Zoltán Péterfi

Comparison of Blocking Agents for an Elisa for Lps

Abstract ELISA is a sensitive, specific, reproducible and fast method for detection of antigen-antibody reactions. in case of non-protein antigens as LPS, problems exist, such as poor proportion of coating to microplates, non-specific binding of antibodies to the plastic wells. These problems were resolved partially by Takahashi and co-workers using poly-L-lysine for coating of LPS antigens. to reduce non-specific binding, blocking agent, such as bovine serum albumin (BSA) or casein is commonly used. We have to choose the blocking agent carefully because LPS can bind proteins non-specifically. This process can inhibit binding of LPS-specific antibody to LPS and decrease the sensitivity of method. in this paper we describe an ELISA test for LPS in which normal goat serum is used for blocking. This modification increases the sensitivity of ELISA. This method is useful for detection of LPS (S, R form) and anti-LPS antibody reaction in serological cross-reaction studies.

Cytokine (IL-10, IL-28B and LT-A) gene polymorphisms in chronic hepatitis C virus infection

Abstract Background Since the clearance of hepatitis C virus (HCV) infection depends on the cytokines which are under genetic control, we have studied genetic polymorphisms of two pro-inflammatory interleukin-28B (IL-28B) (also named as interferon λ-3) and lymphotoxin-A (LT-A) as well as of one anti-inflammatory cytokine interleukin-10 (IL-10) genes in patients with HCV infection. We examined the allele frequencies of these genes in HCV patients as compared with healthy controls, and determined their association with sustained virological response (SVR) on PEG-IFN α-2a + ribavirin (RBV) (P/R) treatment, to assess the predictive value of these genetic variants. A total of 292 chronic HCV genotype 1 infected patients and 104 healthy controls have been studied. The samples were genotyped using PCR-RFLP and ABI Taqman genotyping assay. Results IL-28B — The C/C genotype in HCV patients occurred with lower frequency than in healthy controls (28.11% vs. 51.92%, p = 0.0001, OD 2.76), suggesting a protective role ...

Quantitative microfluidic analysis of S- and R-type endotoxin components with chip capillary electrophoresis

A novel, fast, and sensitive ME method was developed to analyze and differentiate the smooth (S) and rough (R) type bacterial endotoxin components labeled covalently with a fluorescent dye. The quantitative analysis of purified lipopolysaccharides, or partially purified samples from whole‐cell lysates becomes possible with this method. Two groups with three sub‐groups in the first group of S‐type lipopolysaccharides can be classified based on the electrophoretic profiles. The LOD of the endotoxins from S‐ and R‐type Gram‐negative bacteria was found to be 2.6 ng and 6.9 ng, respectively. This method is capable to replace the commonly used SDS‐PAGE combined with silver staining.

IL28B and IL10R −1087 polymorphisms are protective for chronic genotype 1 HCV infection and predictors of response to interferon-based therapy in an East-Central European cohort

BackgroundPrevious studies have shown that single nucleotide polymorphisms (SNP) in IL28B and IL10R are associated with sustained virological response (SVR) in chronic hepatitis C patients treated with pegilated interferon plus ribavirin (P/R). The present study extends our earlier investigations on a large East-Central European cohort. The allele frequencies of IL28B and IL10R in genotype 1 HCV infection were compared with that of healthy controls for the purpose of examining the relationship between the polymorphisms and the SVR to P/R treatment.MethodsA total of 748 chronic HCV1 infected patients (365 male, 383 female; 18–82 years) and 105 voluntary blood donors as controls were enrolled. Four hundred and twenty HCV patients were treated with P/R for 24–72 weeks, out of them 195 (46.4%) achieved SVR. The IL28 rs12979860 SNP was determined using Custom Taqman SNP Genotyping Assays. The IL10R −1087 (also known as IL10R −1082 (rs1800896) promoter region SNP was determined by RT-PCR and restriction fragment length polymorphism analysis.ResultsThe IL28B CC genotype occurred with lower frequency in HCV patients than in controls (26.1% vs 51.4%, p<0.001). P/R treated patients with the IL28B CC genotype achieved higher SVR rate, as compared to patients with CT (58.6% vs 40.8%, p=0.002). The prevalence of IL10R −1087 GG genotype was lower in patients than in controls (31.8 % vs 52.2%, p<0.001). Among patients achieving SVR, the IL10R −1087 GG genotype occurred with higher frequency than the AA (32.0% vs 17.4%, p=0.013). The IL28B T allele plus IL10R A allele combination was found with higher prevalence in patients than in controls (52% vs 20.7%, p<0.001). The IL28B CC plus IL10R A allele combination occurred with higher frequency among patients with SVR than in non-responders (21.3% vs 12.8%, p=0.026). Both the IL28B CC plus IL10R GG and the IL28B CC plus IL10R A allele combinations occurred with lower frequency in patients than in controls.ConclusionsIn our HCV1 patients, both the IL28B CC and IL10R GG genotypes are associated with clearance of HCV. Moreover, distinct IL28B and IL10R allele combinations appear to be protective against chronic HCV1 infection and predictors of response to P/R therapy.

