Study on Cloning and Expression of TNF-α Variants in E. coli: Production, Purification, and Interaction with Anti-TNF-α Inhibitors.

BACKGROUND: TNF-α is a proinflammatory cytokine and plays a role in cell proliferation, differentiation, survival, and death pathways. When administered at high doses, it may cause damage to the tumor vasculature, thereby increasing the permeability of the blood vessels. Therefore, monitoring the dose and the response of the TNF-α molecule is essential for patients' health. OBJECTIVES: This study aimed to clone, express, and purify the active form of the TNF-α protein, which can interact with various anti-TNF-α inhibitors with high efficiency. METHODS: Recombinant DNA technology was used to clone three different versions of codon-optimized human TNF-α sequences to E. coli. Colony PCR protocol was used for verification and produced proteins were analyzed through SDS-PAGE and western blot. Size exclusion chromatography was used to purify sTNF-α. ELISA techniques were used to analyze and compare binding efficiency of sTNF-α against three different standards. RESULTS: Under native condition (25°C), interaction between sTNF-α and anti-TNF-α antibody was 3,970, compared to positive control. The interaction was 0,587, whereas it was 0,535 for TNF- α and anti-TNF-α antibodies under denaturing conditions (37°C). F7 of sTNF-α (920 µg/mL) had the same/higher binding efficiency to adalimumab, etanercept, and infliximab, compared to commercial TNF-α. CONCLUSION: This study was the first to analyze binding efficiency of homemade sTNF-α protein against three major TNF-α inhibitors (adalimumab, etanercept, and infliximab) in a single study. The high binding efficiency of sTNF-α with adalimumab, etanercept, and infliximab, evidenced in this study supports the feasibility of its use in therapeutic applications, contributing to more sustainable, cost-effective, and independent healthcare system.

[Demonstration of ß-1,2 mannan structures expressed on the cell wall of Candida albicans yeast form but not on the hyphal form by using monoclonal antibodies]. / Candida albicans maya hücre duvarinda eksprese edilip hif duvarinda edilmeyen ß-1,2 mannan yapilarinin monoklonal antikorlar ile gösterilmesi.

Candida albicans is a polymorphic fungus that may be observed as both commensal and opportunistic pathogen in humans. As one of the major components of Candida cell wall structure, mannan plays an important role in the fungus-host cell interaction and in virulence. The ability to switch from yeast to hypha form of microorganism is crutial in the development of C.albicans infections. Hyphal form has different antigenic properties compared to yeast form and structural changes occur in the yeast cell wall during transition from yeast to hypha form. Although there are several factors associated with this transition process, sufficient information is not available. The aim of this study was to investigate the change of configuration in mannan structure found in C.albicans cell wall by using monoclonal antibodies. C.albicans (NIHA 207) serotype A strains were used as test strains throughout the study, together with Salmonella choleraesuis 211 and Salmonella infantis as controls with similar cell wall structures to that of C.albicans. Cultures were maintained on YPD-agar medium by incubating at 28°C for yeast forms, and on YPD-broth medium in a shaking incubator at 37°C for 3-4 hours for the growth of hyphal forms. Cells were harvested in the exponential phase, and after being washed, the mannan content from C.albicans were extracted from pellet by heating in 20 mM sodium citrate buffer for 90 minutes at 125°C. Hybridoma technique was used for the production of monoclonal antibodies. After immunizing the Balb/C mice with antigen, the splenocytes were harvested and fusion was performed between spleen cells and F0 myeloma cells. The clones grown in HAT medium were screened for the presence of antibody producing hybrid cells by ELISA method. The antibody isotypes were determined by using a commercial kit (Pierce Biotechnology, ABD). The culture supernatants which contained monoclonal antibodies were collected and purified according to the ammonium sulphate method. Sandwich ELISA and immunofluorescence (IF) methods have been used to detect the experimental reactions. In our study, highly specific class IgM murine monoclonal antibodies (mAb-2B7) against C.albicans yeast cell wall were obtained from clone 2B7. These antibodies cross-reacted with S.choleraesuis 211 and S.infantis bacteria sharing similar cell wall structure of C.albicans. The existence of mannan ß-1,2 bonds on the surface of C.albicans yeast form was confirmed with a commercial monoclonal antibody (mAb-ACMK-1; Matriks Biotek(®), Turkey) specific for those bonds. Besides, mAb-ACMK-1 interacted with C.albicans yeast form and gave intense fluorescence (high positive reaction) in IF method, but no fluorescence (negative) was detected with hyphal form. This data, obtained for the first time with this study, indicates that the mannan ß-1,2 bonds are either found infrequently or none in the fungal hyphal wall. Although both monoclonal antibodies recognize the mannan antigen, mAb-2B7 reacted with S.choleraesuis 211, while mAb-ACMK-1 did not, due to the difference of epitope specificity. In conclusion, monoclonal antibodies may facilitate the characterization of antigenic structures of Candida, which will lead for the identification of new determinants that may increase the sensitivity and specificity of commercial tests used for mannan detection in serum.

