RESUMO
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 host's 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.
Assuntos
Formação de Anticorpos/imunologia , Bactérias/crescimento & desenvolvimento , Bactérias/imunologia , Coinfecção/imunologia , Coinfecção/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Antibacterianos/uso terapêutico , Bactérias/efeitos dos fármacos , Bactérias/patogenicidade , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Sangue/imunologia , Líquidos Corporais/imunologia , Líquidos Corporais/microbiologia , Doença Crônica , Técnicas de Cocultura , Coinfecção/diagnóstico , Coinfecção/diagnóstico por imagem , Técnicas e Procedimentos Diagnósticos , Enterococcus/imunologia , Enterococcus/patogenicidade , Enterococcus faecalis/imunologia , Feminino , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Negativas/imunologia , Bactérias Gram-Negativas/patogenicidade , Infecções por Bactérias Gram-Negativas/diagnóstico , Infecções por Bactérias Gram-Negativas/tratamento farmacológico , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Bactérias Gram-Positivas/imunologia , Bactérias Gram-Positivas/patogenicidade , Humanos , Técnicas In Vitro , Linfócitos , Masculino , Microscopia Eletrônica , Pessoa de Meia-Idade , Especificidade da Espécie , Ressonância de Plasmônio de Superfície/métodos , Úlcera/microbiologiaRESUMO
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.
Assuntos
Biomarcadores/análise , Fezes/química , Gastroenterite/diagnóstico , Fator de Crescimento de Hepatócito/análise , Doenças Inflamatórias Intestinais/diagnóstico , Reação de Fase Aguda/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Diarreia/etiologia , Diarreia/mortalidade , Ensaio de Imunoadsorção Enzimática , Fezes/microbiologia , Fezes/parasitologia , Fezes/virologia , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Doenças Inflamatórias Intestinais/etiologia , Doenças Inflamatórias Intestinais/metabolismo , Masculino , Pessoa de Meia-Idade , Polissacarídeos/metabolismo , Sensibilidade e Especificidade , Índice de Gravidade de Doença , Suécia , Síndrome de Resposta Inflamatória Sistêmica/diagnóstico , Fatores de Tempo , Adulto JovemRESUMO
BACKGROUND: Widely studied for the past 20 years, hepatocyte growth factor (HGF) has been identified as a regenerative marker and an important factor in the development and healing of injuries. Antithrombin III (AT III) is a protein in the blood stream with anti-thrombotic and anti-inflammatory properties and has been used as an adjuvant treatment along with antibiotics in severe sepsis. OBJECTIVE: To study the content and properties of HGF in plasma-derived AT III products, and the regenerative effect in severe deep ulcer infections. METHODS: Commercial AT III products were analyzed for the presence and biological activity of HGF. One AT III product containing biologically active HGF was used to treat 18 cases of critical, deep ulcer infections scheduled for major invasive intervention. The patients were followed up for 6-60 months. RESULTS: The AT III products contained HGF with different biological activity. No adverse reactions were observed after local administration of AT III during the study or follow-up period. In 16 of 18 cases no surgical intervention was needed within the first 6 month of inclusion. CONCLUSION: AT III products containing biologically active HGF may contribute to regeneration and healing in severe deep ulcer infections which do not respond adequately to different combinations of antibiotics alone.
