[Humanization of Murine Monoclonal anti-hTNF Antibody: The F10 Story].

Tumor necrosis factor (TNF) is a proinflammatory cytokine implicated in pathogenesis of multiple autoimmune and inflammatory diseases. Anti-TNF therapy has revolutionized the therapeutic paradigms of autoimmune diseases and became one of the most successful examples of the clinical use of monoclonal antibodies. Currently, anti-TNF therapy is used by millions of patients worldwide. At the moment, fully human anti-TNF antibody Adalimumab is the best-selling anti-cytokine drug in the world. Here, we present a story about a highly potent anti-TNF monoclonal antibody initially characterized more than 20 years ago and further developed into chimeric and humanized versions. We present comparative analysis of this antibody with Infliximab and Adalimumab.

Risk factors of post-operative delirium after elective vascular surgery in the elderly: A systematic review.

BACKGROUND: Postoperative delirium is a common and serious adverse event in the elderly patient and is associated with significant morbidity and mortality. It is of great importance to identify patients at risk for delirium, in order to focus preventive strategies. The aim of this article is to systematically review current available literature on pre-operative risk factors for delirium after vascular surgery. METHODS: A systematic literature search was conducted using PubMed and EMBASE, using the MeSH terms and key words "delirium", "surgery" and "risk factor". Studies were retained for review after meeting strict inclusion criteria that included only prospective studies evaluating risk factors for delirium in patients who had elective vascular surgery. Diagnosis of delirium needed to be confirmed using the Diagnostic and Statistical Manual of Mental Disorders (DSM) or ICD-10. RESULTS: Fifteen articles were selected for inclusion, incidence of delirium across the studies ranged from 5% to 39%. Many factors have been associated with increased risk of delirium, including age, cognitive impairment, comorbidity, depression, smoking, alcohol, visual and hearing impairment, ASA-score, biochemical abnormalities, operative strategies and blood loss. CONCLUSIONS: Delirium is a common complication after elective vascular surgery in elderly. The highest delirium incidence was observed after open aortic surgery as well as after surgery for critical limb ischemia. A picture starts to form of which predisposing factors lead to increased risk of delirium. The leading risk factors consistently identified in this systematic review were advanced age and cognitive impairment. Multi-disciplinary specialist-led interventions in the preoperative phase could decrease incidence and severity of delirium and should be focused on identified high-risk patients.

Postoperative delirium in elderly after elective and acute colorectal surgery: A prospective cohort study.

OBJECTIVE: To assess the frailty and the incidence of delirium in elderly patients undergoing elective and acute colorectal surgery in correlation with morbidity and mortality. METHODS: Patients aged 65 years and older having elective and acute colorectal surgery, between April 2013 and December 2013 were included in a prospective database. Patients diagnosed with a colorectal carcinoma or diverticulitis who were operated on were included. Factors that characterize frailty of patients were noted. The incidence rates of delirium after elective and acute surgery were recorded. Delirium was diagnosed using the Delirium Observation Screening Scale (DOSS). Preoperative evaluation, surgical outcome including morbidity, hospital stay and mortality were analyzed. RESULTS: Patients ≥ 65 years were included, 83 (75%) received elective and 28 (25%) acute surgery. The overall incidence of delirium was 21%, 18% for elective and 29% for patients having urgent surgery (p = 0.24). Patients with delirium were older than the non-delirious patients (median 82 years vs. 74 years; p < 0.001). Delirious patients showed higher incidence of adverse events. Hospital stay, mortality and discharge to a nursing home were significant higher in the delirious compared to the non-delirious group (p = 0.01; 0.01; 0.02 respectively). CONCLUSION: High incidence of delirium was found in both acute and elective colorectal surgery. Delirium was associated with adverse outcomes.

A randomized, placebo-controlled, double-blind phase 2 study of docetaxel compared to docetaxel plus zosuquidar (LY335979) in women with metastatic or locally recurrent breast cancer who have received one prior chemotherapy regimen.