[Characterization of Toxoplasma gondii proteins from various strains]. / Toxoplasma gondii'nin çesitli suslarinin proteinlerinin karakterizasyonu.

OBJECTIVE: Toxoplasma gondii the causative agent of toxoplasmosis, is a worldwide intracellular protozoan parasite that infects all warm blooded animals including humans. It can be devastating to immunocompromised humans and congenital transmission may result in severe clinical spectrum. It causes economic losses due to abortus in animals. Toxoplasmosis diagnosis depends on direct and indirect methods. Besides the Sabin-Feldman test, which is accepted to be the reference test, serologic tests such as ELISA and immunofluorescence antibody tests are means of indirect diagnosis. As detected antibodies in serologic tests are correlated with antigens that cause their synthesis, it is important to know different proteins of different strains. In this study RH, Ankara and TS-4 strains were used and differences between their proteins were examined. METHODS: RH and Ankara strains were inoculated into the peritoneal cavity of mice. TS- 4 strain was produced in Vero cell culture. Tachyzoites collected by peritoneal wash were lysed and lyophilised. This was run on SDS-PAGE gel and protein bands were compared with a standard protein ladder after staining with polychromatic silver stain. RESULTS: It was observed that, while Ankara and RH strains had dense bands between 60-70 kDa and at 15 kDa, the most prominent bands of TS-4 strain were 60 ve 115 kDa bands. CONCLUSION: RH and Ankara strains have the same protein bands while TS-4 strain has different and fewer protein bands than the others.

Evaluation of in vitro Probiotic Potential of Pediococcus pentosaceus OZF Isolated from Human Breast Milk.

This study was conducted to evaluate the probiotic properties of Pediococcus pentosaceus OZF isolated from human breast milk. The results obtained so far suggest that the strain is resistant to low pH, bile salt, pepsin and pancreatin, so it could survive while passing through the upper part of the gastrointestinal tract and reveal its potential probiotic action on host organism. The strain was non-pathogenic (γ-hemolytic), produced anti-Listerial bacteriocin, exhibited a strong autoaggregating phenotype (85.71%) and demonstrated 6.26 and 12.99% coaggregation with Salmonella enterica serotype Typhimurium SL 1344 and Escherichia coli LMG 3083 (ETEC), respectively. The degree of adhesion of Ped. pentosaceus OZF to the human Caco-2 cell line was investigated and when compared to the adhesion of pathogenic strains tested, it was shown to inhibit the growth of human enterotoxigenic E. coli LMG 3083 (ETEC) and of Salm. Typhimurium SL 1344. Ped. pentosaceus OZF seems to adhere to human intestinal cells via mechanisms that involve different combinations of carbohydrate and lipid factors on the bacteria and eukaryotic cell surface. The percentage of adhesion to n-hexadecane was 34% showing that the surface was rather hydrophilic. Higher affinity displayed by Ped. pentosaceus OZF for chloroform demonstrates the basic property of a cell, which may be due to the presence of carboxylic groups on the cell surface.

