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 (5 mg/kg) every 8 weeks 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 5.9 +/- 3.2 infliximab infusions achieved a fall in the PASI score from a mean of 20.4 +/- 8.3 to 5.3 +/- 2.4 in ATI-negative patients, these values changed from 23.3 +/- 11 to 10 +/- 4.9 after 9 +/- 5.2 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.

Homocysteine, fibrinogen and anti-ox-LDL antibody levels as markers of atherosclerosis in prepubertal obese children.

BACKGROUND: Homocysteine, fibrinogen and antibodies to oxidised LDL were shown to be important markers of atherosclerosis in adults. AIM: To investigate the levels of these three risk factors in prepubertal obese children. METHODS: Fasting homocysteine, fibrinogen and antibodies to oxidised LDL, plasma lipids, insulin, HbA1c and blood glucose levels were investigated in 30 prepubertal obese and 28 control children 6-9 years old. Investigations in the obese group were repeated after an oral glucose tolerance test. RESULTS: Fasting fibrinogen levels of the obese children were found to be significantly higher than those in the controls. Anti-ox-LDL antibody levels increased significantly after an oral glucose tolerance test. CONCLUSION: Fasting fibrinogen and postload ox-LDL levels which could act as important markers of coronary heart disease in later life could also be important risk factors in prepubertal obese children.

The relationship between adiponectin levels and proinflammatory cytokines and left ventricular mass in dialysis patients.

INTRODUCTION: Adiponectin is increased in end-stage renal disease. However, efforts to clarify the cause of that increase and its clinical effects have been inconclusive. The aim of this study was to compare serum adiponectin levels of dialysis patients against healthy individuals and evaluate the relationship among adiponectin levels, IL-6, TNF- alpha and left ventricular mass index (LVMI). METHODS: Adiponectin, IL-6 and TNF- alpha measurements and echocardiographic evaluations were performed in 36 hemodialysis, 30 continuous ambulatory peritoneal dialysis (CAPD) patients and 22 healthy volunteers. Adiponectin, IL-6 and TNF- alpha levels were measured by ELISA. RESULTS: Adiponectin was found to be higher in hemodialysis (52.78+/-18.01 ng/mL) and CAPD (52.96+/-17.53 ng/mL) groups than controls (28.36+/-13.20 ng/ mL; p=0.0003, p=0.0003, respectively). No difference was observed between the hemodialysis and CAPD groups. Adiponectin was positively correlated with IL-6 (r=0.293, p=0.02), TNF- alpha (r=0.458, p=0.0003) and LVMI (r=0.283, p=0.02). In the partial correlation analysis, by controlling for body mass index, the correlation between adiponectin and TNF- alpha (r=0.466, p=0.0003) persisted. When IL-6 was controlled with TNF- alpha, the relation between adiponectin and LVMI disappeared (r=0.145, p=0.30). In the linear regression analysis, with adiponectin as the dependent variable, and IL-6, TNF- alpha and body mass index as independent variables, a significant relationship was found between adiponectin and TNF- alpha (beta=0.488, p=0.001). CONCLUSIONS: Increased adiponectin seems to be associated with increased proinflammatory cytokines in dialysis patients, and this relationship suggests adiponectin may have a role in the development of left ventricular hypertrophy.

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 was to 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 oophorectomy and exogenous estrogen replacement on liver injury in experimental obstructive jaundice.

AIM: To investigate the role of estrogen on liver injury in an experimental obstructive jaundice model. METHODS: Three groups of female rats were constituted; group 1 was oophorectomized and given E2 (n = 14), group 2 was oophorectomized and given placebo (n = 14), and group 3 was sham operated (n = 14). Fourteen days following constitution of bile duct ligation, all groups were compared in terms of serum tests, histopathologic parameters, and tissue levels of IFN-gamma and IL-6. RESULTS: The parameters representing both the injury and/or the reactive response and healing were more pronounced in groups 1 and 2 (c2 = 17.2, c2 = 10.20; c2 = 12.4, P < 0.05). In the sham operated or E2 administered groups significantly lower tissue levels of IFN-gamma and higher IL-6 levels were found. In contrast, high IFN-gamma and low IL-6 tissue levels were found in the oophorectomized and placebo group (P < 0.001). Kupffer cell alterations were observed to be more pronounced in the groups 1 and 3 (c2 = 6.13, P < 0.05). CONCLUSION: Our study indicates that E2 impaired liver functions, accelerated both the liver damage and healing. In the conditions of bile duct obstruction, estrogen significantly changed the cytokine milieu in the liver.

Determinants of protection against diphtheria in adult hemodialysis patients.