PURPOSE: To determine if concomitant administration of docetaxel plus zosuquidar.3HC1 can prolong progression-free survival in patients with metastatic breast cancer. METHODS: A randomized, double-blind, multicenter, placebo-controlled clinical trial comparing docetaxel plus 500 mg zosuquidar.3HCl (DZ) with docetaxel plus placebo (DP). RESULTS: A total of 170 patients were enrolled and randomly assigned to treatment. The median age was 53 years (range, 31-74 years). 81.7% of patients had prior chemotherapy in the adjuvant setting and 18.3% in the neoadjuvant setting. The median progression-free survival time was statistically different between groups [7.2 months (DZ) vs. 8.3 months (DP)]. Once the stratification factor relative to progression following prior chemotherapy was considered, no significant treatment difference existed. CONCLUSION: The combination of zosuquidar.3HCl plus docetaxel is safe. The analysis of efficacy data is complex, but it can be concluded that there is no difference in progression-free survival, overall survival, or response rate in the study as a whole.

A comparison of nutritionally-related blood metabolites among Nguni, Bonsmara and Angus steers raised on sweetveld.

The objective of the study was to determine monthly variations in serum glucose, cholesterol, total protein (TP), urea, albumin, globulin, albumin/globulin ratio, creatinine, aspartate aminotransferase (AST), creatinine kinase (CK), alkaline phosphatase (ALP), calcium, phosphorus and magnesium in Nguni, Bonsmara and Angus beef steers raised on sweetveld. Twenty-five Nguni, 15 Aberdeen Angus and 15 Bonsmara 8-month old steers were studied from June 2006 until March 2007. Across the 9 months, Nguni had higher concentrations of glucose (P =0.019) and cholesterol (P =0.001) than the other two breeds. The overall glucose and cholesterol concentrations in the Nguni were 4 and 2.86mmol/L, respectively. There was a breedxmonth interaction on glucose, cholesterol, creatinine, calcium, albumin and phosphorus concentrations. Breed had no effect on TP, urea, globulin and AST concentrations. Breed and month differences obtained could be attributed to changes in environment temperature and nutrient content of the forage.

Relationship between pre-slaughter stress responsiveness and beef quality in three cattle breeds.

The relationship between stress responsiveness and beef quality of 40 Nguni, 30 Bonsmara and 30 Angus steers was determined. The L(∗) values, pHu, cooking loss (CL) and Warner-Bratzler shear force (WBSF) were determined. Catecholamine levels were determined from urine samples collected at slaughter. Bonsmara steers had the highest (P<0.05) levels of catecholamines with respective epinephrine, norepinephrine and dopamine concentrations of 10.8, 9.7 and 14.8nmol/mmol. Nguni steers had the lowest (P<0.05) levels of catecholamines, with respective catecholamine concentrations of 5.1, 4.3 and 4.0nmol/mmol. In the Nguni steers, there were significant (P<0.05) correlations between catecholamines and L(∗) and between dopamine and tenderness in meat aged for two days (WBSF2). In the Bonsmara, dopamine was correlated (P<0.05) pHu, WBSF2 and CL. No significant correlations were found in the Angus. Therefore the relationship between stress responsiveness and certain beef quality traits may not be similar in different breeds.

Meat quality of Nguni, Bonsmara and Aberdeen Angus steers raised on natural pasture in the Eastern Cape, South Africa.

The current study compared meat quality of Nguni, Bonsmara and Angus steers raised on natural pasture. Fifteen seven-month-old weaners of each breed were kept at the University of Fort Hare Farm for 12 months till slaughter. Monthly weights of the steers were recorded. Carcasses were electrically stimulated. The m. longissimus thoracis et lumborum was sampled for the measurement of meat colour, pH, drip loss, sarcomere length, myofibrillar fragmentation length and Warner Bratzler (WB) shear force. The Nguni had the highest (P<0.05) average daily gain. Bonsmara and Angus steers had higher (P<0.05) carcass weight and dressing percentage than the Nguni steers. Meat quality characteristics were similar among all the breeds except that Nguni meat was darker (L(∗)) (P<0.05) than meat from the other two breeds. The respective L(∗) values for Nguni, Bonsmara and Angus steers were 36.5, 38.6 and 39.9. There were significant (P<0.05) correlations among some meat quality traits. There were significant (P<0.05) correlations between WB values of meat aged for 2 and 21 days in Nguni and Bonsmara, but not in Angus. Meat quality from Nguni compares favourably with that from established breeds, when raised on natural pasture.

