Characterization of golimumab,a human monoclonal antibody specific for human tumor necrosis factor α

We prepared and characterized golimumab, a human IgG1 tumor necrosis factor alpha (TNFα) antagonist monoclonal antibody chosen for clinical development based on its molecular properties. Golimumab was compared with infliximab, adalimumab and etanercept for affinity and in vitro TNFα neutralization. The affinity of golimumab for soluble human TNFα, as determined by surface plasmon resonance, was similar to that of etanercept (18 pM versus 11 pM), greater than that of infliximab (44 pM) and significantly greater than that of adalimumab (127 pM, p = 0.018). The concentration of golimumab necessary to neutralize TNFα-induced E-selectin expression on human endothelial cells by 50% was significantly less than those for infliximab (3.2-fold; p = 0.017) and adalimumab (3.3-fold; p = 0.008) and comparable to that for etanercept. The conformational stability of golimumab was greater than that of infliximab (primary melting temperature [Tm] 74.8°C vs. 69.5°C) as assessed by differential scanning calorimetry. In addition, golimumab showed minimal aggregation over the intended shelf life when formulated as a high concentration liquid product (100 mg/mL) for subcutaneous administration. In vivo, golimumab at doses of 1 and 10 mg/kg significantly delayed disease progression in a mouse model of human TNFα-induced arthritis when compared with untreated mice, while infliximab was effective only at 10 mg/kg. Golimumab also significantly reduced histological scores for arthritis severity and cartilage damage, as well as serum levels of pro-inflammatory cytokines and chemokines associated with arthritis. Thus, we have demonstrated that golimumab is a highly stable human monoclonal antibody with high affinity and capacity to neutralize human TNFα in vitro and in vivo.Key words: TNF, golimumab, neutralization, affinity, bioassay, arthritis, stability, solubility     

Characterization of prostate-tissue-directed monoclonal antibody, α-Pro 13

Summary The -Pro 13-secreting hybridoma was produced by immunizing mice with an equal mixture of PC-3, DU145, and LNCaP established prostatic carcinoma cell lines. The specificity of -Pro 13 monoclonal antibody was evaluated by the criteria of differential binding to cultured cells; differential binding to extracts of malignant prostate, nonmalignant prostate, and malignant and nonmalignant tissues of various histiotypes in solid phase radioimmunoassay; and by immunoperoxidase staining of primary surgical tissues of varied histiotypes. The data generated by multiple assay investigation indicate that -Pro 13 exhibits preferential binding to the ductal epithelium of prostate tissue; immunoperoxidase evaluation indicates a considerable heterogeneity of staining of ductal epithelial cells. The most prevalent cross-reactivity of -Pro 13 monoclonal antibody with non-prostate tissue occurs with blood vessel endothelium of restricted tissues. Electrophoretic analysis of immunoprecipitates from radioiodinated prostatic tumor extracts indicates that the molecule recognized by -Pro 13 is of 120,000 dalton apparent nonreduced molecular weight. Under reducing conditions, the antigen (p40) consists of a major component of 40,000 dalton apparent MW and a minor component of 17,000 dalton MW. p40 has an isoelectric point of 3.5–4.5. The antigen is intrinsically stable on the PC-3 cell surface; its release into spent culture medium is negligible. p40 is also stable upon complexation with -Pro 13 antibody in that it is not shed from the cell surface as an immune complex nor is it endocytosed to any extent as an immune complex.Abbreviations MIg anti-mouse immunoglobulin - IEF isoelectric focusing - kD kilodalton - MW molecular weight - PA Staphylococcus protein A - RIA radioimmunoassay - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - SCM spent culture medium     

Effector mechanism of human monocyte-mediated cytotoxicity: role of a new tumor cytotoxic factor distinct from interleukin 1 and tumor necrosis factorα