Assuntos
Antitrombina III/uso terapêutico , Fator de Crescimento de Hepatócito/uso terapêutico , Úlcera/tratamento farmacológico , Idoso , Idoso de 80 Anos ou mais , Animais , Especificidade de Anticorpos/imunologia , Western Blotting , Estudos de Casos e Controles , Células Cultivadas , Estado Terminal , Ensaio de Imunoadsorção Enzimática , Epitopos/imunologia , Feminino , Humanos , Ligantes , Masculino , Camundongos , Pessoa de Meia-Idade , Ressonância de Plasmônio de Superfície , Úlcera/patologia , Adulto JovemRESUMO
Porphyromonas gingivalis is an etiological agent that is strongly associated with periodontal disease, and it correlates with numerous inflammatory disorders, such as cardiovascular disease. Circulating bacteria may contribute to atherogenesis by promoting CD11b/CD18-mediated interactions between neutrophils and platelets, causing reactive oxygen species (ROS) production and aggregation. Lipoxin A4 (LXA4) is an endogenous anti-inflammatory and proresolving mediator that is protective of inflammatory disorders. The aim of this study was to investigate the effect of LXA4 on the P. gingivalis-induced activation of neutrophils and platelets and the possible involvement of Rho GTPases and CD11b/CD18 integrins. Platelet/leukocyte aggregation and ROS production was examined by lumiaggregometry and fluorescence microscopy. Integrin activity was studied by flow cytometry, detecting the surface expression of CD11b/CD18 as well as the exposure of the high-affinity integrin epitope, whereas the activation of Rac2/Cdc42 was examined using a glutathione S-transferase pulldown assay. The study shows that P. gingivalis activates Rac2 and Cdc42 and upregulates CD11b/CD18 and its high-affinity epitope on neutrophils, and that these effects are diminished by LXA4. Furthermore, we found that LXA4 significantly inhibits P. gingivalis-induced aggregation and ROS generation in whole blood. However, in platelet-depleted blood and in isolated neutrophils and platelets, LXA4 was unable to inhibit either aggregation or ROS production, respectively. In conclusion, this study suggests that LXA4 antagonizes P. gingivalis-induced cell activation in a manner that is dependent on leukocyte-platelet interaction, likely via the inhibition of Rho GTPase signaling and the downregulation of CD11b/CD18. These findings may contribute to new strategies in the prevention and treatment of periodontitis-induced inflammatory disorders, such as atherosclerosis.
Assuntos
Infecções por Bacteroidaceae/metabolismo , Plaquetas/metabolismo , Antígeno CD11b/biossíntese , Lipoxinas/metabolismo , Neutrófilos/metabolismo , Porphyromonas gingivalis/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aterosclerose/metabolismo , Aterosclerose/microbiologia , Infecções por Bacteroidaceae/complicações , Infecções por Bacteroidaceae/imunologia , Plaquetas/imunologia , Plaquetas/patologia , Western Blotting , Antígenos CD18/metabolismo , Agregação Celular/fisiologia , Comunicação Celular/fisiologia , Separação Celular , Citometria de Fluxo , Humanos , Microscopia de Fluorescência , Neutrófilos/imunologia , Neutrófilos/patologia , Porphyromonas gingivalis/imunologia , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
The use of conventional antibiotics has substantial clinical efficacy, however these vital antimicrobial agents are becoming less effective due to the dramatic increase in antibiotic-resistant bacteria. Novel approaches to combat bacterial infections are urgently needed and bacteriocins represent a promising alternative. In this study, the activities of the two-peptide bacteriocin PLNC8 αß were investigated against different Staphylococcus spp. The peptide sequences of PLNC8 α and ß were modified, either through truncation or replacement of all L-amino acids with D-amino acids. Both L- and D-PLNC8 αß caused rapid disruption of lipid membrane integrity and were effective against both susceptible and antibiotic resistant strains. The D-enantiomer was stable against proteolytic degradation by trypsin compared to the L-enantiomer. Of the truncated peptides, ß1-22, ß7-34 and ß1-20 retained an inhibitory activity. The peptides diffused rapidly (2 min) through the bacterial cell wall and permeabilized the cell membrane, causing swelling with a disorganized peptidoglycan layer. Interestingly, sub-MIC concentrations of PLNC8 αß substantially enhanced the effects of different antibiotics in an additive or synergistic manner. This study shows that PLNC8 αß is active against Staphylococcus spp. and may be developed as adjuvant in combination therapy to potentiate the effects of antibiotics and reduce their overall use.
