Cemalettin Aybay

Anti‐infliximab antibody status and its relation to clinical response in psoriatic patients: A pilot study

Although the mechanisms underlying the loss of response to infliximab are not completely understood, the formation of antibodies to infliximab (ATI) are thought to play a role. The aim of this study was to investigate the presence of ATI in psoriatic patients and to evaluate its relationship to the clinical response. Fifteen patients with psoriasis were treated with infliximab (5u2003mg/kg) every 8u2003weeks after an initial three‐dose induction treatment. An enzyme linked immunosorbent assay kit was used for analyzing the presence of ATI in sera. Effectiveness assessments included the change in Psoriasis Area and Severity Index (PASI) compared with study entry. Five (33.3%) patients developed ATI. While 5.9u2003±u20033.2 infliximab infusions achieved a fall in the PASI score from a mean of 20.4u2003±u20038.3 to 5.3u2003±u20032.4 in ATI‐negative patients, these values changed from 23.3u2003±u200311 to 10u2003±u20034.9 after 9u2003±u20035.2 infusions in ATI‐positive patients. Our results suggested that ATI measured in psoriatic patients are of clinical importance. Therefore, monitoring for the induction of ATI and rescue strategies should be developed to avoid or to maintain a delay in ATI development.

Differential binding characteristics of protein G and protein A for Fc fragments of papain-digested mouse IgG

It has been previously reported that staphylococcal protein A (SPA) bound only to the Fc region of mouse immunoglobulin G (IgG) and streptococcal protein G (SPG) bound to both Fab and Fc regions of mouse IgG and the binding sites for SPG and SPA on Fc were overlapped. In this study the binding characteristics of SPG and SPA for papain-digested mouse IgG were analysed. Papain digestion of mouse IgG purified from CAy-IFNg99C hybridoma (secreting IgG1 monoclonal antibody specific for human interferon gamma)-induced ascites resulted in Fab and two major Fc fragments referred to as the high molecular weight (HMW) and the low molecular weight (LMW) Fc fragments. SPG bound to Fab and the LMW Fc fragments of the papain-digested IgG. However SPG did not bind to the HMW Fc fragment. SPA showed practically no reactivity with the Fab and the LMW Fc fragments of the papain-digested mouse IgG but only to the HMW Fc fragment. SPG and SPA binding assays showed that papain digestion discriminated the SPG and SPA binding sites in the Fc fragment of mouse IgG. These results demonstrated a clear evidence for the presence of two independent SPG and SPA binding sites in the Fc fragment of mouse IgG.

Demonstration of specific antibodies against infliximab induced during treatment of a patient with ankylosing spondylitis.

Therapeutic proteins, such as infliximab, have revolutionized the treatment of many diseases during the last decade and more than 80 therapeutic proteins are currently approved for clinical use. However, all exogenous proteins have the potential to cause antibody formation. In order to ensure patient safety and the efficacy of therapeutic proteins, careful monitoring of the immunogenicity of therapeutic proteins is therefore necessary not only during preclinical trials, but also during the treatment of patients. Here, we report a clear-cut demonstration of the induction of anti-infliximab antibodies during the treatment of a patient with ankylosing spondylitis (AS). Assessment of anti-infliximab antibodies in sera obtained at various time periods were performed using a highly specific double antigen assay system developed in our laboratory. Immunoreactivity was found to be solely specific for infliximab. Because all sera obtained from the patient were found to be negative for the presence of human anti-mouse antibody (HAMA) and anti-human antibodies. The loss of effect of infliximab, as judged by observing the relapse of signs and symptoms of disease in the patient, seemed to be related with the appearance of antibodies. This study clearly demonstrates that monitoring for the induction of specific antibodies during clinical trials is an important issue for therapeutic proteins.

Endomysium antibodies in the diagnosis of celiac disease in short-statured children with no gastrointestinal symptoms.

Background : Endomysium antibodies (EmAb) are strongly associated with untreated celiac disease and are suggested to be diagnostic. The aim of the present study was to assess the value of anti‐EmAb for celiac disease screening in children with short stature.

Development of a rapid, single-step procedure using protein G affinity chromatography to deplete fetal calf serum of its IgG and to isolate murine IgG1 monoclonal antibodies from supernatants of hybridoma cells.

Fetal calf serum (FCS) was depleted of its immunoglobulin G (IgG) in a rapid procedure using protein G affinity chromatography. 20 ml of FCS was depleted of its IgG in less than 80 min by applying 5 ml of FCS to a 1 ml HiTrap protein G Sepharose column followed by appropriate elution. Various concentrations of IgG-depleted FCS (G-FCS) were used in RPMI-1640 medium to grow the mouse hybridoma cell lines CAy-G (anti-HBs IgG1 mAb producing hybridoma cell) and CAy-M (anti-HBs IgM mAb producing hybridoma cell), which secreted hepatitis B virus surface antigen (HBsAg)-reactive IgG1 and IgM monoclonal antibodies (mAbs), respectively. Antibody production and cell growth were used as indices to compare the efficacy of RPMI/G-FCS with that of RPMI/FCS and serum/protein-free Hybri Max (Sigma, MO, USA) hybridoma medium. MAb production and cell growth of CAy-G and CAy-M hybridoma cell lines in RPMI/G-FCS were similar to culture in RPMI/FCS and significantly better than culture in Hybri Max. We found that G-FCS was superior to whole FCS as a culture supplement for the purification of IgG1 mAbs. IgG1 mAbs were isolated in a single-step procedure using protein G affinity chromatography, from the supernatant of CAy-G hybridoma cells cultured in RPMI/10% G-FCS (RPMI-1640 medium supplemented with 10% G-FCS). SDS-PAGE analysis revealed that the purity of IgG isolated from the supernatant of CAy-G cells cultured in RPMI/10% G-FCS was more than 99%.