Human blood monocytes activated to the tumoricidal state were previously found to release a factor(s) responsible for tumor cell killing. The activity of the tumor cytotoxic factor(s) (TCF) was determined by release assay of radioactivity from human A375 melanoma cells. On fractionation of the supernatant of activated monocytes by Ultrogel AcA34 and TSK-G3000SW gel chromatographies two major peaks of the material with TCF activity with MWs of 30,000 and 15,000, called TCF-I and TCF-11, respectively were obtained. TCF-II could be neutralized by polyclonal anti-IL-1 antiserum, but anti-IL-1 antiserum did not neutralize either factor. TCF-I was separated by ampholine column electrofocusing into three major fractions with TCF activity at pI 5, 6 and 6.8, named TCF-1, TCF-1 and TCF-1, respectively. The cytotoxic and IL-1 activities of TCF-1 were neutralized by anti-IL-1 serum, whereas those of TCF-1 and TCF-1 were not completely neutralized by anti-IL-1 or anti-IL-1 antiserum. On DEAE ion-exchange chromatography (TSK DEAE 5PW) TCF-I gave two peaks with TCF activity (TCF-I1 and TCF-I2). TCF-I1 was slightly neutralized by anti-TNF antibody, but TCF-I2 was not affected by antisera against IL-1 and IL-1, or anti-TNF antibody, thus ruling out the possibility that tumor necrosis factor (TNF) might be involved in tumor cell killing mediated by TCF-I2. These results indicate that human monocyte-mediated cytotoxicity against human A375 melanoma cells is mediated in part by a tumor cytotoxic factor (TCF; MW, 30,000; pI 6), differing from IL-1 and TNF.     

Radioimmunodetection of human tumor xenografts by monoclonal antibody F(ab′)2 fragments

Experimental procedures are described for the radiolocalization of human tumors by murine monoclonal antibodies (MAb) in animal model systems. Visualization of tumor xenografts was clearer in nude mice as compared to experimentally immunosuppressed mice due to the higher viability of the tumors in nude mice. MAb localization in tumor tissue was greatly enhanced when F(ab′)₂ fragments rather than intact antibody molecules were used. Although tumors could be visualized with either ¹³¹I-, ¹²³I- or ¹¹¹In-labeled MAb fragments without using background subtraction, tumor-to-background ratios of radioactivity were highest for ¹³¹I-labeled fragments. ¹³¹I-labeled F(ab′)₂ fragments of eight MAb against human colorectal carcinoma, melanoma or lung carcinoma localized specifically only in those tumors that bound the MAb in vitro and not in unrelated tumors. Radiolabeled fragments of MAb with other specificities (anti-hepatitis virus MAb) did not localize in tumors. All MAb that inhibited tumor growth in nude mice effectively localized these tumors by γ-scintigraphy. On the other hand, some MAb were effective in localizing tumors but ineffective in inhibiting their growth. The ability of the specific radiolabeled F(ab′)₂ fragments to localize in tumor grafts correlated significantly with MAb binding affinity and density of antigenic sites on tumor cells together, but not with either in vitro binding parameter alone. Thus, Scatchard analysis of MAb binding to tumor cells may be an effective means to screen for MAb with tumor radiolocalization potential.     

Establishment of a monoclonal antibody for human LXRα: Detection of LXRα protein expression in human macrophages

Liver X activated receptor alpha (LXRalpha) forms a functional dimeric nuclear receptor with RXR that regulates the metabolism of several important lipids, including cholesterol and bile acids. As compared with RXR, the LXRalpha protein level in the cell is low and the LXRalpha protein itself is very hard to detect. We have previously reported that the mRNA for LXRalpha is highly expressed in human cultured macrophages. In order to confirm the presence of the LXRalpha protein in the human macrophage, we have established a monoclonal antibody against LXRalpha, K-8607. The binding of mAb K-8607 to the human LXRalpha protein was confirmed by a wide variety of different techniques, including immunoblotting, immunohistochemistry, and electrophoretic mobility shift assay (EMSA). By immunoblotting with this antibody, the presence of native LXR protein in primary cultured human macrophage was demonstrated, as was its absence in human monocytes. This monoclonal anti-LXRalpha antibody should prove to be a useful tool in the analysis of the human LXRalpha protein.     