Assuntos
Antibacterianos/farmacologia , Bacteriocinas/farmacologia , Staphylococcus/efeitos dos fármacos , Antibacterianos/química , Bacteriocinas/química , Bacteriocinas/genética , Farmacorresistência Bacteriana , Testes de Sensibilidade Microbiana , Staphylococcus/crescimento & desenvolvimentoRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
There is a strong association between periodontal disease and atherosclerotic cardiovascular disorders. A key event in the development of atherosclerosis is accumulation of modified lipoproteins within the arterial wall. We hypothesise that patients with periodontitis have an altered lipoprotein profile towards an atherogenic form. Therefore, the present study aims at identifying modifications of plasma lipoproteins in periodontitis. Lipoproteins from ten female patients with periodontitis and gender- and age-matched healthy controls were isolated by density-gradient ultracentrifugation. Proteins were separated by 2D gel-electrophoresis and identified by map-matching or by nano-LC followed by MS. Apolipoprotein (Apo) A-I (ApoA-I) methionine oxidation, Oxyblot, total antioxidant capacity and a multiplex of 71 inflammation-related plasma proteins were assessed. Reduced levels of apoJ, phospholipid transfer protein, apoF, complement C3, paraoxonase 3 and increased levels of α-1-antichymotrypsin, apoA-II, apoC-III were found in high-density lipoprotein (HDL) from the patients. In low-density lipoprotein (LDL)/very LDL (VLDL), the levels of apoL-1 and platelet-activating factor acetylhydrolase (PAF-AH) as well as apo-B fragments were increased. Methionine oxidation of apoA-I was increased in HDL and showed a relationship with periodontal parameters. α-1 antitrypsin and α-2-HS glycoprotein were oxidised in LDL/VLDL and antioxidant capacity was increased in the patient group. A total of 17 inflammation-related proteins were important for group separation with the highest discriminating proteins identified as IL-21, Fractalkine, IL-17F, IL-7, IL-1RA and IL-2. Patients with periodontitis have an altered plasma lipoprotein profile, defined by altered protein levels as well as post-translational and other structural modifications towards an atherogenic form, which supports a role of modified plasma lipoproteins as central in the link between periodontal and cardiovascular disease (CVD).
Assuntos
Aterosclerose/sangue , Doenças Cardiovasculares/sangue , Lipoproteínas/sangue , Periodontite/sangue , Apolipoproteína A-I , Apolipoproteínas B/sangue , Citocinas/sangue , Eletroforese em Gel Bidimensional , Feminino , Humanos , Lipoproteínas HDL/sangue , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Espectrometria de Massas/métodos , Pessoa de Meia-IdadeRESUMO
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.
Assuntos
Fator de Crescimento de Hepatócito/fisiologia , Cicatrização , Adulto , Idoso , Idoso de 80 Anos ou mais , Membrana Basal/metabolismo , Linhagem Celular Tumoral , Feminino , Proteoglicanas de Heparan Sulfato/química , Humanos , Masculino , Pessoa de Meia-Idade , Ligação Proteica , Proteínas Recombinantes/química , Ressonância de Plasmônio de Superfície , Úlcera/metabolismoRESUMO
OBJECTIVE: Cough and fever are the initial symptoms of lower respiratory infection. Severe cases might be fatal. Therefore, particularly in the non-equipped centers, the lack of diagnostic methods to identify the severe cases has resulted in overconsumption of antibiotics. On the basis of the knowledge about non-specific immune response at the site of injury, we developed a colorimetric dip-test that shows abrupt, sensitive and quite specific color change upon contact with sputum in the cases of lower respiratory infection. We further explored the mechanism of the test. RESULTS: We detected deoxyribonucleic acid (DNA) and hepatocyte growth factor in the sputum of patients that suffered from respiratory infection (n = 18). The results differed significantly (P < 0.0001) from age-matched patients (n = 18) with other respiratory disorders and highly correlated with the index-test results (Spearman Rank test = 0.84). DNA with a concentration more than 0.03 mg/ml induced a visible and stable color change on index-test within 1 min. The test recognized all of the cases with respiratory infection and the specificity was 72%. With a high negative predictive value. The index test detects, inter alia, cell-free DNA in sputum and might safely rule-out respiratory infection in 2/3 of cases that present symptoms of acute respiratory infection.
