Interaction of Interleukin-17A with a Th2 Response in a Mouse Model of Allergic Airway Inflammation.

BACKGROUND: A total of 262 million people worldwide suffer from asthma and 461000 people died from it in 2019. Asthma is a disease with different endotypes defined by the granulocytes found in the asthmatic lung. In allergic asthma, the eosinophilic endotype is present, driven by a TH2 response. A TH17 immune response leads to the neutrophil endotype. This often causes uncontrolled asthma and is triggered by pollutants, microbes, and oxidative stress. It has been described that a significant number of patients with eosinophilic asthma develop mixed granulocytic asthma over time. The severity of asthma in the mixed endotype is related to the proportion of neutrophils in the lungs. PURPOSE: In this report, we address the question of how a TH2 response interacts with IL-17A in allergic asthma. METHODS: To this end, we used a mouse model to induce allergic asthma followed by an aerosol challenge with ovalbumin. To investigate the role of IL-17A, we administered IL-17A intranasally during the challenge phase. RESULTS: IL-17A alone did not elicit an immune response, whereas in combination with allergic asthma, it resulted in a shift of the asthmatic endotype from eosinophilic to neutrophilic. TGFß1 was increased in these lungs compared to asthmatic lungs without IL-17A, as was the expression of the IL-17A receptor subunits IL-17RA and IL-17RC. In cultures with human cells, we also found that IL-17A increased the expression of its receptors only in combination with IL-13. We also found this effect for IL-8, which attracts neutrophils in humans. CONCLUSIONS: The TH2 response increased the sensitivity to IL-17A in a mouse asthma model as well as in human cell lines.

The molecular mechanisms of remodeling in asthma, COPD and IPF with a special emphasis on the complex role of Wnt5A.

INTRODUCTION: Chronic inflammatory lung diseases are a common cause of suffering and death. Chronic obstructive pulmonary disease (COPD) is the reason for 6% of all deaths worldwide. A total of 262 million people are affected by asthma and 461,000 people died in 2019. Idiopathic pulmonary fibrosis (IPF) is diagnosed in 3 million people worldwide, with an onset over the age of 50 with a mean survival of only 24-30 months. These three diseases have in common that remodeling of the lung tissue takes place, which is responsible for an irreversible decline of lung function. Pathological lung remodeling is mediated by a complex interaction of different, often misguided, repair processes regulated by a variety of mediators. One group of these, as has recently become known, are the Wnt ligands. In addition to their well-characterized role in embryogenesis, this group of glycoproteins is also involved in immunological and structural repair processes. Depending on the combination of the Wnt ligand with its receptors and co-receptors, canonical and noncanonical signaling cascades can be induced. Wnt5A is a mediator that is described mainly in noncanonical Wnt signaling and has been shown to play an important role in different inflammatory diseases and malignancies. OBJECTIVES: In this review, we summarize the literature available regarding the role of Wnt5A as an immune modulator and its role in the development of asthma, COPD and IPF. We will focus specifically on what is known about Wnt5A concerning its role in the remodeling processes involved in the chronification of the diseases. CONCLUSION: Wnt5A has been shown to be involved in all three inflammatory lung diseases. Since the ligand affects both structural and immunological processes, it is an interesting target for the treatment of lung diseases whose pathology involves a restructuring of the lung tissue triggered in part by an inflammatory immune response.

Comparison of Airway Remodeling in Two Different Endotypes of Allergic Asthma.

Different endotypes of asthma were described in human. Atopic asthma is a T-helper 2 (Th2)-mediated disease consisting mainly of an eosinophilic inflammation in the airways. Other endotypes show neutrophilic inflammation of the airways that is probably based on a Th17 response. There are several mouse models described in the literature to study the Th2 polarized eosinophilic disease, however, only a few models are available which characterize the neutrophilic endotype. The aim of this study was to compare both endotypes in relation to the severity of the allergen-induced inflammation. Groups of either Balb/c or DO11.10 mice were sensitized with ovalbumin (OVA) adsorbed to aluminum hydroxide. Mice were subsequently challenged with OVA for different periods of time. They were evaluated for airway hyperreactivity (AHR), cytokine production, airway inflammation, and remodeling of the airways. As expected, Balb/c mice developed a Th2 response with AHR, eosinophilic airway inflammation, and allergen-specific IgE and IgG1. By contrast DO11.10 mice showed a mixed Th1/Th17 response with strong neutrophilic airway inflammation, IgG2a, but only limited induction of AHR. While Balb/c mice showed remodeling of the airways with subepithelial fibrosis and goblet cell metaplasia, airway remodeling in DO11.10 mice was marginal. Both airway inflammation and remodeling resolved after prolonged periods of challenge in both models. In conclusion, strong allergen-induced airway remodeling in mice seems to be triggered by the specific conditions arising from infiltration with eosinophilic granulocytes in the lung. A Th1/Th17 response leading to neutrophilic inflammation does not seem to be sufficient to induce pronounced airway remodeling.