Polyunsaturated fatty acids stimulate superoxide formation in tumor cells: A mechanism for specific cytotoxicity and a model for tumor necrosis factor?

Some neoplastic cell lines are readily killed when incubated in the presence of polyunsaturated fatty acids (PUFA). In an attempt to elucidate this phenomenon, we studied PUFA-driven superoxide (O2-) production by cultured NS-1 cells (murine lymphoid tumor cells). We find: (1) Even in the absence of added PUFA, NS-1 cells generate O2- (i.e., reduce nitroblue tetrazolium). (2) addition of PUFA increases O2- by greater than 50%. (3) Artificial loading of NS-1 cells with liposome encapsulated superoxide dismutase prevents the majority of spontaneous and PUFA-driven NBT reduction. We conclude that PUFA drives O2- generation by tumor cells, that this generation is largely intracellular, and that this phenomenon may help explain toxicity of PUFA for tumor cells.     

Characterization and functional activity of murine monoclonal antibodies specific for α1,6-glucan chain of Helicobacter pylori lipopolysaccharide

Background: The outer core region of H. pylori lipopolysaccharide (LPS) contains α1,6‐glucan previously shown to contribute to colonizing efficiency of a mouse stomach. The aim of the present study was to generate monoclonal antibodies (mAbs) specific for α1,6‐glucan and characterize their binding properties and functional activity. Materials and Methods: BALB/c mice were injected intraperitoneally with 10⁸ formalin‐fixed H. pylori O:3 0826::Kan cells 3× over 56 days to achieve significant titer. Anti‐α1,6‐glucan‐producing hybridomas were screened by indirect ELISA using purified H. pylori O:3 0826::Kan LPS. One clone, 1C4F9, was selected for further characterization. The specificities of mAbs were determined by indirect and inhibition ELISA using structurally defined H. pylori LPS and synthetic oligosaccharides, and whole‐cell indirect ELISA (WCE) of clinical isolates. They were further characterized by indirect immunofluorescent (IF) microscopy and their functional activity in vitro determined by serum bactericidal assays against wild‐type and mutant strains of H. pylori. Results: The generated anti‐α1,6‐glucan IgM, 1C4F9, has demonstrated an excellent specificity for the glucan chain containing 5 to 6 α1,6‐linked glucose residues and showed surface accessibility by IF microscopy with H. pylori cells adherent to gastric adenocarcinoma cells monolayers. Of 38 isolates from Chile, 17 strains reacted with antiglucan mAbs in WCE (OD₄₅₀ ≥ 0.2). Bactericidal activity was observed against selective wild‐type and mutant H. pylori strains exhibiting OD₄₅₀ values of ≥0.45 in WCE. Conclusions: Anti‐α1,6‐glucan mAbs could have potential application in typing and surveillance of H. pylori isolates as well as offer insights into structural requirements for the development of LPS‐based vaccine against H. pylori infections.     

Inhibitory effects of tamoxifen and tumor necrosis factor α on human glioblastoma cells

We reported previously that tumor necrosis factor α (TNFα) inhibited proliferation and invasiveness of human malignant glial cells. Because tamoxifen, an estrogen antagonist, has also been shown to inhibit growth of such cells, we hypothesized that a combination of tamoxifen and TNFα might be more effective than either reagent alone. TNFα (1–100 ng/ml) or tamoxifen (80 ng/ml-2 μg/ml) alone inhibited proliferation of a human glioblastoma cell line (WITG3) in a dose-dependent fashion; in combination, tamoxifen and TNFα yielded additive growth inhibition. Apoptotic cells characterized by nuclear fragmentation were detectable after 48 h of TNFα or tamoxifen exposure and were significantly increased by combination treatment. In non-neoplastic human astroglia and fibroblasts, proliferation was unaffected by tamoxifen, and enhanced by TNFα as previously reported. Staurosporine (2–50 nM), which has been reported to augment the effects of TNFα, was less effective than tamoxifen against WITG3 and, in addition, was markedly inhibitory to non-neoplastic glial cells. Binding studies yielded no evidence of WITG3 estrogen or progesterone receptors, nor of tamoxifen effects on TNFα receptors. Data suggest that TNFα and tamoxifen in combination display growth-regulatory properties, which (a) are more inhibitory to human glioblastoma cells than either agent alone, (b) do not affect non-neoplastic glia, (c) do not require either estrogen/ progesterone receptors or alteration of external TNFα receptors, and (d) may involve apoptosis.     

