Concentration of antibodies against Porphyromonas gingivalis is increased before the onset of symptoms of rheumatoid arthritis.

BACKGROUND: The periodontal pathogen Porphyromonas gingivalis is hypothesized to be important in rheumatoid arthritis (RA) aetiology by inducing production of anti-citrullinated protein antibodies (ACPA). We have shown that ACPA precede RA onset by years, and that anti-P. gingivalis antibody levels are elevated in RA patients. The aim of this study was to investigate whether anti-P. gingivalis antibodies pre-date symptom onset and ACPA production. METHODS: A case-control study (251 cases, 198 controls) was performed within the Biobank of Northern Sweden. Cases had donated blood samples (n = 422) before the onset of RA symptoms by 5.2 (6.2) years (median (interquartile range)). Blood was also collected from 192 RA patients following diagnosis. Antibodies against P. gingivalis virulence factor arginine gingipainB (RgpB), and a citrullinated peptide (CPP3) derived from the P. gingivalis peptidylarginine deiminase enzyme, were analysed by ELISA. RESULTS: Anti-RgpB IgG levels were significantly increased in pre-symptomatic individuals (mean ± SEM; 152.7 ± 14.8 AU/ml) and in RA patients (114.4 ± 16.9 AU/ml), compared with controls (p < 0.001). Anti-CPP3 antibodies were detected in 5 % of pre-symptomatic individuals and in 8 % of RA patients, with elevated levels in both subsets (4.33 ± 0.59 and 9.29 ± 1.81 AU/ml, respectively) compared with controls (p < 0.001). Anti-CPP3 antibodies followed the ACPA response, with increasing concentrations over time, whilst anti-RgpB antibodies were elevated and stable in the pre-symptomatic individuals with a trend towards lower levels after RA diagnosis. CONCLUSIONS: Anti-P. gingivalis antibody concentrations were significantly increased in RA patients compared with controls, and were detectable years before onset of symptoms of RA, supporting an aetiological role for P. gingivalis in the development of RA.

Design of an optimized scaffold for affibody molecules.

Affibody molecules are non-immunoglobulin-derived affinity proteins based on a three-helical bundle protein domain. Here, we describe the design process of an optimized Affibody molecule scaffold with improved properties and a surface distinctly different from that of the parental scaffold. The improvement was achieved by applying an iterative process of amino acid substitutions in the context of the human epidermal growth factor receptor 2 (HER2)-specific Affibody molecule Z(HER2:342). Replacements in the N-terminal region, loop 1, helix 2 and helix 3 were guided by extensive structural modeling using the available structures of the parent Z domain and Affibody molecules. The effect of several single substitutions was analyzed followed by combination of up to 11 different substitutions. The two amino acid substitutions N23T and S33K accounted for the most dramatic improvements, including increased thermal stability with elevated melting temperatures of up to +12 degrees C. The optimized scaffold contains 11 amino acid substitutions in the nonbinding surface and is characterized by improved thermal and chemical stability, as well as increased hydrophilicity, and enables generation of identical Affibody molecules both by chemical peptide synthesis and by recombinant bacterial expression. A HER2-specific Affibody tracer, [MMA-DOTA-Cys61]-Z(HER2:2891)-Cys (ABY-025), was produced by conjugating MMA-DOTA (maleimide-monoamide-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) to the peptide produced either chemically or in Escherichia coli. ABY-025 showed high affinity and specificity for HER2 (equilibrium dissociation constant, K(D), of 76 pM) and detected HER2 in tissue sections of SKOV-3 xenograft and human breast tumors. The HER2-binding capacity was fully retained after three cycles of heating to 90 degrees C followed by cooling to room temperature. Furthermore, the binding surfaces of five Affibody molecules targeting other proteins (tumor necrosis factor alpha, insulin, Taq polymerase, epidermal growth factor receptor or platelet-derived growth factor receptor beta) were grafted onto the optimized scaffold, resulting in molecules with improved thermal stability and a more hydrophilic nonbinding surface.

Selection of affibody molecules to the ligand-binding site of the insulin-like growth factor-1 receptor.

Affibody molecules binding to the site of hormone interaction in IGF-1R (insulin-like growth factor-1 receptor) were successfully selected by phage-display technology employing a competitive-elution strategy during biopanning, whereby release of receptor-bound phagemids was accomplished by competition with IGF-1 (insulin-like growth factor-1). In non-competitive selections, the elution of receptor-bound phagemids was performed by imidazole or low-pH incubation, which also resulted in the isolation of affibody molecules that could bind to the receptor. An ELISA-based assay showed that the affibody molecules generated by IGF-1 competition during elution, in addition to affibody molecules generated in the non-competitive selections, could compete with IGF-1 for binding to the receptor. The affinities of the isolated variants to IGF-1R-overexpressing MCF-7 cells were determined and ranged from high nanomolar to 2.3 nM. The most promising variant, Z4:40, was shown to recognize IGF-1R efficiently in several different contexts: in analyses based on flow cytometry, fluorescence microscopy and receptor pull-down from cell extracts. In addition, when Z4:40 was added to the medium of MCF-7 cells that were dependent on IGF-1 for efficient growth, it was found to have a dose-dependent growth-inhibitory effect on the cells. Applications of affibody-based reagents for quantitative and qualitative analyses of IGF-1R status, as well as applications of affibody-based reagents for therapy, are discussed.