The Pathogenic Role of Smooth Muscle Cell-Derived Wnt5a in a Murine Model of Lung Fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a disease characterized by extensive fibrosis of the lung tissue. Wnt5a expression was observed to be upregulated in IPF and suggested to be involved in the progression of the disease. Interestingly, smooth muscle cells (SMC) are a major source of Wnt5a in IPF patients. However, no study has been conducted until now to investigate the precise role of smooth muscle-derived Wnt5a in IPF. Here, we used the bleomycin-induced lung fibrosis model in a conditional gene-deficient mouse, where the Wnt5a gene was excised from SMC. We show here that the excision of the Wnt5a gene in SMC led to significantly improved health conditions with minimized weight loss and improved lung function. This improvement was based on a significantly lower deposition of collagen in the lung with a reduced number of fibrotic foci in lung parenchyma. Furthermore, the bleomycin-induced cellular infiltration into the airways was not altered in the gene-deficient mice compared with wild-type mice. Thus, we demonstrate that the Wnt5a expression of SMC of the airways leads to aggravated fibrosis of the lung with poor clinical conditions. This aggravation was not an influence in the bleomycin-induced inflammatory processes but on the development of fibrotic foci in lung parenchyma and the deposition of collagen.

The Role of Lectin Receptors and Their Ligands in Controlling Allergic Inflammation.

More than fifty c-type lectin receptors (CLR) are known and have been identified so far. Moreover, we know the group of galectins and sialic acid-binding immunoglobulin-type lectins that also belong to the carbohydrate-binding receptors of the immune system. Thus, the lectin receptors form the largest receptor family among the pathogen recognition receptors. Similar to the toll-like receptors (TLRs), the CLR do not only recognize foreign but also endogenous molecules. In contrast to TLRs, which have a predominantly activating effect on the immune system, lectin receptors also mediate inhibitory signals. They play an important role in innate and adaptive immunity for the induction, regulation and shaping of the immune response. The hygiene hypothesis links enhanced infection to protection from allergic disease. Yet, the microbial substances that are responsible for mediating this allergy-protective activity still have to be identified. Microbes contain both ligands binding to TLRs and carbohydrates that are recognized by CLR and other lectin receptors. In the current literature, the CLR are often recognized as the 'bad guys' in allergic inflammation, because some glycoepitopes of allergens have been shown to bind to CLR, facilitating their uptake and presentation. On the other hand, there are many reports revealing that sugar moieties are involved in immune regulation. In this review, we will summarize what is known about the role of carbohydrate interaction with c-type lectins and other sugar-recognizing receptors in anti-inflammation, with a special focus on the regulation of the allergic immune response.

Total Synthesis of a Partial Structure from Arabinogalactan and Its Application for Allergy Prevention.

Arabinogalactan, a microheterogeneous polysaccharide occurring in plants, is known for its allergy-protective activity, which could potentially be used for preventive allergy treatment. New treatment options are highly desirable, especially in a preventive manner, due to the constant rise of atopic diseases worldwide. The structural origin of the allergy-protective activity of arabinogalactan is, however, still unclear and isolation of the polysaccharide is not feasible for pharmaceutical applications due to a variation of the activity of the natural product and contaminations with endotoxins. Therefore, a pentasaccharide partial structure was selected for total synthesis and subsequently coupled to a carrier protein to form a neoglycoconjugate. The allergy-protective activity of arabinogalactan could be reproduced with the partial structure in subsequent in vivo experiments. This is the first example of a successful simplification of arabinogalactan with a single partial structure while retaining its allergy-preventive potential.

Neutralisation of factor VIII inhibitors by anti-idiotypes isolated from phage-displayed libraries.