Recognition of human tumor necrosis factor α (TNF-α) by therapeutic antibody fragment: energetics and structural features

Human tumor necrosis factor α (TNF-α) exists in its functional state as a homotrimeric protein and is involved in inflammation processes and immune response of a human organism. Overproduction of TNF-α results in the development of chronic autoimmune diseases that can be successfully treated by inhibitors such as monoclonal antibodies. However, the nature of antibody-TNF-α recognition remains elusive due to insufficient understanding of its molecular driving forces. Therefore, we studied the energetics of binding of a therapeutic antibody fragment (Fab) to the native and non-native forms of TNF-α by employing calorimetric and spectroscopic methods. Global thermodynamic analysis of data obtained from the corresponding binding and urea-induced denaturation experiments has been supported by structural modeling. We demonstrate that the observed high affinity binding of Fab to TNF-α is an enthalpy-driven process due mainly to specific noncovalent interactions taking place at the TNF-α-Fab binding interface. It is coupled to entropically unfavorable conformational changes and accompanied by entropically favorable solvation contributions. Moreover, the three-state model analysis of TNF-α unfolding shows that at physiological concentrations, TNF-α may exist not only as a biologically active trimer but also as an inactive monomer. It further suggests that even small changes of TNF-α concentration could have a considerable effect on the TNF-α activity. We believe that this study sets the energetic basis for understanding of TNF-α inhibition by antibodies and its unfolding linked with the concentration-dependent activity regulation.     

Effects of natural interferon α, natural tumor necrosis factor α and their combination on human mesothelioma xenografts in nude mice

Effects of human natural interferon (nIFN) alone, human natural tumor necrosis factor (nTNF) alone and their combination (OH-1) were tested on three human mesothelioma lines implanted in nude mice. Tumors were transplanted subcutaneously by trocar on treatment day –12. nIFN was given intraperitoneally (i.p.) at a dose of 2 × 10⁷ or 2 × 10⁸ IU kg^–1 day^–1, 5 days a week for 3 weeks. nTNF was given i.p. at a dose of 2 × 10⁷ or 2 × 10⁸ U kg^–1 day^–1 in the same schedule as that of nIFN. Tumor diameters were serially measured and tumor volumes were calculated. Antitumor effects were assessed by two methods: comparison of final tumor volumes in treated and control groups (T/C), and changes in median average total tumor volume. The treatment produced no clinically discernible toxicities. nIFN had strong inhibitory activity against all three human mesothelioma lines. nTNF alone had modest activity only at the high dose used. The combination of the two produced activity essentially similar to that produced by nIFN alone. High-dose nIFN may have a role as an active agent in the treatment of patients with mesothelioma.     

Enhancement of epidermal growth factor receptor expression on glioma cells by recombinant tumor necrosis factor α

Summary Recombinant tumor necrosis factor (rTNF; optimal dose 1000 U/ml) significantly increased the density of epidermal growth factor receptor (EGF-R) in three of four glioma cell lines in culture as determined by binding analysis of anti-EGF-R monoclonal antibody (mAb) 425. Since enhancement of EGF-R expression by rTNF- was inhibited when cells were treated with the protein synthesis inhibitor cycloheximide, the effects of rTNF may be protein-synthesis-dependent. The dose of rTNF that was optimal for up-regulation of EGF-R on glioma cells did not inhibit the growth of these cells.¹²⁵I-labeled mAb 425 lysed glioma cells in culture following its internalization into the cells. After glioma cells had been treated with rTNF, the growth-inhibitory effects of the mAb were significantly enhanced, probably a reflection of the increase in EGF-R density on the tumor cell surfaces. The rTNF effects were specific to the EGF-R and did not affect unrelated glioma-associated antigens. In our previous clinical trials,¹²⁵I-labeled mAb 425 showed immunotherapeutic effects in glioma patients. The present study provides the basis for considerations of combined immunotherapy of glioma patients with¹²⁵I-labeled mAb 425 and rTNF.     