Sensory evaluation and its relationship to physical meat quality attributes of beef from Nguni and Bonsmara steers raised on natural pasture.

The current study compared sensory characteristics and their relationships with physical meat characteristics of beef from Nguni and Bonsmara steers. Nguni beef was more (P < 0.05) tender than Bonsmara beef after ageing for 2 and 21 days, and had higher (P < 0.05) intramuscular fat (IMF; 1.12%) than Bonsmara beef (1.07%). Nguni beef had higher (P < 0.05) sensory scores than Bonsmara beef after ageing for 2 days. There were no (P > 0.05) relationships between IMF and sensory characteristics. Aroma intensity, impression on juiciness and tenderness-related scores were affected (P < 0.05) by pH. There were significant (P < 0.05) correlations between most physical meat characteristics and sensory characteristics. Nguni beef had better sensory scores than Bonsmara beef for beef aged for 2 days. While most physical meat characteristics were correlated to sensory scores, all sensory scores were not significantly correlated to IMF.

Tick susceptibility and its effects on growth performance and carcass characteristics of Nguni, Bonsmara and Angus steers raised on natural pasture.

The objective of the current study was to compare tick loads, growth and carcass characteristics of dipped and non-dipped Nguni, Bonsmara and Angus steers raised on natural pasture. One hundred 7-month-old castrated weaners were kept at the University of Fort Hare Farm for 12 months. There were 30 weaners each of Angus and Bonsmara, and 40 weaners of the Nguni breed. Half the Bonsmara, Angus and 14 Nguni weaners were dipped every fortnight. The rest were not dipped. Monthly weights and tick counts under the tail, on scrotum, belly, sternum and ears of the steers were recorded. The dipped Nguni steers had lowest (P < 0.05) tick counts, and the non-dipped Angus steers had the highest (P < 0.05) tick counts. There were more ticks (P < 0.05) during the warm wet season than during the cool dry season. Ears had the highest (P < 0.05) tick infestation. Average daily gain (ADG) was similar (P > 0.05) among the three breeds. The non-dipped Bonsmara steers had the heaviest (P < 0.05) carcasses (142 ± 5.4) while the non-dipped Nguni steers had the lightest (P < 0.05) carcasses (111 ± 4.5 kg). The non-dipped Bonsmara had the highest (P < 0.05) eye muscle area (3996 ± 120.8 mm2) while the non-dipped Angus had the smallest (P < 0.05) eye muscle area (3291 ± 210.6 mm2). The non-dipped Bonsmara also had the highest (P < 0.05) dressing percentage (53.8 ± 1.01) while the non-dipped Nguni had the lowest (P < 0.05) dressing percentage (50.3 ± 0.84). The current study has shown that while the non-dipped steers had higher (P < 0.05) tick loads than the dipped steers, their growth and carcass characteristics were similar (P > 0.05). The study has also shown that, despite being a small-framed breed, the Nguni steers had similar (P > 0.05) ADG to the large-framed Bonsmara and Angus steers. Therefore, the Nguni cattle have the potential to produce organic beef. However, a reasonable assessment of organic beef production potential of the Nguni requires an evaluation of its meat quality traits under natural pasture.

Multiple-antigen immunization of chickens facilitates the generation of recombinant antibodies to autoantigens.