Assuntos
Infecções Respiratórias/diagnóstico , Escarro/microbiologia , Antibacterianos , Tosse , DNA/análise , Humanos , Sensibilidade e Especificidade , Fatores de TempoRESUMO
BACKGROUND: Eosinophilic inflammation of the airways is a key characteristic of asthma. A defect in eosinophil apoptosis might contribute to the chronic tissue eosinophilia associated with asthma. OBJECTIVE: Our purpose was to examine whether the occurrence of apoptotic eosinophils in induced sputum from asthmatic patients correlate with interleukin (IL)-5 and eotaxin. METHODS: Thirty stable and 30 exacerbated asthmatic patients were recruited. Twenty healthy subjects were enrolled as a control group. Induced sputum was obtained from asthmatic patients and from control subjects. The number of apoptotic eosinophils in sputum was assessed by flow cytometry. In sputum supernatant, eosinophil cationic protein (ECP) was measured by sensitive radioimmunoassay, and IL-5 and eotaxin by sandwich enzyme linked immunosorbant assay. RESULTS: Levels of eosinophils, apoptotic eosinophils, IL-5, ECP and eotaxin from asthmatic patients were higher than those from healthy subjects. Thirty exacerbated asthmatics showed higher proportions of eosinophils (median 29.3%, range 13.4%-40.9%), more detectable levels of IL-5 (50.44, 32.99-67.01 pg/ml) and eotaxin (644.6, 197.4-937.7 pg/ml) in their sputum than the patients with stable asthma (P<0.05). There were significant inverse correlations between the levels of sputum IL-5 and the proportion of sputum eosinophil apoptosis in patients with exacerbated and stable asthma (r=-0.85 and -0.79, P<0.01 and P<0.05, respectively). Also inverse correlations were found between the levels of eotaxin and the proportion of sputum eosinophil apoptosis in exacerbated (r=-0.85, P<0.01), or stable asthma (r=-0.69, P<0.05). Additional positive correlations between the levels of sputum IL-5 and eotaxin in either exacerbatated (r=0.93, P<0.01) or stable asthma (r=0.82, P<0.05) were observed. CONCLUSIONS: Apoptosis of eosinophils might be suppressed by proinflammatory cytokines and chemokines such as IL-5 and eotaxin leading to their accumulation in the lung. Stimulation of eosinophils in airway with IL-5 and eotaxin may play a crucial role in allergic inflammation.
