Detection of Bacterial Flora in Biological Secretions Using Antibodies Developed In Vitro and Immobilized in a Surface Plasmon Resonance System

Identification of pathogens living in biofilms of chronic infections has been difficult with PCR, serological, biochemical and culture techniques. The study aims at the detection of bacterial pathogens in biofilms of biological secretions using SPR analysis Biacore. The antibodies were developed by isolating mononuclear lymphocytes from the blood of the patients who sustained systemic infection. The isolated lymphocytes had antibody secreting B cells (plasma cells) which were identified using flow cytometry analysis. The antibodies produced (n=4) were used to immobilize CM5 chip of Biacore to detect the bacteria in ulcer secretions with wound secretions of healthy volunteers as controls. The results from Surface Plasmon Resonance (SPR) analysis and culture technique were compared and statistically there was no significant difference obtained. The results from present study suggest that SPR analysis could be used as an alternative system for detection of bacteria in poly-microbial samples and detect the organisms that might not be discovered by culture or PCR method

Development of novel tools for prevention and diagnosis of Porphyromonas gingivalis infection and periodontitis

Periodontitis is a chronic inflammatory disease caused by exaggerated host immune responses to dysregulated microbiota in dental biofilms leading to degradation of tissues and alveolar bone loss. Porphyromonas gingivalis is a major periodontal pathogen and expresses several potent virulence factors. Among these factors, arginine and lysine gingipains are of special importance, both for the bacterial survival/proliferation and the pathological outcome. The major aim of this thesis was to develop and test novel methods for diagnosis and prevention of P. gingivalis infection and periodontitis. In study I, anti-P. gingivalis antibodies were developed in vitro for immunodetection of bacteria in clinical samples using a surface plasmon resonance (SPR)-based biosensor. Specific binding of the antibodies to P. gingivalis was demonstrated in samples of patients with periodontitis and the results were validated using real-time PCR and DNA-DNA checkerboard analysis. In study II, we elucidated the properties and antimicrobial effects of different lactobacillus species and the two-peptide bacteriocin PLNC8 αβ on P. gingivalis. L. plantarum NC8 and 44048 effectively inhibited P. gingivalis growth and pure PLNC8 αβ induced bacterial lysis by damaging P. gingivalis membrane. In study III, we demonstrated that PLNC8 αβ dose-dependently induces proliferation and release of growth factors in gingival epithelial cells (GECs). Furthermore, PLNC8 αβ decreased P. gingivalis-induced cytotoxic effects in GECs but did not alter the effect of gingipains on cytokine expression. In study IV, we elucidated the effects of anti-P. gingivalis antibodies and PLNC8 αβ in regulating cellular responses during P. gingivalis infection. Both antibodies and PLNC8 αβ modulated P. gingivalis-induced expression of growth factors in GECs, however, their effects were diminished when used in combination. The results of this thesis demonstrate a possible role of anti-P. gingivalis antibodies and PLNC8 αβ in prevention and treatment of P. gingivalis infection and periodontitis with no cytotoxic effects on human cells

A methachromatic-based experimental model for identification of bowel as the focus of an acute inflammation

Diarrhea is the most common symptom of acute inflammation in gastrointestinal tract and the patients are isolated in order to inhibit transmission and to conduct investigations. Yet there is no standard test to distinguish gastrointestinal infection from more generalized diseases at admittance which might cause delay in therapy. Hepatocyte growth factor (HGF) is produced upon injury by mesenchymal cells. On the contrary to chronic inflammation, HGF produced in the course of acute inflammation is biologically active and shows binding affinity to heparan sulphate proteoglycan (HSPG) and dextran sulphate (DS). Based on this phenomenon, an agarose gel containing DS was prepared and immobilized on loops to investigate the feces samples for the presence or absence of growth factors such as HGF with affinity to DS. The study is conducted as a clinical evaluation of an experimental model to distinguish acute infectious gastroenteritis from other causes of diarrhea. 656 fecal samples gathered consequently from patients seeking for bowel disturbances and healthy were tested by the test and the medical reports were investigated. Upon interaction with DS, methylene blue changes color to pink. This phenomenon was inhibited by HGF and converted by addition of anti-HGF antibodies to the samples. The test distinguished acute infectious gastroenteritis with high sensitivity and specificity (96% and 92% respectively) from other causes of diarrhea. We introduce a metachromatic experimental model that might distinguish acute inflammation in alimentary tract from other causes of diarrhea. This model might be used in developing rapid diagnostic tests