Capillary electrophoresis chips for screening of endotoxin chemotypes from whole-cell lysates.

A fast microchip electrophoresis method was developed to analyze and differentiate bacterial endotoxins directly from whole-cell lysates after removal of the proteinaceous components with proteinase K digestion and a precipitation of the endotoxin components. The partially purified endotoxin components were visualized by the interaction with dodecyl sulphate and then a fluorescent dye. The lipopolysaccharide (LPS) profiles can be directly evaluated from digested bacterial cells, and the electrophoresis patterns very closely resembled to those of pure LPSs, and the R and S chemotypes can be used to assign the strains. The method has been found to be useful in the screening of a large number of bacterial mutants and the structural characterization of endotoxins extracted only from 1 ml cultures.

Anti-lipopolysaccharide antibodies in acute appendicitis detected by ELISA.

In acute appendicitis the bowel transmissibility of the intestinal flora increases in relation to inflammation and edema formation. We can therefore observe an immunologic response in patients, which is detectable using different bacteria isolated from the normal intestinal flora. Our aim was to measure this immunologic reaction and to detect the relationship between this response and histologic types of acute appendicitis. Sera from 47 cases, comprising 38 patients suffering from appendicitis and 9 healthy controls, were examined. The sera were taken shortly before appendectomy and 14 days after operation. The antigens were lipopolysaccharides (LPS) extracted from bacteria of normal intestinal flora: Escherichia coli O21, O22, O33, O61, O68, Bacteroides fragilis and an absolute rough mutant: Shigella sonnei Re 4350. Antibodies were detected by ELISA. We showed a direct relationship between severity of appendicitis and registered antibody titer. Both aerobic and anaerobic bacteria play a role in infection in appendicitis. According to our serologic results the synergy of B. fragilis with E. coli from normal flora is more important in the initiation of inflammation, but in the perforation process the role of E. coli seems more important compared to that of B. fragilis.

[IL28B CC genotype: a protective factor and predictor of the response to interferon treatment in chronic hepatitis C virus infection].