Assuntos
Asma/patologia , Quimiocinas CC/análise , Eosinófilos/patologia , Interleucina-5/análise , Escarro/citologia , Adolescente , Adulto , Idoso , Apoptose , Asma/imunologia , Asma/fisiopatologia , Biomarcadores/análise , Quimiocina CCL11 , Proteína Catiônica de Eosinófilo/análise , Feminino , Volume Expiratório Forçado , Humanos , Masculino , Fluxo Máximo Médio Expiratório , Pessoa de Meia-Idade , Manejo de Espécimes/métodos , Escarro/química , Escarro/imunologiaRESUMO
BACKGROUND: Hepatocyte growth factor (HGF) is a potent regenerative factor involved in wound healing. Previous studies have shown that mesenchymal cells produce HGF, stimulating epithelial cells in a paracrine fashion. OBJECTIVE: To examine whether autocrine HGF production by keratinocytes can occur upon skin injury. METHODS: A 31-year-old male patient sustained a burn affecting 80% of his total body surface area. Biopsies were taken from intact skin near the injured area, and skin keratinocytes were separated and cultured. Conditioned medium from keratinocytes was analyzed for HGF by ELISA, surface plasmon resonance (SPR), and dot blotting. Binding of HGF from conditioned medium to its receptor, c-Met, was compared with recombinant HGF by SPR. Finally, we examined the motogenic effect on mouse transformed skin epithelial cells (CCL-53.1) of HGF from conditioned medium. RESULTS: HGF was detected in the cultured keratinocyte medium. Similar to recombinant HGF, HGF from conditioned medium had a high affinity for dextran sulfate and albumin, and the same epitopes were engaged by the interaction of HGF with the c-Met receptor. The conditioned medium from keratinocytes obtained from the burn patient, but not medium from keratinocytes obtained from healthy volunteers, accelerated the motogenesis of CCL-53.1 cells. Unexpectedly, anti-HGF antibodies did not prevent this effect. However, anti-c-Met antibodies completely inhibited the motogenic effect. CONCLUSION: Upon injury, human skin keratinocytes might produce biologically active HGF in an autocrine fashion. This HGF might have different structural and/or biological properties from HGF produced by mesenchymal cells.
Assuntos
Fator de Crescimento de Hepatócito/biossíntese , Pele/lesões , Pele/metabolismo , Adulto , Animais , Comunicação Autócrina , Células Cultivadas , Meios de Cultivo Condicionados , Humanos , Técnicas In Vitro , Queratinócitos/metabolismo , Masculino , Camundongos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-met/metabolismoRESUMO
BACKGROUND: Topical occlusive/semi-occlusive dressings that induce a damp and trapped environment are widely used in wound treatment. Subjecting the wound with impaired circulation to such trapped/air-free environment potentiates the growth of anaerobic bacteria and risk for serious infection. CASE PRESENTATION: We present a case of previously healthy Swedish male that had a muscle contusion after heavy trauma that induced impaired circulation. The application of an occlusive bandage to the post-traumatic wound on the patient resulted in a poly-microbial anaerobic infection and necrosis. These complications were treated successfully with antibiotics and open dressing of the wound. CONCLUSION: The pathophysiology of difficult- to- treat ulcers should be reviewed by the physician and occlusive dressing should be avoided when treating wounds with impaired circulation.
Assuntos
Bactérias Anaeróbias/crescimento & desenvolvimento , Circulação Sanguínea , Curativos Oclusivos , Ferimentos e Lesões/microbiologia , Ferimentos e Lesões/patologia , Tecido de Granulação/patologia , Hemorragia/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Necrose , Cicatrização , Ferimentos e Lesões/diagnóstico por imagem , Raios XRESUMO
BACKGROUND: Hepatocyte growth factor (HGF) is a multifunctional cytokine that is involved in recovery process after organ injuries. OBJECTIVE: We studied HGF and the membrane bound receptor, c-met locally in patients who suffered from chronic leg ulcers (> or =1 year) caused by venous insufficiency. METHODS: Skin biopsies from the edge of the ulcers were taken from patients (n=13) and studied by immunohistochemical staining for detection of HGF and c-met. Skin biopsies from healthy volunteers (n=10) were used as the control material. Ulcer secretion from chronic ulcers (n=11) was examined for the presence of HGF by ELISA and the concentration of HGF was compared with acute ulcers in healthy controls (n=10) and in patients operated for a non-invasive breast cancer (n=12). RESULTS: We observed that c-met expression in the ulcer area increased significantly in chronic ulcers compared to controls (p=0.005). Concentration of ulcer-HGF in the patients with chronic ulcer was significantly higher than acute ulcers (p<0.01). The biological activity of HGF in ulcer secret was assessed in-vitro in transferred, mouse skin epithelial cell monolayer. Enhanced migration and morphologic changes were seen after adding ulcer secret from acute ulcers (> 1 ng/mL) that was inhibited by anti-HGF antibodies. No biological activity was observed by adding ulcer secret from chronic ulcers irrespective HGF concentration. CONCLUSION: We conclude that in chronic skin ulcers decreased biological activity of endogenous HGF and overexpression of c-met is seen which might explain fibrosis and delayed recovery. Administration of exogenous active HGF might contribute to accelerated healing in these patients.