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

Antibacterial effects of Lactobacillus and bacteriocin PLNC8 alpha beta on the periodontal pathogen Porphyromonas gingivalis

Background: The complications in healthcare systems associated with antibiotic-resistant microorganisms have resulted in an intense search for new effective antimicrobials. Attractive substances from which novel antibiotics may be developed are the bacteriocins. These naturally occurring peptides are generally considered to be safe and efficient at eliminating pathogenic bacteria. Among specific keystone pathogens in periodontitis, Porphyromonas gingivalis is considered to be the most important pathogen in the development and progression of chronic inflammatory disease. The aim of the present study was to investigate the antimicrobial effects of different Lactobacillus species and the two-peptide bacteriocin PLNC8 alpha beta on P. gingivalis. Results: Growth inhibition of P. gingivalis was obtained by viable Lactobacillus and culture media from L. plantarum NC8 and 44048, but not L. brevis 30670. The two-peptide bacteriocin from L. plantarum NC8 (PLNC8 alpha beta) was found to be efficient against P. gingivalis through binding followed by permeabilization of the membranes, using Surface plasmon resonance analysis and DNA staining with Sytox Green. Liposomal systems were acquired to verify membrane permeabilization by PLNC8 alpha beta. The antimicrobial activity of PLNC8 alpha beta was found to be rapid (1 min) and visualized by TEM to cause cellular distortion through detachment of the outer membrane and bacterial lysis. Conclusion: Soluble or immobilized PLNC8 alpha beta bacteriocins may be used to prevent P. gingivalis colonization and subsequent pathogenicity, and thus supplement the host immune system against invading pathogens associated with periodontitis.Funding Agencies|Swedish Heart-Lung Foundation; Foundation of Magnus Bergvall; Foundation of Olle Engkvist; Knowledge Foundation, Sweden</p

Profiling inflammatory response in lesions of cutaneous leishmaniasis patients using a non-invasive sampling method combined with a high-throughput protein detection assay

To access publisher's full text version of this article click on the hyperlink belowBackground: Cutaneous leishmaniasis (CL) is an infection caused by Leishmania (L.) protozoa transmitted through the bite of infected sand fly. Previously, invasive sampling of blood and skin along with low throughput methods were used for determination of inflammatory response in CL patients. Aims/methodology: We established a novel approach based on a non-invasive adhesive tape-disc sampling combined with a powerful multiplexing technique called proximity extension assay for profiling 92 inflammatory cytokines, chemokines and surface molecules in the lesions of CL patients infected with L. tropica. Sample collection was done non-invasively by using adhesive tape-discs from lesion and normal skin of 33 L. tropica positive patients. Results: Out of 92 inflammatory proteins, the level of 34 proteins was significantly increased in the lesions of CL patients compared to their normal skin. This includes the chemokines CCL2, CCL3, CCL4, CXCL1, CXCL5, CXCL9, CXCL10 and CXCL11, together with the interleukins IL-6, IL-8, IL-18, LIF and OSM. The remaining significantly changed inflammatory proteins include 7 surface molecules and receptors: CD5, CD40, CDCP1, 4E-BP1, TNFRSF9, IL-18R1 and OPG as well as 16 other cytokines and proteins: MMP-1, CSF-1, VEGFA, uPA, EN-RAGE, LAP TGF-β1, HGF, MMP-10, CASP-8, TNFSF14, STAMPB, ADA, TRAIL and ST1A1. Further, 13 proteins showed an increasing trend, albeit not statistically significant, in the CL lesions, including TGF-α, CCL23, MCP-2, IL-12B, CXCL6, IL-24, FGF-19, TNFβ, CD6, TRANCE, IL10, SIR2 and CCL20. Conclusion: We herein report a novel approach based on a non-invasive sampling method combined with the high-throughput protein assay for profiling inflammatory proteins in CL lesions. Using this approach, we could profile inflammatory proteins in the lesions from CL patients. This new non-invasive approach may have implications for studying skin inflammatory mediators in CL and other skin disorders.European Commission under the VASA SHIGETECVAX consortia Innovative Medicines Initiative European Commission under the VSV-EBOPLUS consortium European Union (EU) Iran National Science Foundation (INSF) Pasteur Institute of Ira