Antibody phage display is a powerful tool for the generation of monoclonal antibodies against virtually any given antigen. Chickens are phylogenetically more distant from humans compared to other laboratory animals, such as mice and rats. Therefore, the use of chickens is especially beneficial when generating recombinant antibodies against human autoantigens, which are often highly conserved among mammals. Another advantage of using chickens in antibody phage display is that the preparation of single chain variable fragment (scFv) antibody libraries is faster and easier compared to preparing such libraries from other species, as only two primer sets are needed for amplification of the chicken variable heavy chain (V(H)) and variable light chain (V(L)) genes. In the present study we explored the possibility to immunize chickens with antigen cocktails for the generation of recombinant antibody fragments directed to a range of human autoantigens. Two pairs of chickens were immunized with two cocktails of seven recombinant autoantigenic proteins, libraries were prepared and panned on the individual proteins. The polyclonal chicken sera reacted strongly with most of the antigens used for immunization. By creating and screening single-chain variable fragment antibody phage display libraries, recombinant monoclonal antibody fragments were isolated successfully against the autoantigens annexin XI, centromere protein B, heat shock protein B3, DNA topoisomerase I, histidyl tRNA synthetase, Ro52, Ro60, Rpp30 and U1A. In conclusion, the immunization of only four chickens with two distinct pools of a total of 14 autoantigenic proteins allowed the isolation of scFvs against nine of these antigens.

Structurally derived mutations define congenital heart block-related epitopes within the 200-239 amino acid stretch of the Ro52 protein.

Congenital heart block is a passively transferred autoimmune condition, which affects the children of mothers with Ro/SSA autoantibodies. During pregnancy, the antibodies are transported across the placenta and affect the fetus. We have previously demonstrated that antibodies directed to the 200-239 amino acid (aa) stretch of the Ro52 component of the Ro/SSA antigen correlate with the development of congenital heart block. In this report, we investigated the antibody-antigen interaction of this target epitope in detail at a molecular and structural level. Peptides representing aa 200-239 (p200) with structurally derived mutations were synthesized to define the epitopes recognized by two Ro52 human monoclonal antibodies, S3A8 and M4H1, isolated from patient-derived phage display libraries. Analyses by ELISA, circular dichroism and MALDI-TOF-MS demonstrate that the antibody recognition is dependent on a partly alpha-helical fold within the putative leucine zipper of the 200-239 aa stretch and that the two human anti-p200 monoclonal antibodies, M4H1 and S3A8, recognize different epitopic structures within the p200 peptide. In addition, we investigated the representation of each fine specificity within the sera of mothers with children born with congenital heart block, and in such sera, antibodies of the S3A8 idiotype were more commonly detected and at higher levels than M4H1-like antibodies.

Technical and clinical evaluation of anti-ribosomal P protein immunoassays.

Autoantibodies to the three ribosomal phospho (-P) proteins P0, P1, P2, referred to as Rib-P, are specifically found in 10-40% of patients with systemic lupus erythematosus. The variations in the observed frequency of these autoantibodies is related to a number of factors such as the test system used to detect the antibodies. Several immunoassays that were designed for research and diagnostic laboratory use have been developed. The autoantigens employed in these tests include native proteins, recombinant polypeptides, and synthetic peptides. In this study, we compared the technical and clinical accuracy of anti-Rib-P antibody assays from different commercial suppliers including ELISA systems and a novel addressable laser bead assay (from Euroimmun, MBL, Pharmacia Diagnostics, INOVA). Although the assays from all suppliers used in this study performed well in the technical part of the study, relatively poor correlations and significant differences in the clinical accuracy were found. Based on the results, we conclude that the detection of anti-Rib-P antibodies strongly depends on both the nature of the antigen and the detection system. We recommend that anti-Rib-P assays should be standardized on an international level. The Varelisa Rib-P profile and the addressable laser bead Rib-P assays represent promising tools and platforms for the detection of anti-Rib-P antibodies in the future.

Recombinant anti-P protein autoantibodies isolated from a human autoimmune library: reactivity, specificity and epitope recognition.