Site-specifically conjugated anti-HER2 Affibody molecules as one-step reagents for target expression analyses on cells and xenograft samples.

Affibody molecules are a class of small and robust affinity proteins that can be generated to interact with a variety of antigens, thus having the potential to provide useful tools for biotechnological research and diagnostic applications. In this study, we have investigated Affibody-based reagents interacting specifically with the tyrosine kinase receptor HER2. A head-to-tail dimeric construct was site-specifically conjugated with different fluorescent and enzymatic groups resulting in reagents that were used for detection and quantification. The amount of cell surface expressed HER2 on eleven (11) well characterized cell lines was quantified relative to each other by flow cytometry and shown to correlate well with results from parallel analyses of HER2 mRNA levels measured by real-time PCR. Further, immunofluorescence microscopy studies of the cell lines and immunohistochemical analyses of cryosections of HER2 expressing SKOV-3 xenografts showed strong staining of the plasma membrane of tumor cells with little background staining. Full-length HER2 protein could also be efficiently recovered from a cell extract by an immunoprecipitation procedure, using an Affibody ligand-based resin. These novel non-IgG derived reagents could be used to detect and quantify HER2 expression. By adapting the methods for use with Affibody molecules binding to other cell surface receptors, it is anticipated that also these receptors can be detected and quantified in a similar manner.

Tumor imaging using a picomolar affinity HER2 binding affibody molecule.

The detection of cell-bound proteins that are produced due to aberrant gene expression in malignant tumors can provide important diagnostic information influencing patient management. The use of small radiolabeled targeting proteins would enable high-contrast radionuclide imaging of cancers expressing such antigens if adequate binding affinity and specificity could be provided. Here, we describe a HER2-specific 6 kDa Affibody molecule (hereinafter denoted Affibody molecule) with 22 pmol/L affinity that can be used for the visualization of HER2 expression in tumors in vivo using gamma camera. A library for affinity maturation was constructed by re-randomization of relevant positions identified after the alignment of first-generation variants of nanomolar affinity (50 nmol/L). One selected Affibody molecule, Z(HER2:342) showed a >2,200-fold increase in affinity achieved through a single-library affinity maturation step. When radioiodinated, the affinity-matured Affibody molecule showed clear, high-contrast visualization of HER2-expressing xenografts in mice as early as 6 hours post-injection. The tumor uptake at 4 hours post-injection was improved 4-fold (due to increased affinity) with 9% of the injected dose per gram of tissue in the tumor. Affibody molecules represent a new class of affinity molecules that can provide small sized, high affinity cancer-specific ligands, which may be well suited for tumor imaging.

Surface electromyography and peak torque of repetitive maximum isokinetic plantar flexions in relation to aspects of muscle morphology.

This study investigates the relationships between surface electromyography (EMG [Mean frequency of the power spectrum (MNF)]) and peak torque variables obtained during 100 maximum concentric plantar flexions with the right limb at 60 degrees s(-1) and different muscle morphological variables. Surface EMG was recorded from the right gastrocnemius lateralis and muscle biopsies were taken from the same site as the EMG electrodes were positioned. Muscle fibre area and fibre type composition were determined on serial muscle cross sections using both histochemistry (myofibrillar adenosine triphosphatase) and immunohistochemistry (monoclonal antibodies against specific myosin heavy chain isoforms). Forty-three female and nine male students participated in the study. Gastrocnemius lateralis contained predominantly type I fibres (50%) and type IIA fibres (40%) in both sexes and large individual differences were found. Principal component analysis (PCA) was used for the intercorrelation analyses, and projection to latent structures (PLS) was used for the multivariate regression analysis. MNF correlated positively with different fibre areas and with the proportion of type I fibres. Fibre areas and sex were the most important factors in the regression of maximum peak torque. High proportion of type I fibres and sex were the most important regressors of peak torque endurance normalised for lean body mass. More studies are needed to understand the complex interrelationships between intrinsic muscle properties and the frequency content of the surface EMG before theoretical models can be formulated that incorporate both fibre areas and fibre type proportions.

Evaluation of ((4-hydroxyphenyl)ethyl)maleimide for site-specific radiobromination of anti-HER2 affibody.

Affibody molecules are a new class of small phage-display selected proteins using a scaffold domain of the bacterial receptor protein A. They can be selected for specific binding to a large variety of protein targets. An affibody molecule binding with high affinity to a tumor antigen HER2 was recently developed for radionuclide diagnostics and therapy in vivo. The use of the positron-emitting nuclide (76)Br (T(1/2) = 16.2 h) could improve the sensitivity of detection of HER2-expressing tumors. A site-specific radiobromination of a cysteine-containing variant of the anti-HER2 affibody, (Z(HER2:4))(2)-Cys, using ((4-hydroxyphenyl)ethyl)maleimide (HPEM), was evaluated in this study. It was found that HPEM can be radiobrominated with an efficiency of 83 +/- 0.4% and thereafter coupled to freshly reduced affibody with a yield of 65.3 +/- 3.9%. A "one-pot" labeling enabled the radiochemical purity of the conjugate to exceed 97%. The label was stable against challenge with large excess of nonlabeled bromide and in a high molar strength solution. In vitro cell tests demonstrated that radiobrominated affibody binds specifically to the HER2-expressing cell-line, SK-OV-3. Biodistribution studies in nude mice bearing SK-OV-3 xenografts have shown tumor accumulation of 4.8 +/- 2.2% IA/g and good tumor-to-normal tissue ratios.