Assuntos
Fator de Crescimento de Hepatócito/biossíntese , Fator de Crescimento de Hepatócito/fisiologia , Úlcera da Perna/metabolismo , Proteínas Proto-Oncogênicas c-met/biossíntese , Pele/patologia , Adulto , Animais , Biópsia , Western Blotting , Neoplasias da Mama/metabolismo , Estudos de Casos e Controles , Linhagem Celular , Movimento Celular , Citocinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Células Epiteliais/citologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Fatores de Tempo , Fator de Crescimento Transformador beta/metabolismo , Fator de Crescimento Transformador beta1 , Úlcera/metabolismo , Insuficiência Venosa/patologia , CicatrizaçãoRESUMO
BACKGROUND: The development of biosensors, based on surface plasmon resonance (SPR) technology, enables monitoring of a variety of biospecific interactions without the need for chemical-, biological- or radiological-labelled reagents. METHOD: We utilised SPR to detect hepatocyte growth factor (HGF) in reconstituted faecal samples and studied samples from patients with infectious gastroenteritis (n = 20) and normal controls (n = 10). Mouse anti-human HGF monoclonal antibodies and recombinant human HGF receptor (c-Met)/Fc chimera were immobilised in flow cells of a CM5 biosensor chip. RESULTS: We found that infectious gastroenteritis produced a higher signal response compared to controls, due to binding of HGF to monoclonal anti-HGF antibody as well as binding of HGF to c-Met receptor (p < 0.01). The SPR signal response correlated with results from ELISA (r = 72%, p > 0.001). The signal response decreased significantly (p < 0.05) when samples were diluted with dextran, because of reduction in both specific as well as unspecific binding of HGF to dextran. The decrease in the specific response might imply that the dextran- binding site for HGF overlaps with the antibody binding epitope, or that dextran binding induces a conformational change of the HGF molecule. Bands corresponding to HGF were found by gel electrophoresis of purified faeces in an affinity chromatography column immobilised by HGF ligands. CONCLUSION: Determination of HGF by SPR might be beneficial in diagnosis of acute situations that present with symptoms of gastroenteritis and may, possibly, guide appropriate medical treatments. This is to our knowledge the first report on the use of SPR for detection of HGF in faeces samples.
Assuntos
Fezes/química , Gastroenterite/metabolismo , Gastroenterite/microbiologia , Fator de Crescimento de Hepatócito/análise , Infecções , Ressonância de Plasmônio de Superfície , Adulto , Idoso , Idoso de 80 Anos ou mais , Envelhecimento/metabolismo , Estudos de Casos e Controles , Dextranos/metabolismo , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Feminino , Fator de Crescimento de Hepatócito/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Inibidores de Proteases/farmacologia , Reprodutibilidade dos Testes , Ressonância de Plasmônio de Superfície/normas , Fatores de TempoRESUMO
After an episode of diarrhoea, a previously healthy young man developed a splenic abscess due to invasive non-typhoidal Salmonella. The patient was presented with >1 month of fever, diffuse abdominal pain, raised C reactive protein and increased white cell count. Ultrasonography revealed a 5 × 5 cm abscess in the spleen. After an unsuccessful treatment attempt with percutaneous drainage and antibiotics, the patient was successfully treated with splenectomy and antibiotics. This case highlights the difficulties inherent in making a correct diagnosis of splenic abscess in patients without risk factors. Splenic abscess is rare in previously healthy individuals. Antibiotics are inadequate as a sole treatment, and percutaneous drainage is usually only a temporary solution. Splenectomy is still the standard treatment in most cases.