The ribosomal P proteins are specific and important autoantigens in patients affected by systemic lupus erythematosus. In this study, we describe for the first time the selection and characterization of recombinant human monoclonal anti-P protein (auto)-antibody fragments from an autoimmune patient-derived phage display antibody library. The selected recombinant anti-P antibodies specifically recognize the P proteins in immunofluorescence assays on HEp-2 cells and in immunoblotting assays, and they immunoprecipitate the P proteins under native conditions. Using both anti-P-positive patient sera and the selected recombinant anti-P antibodies, the immunodominant epitope was determined and shown to be located at the C-terminal end of the P proteins (amino acids 111-115). Inhibition of in vitro protein translation demonstrated that interaction of the monoclonal patient-derived anti-P antibodies with their native epitope functionally inhibits the activity of the P proteins on the ribosome, confirming the notion that patient autoantibodies are often directed to the functional centre of their autoantigenic target.

Characterization of the human autoimmune response to the major C-terminal epitope of the ribosomal P proteins.

Autoantibodies to the ribosomal phospho (-P) proteins P0, P1, and P2, collectively referred to as Rib-P, are specifically found in 10-40% of patients with systemic lupus erythematosus (SLE). These antibodies are believed to be correlated with lupus nephritis, hepatitis, and central nervous system involvement. The major immunoreactive epitope of these ribosomal antigens has been localized to the carboxy terminus, which is a highly conserved domain of all three proteins and contains two phosphorylated serine residues. The phosphorylated amino acids of the P proteins are known not to be critical epitope determinants. Furthermore, epitope-mapping studies have shown that the major epitope is located within the last 11 C-terminal amino acids. Using peptide arrays we identified more precisely this shared epitope as the six C-terminal amino acids GFGLFD and elucidated the molecular recognition events of anti-Rib-P antibodies at the amino acid level. We identified Phe(111) and Phe(114) of Rib-P2 as the key residues for the interaction, with further contributions of Gly-112 and Asp-115. This amino acid stretch is also present in proteins of several pathogenic micro-organisms such as Trypanosoma cruzi, Brugia malayi, Pseudomonas aeruginosa, Candida albicans, several Leishmania species, and Bartonella henselae. Using newly developed ELISA systems with a C-terminal peptide (C22) and the recombinant proteins (P0, P1, and P2) as antigens we found a high specificity of anti-Rib-P antibodies for SLE and demonstrated positive correlations with anti-U1-C, anti-Sm-B/B' and anti-D and anti-dsDNA antibodies. The sensitivity and specificity in the peptide (C22) based assay varied between 12.8%/100% and 23.4%/96.7% for SLE, depending on the assigned cutoff. In contrast to other studies, we found no significant correlation of anti-Rib-P reactivity with central nervous system manifestations or renal involvement in SLE patients. We conclude that the epitope motif GFGLFD in the C-termini of the ribosomal P proteins is the key determinant of anti-Rib-P antibodies, and that the C22 peptide and the recombinant proteins can be used equally well for the detection of anti-Rib-P antibodies. The role of the major Rib-P epitope in the development of anti-ribosomal P antibodies and in the pathogenesis of SLE remains a subject of further investigation.

Recombinant human antibodies specific for the Pfs48/45 protein of the malaria parasite Plasmodium falciparum.

We report the first construction of two combinatorial human phage display libraries derived from malaria-immune patients. Specific single-chain Fv fragments (scFv) against Pfs48/45, a gamete surface protein of the sexual stages of Plasmodium falciparum, were selected and analyzed extensively. The selected scFv reacted with the surface of extracellular sexual forms of the parasite and showed Pfs48/45 reactivity on immunoblot. The scFv inhibit binding of human malaria sera to native Pfs48/45 from gametocytes. Moreover, the scFv bind to target epitopes of Pfs48/45 exposed in natural infections. Sequence analysis of eight scFv clones specific for epitope III of Pfs48/45 revealed that these clones could be divided into one V(H) family-derived germ-line gene (V(H)1) and two V(L) family segments (V(L)2 and V(K)I).

Characterization of recombinant human autoantibody fragments directed toward the autoantigenic U1-70K protein.

