ABSTRACT
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.
Subject(s)
Activin Receptors, Type I/antagonists & inhibitors , Leukocytes, Mononuclear/metabolism , Neoplasms/metabolism , Protein Kinase Inhibitors/pharmacology , Receptors, Transforming Growth Factor beta/antagonists & inhibitors , Smad Proteins/metabolism , Transforming Growth Factor beta1/pharmacology , Animals , Biomarkers/analysis , Biomarkers/metabolism , Blotting, Western , Cell Line, Tumor , Data Interpretation, Statistical , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical/methods , Enzyme-Linked Immunosorbent Assay , Female , Humans , Leukocytes, Mononuclear/drug effects , Mice , Mice, Nude , Neoplasms/drug therapy , Neoplasms/pathology , Phosphorylation/drug effects , Protein Serine-Threonine Kinases , Rats , Rats, Inbred F344 , Receptor, Transforming Growth Factor-beta Type I , Reproducibility of Results , Smad Proteins/analysis , Smad2 Protein/analysis , Smad2 Protein/metabolism , Smad3 Protein/analysis , Smad3 Protein/metabolism , Xenograft Model Antitumor Assays/methodsABSTRACT
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.
Subject(s)
Biomarkers/urine , Desmosine/urine , Aged , Aged, 80 and over , Female , Humans , Male , Middle Aged , Radioimmunoassay , Reference Standards , Reproducibility of ResultsABSTRACT
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.