Diphtheria is of great epidemiological concern. Although mainly observed during childhood, unvaccinated adults and relatively immunocompromised patients are at increased risk for acquiring diphtheria. We aimed to determine the rates and certain determinants of protection against diphtheria in adult hemodialysis (HD) patients. Protection rates of 322 HD patients were compared with 65 diabetes mellitus type 2 (DM) patients and 65 healthy controls. A questionnaire was held in regard to smoking habits and alcohol intake. Antibody levels against diphtheria were assessed by an in-house ELISA and a concentration of >or=0.1 IU/mL was regarded as protective. Effects of age, gender and time being on dialysis on protection were assessed by logistic regression. Ratios of individuals with protective antibody levels were found to be 36% (116/322), 27.7% (18/65), and 52.3% (34/65) for HD, DM, and control groups, respectively. Hemodialysis patients had a significantly (p < 0.05) lower protection rate than healthy controls. In all study groups, there was a tendency of higher protection rate with increasing age. These low ratios of protected individuals in both HD and DM patient groups are alarming, as these patients generally have defects in vaccine responses, and carriage is important in the perpetuation of diphtheria. The protection status of these patient groups might be improved with additional vaccinations.

Investigation of the functional characteristics of antibodies to therapeutic anti-human tumor necrosis factor alpha monoclonal antibody.

Humanized antibody-based treatment modalities represent an active area of investigation. Included in these strategies are passive administrations of monoclonal antibodies, which recognize tumor necrosis factor alpha (TNF-alpha). However, several problems associated with these types of treatment strategies have been reported in the literature. We attempted to address the issue related to unresponsiveness to infliximab that might be induced by anti-idiotype response to the passively administered humanized monoclonal antibody. The characteristics and functional importance of antibodies to infliximab (ATI) were investigated in human sera. We studied the binding characteristics of ATI to infliximab, TNF-alpha Receptor-I (RI, p55) and Receptor-II (RII, p75). In addition, cytotoxicity effect on L929 cells and blocking effects on the binding of TNF-alpha with infliximab and etanercept were also analyzed. On the basis of the results obtained from the experiments, it seems that the target epitope for ATI is related with somewhere else not residing in the region capable of generating "mirror image". The results presented indicate that ATI does not mimic the functional characteristics of TNF-alpha. However, ATI inhibited the binding properties of infliximab to TNF-alpha.

Characterization of human epidermal growth factor in human serum and urine under native conditions.

The objective of this study was to investigate the molecular nature of the human epidermal growth factor (EGF) in serum and urine samples of normal subjects. Recombinant EGF emerged as a single peak and did not interact with human IgG1 and albumin up to the concentration of 12 microg/ml. Freshly separated human serum contained only trace amounts of EGF. However, EGF appeared and increased in serum separated from blood after spontaneous overnight clotting. The authentic 6 kDa form of EGF made up nearly 40% of the total EGF in serum and revealed relatively homogeneous feature. The remaining immunoreactive fractions corresponded to 160 kDa proEGF. Immunoreactive EGF in blood seemed to be associated with the EGF release from platelets. TSKgel G3000SW chromatography of freshly-voided morning and day urines revealed that urine samples mainly contained two major form of EGF; a high-molecular-weight (HMW) and low-molecular-weight (LMW) forms. In the sense of molecular nature of EGF contents, morning urine was more heterogeneous than day urine of the same individuals. The LMW form of EGF in morning urine, in which its proportion was more than 90% of the total EGF, revealed further heterogeneous feature generally containing three to four different components.

A novel mutation in the vascular Ehlers-Danlos syndrome: a case presenting with colonic perforations.

A 15-year-old girl who had chronic constipation presented with peritonitis caused by sigmoid colon perforation. After her sigmoid colon was resected and an end colostomy performed, as there were no apparent causes for perforation, she was followed-up. After the second colonic perforation proximal to the end colostomy, as the pathologic findings revealed myopathic changes, the connective tissue disorders were evaluated. Her molecular biology studies revealed an undefined missense mutation in the COL3A1 gene, confirming the diagnosis of vascular Ehlers-Danlos syndrome (EDS). As she refused a permanent stoma, total colectomy and ileorectal anastomosis were performed, but the postoperative complications resulted in a fatal progression. The typical progression of vascular EDS will be discussed with the presented case by means of a review of the English medical literature on children diagnosed with vascular EDS.

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.

Endothelial activation and inflammation in prepubertal obese Turkish children.