Mutational analysis of human tumor necrosis factor-α

To understand the structure-function relationship of human tumor necrosis factor- (TNF-), mutational analysis was carried out on the lower regions (regions 1–6) of the molecule. The muteins were prepared as a soluble form by using a chaperonin co-expression system and the cytotoxic activities of the purified muteins were evaluated on TNF-sensitive murine fibrosarcoma L929 cells. Three regions (regions 1, 2 & 4) were found where mutations significantly influenced the bioactivity. In region 1 (residues 1–10), the number of deleted residues and the positioning of positive charges are important to achieve a maximum activity and in region 4 (residues 84–88), introduction of charged residues in one of the positions 86–88 significantly increased the cytotoxic activity. On the other hand, any mutation introduced in region 2 (residues 37–41) had a deleterious effect. The present study provides a structural basis for the design of highly potent TNF- as a therapeutic agent.Revisions requested 18 October 2004; Revisions received 22 November 2004     

Grape powder extract attenuates tumor necrosis factor α-mediated inflammation and insulin resistance in primary cultures of human adipocytes

Grapes are rich in phenolic phytochemicals that possess anti-oxidant and anti-inflammatory properties. However, the ability of grape powder extract (GPE) to prevent inflammation and insulin resistance in human adipocytes caused by tumor necrosis factor α (TNFα), a cytokine elevated in plasma and white adipose tissue (WAT) of obese, diabetic individuals, is unknown. Therefore, we examined the effects of GPE on markers of inflammation and insulin resistance in primary cultures of newly differentiated human adipocytes treated with TNFα. We found that GPE attenuated TNFα-induced expression of inflammatory genes including interleukin (IL)-6, IL-1β, IL-8, monocyte chemoattractant protein (MCP)-1, cyclooxygenase (COX)-2 and Toll-like receptor (TLR)-2. GPE attenuated TNFα-mediated activation of extracellular signal-related kinase (ERK) and c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1, i.e., c-Jun). GPE also attenuated TNFα-mediated IκBα degradation and nuclear factor-kappa B (NF-κB) activity. Finally, GPE prevented TNFα-induced expression of protein tyrosine phosphatase (PTP)-1B and phosphorylation of serine residue 307 of insulin receptor substrate-1 (IRS-1), which are negative regulators of insulin sensitivity, and suppression of insulin-stimulated glucose uptake. Taken together, these data demonstrate that GPE attenuates TNFα-mediated inflammation and insulin resistance in human adipocytes, possibly by suppressing the activation of ERK, JNK, c-Jun and NF-κB.     

Phase I study of recombinant human tumor necrosis factor α in advanced malignant disease

Summary A phase I study with recombinant human tumor necrosis factor (rhuTNF-; Knoll AG, Ludwigshafen, FRG) in patients with advanced malignant disease was undertaken to evaluate drug toxicity (organ specifity, time course, predictability, reversibility, maximal tolerated dose), effectiveness, antigenicity and pharmacokinetics. TNF was administered as a test dose followed by daily i.v. infusions for 5 days, every 3 weeks (single i.v. infusion lasting 10 min, TNF dissolved in 50 ml 5% human albumin). Dosage was increased in groups of 3 or 4 patients from 0.04 mg/m² to 0.28 mg/m². A total of 19 patients with different cancers, including seven large-bowel carcinomas, three chronic myelogenous leukemias, three hypernephromas, two small-cell lung cancers, one malignant melanoma, one malignant lymphoma, one rhabdomyosarcoma and one fibrosarcoma were treated. Major side-effects were chills and fever (maximum 40.4°C, median 38.7°C, 19/19), headache (12/19), nausea and vomiting (12/19) and pronounced (>20%) hypotension (4/19). Acute side-effects could be diminished by paracetamol or indomethacin pretreatment, and with one possible exception no tachyphylaxis to TNF was noted. Mild renal toxicity was seen during TNF treatment. Pharmacokinetic studies showed a serum half-life (t _1/2) ranging from 11 min to 17 min for doses from 0.04 mg/m² to 0.16 mg/m² and prolonged clearance with t _1/2 ranging from 54 min to 70 min in the 0.20–0.28 mg/m² dose range. No objective antitumor effects were observed in this phase I study.     