Aflatoxin exposure in viral hepatitis patients in Turkey.

BACKGROUND/AIMS: Hepatocellular carcinoma is the fifth most common cancer and a major public health problem worldwide. Differences in distribution of hepatocellular carcinoma incidence are probably due to different levels of exposure to hepatocellular carcinoma risk factors: chronic infections with hepatitis B virus (HBV) and aflatoxin exposure in developing countries, and smoking and alcohol abuse in developed countries. Aflatoxin is one of the most important of the environmental toxins that contribute to the pathogenesis of hepatocellular carcinoma, especially in the regions where dietary foodstuffs (peanuts, corn, Brazil nuts, pistachios, spices and figs) are highly contaminated. High aflatoxin levels have been shown in the foodstuffs that are produced in our country. The specific aim of this study was to assess the rate of aflatoxin exposure and to determine some clues about aflatoxin metabolism by measuring and comparing the levels of carcinogenic forms in healthy subjects, in different stages of viral disease, and in different viral hepatitis types. METHODS: This was a cross-sectional observational, single-center study. A total of 203 (male/female: 119/84) viral hepatitis patients who were consecutively admitted to Ankara University, School of Medicine, Gastroenterology Clinic, between January 2006 and June 2007 were enrolled into the study. Sixty-two healthy subjects (male/female: 33/29) with normal blood chemistry and negative viral serology served as controls. Chemical forms AFB1, AFB2, AFG1, and AFG2 were assessed in plasma of study participants by high-performance liquid chromatography. RESULTS: AFB1, AFB2, AFG1, and AFG2 were detected in 24.6%, 17.2%, 22.7%, 18.2% of the 203 patients, respectively, and were significantly higher than in the control group for all chemical forms. Percentage of AFB1-positive patients was significantly higher than in the control group irrespective of disease stage. There was no significant difference between chronic infected patients, cirrhotic patients and patients with Hepatocellular carcinoma with respect to percentage of aflatoxin-positive individuals. CONCLUSIONS: With this study, we have documented that in viral hepatitis patients, aflatoxin exposure is significantly higher than in healthy subjects in Turkey and it may play an important role in the development of hepatocellular carcinoma. Thus, large studies exploring the relation between aflatoxin exposure, viral hepatitis status, and risk of hepatocellular carcinoma development are needed.

Erythropoietin reduces lipopolysaccharide-induced cell Damage and midkine secretion in U937 human histiocytic lymphoma cells.

INTRODUCTION: Erythropoietin (EPO) is a haematopoietic stimulatory protein that is used to treat anaemia in patients on dialysis. In addition, EPO has been shown to have anti-inflammatory properties, which may be important as dialysis patients tend to exist within a chronic, low-grade inflammatory state, and tend to be more susceptible to infections. It has been suggested that EPO has direct immunomodulatory potency on monocytes/macrophages. METHODS: In this study, we aimed to clarify the effects of EPO during the inflammatory processes in human mononuclear phagocytic cells by monitoring the secretion of the following cytokines; midkine, tumour necrosis factor alpha (TNF-alpha), and interleukin-6 (IL-6). For this purpose, U937 human histiocytic lymphoma cell lines were used. Time-dependent effects of varying doses of EPO (0.1 to 50 IU/ml) treatment during lipopolysaccharide (LPS)-mediated cytotoxicity was measured by 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide test. LPS-stimulated midkine secretion was measured immunohistochemically and quantification of TNF-alpha, IL-6 and midkine secretion was achieved by ELISA. RESULTS: EPO treatment prevented the direct toxic effects of LPS on the U937 cells. TNF-alpha, IL-6 and midkine secretions were found to increase in the U937 cells in response to LPS treatment. Interleukin-6 response was varied in a doseand time-dependent manner. CONCLUSION: Treatment with EPO significantly inhibits the LPS-induced secretion of midkine and TNF-alpha regardless of the dosage. The data presented here provide the first evidence to indicate that EPO treatment directly reverses some of the cytotoxic and secretory effects of LPS in mononuclear cells. Inhibition of midkine secretion might be responsible for the anti-inflammatory role of EPO.

