Clinical impact of real-time evaluation of the biological activity and degradation of hepatocyte growth factor

Hepatocyte growth factor (HGF) is essential for injury repair. Despite high HGF levels in chronic ulcers, up-regulation of HGF receptor in ulcer tissue and decreased biological activity of HGF in ulcer secretions have been observed. With a surface plasmon resonance-based method, we assessed the binding of HGF to antibodies, receptors, and the basement membrane and identified binding interactions that are indispensable for the biological activity of HGF. Recombinant HGF (rHGF) lots were tested for activity, structural integrity, and degradation, and the results were verified in an in vitro model of cell injury. Biologically active rHGF, as well as plasma from healthy volunteers, bound to heparan sulphate proteoglycan (HSPG) and to anti-HGF antibodies. Decreased binding to HSPG was the first event in rHGF degradation. This study established the feasibility of identifying patients with chronic inflammation who need exogenous HGF and of using ligand-binding assessment to evaluate rHGF lots for biological activity

Evaluation of hepatocyte growth factor as a local acute phase response marker in the bowel : The clinical impact of a rapid diagnostic test for immediate identification of acute bowel inflammation

BACKGROUND: There are no rapid tests that can distinguish contagious gastroenteritis, which requires isolation at its onset, from exacerbation of chronic inflammatory bowel disease (IBD) or bowel engagement in the course of systemic inflammatory response syndrome (SIRS). Hepatocyte growth factor (HGF) is an acute phase cytokine that is produced at the site of injury. It has high affinity to sulfated glycan, and this binding affinity is lost during chronic inflammation. The fecal pH strongly impacts the prognosis for severe bowel disease. We developed a strip test to evaluate HGF as a local acute phase response marker in the bowel. This test assessed the binding affinity of HGF to sulfated glycans in fecal samples and determined fecal pH as an indicator of illness severity. METHODS: Fresh feces from patients with diarrhea (n=513) were collected and tested blindly, and information about patient illness course and outcome was collected. Patients were classified based on the focus of inflammation and the cause of the symptoms. Objectively verified diagnoses of infectious gastroenteritis (n=131) and IBD onset/exacerbation and bowel cancer (n=44) were used to estimate the performance of the test strip. ELISA was performed on 101 freeze-thawed feces samples to determine the fecal HGF levels. RESULTS: The test rapidly distinguished infectious gastroenteritis from non-infectious inflammatory causes of diarrhea (sensitivity, 87.96%; specificity, 90.9%; positive predictive value, 96.6%; negative predictive value, 71.4%; accuracy, 89.1%). Fecal pH (p<0.0001) and mortality within 28days of sampling (p<0.04) was higher in patients with sepsis/SIRS and diarrhea. The concentration of HGF was higher in strip test-positive stool samples (p<0.01). CONCLUSIONS: HGF is a good local acute phase response marker of acute bowel inflammation. Test-strip determination of the binding affinity of fecal HGF to sulfated glycan was a rapid, equipment-free way to assess patients with diarrhea and to guide the diagnostic and therapeutic approaches on admission

Evaluation of hepatocyte growth factor as a local acute phase response marker in the bowel : The clinical impact of a rapid diagnostic test for immediate identification of acute bowel inflammation