Murine monoclonal antibody recognizing human α(1,3/1,4)fucosyltransferase

We prepared a mouse monoclonal antibody, FTA1-16, that specifically recognizes human (1,3/1,4)fucosyltransferase without crossreactivity to any other members of the (1,3)fucosyltransferase family. The specificity was confirmed by both immunofluorescense staining of native antigens in the Golgi apparatus and Western blotting analysis, using stable transformant cells transfected with each gene of the (1,3)fucosyltransferase family. Western blotting analysis on a series of human tumour cell lines from various tissues revealed that some epithelial cancer cell lines from digestive organs expressed an amount of (1,3/1,4)fucosyltransferase in good correlation with expression of sialyl Lewis a antigen. Immunohistochemical staining by FTA1-16 on colon cancer tissues revealed enhanced expression of the enzyme in cancer cells in comparison to normal cells. Finally, the antigenic epitope recognized by FTA1-16 was determined using truncated recombinant peptides which were expressed inE. coli. A minimal length determined was a fragment, amino acid positions 132–153, of the (1,3/1,4)fucosyltransferase.     

Five birds,one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ～26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.     

Five birds,one stone: Neutralization of α-hemolysin and 4 bi-component leukocidins of Staphylococcus aureus with a single human monoclonal antibody

Staphylococcus aureus is a major human pathogen associated with high mortality. The emergence of antibiotic resistance and the inability of antibiotics to counteract bacterial cytotoxins involved in the pathogenesis of S. aureus call for novel therapeutic approaches, such as passive immunization with monoclonal antibodies (mAbs). The complexity of staphylococcal pathogenesis and past failures with single mAb products represent considerable barriers for antibody-based therapeutics. Over the past few years, efforts have focused on neutralizing α-hemolysin. Recent findings suggest that the concerted actions of several cytotoxins, including the bi-component leukocidins play important roles in staphylococcal pathogenesis. Therefore, we aimed to isolate mAbs that bind to multiple cytolysins by employing high diversity human IgG1 libraries presented on the surface of yeast cells. Here we describe cross-reactive antibodies with picomolar affinity for α-hemolysin and 4 different bi-component leukocidins that share only ∼26% overall amino acid sequence identity. The molecular basis of cross-reactivity is the recognition of a conformational epitope shared by α-hemolysin and F-components of gamma-hemolysin (HlgAB and HlgCB), LukED and LukSF (Panton-Valentine Leukocidin). The amino acids predicted to form the epitope are conserved and known to be important for cytotoxic activity. We found that a single cross-reactive antibody prevented lysis of human phagocytes, epithelial and red blood cells induced by α-hemolysin and leukocidins in vitro, and therefore had superior effectiveness compared to α-hemolysin specific antibodies to protect from the combined cytolytic effect of secreted S. aureus toxins. Such mAb afforded high levels of protection in murine models of pneumonia and sepsis.     

Risk factors for total joint arthroplasty infection in patients receiving tumor necrosis factor α-blockers: a case-control study

Introduction

The objective of this study was to assess natural microbial agents, history and risk factors for total joint arthroplasty (TJA) infections in patients receiving tumor necrosis factor (TNF)α-blockers, through the French RATIO registry and a case-control study.