Midkine secretion protects Hep3B cells from cadmium induced cellular damage.

AIM: To evaluate role of midkine secretion during Cadmium (Cd) exposure in the human hepatocyte cell line Hep3B cells. METHODS: Different dosages of Cd (0.5-1-5-10 microg/mL) were applied to Hep3B cells and their effects to apoptosis, lactate dehydrogenase (LDH) leakage and midkine secretion were evaluated as time dependent manner. Same experiments were repeated with exogenously applied midkine (250-5000 pg/mL) and/or 5 microg/mL Cd. RESULTS: Cd exposure induced prominent apoptosis and LDH leakage beginning from lower dosages at the 48th h. Cd induced midkine secretion with higher dosages (P < 0.001), (control, Cd 0.5-1-5-10 microg/mL respectively: 1123 +/- 73, 1157 +/- 63, 1242 +/- 90, 1886 +/- 175, 1712 +/- 166 pg/mL). Exogenous 500-5000 pg/mL midkine application during 5 microg/mL Cd toxicity prevented caspase-3 activation (control, Cd toxicity, 250, 500, 1000, 2500, 5000 pg/mL midkine+ Cd toxicity, respectively: 374 +/- 64, 1786 +/- 156, 1545 +/- 179, 1203 +/- 113, 974 +/- 116, 646 +/- 56, 556 +/- 63 cfu) LDH leakage and cell death in Hep3B cells (P < 0.001). CONCLUSION: Our results showed that midkine secretion from Hep3B cells during Cd exposure protects liver cells from Cd induced cellular damage. Midkine has anti-apoptotic and cytoprotective role during Cd toxicity. Further studies are needed to explain the mechanism of midkine secretion and cytoprotective role of midkine during Cd exposure. Midkine may be a promising therapeutic agent in different toxic hepatic diseases.

The effect of K-ATP channel blockage during erythropoietin treatment in renal ischemia-reperfusion injury.

ATP dependent K channels (K-ATP) take part in the Erythropoietin (EPO) induced cardioprotection but these channel activations have role in cytoprotective role of EPO in the renal ischemia reperfusion (IR) damage is still unknown. For this purpose rats were pretreated with EPO (500 IU/kg) and/or K-ATP channel blocker glibenclamide (40 mM/kg) i.p. before bilateral renal IR damage. Renal tissues were used for histological examination and measurement of caspase-3 and TNF-alpha levels. Renal functions were evaluated by glomerular filtration rate (GFR) fractional excretion of sodium (FENa) and potassium (FEK). Renal TNF-alpha and caspase-3 levels were decreased in both glibenclamide and EPO-treated IR rats compared to untreated rats. The protection afforded by the pretreatment with EPO alone was greater than that of administering glibenclamide alone. Application of glibenclamide at the same time partly abolished the cytoprotective effect of EPO treatment. K-ATP mediated cytoprotection is not the main mechanism of protective effect of EPO.

Erythropoietin attenuates hydrogen peroxide-induced damage of hepatocytes.