Tumor Necrosis Factor (TNF) Induction from Monocyte/Macrophages by Candida Species

Candida albicans was studied for its capacity to induce TNF production from mouse peritoneal macrophages (PM phi). TNF activities in the culture supernatants of Candida-stimulated PM phi and human peripheral blood monocytes were assessed by L 929 bioassay and ELISA respectively. C. albicans induced TNF production from PM phi and human peripheral blood monocytes in a dose-dependent manner. Although the capacity was lesser than live form, heat-killed C. albicans was also found to be capable of stimulating PM phi to induce TNF. The filtered supernatant of 24 h cultured live C. albicans had no effects on TNF production from PM phi. Saccharomyces cerevisiae-extracted mannan, a yeast cell wall antigen, induced TNF production from PM phi in a dose-dependent manner. Thus, the effect of C. albicans on TNF production from PM phi was seemed to be directly related to the presence of the yeast cell wall itself. Compatible with these data, when various candida species (C. albicans, C. tropicalis, C. pseudotropicalis. C. lusitaniae, C. krusei, C. parapsilosis, C. guilliermondii, C. stellatoidea, C. glabrata) and S. cerevisiae were compared to each other, at a concentration of 2 x 10(6) yeast cells/ml from each species, it was observed that TNF inducing capacities varied. Among the species used in this study, C. guilliermondii and C. glabrata, of which the yeast cell size were the smallest ones, were found to be less potent than that of others to induce TNF from PM phi.

Salivary Epidermal Growth Factor Levels in Behçet’s Disease and Recurrent Aphthous Stomatitis

Background: Epidermal growth factor (EGF) in saliva is cytoprotective against injuries and contributes to the maintenance of the integrity of the gastrointestinal mucosa. Low salivary EGF levels have been observed in patients with various forms of oral mucosal disease. Objective: Our aim wasto determine whether salivary EGF is low in patients with recurrent aphthous stomatitis (RAS) or those with Behçet’s disease (BD) when compared with healthy controls. Methods: The study population consisted of 33 BD and 16 RAS patients and 60 healthy controls. Measurement of EGF concentration in human saliva was performed with an enzyme-linked immunosorbent assay using an antibody-coated solid phase. Results: The mean salivary EGF levels (±SD) of active (with oral ulceration) and inactive stages (absence of oral ulceration) of BD (1,939.7 ± 1,561.5 and 2,305.7 ± 1,481.6 pg/ml, respectively) and RAS patients (1,650.5 ± 704.7 and 1,069.9 ± 539.2 pg/ml, respectively) were both lower than those of the healthy controls (2,758.7 ± 1,657.9 pg/ml) (p < 0.05 for each). Conclusions: BD and RAS patients have reduced salivary EGF levels even in the absence of oral ulcerations. EGF could be involved in the pathogenesis of BD and RAS by disturbing the mucosal integrity that may result in a susceptibility to the development of oral ulcers in these diseases.

Effect of Blood Collection Tube Types on the Measurement of Human Epidermal Growth Factor

Abstract We observed significant differences in measured human epidermal growth factor (hEGF) levels for the same individuals serum/plasma samples between different tube types (glass, polystyrene, plastic with clot activator, plastic without clot activator, plastic with EDTA, polypropylene tubes). For all individuals, hEGF levels in plasma were found to be below the detection limit. The discrepancy of the hEGF levels in serum and plasma was attributed to the platelet derived EGF by analyzing platelet lyzate with size exclusion chromotography and demonstrating the immunoreactivity of the fractions corresponding to the pre‐proEGF and/or proEGF elution time. Besides, samples of females showed much higher EGF levels than those of males in certain test tube types. As a conclusion, all blood samples should be taken and stored in the same type of test tubes in order to make precise measurements for hEGF. And, the measured hEGF level in blood is susceptible to changes with blood clotting.

Determination of tetanus antibodies by a double-antigen enzyme-linked immunosorbent assay in individuals of various age groups

In this study, tetanus immunity was determined in 549 randomly chosen individuals of various age groups in Ankara, Turkey. Antibody levels in sera of the individuals were measured using a double-antigen enzyme-linked immunosorbent assay. Overall, 66.5% (95%CI, 62.4–70.4) of the population studied was found to have basic protection (≥0.01xa0IU/ml) against tetanus. Protective levels of tetanus antibodies declined progressively with age. The rate of protection in children and adolescents (aged <20 years) exceeded 90%, while only 16.3% (95%CI, 8.9–26.2) of those over 60 years of age were protected. Females over 60 years of age were less immune than males of the same age group (p=0.034). Although the rates of protection in children and adolescents are regarded as satisfactory, the rates among adults are low. Preventive measures against tetanus should therefore focus on scheduled booster immunization for adults as well as children.

Effect of monophosphoryl lipid A on antibody response to diphtheria toxin and its subunits

Monophosphoryl lipid A (MPL) was evaluated for its ability to enhance the antibody response to diphtheria toxin and its fragment A and fragment B subunits. BALB/c mice were immunized subcutaneously with 1 Lf of diphtheria toxoid in the presence of 25 μg of MPL on days 0 and 14. Two weeks after the second immunization, sera were obtained from the mice and analysed for antibody response to diphtheria toxin and its subunits. A new ELISA method, developed in our laboratory, was used to measure antibody levels against the toxin, fragment A, and fragment B. It was observed that MPL significantly enhanced antibody responses to diphtheria toxin and its subunits. However, there was no statistical difference between anti‐A and anti‐B responses. The results indicated that MPL seems to be a potential candidate as an adjuvant for future diphtheria vaccine formulation.