To investigate the degree of endothelial activation and inflammation in prepubertal obese children and to determine the relationship between the markers of endothelial activation, inflammation, and cardiovascular risk factors. In 30 obese and 28 healthy prepubertal children, soluble intercellular adhesion molecule-1 and endothelial leukocyte adhesion molecule-1 (sE-selectin) as markers of endothelial activation and soluble vascular cell adhesion molecule-1 (sVCAM-1) and C-reactive protein (CRP) as markers of endothelial inflammation in addition to cardiovascular risk factors including blood lipids, glucose, insulin, hemoglobin A1c, and systolic and diastolic blood pressure were investigated and compared. The tests were repeated after an oral glucose tolerance test in the obese group. Fasting CRP levels were found to be significantly higher in obese children. Vascular cell adhesion molecule-1 levels were found to be significantly increased in obese children after oral glucose tolerance test. Fasting CRP was positively correlated with body mass index (BMI) and low-density lipoprotein, whereas sE-selectin was positively correlated with total cholesterol. In the obese group, postload levels of soluble sE-selectin was positively correlated with low-density lipoprotein; sVCAM-1 was positively correlated with insulin and homeostasis model assessment values. Postload soluble intercellular adhesion molecule-1, sVCAM-1, and soluble sE-selectin levels were also positively correlated with each other. In the fasting state, BMI was the significant independent risk factor for CRP, and total cholesterol was the significant risk factor for soluble sE-selectin. Insulin resistance was the significant independent risk factor for postload sVCAM-1, and postload low-density lipoprotein stood as the significant independent risk factor for postload soluble sE-selectin. Endothelial inflammation is present in obese prepubertal children and is mainly associated with insulin resistance and lipid levels as well as BMI.

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 microg 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.

A new efficient method for eliminating the interference effect of human serum and increasing the sensitivity and recovery rate of enzyme immunoassay.

A new, very simple method for increasing the sensitivity and recovery rate of enzyme-linked immunosorbent assay (ELISA) for the precise quantification of antigen in human serum is described. The assay design uses CATNF6A4c IgG2a monoclonal antibody and biotinylated anti-human tumor necrosis factor-alpha (hTNF-alpha) polyclonal mouse IgG as the capture and tracer antibodies, respectively. The assay is completed within 4 hours at room temperature and is capable of detecting both recombinant and native human TNF-alpha. The assay incorporates the use of saturated ammonium sulfate (SAS) as a component of the dilution buffer to amplify the resultant signal from antigen containing human serum and eliminating the endogenous interference of native human serum. SAS worked optimally at the final concentrations, ranging from 1.2% to 11%. The addition of SAS to the dilution buffer resulted in a dramatic increase in both sensitivity and recovery rate of the ELISA. The results demonstrated that 50 microL of dilution buffer, containing SAS, enabled the precise quantification of human TNF-alpha in 100 microL of human serum samples and eliminated the interference of native serum, which seemed to be related to complement proteins. Therefore, dilution buffer containing SAS, at a defined concentration, seemed to be a potential candidate for resolving sensitivity and recovery problems usually encountered in immunoassays when measurement was performed with native serum samples. The proposed technique provides an easy, practical, and consistent method for ELISA when using human native serum.

Fc-dependent monoclonal IgG1-mediated suppression of antibody response.

It has been known for a long time that passively administered antibodies (Abs) or immune complexes regulate the immune response to their specific antigen (Ag). IgG may sometimes suppress the humoral immune response against soluble antigens. The exact mechanism behind this phenomenon has not been understood yet and the requirement for the Fc part is still a matter of controversy. The present study was undertaken to clarify whether there is a true IgG-mediated Fc-dependent suppression of the immune response. Antigen and monoclonal antibody (mAb) used in this study were recombinant human interferon gamma (r-hIFN-gamma) and mouse monoclonal antibodies specific for human IFN-gamma [anti-hIFN-gamma mAb (CAy-IFNgamma38)] respectively. An intact IgG-free preparation of Fab plus various Fc fragments was prepared from papain-digested CAy-IFNgamma38. Ag/IgG and Ag/Fab complexes were prepared at various molar ratios. Keeping the Ag doses constant, mice were immunized either with Ag, Ag/IgG or Ag/Fab complexes. Primary immunization and the boosting were performed with the samples in complete and incomplete Freund's adjuvants respectively. Specific antibody levels were measured by an ELISA. Immunization performed with Ag/Fab complexes even at a molar ratio of 1:1.36 did not result in marked suppression of the response when compared to that of Ag only-immunization. In contrast, Ag/IgG complexes resulted in nearly 90% suppression of the antibody response. Our observations suggest that Fc part of IgG molecule plays a crucial role in suppression of the in vivo antibody response against the Ag when complexed with intact IgG.

Development of an enzyme-linked immunoassay for sensitive detection of native and recombinant human interferon-gamma using whole IgG fraction as polyclonal tracer.

