Development and validation of a phosphorylated SMAD ex vivo stimulation assay.

Assessing the pharmacodynamics (PD) of a potential therapeutic through the use of a downstream biomarker is essential. This is traditionally performed in the target tissue but limited volume and invasiveness of sampling pose challenges with solid tumours. Currently, there are several small molecule receptor kinase inhibitors and large molecule therapeutic antibodies in clinical trials that interfere with TGFbeta signalling to treat various forms of cancer. With the advent of these new therapies, there is a need for a surrogate tissue that is easily accessible and indicative of tumour response. We propose the use of an ex vivo TGFbeta1 stimulation of peripheral blood mononuclear cells (PBMCs) coupled with the measurement of phosphorylated SMAD2 (Sma/Mothers Against dpp, a downstream transcriptional activator) using a sandwich ELISA. TGFbeta is involved in many different cellular responses, such as proliferation, angiogenesis, migration, invasion and immunomodulation. SMAD2 and SMAD3 are phosphorylated as a result of the canonical cascade through ligand binding and receptor kinase activation. These phosphorylated SMADs (pSMAD) associate with SMAD4, a co-SMAD, and transcriptionally activate TGFbeta-mediated genes. This paper describes the novel method for measuring the downstream effects of inhibiting canonical TGFbeta signalling using ex vivo stimulation of surrogate tissue to predict tumour response. In addition, we present the assay validation rationale and data. This novel, validated assay can be used to gain insight into clinical trials regarding TGFbeta signal modulation by multiple inhibitor platforms for both large and small molecules.

Urinary desmosine as a biomarker in acute lung injury.

Acute lung injury (ALI) is a complex disorder associated with an acute inflammatory response thought to contribute to tissue injury. Desmosine, a cross-linking amino acid present in elastin, is released during matrix degradation and cleared by the kidney. Results from animal models and human disease studies have suggested that ALI is associated with the release of desmosine, resulting in increased urinary desmosine. A radioimmunoassay was used to monitor urinary desmosine levels over 10 days in ten patients with ALI. The concentration of desmosine was measured with and without acid hydrolysis. Baseline urinary desmosine was increased in two of ten patients. The concentration of desmosine at baseline did not appear to be related to age, gender, neutrophil elastase (NE)/alpha(1)-antiprotease complex concentration or P(a)O(2)/F(i)O(2) ratio. No meaningful changes in desmosine levels were noted after removal from mechanical ventilation. Baseline desmosine concentrations did not appear to correlate with the risk of death. The limited sensitivity, predictive correlations and dynamic modulation would suggest that urine desmosine has a limited role as a biomarker for ALI. Hydrolysis of urine samples appears necessary for optimal measurement of urine desmosine.

Multicenter evaluation of the Clostridium difficile TOX A/B TEST.

Clostridium difficile, the primary cause of nosocomial diarrhea in the United States and many other industrialized countries, is recognized as a major health concern because of its ability to cause severe intestinal disease leading to complications such as relapses and infections due to vancomycin-resistant enterococci. The disease results from two toxins, toxins A and B, produced by this pathogen. In this study, we evaluated the TOX A/B TEST, a new 1-h enzyme immunoassay (EIA) that detects toxins A and B. We compared the test with the tissue culture assay, which is recognized as the "gold standard" for C. difficile testing. Evaluations were performed in-house at TechLab, Inc. (Blacksburg, Va.) and off-site at four clinical laboratories. Of 1,152 specimens tested, 165 were positive by the TOX A/B TEST and tissue culture and 973 were negative by both tests. The sensitivity and specificity were 92.2 and 100%, respectively. The positive and negative predictive values were 100 and 98.6%, respectively, and the correlation of the TOX A/B TEST with tissue culture was 98.8%. When discrepant samples were resolved by culture, the sensitivity and specificity were 93.2 and 98.9%, respectively. The positive and negative predictive values were 100 and 98.8%, respectively, with a correlation of 99.0%. There were no specimens that were positive by the TOX A/B TEST and negative by tissue culture. Fourteen specimens were negative by the TOX A/B TEST but positive by tissue culture. Of these, two were negative by toxigenic culture, five were positive by toxigenic culture, and seven were not available for further testing. There were no indeterminate results, since the test does not have an indeterminant zone. In a separate study, 102 specimens that were positive by tissue culture and the TOX A/B TEST were examined in toxin A-specific EIAs. Two specimens that presumptively contained toxin A-negative, toxin B-positive (toxA-/toxB+) isolates were identified. One specimen was from a patient with a clinical history consistent with C. difficile infection. Isolates obtained from these specimens by selective culture on solid media and in broth tested toxA-/toxB+ when grown in brain heart infusion dialysis flasks, which stimulate in vitro production of both toxins. Our findings show that the TOX A/B TEST is suitable as a diagnostic aid for C. difficile disease because it correlates well with tissue culture and detects isolates that may be missed with toxin A-specific EIAs.

Suicide: qualitative data from focus group interviews with youth.

Suicide is a leading cause of morbidity and mortality among people aged 15-24 years of age. This paper illustrates the use of focus groups with young people to enhance knowledge of ways to address youth suicide. Analysis of the findings identified three themes perceived by participants as being warning signs of a suicidal friend (personality changes, risk-taking behaviour and unusual actions). An important finding, which has implications for the planning of further suicide prevention strategies, was that young people would either cope alone or turn to a friend if they were feeling suicidal. The fact that a lack of knowledge was identified as the major barrier to youth using existing services/resources suggests that health promotion awareness campaigns which provide information on where young people could access help need to be developed. The use of focus groups with young people has provided valuable insights into ways to address youth suicide. We urge other researchers to incorporate similar methodologies.

Cellular fatty acid analysis in the differentiation of Clostridium in the clinical microbiology laboratory.

The identification of Clostridium species can be problematic for the diagnostic microbiology laboratory. The introduction of the MIDI Microbial Identification System (MIS) has enabled personnel in diagnostic laboratories to perform cellular fatty acid analyses on a variety of microorganisms. We used the Virginia Polytechnic Institute (Blacksburg, VA) Anaerobe Library (Moore Version 3.8) to perform analyses on 216 strains representing 18 species of Clostridium. The organisms were characterized with use of traditional biochemical methods that employ prereduced anaerobically sterilized media and other reference diagnostic methods. The MIS identified 86% of the strains correctly to the species level without the need for supplemental tests, and identified an additional 6% of the strains to species levels with the aid of a few supplemental tests. Only 3% of strains were identified by genus incorrectly. The cellular fatty acid patterns of selected, medically important clostridia were so distinctive that 100% of these species were identified correctly. The MIS has many practical benefits, including speed of identification, and few disadvantages (such as equipment cost) for the clinical microbiology laboratory.