The U1-70K protein is specifically bound to stemloop I of the U1 small nuclear RNA contained in the U1 small nuclear ribonucleoprotein complex (U1 snRNP), which is involved in the splicing of pre-mRNA. All components of the U1 snRNP complex, including the U1-70K protein, are important autoantigens in patients with systemic lupus erythematosus (SLE) and mixed connective tissue disease (MCTD). Here we describe for the first time the selection and characterization of recombinant human anti-U1-70K single chain autoantibody fragments (anti-hU1-70K scFv) from autoimmune patient-derived phage display antibody libraries. All scFv specifically recognize parts of the hU1-70K protein and its apoptotic 40-kDa cleavage product. In Western blotting assays a number of scFv preferentially recognize the 40-kDa apoptotic cleavage fragment of the U1-70K protein, suggesting a possible involvement of this apoptotic cleavage product in the autoimmune response of patients. The germline gene usage of these recombinant autoantibodies was also determined. Using several U1-70K deletion and point mutants of both human (h) and Drosophila melanogaster (Dm) origin, it was established that the U1-70K epitope that is recognized by the anti-hU1-70K scFv is located within the RNA binding domain.

Analysis of autoimmune bone marrow by antibody-phage display: somatic mutations and third complementarity-determining region arginines in anti-DNA gamma and kappa V genes.

OBJECTIVE: To examine anti-double-stranded DNA (anti-dsDNA) IgG autoantibodies from the bone marrow of individuals with systemic lupus erythematosus (SLE). METHODS: A library of single-chain variable fragments (scFv) was constructed from SLE bone marrow complementary DNA of gamma, kappa, and lambda isotype by cloning into the pHENIX phagemid vector. The library was screened with dsDNA in solution, and 2 anti-DNA phage, DNA1 and DNA4, were isolated and their Ig V genes sequenced. Soluble scFv corresponding to DNA1 and DNA4, and their heavy (H)- and light (L)-chain recombinants, were prepared, purified, and analyzed for binding to DNA by enzyme-linked immunosorbent assay. RESULTS: DNA1 and DNA4 used different Ig H-chain (3-30 and 5-51, respectively) and L-chain (DPK15 and DPK22, respectively) V genes. The ratios of replacement mutations to silent mutations in DNA1 and DNA4 suggest that their V genes were selected for improved antigen binding in vivo. The recombinant between DNA4VH and DNA1VL showed the highest relative affinity for both single-stranded DNA and dsDNA. These 2 Ig subunits contained third complementarity-determining region arginines and had acquired the majority of replacement mutations. CONCLUSION: Anti-dsDNA IgG autoantibodies from the bone marrow of SLE patients exploit diverse V genes and cationic V-D-J and V-J junctions for DNA binding, and accumulate replacement mutations that enhance binding.

Cell death: a trigger of autoimmunity?

Systemic autoimmune diseases are characterized by the production of antibodies against a broad range of self-antigens. Recent evidence indicates that the majority of these autoantigens are modified in various ways during cell death. This has led to the hypothesis that the primary immune response in the development of autoimmunity is directed to components of the dying cell. In this article, we summarize data on the modification of autoantigens during cell death and the possible consequences of this for autoimmunity.

Caspase-dependent cleavage of nucleic acids.

Autoimmune diseases are frequently characterized by the presence of autoantibodies directed against nucleic acid-protein complexes present in the nucleus of the cell. The mechanisms by which these autoantigenic molecules escape immunological tolerance are largely unknown, although a number of recent observations suggest that modified self-proteins generated during apoptosis may play an important role in the development of autoimmunity. It has been hypothesized that the recognition of these modified self-proteins by the immune system may promote autoantibody production. While apoptosis is specifically characterized by posttranslational modification of proteins, recent findings also show that nucleic acids are modified. This review summarizes the specific cleavages of some of these key nucleic acids, i.e. chromosomal DNA, ribosomal RNA and small structural RNAs (U1 snRNA, Y RNA), in apoptotic cells.