Abstract
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Introduction: Foot infection in persons with diabetes is a major problem, affecting millions of persons worldwide and costing adding a total healthcare of $17 billion annually for foot care in the US alone. In cases not requiring surgery antibiotic treatment duration is usually based on convention, e.g. 6 weeks without repeat biopsy. Biochemical markers, such ESR or CRP, lack adequate predictive value. Tc-99m radiolabeled white blood cell scanning (WBC scan) is commonly used at our institution at the initial phase for diagnostic purposes and can be used to direct biopsy. Studying the proteome from these directed biopsies offers the potential for identifying key proteins and pathways increased in abundance with the infection and after standard treatment course.
Methods: Subjects with diabetes and foot wounds with suspected osteomyelitis were enrolled in an IRB-approved prospective study comparing Tc-99m radiolabeled WBC scans (WBC scan) to MRI diagnostic accuracy for osteomyelitis and response to treatment. Baseline WBC scans were performed followed by biopsy and then again after a standard 6 weeks of antibiotic therapy. The WBC uptake identified the region of biopsy interest (Figure 1). A total of 20 subjects completed pre- and post-treatment scans and pre- and post-treatment biopsies. Sections from paraffin embedded blocks were subjected to liquid chromatography mass spec /mass spec proteomic analysis. An internal control was comprised of a pooled tryptic digest from all subjects. The resulting peptide profiles were subjected to imputation to deal with missing value and harmonization with the R-package HarmonizR'. The resulting data was subjected to gene ontology pathway software and reactome analysis.
Results: Forty six proteins were identified as differentially abundant in post-treatment samples (Figure 2). The most upregulated was periostin (POSTN), also known as osteoblast-specific factor 2, is a secreted matricellular matrix produced by osteoblast and involved in wound healing and mesenchymal cell proliferation, epithelial cell migration, and bone regeneration. The most significantly decreased protein was orosomucoid (ORM1), which is an acute-phase reactant to acute inflammation. It appears to regulate the innate immune system during the acute phase and belongs to the platelet and neutrophil degranulation reactome.
Conclusions: Image guided biopsies pre- and post-treatment identifies proteins that change in differential abundance with treatment. These may become targets for radiotracer development to inform treatment efficacy at the tissue level. Our study is limited by as no secondary validation of the differentially abundant proteins, such as immunohistochemistry or western blot and small size. The identified proteins maybe tested in prospective larger studies.
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