INTRODUCTION In chronic hepatitis C-virus infection the possible role of gene variants encoding cytokines has become the focus of interest. AIM The aim of the study was to investigate the effect of IL28B polymorphisms on the outcome of chronic hepatitis C-virus genotype 1 infection in the Hungarian population. In addition, the association between IL28B genotypes and the Th1/Th2 cytokine production of activated peripheral blood monocytes and lymphocytes was evaluated. METHOD Total of 748 chronic hepatitis C-virus genotype 1 positive patients (365 males and 383 females, aged between 18 and 82 years; mean age, 54±10 years) were enrolled, of which 420 patients were treated with pegylated interferon plus ribavirin for 24-72 weeks. Of the 420 patients, 195 patients (46.4%) achieved sustained virological response. The IL28B rs12979860 polymorphism was determined using Custom Taqman SNP Genotyping Assays (Applied Biosystems, Life Technologies, Foster, CA, USA). For cytokine studies, tumour necrosis factor-α, interleukin-2, interferon-γ, interleukin-2 and interleukin-4 production by LPS-stimulated monocytes and PMA-ionomycine activated lymphocytes were measured from the supernatant of the cells obtained from 40 hepatitis C-virus infected patients, using FACS-CBA Becton Dickinson test. The cytokine levels were compared in patients with different (CC, CT, TT) IL28B genotypes. RESULTS The IL28B rs12979860 CC genotype occurred in lower frequency in hepatitis C-virus infected patients than in healthy controls (26.1% vs 51.4%, OR 0.333, p<0.001). Patients carried the T allele with higher frequency than controls (73.9%, vs 48.6%, OR 3.003, p<0.001). Pegylated interferon plus ribavirin treated patients with the IL28B CC genotype achieved higher sustained virological response rate than those with the CT genotype (58.6% vs 40.8%, OR 2.057, p = 0.002), and those who carried the T allele (41.8%, OR1.976, p = 0.002). LPS-induced TLR-4 activation of monocytes resulted in higher tumour necrosis factor-α production in patients with the IL28B CC genotype compared to non-CC individuals (p<0.01). Similarly, increased tumour necrosis factor-α, interleukin-2 and interferon-γ production by lymphocytes was found in the IL28B CC carriers (p<0.01) CONCLUSIONS: The IL28B CC genotype exerts protective effect against chronic hepatitis C-virus infection and may be a pretreatment predictor of sustained virological response during interferon-based antiviral therapy. The IL28B CC polymorphism is associated with increased Th1 cytokine production of activated peripheral blood monocytes and lymphocytes, which may play a role in interferon-induced rapid immune control and sustained virological response of pegylated interferon plus ribavirin treated patients.

Structural background for serological cross‐reactivity between bacteria of different enterobacterial serotypes

The structure of the oligosaccharide repeating units of endotoxins from Gram‐negative bacteria is characteristic for the different serogroups and serotypes of bacteria. Detailed examination of the cross‐reactions of three enterobacterial serotypes, Proteus morganii O34, Escherichia coli O111, and Salmonella enterica sv. Adelaide O35, was performed using sensitive tests (ELISA, immunoblotting). Fine differences between the endotoxins of the bacteria were detected using silver staining of SDS‐PAGE gels and chip‐technology for the intact lipopolysaccharides (LPSs). The compositions of the O‐specific polysaccharides of LPSs extracted from the bacteria were studied, and it was proven that the three cross‐reacting bacteria contain O‐antigens built from the same monosaccharides, namely colitoses linked to glucose, galactose, and N‐acetyl‐galactosamine. The NMR and GC‐MS studies revealed that the most probable component for the cross‐reaction is the rare sugar, colitose.

Experience with fecal transplantation in the treatment of Clostridium difficile infection