Methods

Cases were TJA infections during TNFα-blocker treatments. Each case was compared to two controls (with TJA and TNFα-blocker therapy, but without TJA infection) matched on age (±15 years), TJA localization, type of rheumatic disorder and disease duration (±15 years). Statistical analyses included univariate and multivariate analyses with conditional logistic regression.

Results

In the 20 cases (18 rheumatoid arthritis), TJA infection concerned principally the knee (n = 12, 60%) and the hip (n = 5, 25%). Staphylococcus was the more frequent microorganism involved (n = 15, 75%). Four patients (20%) were hospitalized in an intensive care unit and two died from infection. Eight cases (40%) versus 5 controls (13%) had undergone primary TJA or TJA revision for the joint subsequently infected during the last year (P = 0.03). Of these procedures, 5 cases versus 1 control were performed without withdrawing TNFα-blockers (P = 0.08). In multivariate analysis, predictors of infection were primary TJA or TJA revision for the joint subsequently infected within the last year (odds ratio, OR = 88.3; 95%CI 1.1-7,071.6; P = 0.04) and increased daily steroid intake (OR = 5.0 per 5 mg/d increase; 1.1-21.6; P = 0.03). Case-control comparisons showed similar distribution between TNFα-blockers (P = 0.70).

Conclusions

In patients receiving TNFα-blockers, TJA infection is rare but potentially severe. Important risk factors are primary TJA or TJA revision within the last year, particularly when TNFα-blockers are not interrupted before surgery, and the daily steroid intake.     

Regulation of sheep α-TTP by dietary vitamin E and preparation of monoclonal antibody for sheep α-TTP

α-Tocopherol transfer protein (α-TTP) is a cytosolic protein that plays an important role in regulating concentrations of plasma α-tocopherol (the most bio-active form of vitamin E). Despite the central roles that α-TTP plays in maintaining vitamin E adequacy, we have only recently proved the existence of the α-TTP gene in sheep and, for the first time, cloned its full-length cDNA. However, the study of sheep α-TTP is still in its infancy. In the present study, thirty-five local male lambs of Tan sheep with similar initial body weight were randomly divided into five groups and fed with diets supplemented with 0 (control group), 20, 100, 200, 2000 IU·sheep^− 1·d^− 1 vitamin E for 120 days. At the end of the experiment, the plasma and liver vitamin E contents were analyzed first and then α-TTP mRNA and protein expression levels were determined by quantitative real-time PCR (qRT-PCR) and Western-blot analysis, respectively. In addition, as no sheep α-TTP antibody was available, a specific monoclonal antibody (McAb) against the ovine α-TTP protein was prepared. The effect of vitamin E supplementation was confirmed by the significant changes in the concentrations of vitamin E in the plasma and liver. As shown by qRT-PCR and Western-blot analysis, dietary vitamin E does not affect sheep α-TTP gene expression, except for high levels of vitamin E supplementation, which significantly increased expression at the protein level. Importantly, the specific sheep anti-α-TTP McAb we generated could provide optimal recognition in ELISA, Western-blot and immunohistochemistry assays, which will be a powerful tool in future studies of the biological functions of sheep α-TTP.     

Complete nucleotide sequence of a CDNA encoding porcine tumor necrosis factor‐alpha

Abstract

Tumor necrosis factor‐alpha (TNF‐α), produced by immune cells, is a cytokine with a central role in the mediation of inflammatory responses to infection and injury. We report the nucleotide and corresponding amino acid sequence of a full length porcine TNF‐α cDNA. The complete cDNA nucleotide sequence is 1650 bp in length (not including the poly A tail) and has an open reading frame of 696 bp encoding a 232 amino acid protein. The porcine TNF cDNA sequence shows homologies of 86, 77, and 82% to human, murine, and lapine TNF cDNA sequences in the coding regions, respectively. The 5’ untranslated region of the cDNAs shows little sequence similarity. However, the 3´ untranslated region contains highly conserved sequences among all species, especially the TTATTTAT motif characteristic of cytokine messages.