BACKGROUND: There are no rapid tests that can distinguish contagious gastroenteritis, which requires isolation at its onset, from exacerbation of chronic inflammatory bowel disease (IBD) or bowel engagement in the course of systemic inflammatory response syndrome (SIRS). Hepatocyte growth factor (HGF) is an acute phase cytokine that is produced at the site of injury. It has high affinity to sulfated glycan, and this binding affinity is lost during chronic inflammation. The fecal pH strongly impacts the prognosis for severe bowel disease. We developed a strip test to evaluate HGF as a local acute phase response marker in the bowel. This test assessed the binding affinity of HGF to sulfated glycans in fecal samples and determined fecal pH as an indicator of illness severity. METHODS: Fresh feces from patients with diarrhea (n=513) were collected and tested blindly, and information about patient illness course and outcome was collected. Patients were classified based on the focus of inflammation and the cause of the symptoms. Objectively verified diagnoses of infectious gastroenteritis (n=131) and IBD onset/exacerbation and bowel cancer (n=44) were used to estimate the performance of the test strip. ELISA was performed on 101 freeze-thawed feces samples to determine the fecal HGF levels. RESULTS: The test rapidly distinguished infectious gastroenteritis from non-infectious inflammatory causes of diarrhea (sensitivity, 87.96%; specificity, 90.9%; positive predictive value, 96.6%; negative predictive value, 71.4%; accuracy, 89.1%). Fecal pH (p<0.0001) and mortality within 28days of sampling (p<0.04) was higher in patients with sepsis/SIRS and diarrhea. The concentration of HGF was higher in strip test-positive stool samples (p<0.01). CONCLUSIONS: HGF is a good local acute phase response marker of acute bowel inflammation. Test-strip determination of the binding affinity of fecal HGF to sulfated glycan was a rapid, equipment-free way to assess patients with diarrhea and to guide the diagnostic and therapeutic approaches on admission

In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments

Background: There is a rapid emergence of multiple resistant gram-negative bacteria due to overuse of antibiotics in the treatment of infections. Biofilms consist of polymicrobial communities that survive the hosts defense system. The key bacteria in biofilms are slow growing and support an attachment and rapid growth of other microorganisms. Current antimicrobial strategies often fail due to poor diagnosis of key pathogens in biofilms. The study aims to develop anti-bacterial human antibodies in vitro from patients who had recently undergone a systemic infection by pathogenic bacteria and to use these antibodies as a tool for detecting bacteria in biofilms. Methods: Lymphocytes were separated from whole blood of patients (n = 10) and stimulated with heat-killed bacteria to produce antibodies in vitro. The specificity of antibodies in recognizing the bacteria against which they were directed was evaluated by surface plasmon resonance system (SPR) and electron microscopy. The ulcer secretions from patients with chronic and acute leg ulcers and healthy controls were analyzed by the SPR system and the results were compared with culture studies. Results: The produced antibodies recognized bacteria with high sensitivity (SPR). The antibodies against Enterococcus fecalis bound specifically to the microorganism in a bacterial co-culture that was visualized by electron microscopy. Conclusion: In the present work, a method for producing specific antibodies against bacteria is introduced to recognize bacterial components in body fluids of patients suffering from pathogenic biofilms. This diagnostic technique may be most useful in clinical microbiology and in the choice of antibiotics in the treatment of serious infections.Funding Agencies|PEAS Institut; FORSS [F2004-212]; ALF grants [LIO-206241]</p

Additional file 1: of In vitro model of production of antibodies; a new approach to reveal the presence of key bacteria in polymicrobial environments

Microscopic analysis of host lymphocytes after stimulation with heat killed E. fecalis. Lymphocytes cultured in L-15 medium with 10 % FBS were stimulated with the same bacteria which had previously infected the donor and immune response to pathogenic stimulation was observed at 40X resolution in light microscopy. Morphological changes were observed in activated lymphocytes. (JPG 375 kb

Histogram showing the effects of increasing the immobilisation levels by increasing the contact time during immobilisation (1–3 representing 1, 5, and 10 minutes) (Friedman p 0

<p><b>Copyright information:</b></p><p>Taken from "Hepatocyte growth factor (HGF) in fecal samples: rapid detection by surface plasmon resonance"</p><p>BMC Gastroenterology 2005;5():13-13.</p><p>Published online 12 Apr 2005</p><p>PMCID:PMC1090571.</p><p>Copyright © 2005 Nayeri et al; licensee BioMed Central Ltd.</p>01, Wilcoxon signed ranks test between 1 and 5 minutes p = 0.058, between 5 and 10 minutes p = 0.003, and between 1 and 10 minutes p = 0.003). The monoclonal anti-HGF (500 μg/ml) was diluted 1:10 in 10 mM acetate buffer pH 4.5. The activation time was 7 min, followed by a 1,5 and 10 min ligand injection respectively. Deactivation of remaining active esters was performed by a 7 min injection of ethanolamine/hydrochloride at pH 8.5. A flow rate of 5 μl/min was used during immobilisation