Monoclonal antibodies (mAbs) and polyclonal antibodies (pAbs) against human interferon gamma (IFN-gamma) were produced and used for development of a sensitive enzyme-linked immunosorbent assay (ELISA) for the detection and quantitation of native and recombinant human IFN-gamma in tissue culture fluid and human sera. The human IFN-gamma ELISA was constructed using mAb CAy-IFNg111 as the capture antibody (Ab) and biotinylated polyclonal mouse immunoglobulin G (IgG) as the tracer Ab. The assay is completed within 4 hr at room temperature (RT). The human IFN-gamma ELISA worked in tissue culture medium and human serum and was capable of detecting both recombinant and native human IFN-gamma. The assay dynamic range extended from 16 to 1000 pg/mL and the sensitivity level was less than 3 pg/mL of human IFN-gamma with averaged intra- and inter-assay variation coefficients less than 8% for both. The results demonstrated that without the need of an antigen-affinity purification, biotinylation of protein G-purified pAb, obtained from 1 mL of mouse blood, was sufficient for constructing the tracer reagent for the establishment of a highly sensitive ELISA (40,000 test) for the quantitative detection of native and recombinant human IFN-gamma in culture supernatant and human sera.

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.

Determination of intracellular efficacies of azithromycin against Leishmania major infection in human neutrophils in vitro.

Azithromycin is one of a new class of antibiotics known as azalides. Azithromycin has high tissue affinity and this feature is thought to be due to the presence of two basic tertiary amine groups. Leishmania major, one of the causative agents of cutaneous leishmaniosis, is an obligate intracellular parasite. In this in vitro study, the potential anti-leishmanial effect of azithromycin upon intracellular forms namely the amastigote of L. major in mice peritoneal macrophages was investigated. L. major promastigotes were propagated in RPMI-1640 supplemented with 20% fetal calf serum in the log phase. The percentage of phagocytosis and microbiacidal activity of azithromycin on macrophages was assessed in the control and study groups by fluorescence microscopy, using acridine orange. Our results showed that at all the concentrations used (0.05, 0.1, 0.3, 0.6 microg ml(-1)) azithromycin had no inhibitory effect on the phagocytic capacity of mouse peritoneal macrophages. Although no significant difference was observed for leishmaniacidal activity between the study and the control groups at a concentration of 0.05 microg ml(-1) (p>0.05), a significant (p<0.05) increase in leishmaniacidal activity was detected at 0.1, 0.3 and 0.6 microg ml(-1). As a result, azithromycin does not provide any contribution to the phagocytosis of L. major promastigotes in macrophages in vitro, but it increases the intracellular killing rates of amastigotes. These results suggest that it has a potential anti-leishmanial effect, and may provide a significant advantage in the treatment of the disease.

Effect of mucosal immunomodulation with fed cholera toxin on healing of experimental colonic anastomosis.

PURPOSE: The aim of this study was to investigate in rats whether preoperative orogastric administration of low doses of cholera toxin would influence the mechanical strength of experimental colonic anastomosis on the basis of the gut mucosal immunomodulation effect of this antigen. METHODS: The cholera toxin group (n = 14) was fed 10 microg of cholera toxin in phosphate-buffered saline three times before surgery at 10-day intervals, whereas the controls (n = 14) received phosphate-buffered saline only. Twenty-four hours after the last dose of cholera toxin (or placebo in control group), the animals underwent left colonic transection and anastomosis. Seven days after colonic transection-anastomosis, the bursting pressure of the anastomotic segment was recorded in situ. Perianastomotic and extra-anastomotic tissue samples were obtained for measurements of tissue transforming growth factor-beta, interleukin-6, and interferon-gamma levels with enzyme-linked immunosorbent assay. RESULTS: Cholera toxin administration resulted in a significantly higher bursting pressure than in the control group (165.78 +/- 12.37 vs. 138.4 +/- 7.87 mmHg; P < 0.001). Compared with the control group, the heightened mechanical strength of colonic anastomosis provided by cholera toxin was associated with significant increases in the perianastomotic tissue levels of transforming growth factor-beta (199.34 +/- 24.85 vs. 70.66 +/- 10.63 pg/ml; P < 0.001) and interleukin-6 (439.31 +/- 95.14 vs. 289.57 +/- 96.59 pg/ml; P = 0.001), whereas interferon-gamma was significantly lower (174.04 +/- 44.82 vs. 219.00 +/- 31.35 pg/ml; P < 0.05). This cytokine pattern induced by cholera toxin in the wound milieu was also found to be similar in the extra-anastomotic colon. CONCLUSION: The mechanical strength of uncomplicated experimental colonic anastomosis increased significantly with gut mucosal immunomodulation with repeated low preoperative doses of cholera toxin. This enhanced healing had significant positive correlation with the colonic tissue level of transforming growth factor-beta and inverse correlation with interferon-gamma. If the relevant dose regimen is identified and its safety is assured in humans, gut mucosal immunomodulation might provide an efficient, safe, and inexpensive tool to improve surgical outcome in colorectal surgery, particularly in high-risk situations.