BACKGROUND/AIMS: High levels of hydrogen peroxide (H2O2) are observed during inflammatory and ischemic states of the liver and usually lead to cellular dysfunction and cytotoxicity. Recently, it has been reported that erythropoietin and mitochondrial K (ATP) channel openers have a protective effect via a pharmacological preconditioning action during ischemia reperfusion injury of the liver and heart. However, it remains unclear as to whether K (ATP) channel blockers can reduce the protective effect of erythropoietin in the H2O2-induced injury of hepatocytes. METHODS: To determine whether erythropoietin treatment decreases H2O2-induced toxicity, we used human hepatocyte cell line Hep3B for assays. Cells were pretreated with different dosages of erythropoietin (0.1-1-10-50 IU/ml) 2 h before H2O2 application. For determination of effects of blockage of mitochondrial K (ATP) channels during erythropoietin treatment, glibenclamide treatment was applied to the medium 2 h before H2O2 toxicity. Cell number, lactate dehydrogenase and caspase- 3 levels were measured in erythropoietin, glibenclamide and/or H2O2-treated groups. RESULTS: Erythropoietin treatment significantly increased cell number at the 24th and 48th h compared to the control group. H2O2 application induced apoptosis and lactate dehydrogenase release from Hep3B cells and decreased cell number. Erythropoietin prevents H2O2 toxicity in hepatocytes. The K channel inhibitor glibenclamide decreased the cytoproliferative and cytoprotective effect of erythropoietin during H2O2 toxicity of Hep3B cells. CONCLUSIONS: Erythropoietin treatment may be considered as a therapeutic agent during oxidative injuries of hepatocytes and its cytoprotective effect is abolished by glibenclamide.

Protective role of erythropoietin during testicular torsion of the rats.

Testicular torsion is an important clinical urgency. Similar mechanisms occurred after detorsion of the affected testis as in the ischemia reperfusion (I/R) damage. This study was designed to investigate the effects of erythropoietin (EPO) treatment after unilateral testicular torsion. Fifty male Sprague-Dawley rats were divided into five groups. Group 1 underwent a sham operation of the right testis under general anesthesia. Group 2 was same as sham, and EPO (3,000 IU/kg) infused i.p., group 3 underwent a similar operation but the right testis was rotated 720 degrees clockwise for 1 h, maintained by fixing the testis to the scrotum, and saline infused during the procedure. Group 4 underwent similar torsion but EPO was infused half an hour before the detorsion procedure, and in group 5, EPO was infused after detorsion procedure. Four hours after detorsion, ipsilateral and contralateral testes were taken out for evaluation. Treatment with EPO improved testicular structures in the ipsilateral testis but improvement was less in the contralateral testis histologically, but EPO treatment decreased germ cell apoptosis in both testes following testicular IR. TNF-alpha, IL-1beta, IL-6 and nitrite levels decreased after EPO treatment especially in the ipsilateral testis. We conclude that testicular I/R causes an increase in germ cell apoptosis both in the ipsilateral and contralateral testes. Erythropoietin has antiapoptotic and anti-inflammatory effects following testicular torsion.

Erythropoietin changes contractility, cAMP, and nitrite levels of isolated rat hearts.

There is enough evidence that erythropoietin (EPO) may be involved in cardiovascular function. Therefore we have investigated the possible effects of EPO on left ventricular developed pressure, +dP/dt(max), heart rate, tissue cAMP, and nitrite levels. Isolated rat hearts were perfused under constant flow (10 ml/min) conditions with modified Krebs-Henseleit solution and recombinant human erythropoietin at doses of 100, 200, 500, and 1,000 IU/kg was administered as bolus injections. EPO at 100 IU/kg decreased, but higher doses (500 and 1,000 IU/kg) raised the developed pressure and +dP/dt(max). However, it did not affect heart rate or coronary perfusion pressure when all the respective doses were applied. EPO at 100 IU/kg increased nitrite, and at 1,000 IU/kg it raised cAMP. Our results suggest that EPO may produce dose-dependently negative and positive inotropic effects on myocardial contractility in isolated rat hearts. NO and cAMP may be involved in negative and positive inotropic effects of EPO, respectively.

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.

Characterization of the hypothermic component of LPS-induced dual thermoregulatory response in rats.