Following replacement therapy with coagulation factor VIII (FVIII), up to 30 % of haemophilia A patients develop FVIII-specific inhibitory antibodies (FVIII inhibitors). Immune tolerance induction (ITI) is not always successful, resulting in a need for alternative treatments for FVIII inhibitor-positive patients. As tolerance induction in the course of ITI appears to involve the formation of anti-idiotypes specific for anti-FVIII antibodies, such anti-idiotypes might be used to restore haemostasis in haemophilia A patients with FVIII inhibitors. We isolated anti-idiotypic antibody fragments (scFvs) binding to murine FVIII inhibitors 2-76 and 2-77 from phage-displayed libraries. FVIII inhibitor/anti-idiotype interactions were very specific as no cross-reactivity with other FVIII inhibitors or isotype controls was observed. ScFvs blocked binding of FVIII inhibitors to FVIII and neutralised their cognate inhibitors in vitro and a monoclonal mouse model. In addition, scFv JkH5 specific for FVIII inhibitor 2-76 stained 2-76-producing hybridoma cells. JkH5 residues R52 and Y226, located in complementary determining regions, were identified as crucial for the JkH5/2-76 interaction using JkH5 alanine mutants. SPR spectroscopy revealed that JkH5 interacts with FVIII inhibitor 2-76 with nanomolar affinity. Thus, FVIII inhibitor-specific, high-affinity anti-idiotypes can be isolated from phage-displayed libraries and neutralise their respective inhibitors. Furthermore, we show that anti-idiotypic scFvs might be utilised to specifically target inhibitor-specific B cells. Hence, a pool of anti-idiotypes could enable the reestablishment of haemostasis in the presence of FVIII inhibitors in patients or even allow the depletion of inhibitors by targeting inhibitor-specific B cell populations.

Allergy-Protective Arabinogalactan Modulates Human Dendritic Cells via C-Type Lectins and Inhibition of NF-κB.

Arabinogalactan (AG) isolated from dust of a traditional farm prevents disease in murine models of allergy. However, it is unclear whether this polysaccharide has immune regulatory properties in humans. The aim of this study was to test the influence of AG on the immune-stimulating properties of human dendritic cells (DCs). Moreover, we sought to identify the receptor to which AG binds. AG was produced from plant callus tissue under sterile conditions to avoid the influence of pathogen-associated molecular patterns in subsequent experiments. The influence of AG on the human immune system was investigated by analyzing its impact on monocyte-derived DCs. To analyze whether the T cell stimulatory capacity of AG-stimulated DCs is altered, an MLR with naive Th cells was performed. We revealed that AG reduced T cell proliferation in a human MLR. In the search for a molecular mechanism, we found that AG binds to the immune modulatory receptors DC-specific ICAM-3 -: grabbing non integrin (DC-SIGN) and macrophage mannose receptor 1 (MMR-1). Stimulation of these receptors with AG simultaneously with TLR4 stimulation with LPS increased the expression of the E3 ubiquitin-protein ligase tripartite motif -: containing protein 21 and decreased the phosphorylation of NF-κB p65 in DCs. This led to a reduced activation profile with reduced costimulatory molecules and proinflammatory cytokine production. Blocking of MMR-1 or DC-SIGN with neutralizing Abs partially inhibits this effect. We conclude that AG dampens the activation of human DCs by LPS via binding to DC-SIGN and MMR-1, leading to attenuated TLR signaling. This results in a reduced T cell activation capacity of DCs.

Ultrasound-Guided Versus Blind Subacromial Corticosteroid Injections for Subacromial Impingement Syndrome: A Randomized, Double-Blind Clinical Trial.

BACKGROUND: Subacromial corticosteroid injections are frequently performed for impingement syndrome of the shoulder. To improve the accuracy of injections, ultrasound can be used. PURPOSE: To assess the clinical outcome of ultrasound-guided subacromial injections compared with blind subacromial injections for subacromial impingement syndrome. STUDY DESIGN: Randomized controlled clinical trial; Level of evidence, 1. METHODS: A total of 56 shoulders with subacromial impingement syndrome were randomized into 2 groups: 28 shoulders received a subacromial corticosteroid injection with ultrasound guidance (ultrasound group), and 28 shoulders received a subacromial corticosteroid injection without ultrasound guidance (blind group). The visual analog scale (VAS) for pain with overhead activities and the American Shoulder and Elbow Surgeons (ASES) score were obtained before the injection and at 6 weeks after the injection. RESULTS: The VAS score for pain with overhead activities decreased from 59 ± 5 mm (mean ± SEM) before the injection to 33 ± 6 mm at 6 weeks after the injection in the ultrasound group (P < .001) and from 63 ± 4 mm to 39 ± 6 mm, respectively, in the blind group (P < .001). The decrease in the VAS score was not significantly different between the groups (P > .999). The ASES score increased from 57 ± 2 before the injection to 68 ± 3 at 6 weeks after the injection in the ultrasound group (P < .01) and from 54 ± 3 before the injection to 65 ± 4 after the injection in the blind group (P < .01), with no significant difference between the groups (P = .7). Four shoulders (14%) in the ultrasound group and 6 shoulders (21%) in the blind group eventually needed surgery (P = .7). CONCLUSION: No significant differences were found in the clinical outcome when comparing ultrasound-guided subacromial injections to blind subacromial injections for subacromial impingement syndrome. CLINICAL TRIAL REGISTRATION: Australian New Zealand Clinical Trials Registry: ACTRN12615000562572.