Assuntos
Abscesso/microbiologia , Infecções por Salmonella/diagnóstico , Esplenopatias/microbiologia , Abscesso/tratamento farmacológico , Abscesso/cirurgia , Antibacterianos/uso terapêutico , Gastroenterite/microbiologia , Humanos , Indonésia , Masculino , Salmonella , Infecções por Salmonella/tratamento farmacológico , Infecções por Salmonella/cirurgia , Esplenectomia , Esplenopatias/tratamento farmacológico , Esplenopatias/cirurgia , Viagem , Resultado do Tratamento , Adulto JovemRESUMO
BACKGROUND: As West Africa continues to suffer from a deadly Ebola epidemic, the national health sectors struggle to minimize the damages and stop the spread of disease. METHODS: A cohort of inhabitants of a small village and an Ebola hot zone in Sinoe County of Liberia was followed on a day-by-day basis to search for new cases and to minimize the spread of Ebola to the other community members or to other regions. Technical, clinical, and humanistic aspects of the response are discussed in this report. RESULTS: Of the 22 confirmed Ebola cases in Sinoe County since the beginning of outbreak (June 16, 2014), 7 cases were inhabitants of Polay Town, a small village 5.5 miles east of Greenville, the Sinoe County capital. After the last wave of outbreak at the beginning of December, enhanced response activity provided essential coordination and mobilized the resources to stop the epidemic. Despite unprotected contacts in crowded houses, no new cases were detected among the contact families, or in the surrounding houses or communities. CONCLUSIONS: Strong national mobilization in a decentralized but harmonized system at the community level has been of great value in controlling the epidemic in Liberia. The major interventions include epidemiological surveillance, public information dissemination, effective communication, case management, and infection control.
Assuntos
Transmissão de Doença Infecciosa/prevenção & controle , Doença pelo Vírus Ebola/diagnóstico , Doença pelo Vírus Ebola/prevenção & controle , Controle de Infecções/métodos , Adolescente , Criança , Feminino , Doença pelo Vírus Ebola/transmissão , Humanos , Lactente , Libéria/epidemiologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Fatores de TempoRESUMO
Porphyromonas gingivalis (P. gingivalis) is a major etiological agent associated with periodontitis. This study aims to develop antibodies to P. gingivalis in vitro for real-time detection of bacteria in clinical samples. Lymphocytes were isolated from whole blood of patient treated for periodontitis and were stimulated with P. gingivalis ATCC 33277. B-cell maturation to long-living antibody secreting-plasma cells was studied using flow cytometry and immunofluorescence staining. The antibodies developed in vitro were immobilized onto a CM-5 sensor chip of a biosensor to detect the presence of P. gingivalis in the gingival crevicular fluid of patients with periodontitis compared to periodontally healthy controls (n = 30). Surface plasmon resonance (SPR) analysis was performed to evaluate specific interactions of bacteria in samples with the immobilized antibodies. The results of SPR analysis were compared to the detection of P. gingivalis in the samples using DNA-DNA checkerboard hybridization technique. A clear and distinct change in lymphocyte morphology upon stimulation with P. gingivalis was observed. Anti-P. gingivalis antibodies secreted by CD38+ plasma cells showed the presence of all the four IgG subclasses. The results of DNA-DNA checkerboard analysis were in agreement with that of SPR analysis for the detection of P. gingivalis in patient samples. Furthermore, incubation with anti-P. gingivalis attenuated the bacterial response in SPR. The in vitro method for antibody production developed during this study could be used for an efficient real-time detection of periodontitis, and the attenuating effects of in vitro antibodies suggest their role in passive immunization to prevent periodontitis and their associated risk factors.
