fractions of nasal secretions.