Revision versus primary arthroscopic rotator cuff repair: a 2-year analysis of outcomes in 360 patients.

BACKGROUND: Symptomatic rotator cuff tears are often treated surgically. However, there is a paucity of information regarding the outcomes of revision arthroscopic rotator cuff repairs. PURPOSE: To evaluate the outcome of revision arthroscopic rotator cuff surgery when compared with primary arthroscopic rotator cuff surgery in a large cohort of patients. STUDY DESIGN: Cohort study; Level of evidence, 3. METHOD: A consecutive series of 50 revision arthroscopic rotator cuff repairs performed by a single surgeon, with minimum 2-year follow-up, were retrospectively reviewed using prospectively collected data. As a comparison, 3 primary arthroscopic rotator cuff repair cases (primary group; n = 310) were chosen immediately before each revision case, and 3 were chosen after. Standardized patient-ranked outcomes, examiner-determined assessments, and ultrasound-determined rotator cuff integrity were assessed preoperatively at 6 months and at a minimum of 2 years after surgery. RESULTS: The revision group was older (mean age, 63 years; range, 43-80 years) compared with the primary group (mean age, 60 years; range, 18-88 years) (P < .05) and had larger tear size (mean ± SEM) (4.1 ± 0.5 cm(2)) compared with the primary group (3.0 ± 0.2 cm(2)) (P < .05). Two years after surgery, the primary group reported less pain at rest (P < .02), during sleep (P < .05), and with overhead activity (P < .01) compared with the revision group. The primary group had better passive forward flexion (+13°; P < .05), abduction (+18°; P < .01), internal rotation (+2 vertebral levels; P < .001) and also significantly greater supraspinatus strength (+15 N; P < .001), lift-off strength (+9.3 N; P < .05), and adduction strength (+20 N; P < .01) compared with the revision group at 2 years. When compared with the primary group, the revision group was more satisfied with the overall shoulder function before surgery but was less satisfied with their shoulder function than the primary group at 2 years (P < .005). The retear rate for primary rotator cuff repair was 16% at 6 months and 21% at 2 years, while the retear rate for revision rotator cuff repair was 28% at 6 months and deteriorated to 40% at 2 years (P < .05). CONCLUSION: The short-term clinical outcomes of patients undergoing revision rotator cuff repair were similar to those after primary rotator cuff repair. However, these results did not persist, and by 2 years patients who had revision rotator cuff repair were twice as likely to have retorn compared with those undergoing primary repair. The increase in retear rate in the revision group at 2 years was associated with increased pain, impaired overhead function, less passive motion, weaker strength, and less overall satisfaction with shoulder function.

Epitope mapping via selection of anti-FVIII antibody-specific phage-presented peptide ligands that mimic the antibody binding sites.

The most serious complication in today's treatment of congenital haemophilia A is the development of neutralising antibodies (inhibitors) against factor VIII (FVIII). Although FVIII inhibitors can be eliminated by immune tolerance induction (ITI) based on repeated administration of high doses of FVIII, 20-30% of patients fail to become tolerant. Persistence of FVIII inhibitors is associated with increased morbidity and mortality. Data from recent studies provide evidence for a potential association between ITI outcome and epitope specificity of FVIII inhibitors. Nevertheless the determination of epitopes and their clinical relevance has not yet been established. In this study a general strategy for the identification of anti-FVIII antibody epitopes in haemophilia A patient plasma was to be demonstrated. Phage-displayed peptide libraries were screened against anti-FVIII antibodies to isolate specific peptides. Peptide specificity was confirmed by FVIII-sensitive ELISA binding. Peptide residues essential for antibody binding were identified by mutational analysis and epitopes were predicted via FVIII homology search. The proposed mapping strategy was validated for the monoclonal murine antibody (mAb) 2-76. Binding studies with FVIII variants confirmed the location of the predicted epitope at the level of individual amino acids. In addition, anti-FVIII antibody-specific peptide ligands were selected for 10 haemophilia A patients with FVIII inhibitors. Detailed epitope mapping for three of them showed binding sites on the A2, A3 and C2 domains. Precise epitope mapping of anti-FVIII antibodies using antibody-specific peptide ligands can be a useful approach to identify antigenic sites on FVIII.