RESUMO
BACKGROUND: Due to anatomical restrictions, the inflammatory response to intracerebral bacterial infections exposes swollen brain tissues to pressure and ischemia, resulting in life-threatening damage. Rapid diagnosis and immediate empirical antibiotic therapy is highly important. However, diagnosing meningitis in patients after neurosurgery is complicated, due to brain tissue damage and changes in cerebrospinal fluid (CSF) caused by surgery. Hepatocyte growth factor (HGF) is a local, acute-phase protein with healing properties. Previous studies on community-acquired septic meningitis reported high levels of intrathecally produced HGF. The present study focused on nosocomial meningitis in assessing the levels of HGF in the CSF. METHODS: HGF concentrations (ELISA) and HGF binding to receptors; c-Met receptor and heparan sulfate proteoglycan were determined in CSF samples (surface plasmon resonance). CSF samples from patients with community-acquired or nosocomial meningitis (217 samples from 135 patients) were compared to those from controls without signs of cerebral nervous system involvement (N = 36) and patients with Alzheimer's disease (N = 20). RESULTS: Compared to samples from patients that had undergone neurosurgery and had other infectious diseases, CSF samples from patients with nosocomial meningitis had significantly higher HGF concentrations (p < 0.001) and binding affinity to c-Met (p < 0.001) and HSPG (p = 0.043) receptors. The sensitivity and specificity to identify nosocomial septic meningitis were 69.7 and 93.4%, respectively. The HGF concentration and binding affinity to HGF receptors were significantly higher in CSF from patients with community-acquired septic meningitis compared to patients with aseptic (viral and subacute) meningitis as well as controls (p < 0.001). The sensitivity and specificity to identify community-acquired septic meningitis were 95.4 and 95.7%, respectively. DISCUSSION: In febrile nosocomial infections that occurred post neurosurgery, HGF assessment could substantially improve the differentiation of meningitis from other infections and therefore might be a tool for rapid diagnosis, limiting injuries and guiding antibiotic therapy.
Assuntos
Infecção Hospitalar/líquido cefalorraquidiano , Fator de Crescimento de Hepatócito/análise , Leucocitose/líquido cefalorraquidiano , Meningite Asséptica/líquido cefalorraquidiano , Meningites Bacterianas/líquido cefalorraquidiano , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Pré-Escolar , Infecção Hospitalar/diagnóstico , Feminino , Humanos , Lactente , Leucocitose/diagnóstico , Masculino , Meningite Asséptica/diagnóstico , Meningites Bacterianas/diagnóstico , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas c-met/metabolismo , Sensibilidade e Especificidade , Adulto JovemRESUMO
BACKGROUND: High levels of hepatocyte growth factor (HGF), a healing factor with regenerative and cytoprotective effects, are associated with inflammatory diseases, including periodontitis. HGF biologic activity requires binding to its receptors, the proto-oncogene c-Met and heparan sulfate proteoglycan (HSPG). This study investigates HGF expression and its relationship to subgingival microbiota in medically healthy individuals with and without periodontitis. METHODS: Saliva, gingival crevicular fluid (GCF), and blood samples from 30 patients with severe periodontitis and 30 healthy controls were analyzed for HGF concentration using enzyme-linked immunosorbent assay and binding affinity for HSPG and c-Met using surface plasmon resonance. The regenerative effects of saliva from three patients and controls were analyzed in an in vitro model of cell injury. Subgingival plaques were analyzed for the presence of 18 bacterial species. RESULTS: Patients with periodontitis showed higher HGF concentrations in saliva, GCF, and serum (P <0.001); however, the binding affinities for HSPG and c-Met were reduced in GCF and saliva (P <0.002). In contrast to the controls, saliva from patients showed no significant regenerative effect over time on gingival epithelial cells. Compared with controls, patients had a higher prevalence of periodontally related bacteria. CONCLUSIONS: Higher circulatory HGF levels indicate a systemic effect of periodontitis. However, the HGF biologic activity at local inflammation sites was reduced, and this effect was associated with the amount of periodontal bacteria. Loss of function of healing factors may be an important mechanism in degenerative processes in periodontally susceptible individuals.