We have previously shown that Escherichia coli O111:B4 serotype lipopolysaccharide (LPS) produced a dual change in rectal temperature (Tb), in which hypothermia preceded fever at subthermoneutral ambient temperature (Tamb; 24-26 degrees C) in rats. In this study, the characteristics of the initial hypothermic response were evaluated. Hypothermia was significant when LPS (50 microg/kg, i.p.) was injected at thermoneutral Tamb (30 degrees C). There was no heat loss through tail skin during hypothermia. The open field activity of the rats did not change during this period. However, serum levels of tumor necrosis factor-alpha (TNF-alpha) elevated at the beginning of the hypothermia, whereas serum levels of interleukin (IL)-1beta and interferon (IFN)-gamma remained unchanged. A nonselective cyclooxygenase inhibitor (indomethacin, 5 mg/kg, s.c.) inhibited hypothermia and serum TNF-alpha elevation, which resulted in an acceleration of the subsequent pyrogenic response. Moreover, a nonselective inhibitor of nitric oxide synthase (nitro L-arginine methyl ester (L-NAME), 10 mg/kg, s.c.) not only abolished fever but also prolonged the initial hypothermic response. These data suggest that the hypothermic component of low dose LPS-induced dual response is a regulated decrease in Tb. The data also suggest that hypothermia and fever may occur independently as two different thermoregulatory strategies against immune challenge in rats.

Effects of selective cyclooxygenase enzyme inhibitors on lipopolysaccharide-induced dual thermoregulatory changes in rats.

The effects of selective cyclooxygenase-1 and cyclooxygenase-2 inhibitors (valeryl salicylate and SC-58236, respectively) on Escherichia coli O111:B4 lipopolysaccharide (LPS)-induced dual thermoregulatory changes and serum tumor necrosis factor-alpha elevation were investigated in rats. LPS (50 microg/kg, intraperitoneal) produced an initial hypothermia that was then followed by fever. Serum tumor necrosis factor-alpha levels elevated at the initial phase of hypothermia. Valeryl salicylate injections (20, 40, and 80 mg/kg, subcutaneous [s.c.]) completely inhibited hypothermia without any effect on the elevated serum tumor necrosis factor-alpha levels and on the subsequent fever. On the other hand, SC-58236 injections (10, 20, and 40 mg/kg, s.c.) only partially abolished the hypothermia. SC-58236 had no effect on the initiation of fever, however completely inhibited the maintenance of fever. The serum tumor necrosis factor-alpha elevation was not reduced by SC-58236 treatment. The combination of valeryl salicylate and SC-58236 also failed to inhibit the initiation of fever. These findings suggest that cycloxygenase-1 may have a predominant role for the development of LPS-induced hypothermia, but cyclooxygenase-1 does not seem to be involved in the mediation of LPS-induced fever. Meanwhile, cyclooxgenase-2 may be critical for the late phase rather than the initiation of the fever response in rats.

Nimesulide and diclofenac inhibit lipopolysaccharide-induced hypothermia and tumour necrosis factor-alpha elevation in rats.

The effects of nimesulide and diclofenac on lipopolysaccharide (LPS)-induced rectal temperature changes and serum tumour necrosis factor (TNF)-alpha elevation were investigated in rats. LPS (Escherichia coli O111:B4; 50 microg/kg, intraperitoneally) produces a dual body temperature response, in which initial hypothermia precedes fever. Serum TNF-alpha levels rise during the initial phase of the induced hypothermia. Nimesulide, a preferential inhibitor of cyclooxygenase-2 (0.05, 0.5 or 1 mg/kg, subcutaneously) completely abolished the hypothermia, resulting in an acceleration of the fever phase. However, the peak and plateau phases of fever were not changed by nimesulide treatment. Nimesulide (0.5 mg/kg) partially prevented serum TNF-alpha elevation. The non-selective cyclooxygenase inhibitor diclofenac inhibited hypothermia at all doses tested (0.03, 0.3 or 3 mg/kg, subcutaneously) although fever was completely abolished at the 3 mg/kg dose only. Diclofenac also partially abolished the elevation in serum TNF-alpha levels, but at the highest dose only (3 mg/kg). These data suggest that nimesulide and diclofenac can preferentially inhibit LPS-induced hypothermia at doses that do not abolish fever in rats. Both these drugs also reduced elevated TNF-alpha levels, a fact which may, at least partly, explain the antihypothermic effect of nimesulide.