Bevezetes: Az elmult evtizedben dramai valtozasokat tapasztalhattunk a Clostridium difficile-fertőzesek epidemiologiajaban. Celkitűzes: A szerzők arra a kerdesre kerestek valaszt, van-e kulonbseg a ket, felső gastrointestinalis bejuttatasi modszer eredmenyessege kozott. Modszer: 100 ml szűrletet 15 esetben nasoduodenalis szondan keresztul adtak be, 15 esetben pedig nasogastricus szondan keresztul. Az elsődleges gyogyulasi rata alatt az esetek azon hanyadat ertettek, ahol a tunetek megszűntek a szeklettranszplantacio utan es nem tapasztaltak recidivat sem. Masodlagos gyogyulasi rata alatt pedig a betegek azon aranyat ertelmeztek, akiknel egy ismetelt beavatkozas utan szűntek meg a panaszok es nem tapasztaltak recidivat a kovetkező hat hetben sem. Eredmenyek: Nasojejunalis szondat hasznalva az elsődleges sikerrata 100%-os volt. Nasogastricus szondat hasznalva az elsődleges sikerrata 80%-os, a masodlagos sikerrata 93,3%-os volt. Osszessegeben a felső gastrointestinalis rendszeren keresztul vegzett szeklettranszplantacio elsődleges gyogyulasi rataja 90,0%, a masodlagos gyogyulasi rata 96,7% volt. Kovetkeztetesek: A szeklettranszplantacio igen hatasos modszer, kulonosen a terapiarefrakter esetekben Orv. Hetil., 2014, 155(44), 1758–1762. | Introduction: During the past years a dramatic change has been observed in the epidemiology of Clostridium difficile infections. Aim: The aim of the authors was to investigate the possibility of the fecal microbiota transplantation and study differences, if any, in the success rate of the two different upper gastrointestinal tract method. Method: 100 ml of fecal microbiota solution was instilled via a nasoduodenal tube in 15 cases and a nasogastric tube in 15 cases. The authors defined the primary cure rate as the percentage of cases in which the symptoms disappeared without recurrence within 6 weeks after the first fecal microbiota transplantation, while secondary cure rate was calculated as the percentage of cases in which the symptoms resolved after the second fecal microbiota transplantation. Results: It was found that fecal microbiota transplantation applied via the nasoduodenal tube resulted in a 100% primary cure rate. With the use of the nasogastric tube, the primary and secondary cure rate were 80% and 93.3%, respectively. Fecal microbiota transplantation via the upper gastrointestinal tract was found to have an overall primary cure rate of 90.0% and a secondary cure rate of 96.7%. Conclusions: Fecal microbiota transplantation proved to be very effective, particularly in recurrent infections and cases where conventional treatment failed. Orv. Hetil., 2014, 155(44), 1758–1762.INTRODUCTION During the past years a dramatic change has been observed in the epidemiology of Clostridium difficile infections. AIM The aim of the authors was to investigate the possibility of the fecal microbiota transplantation and study differences, if any, in the success rate of the two different upper gastrointestinal tract method. METHOD 100 ml of fecal microbiota solution was instilled via a nasoduodenal tube in 15 cases and a nasogastric tube in 15 cases. The authors defined the primary cure rate as the percentage of cases in which the symptoms disappeared without recurrence within 6 weeks after the first fecal microbiota transplantation, while secondary cure rate was calculated as the percentage of cases in which the symptoms resolved after the second fecal microbiota transplantation. RESULTS It was found that fecal microbiota transplantation applied via the nasoduodenal tube resulted in a 100% primary cure rate. With the use of the nasogastric tube, the primary and secondary cure rate were 80% and 93.3%, respectively. Fecal microbiota transplantation via the upper gastrointestinal tract was found to have an overall primary cure rate of 90.0% and a secondary cure rate of 96.7%. CONCLUSIONS Fecal microbiota transplantation proved to be very effective, particularly in recurrent infections and cases where conventional treatment failed.

INTERACTION OF CONCANAVALIN A WITH BACTERIAL LIPOPOLYSACCHARIDES IN AGAROSE GEL

Binding of fluorescein isothiocyanate-labeled concanavalin A to a series of molecular species of lipopolysaccharide (LPS), purified from pathogenic bacteria, was studied via agarose gel precipitation experiments and the results were compared with available structural data.The LPS species could be divided into ConA-reactive and non-reactive ones. Reactivity resided in the O-specific chain of LPS, and binding to the lipid A or core moieties of LPS could not be demonstrated by the present methods. The α-D-glucose or α-D-mannose residues of the repeating O-specific oligosaccharide units appeared to be recognized by ConA, except when blocked by steric hindrance. Specificity of the reaction was verified by inhibition with 2% D-glucose. Binding by bacterium-specific sugar-residues could not be demonstrated.For precipitation to occur, polyvalency was required both for LPS and ConA, and the resulting precipitation appeared to be promoted by hydrophobic interactions between the lipid A moieties of LPS molecules. The LPS species were differently retained by the agarose gel, which can be explained by differences in their micellar structure in aqueous solution. E. coli O83 LPS did not readily diffused in 1% agarose gel, but its precipitation with ConA could be demonstrated either at elevated temperature or mixing it previously with molten agarose (Mancinis arrangement).