A comparison of outcomes after arthroscopic repair of partial versus small or medium-sized full-thickness rotator cuff tears.

BACKGROUND: Little is known about the outcomes after repair of partial-thickness rotator cuff tears. The aim of this study was to assess the outcome after repair of partial-thickness rotator cuff tears compared with full-thickness tears. Our hypothesis was that repair of partial-thickness tears leads to more shoulder stiffness but fewer retears compared with repair of full-thickness tears. METHODS: A group of 105 consecutive patients who had a full-thickness tear measuring <3 cm2 was compared with a group of sixty-four patients who had a partial-thickness tear. All tears were repaired with use of a knotless single-row arthroscopic repair. The American Shoulder and Elbow Surgeons (ASES) score and standardized patient and examiner-determined outcomes were obtained preoperatively and at six, twelve, and twenty-four weeks and at two years after surgery. Rotator cuff integrity was determined by ultrasound examination at six months and two years after surgery. RESULTS: Examiner-determined postoperative stiffness at six weeks was common in both groups (50% of those with a partial-thickness tear and 47% of those with a full-thickness tear) but was decreased compared with preoperative findings in both groups to 21% and 19%, respectively, at three months and to 15% and 14% at six months. The ultrasound-determined retear rate was small (5% in the partial-thickness group and 10% in the full-thickness group) at six months, but increased to 10% and 20%, respectively, at twenty-four months. The ASES score, patient-determined overall shoulder function, and all pain scores were superior to preoperative scores at six months (p < 0.001) and at twenty-four months (p < 0.001) in both groups. CONCLUSIONS: Arthroscopic repair of partial-thickness and small and medium-sized full-thickness rotator cuff tears was associated with excellent medium-term clinical outcomes with low retear rates. The data did not support our hypothesis: the differences in retear rate and postoperative shoulder stiffness rate found between the two groups did not reach significance.

Carbon, nitrogen, and phosphorus distribution in particle size-fractionated separated pig and cattle slurry.

Solid liquid separation of animal slurry is a method to reduce the excess nutrient loads from intensive livestock production. Five different separation technologies (sedimentation, centrifugation, pressurized filtration, polymer flocculation and drainage, and iron chloride addition + polymer flocculation and drainage) were applied to pig and cattle slurry in a laboratory study. Separation efficiencies of mass, dry matter (DM), N, and P were measured. Particle size fractionation of the solid fractions was performed by subjecting them to wet fractionation and C, organic N (N(org)), and P contents were subsequently measured. Chemical pretreatment with polymer before gravity drainage separated DM, total N, and P from raw pig and cattle slurry with the highest efficiencies. Sedimentation and centrifugation separated P from pig and cattle slurries with higher simple separation efficiencies (0.77 and 0.70, respectively) compared with pressurized filtration (0.15 and 0.37). Pressurized filtration transferred the lowest masses (14 and 18%) to the solid fractions. Solid fractions from pig slurry generally contained higher concentrations of P and C compared with cattle slurry solid fractions. The majority of C in solid fractions was present in particles > 25 microm, whereas N and P were present in larger proportions in particles < 25 microm. Chemical pretreatment increased the capture of smaller N(org)- and P-rich particles into larger particles between 25 and 1000 microm.

Repair of partial-thickness rotator cuff tears: a biomechanical analysis of footprint contact pressure and strength in an ovine model.

PURPOSE: The purpose of this study was to determine whether transtendon repair by use of a novel small-diameter knotless anchor showed enhanced mechanical properties compared with tear completion and repair. METHODS: Articular-sided partial-thickness tears were created ex vivo in the infraspinatus of 24 ovine shoulders. The specimens were randomized into 4 groups of 6 each: (1) no repair, (2) transtendon repair, (3) completion of tear with tension-band single-row repair, and (4) completion of tear with double-row repair. Footprint contact pressure and ultimate load to failure were measured in each specimen. RESULTS: Technical failure of the transtendon anchors occurred in 3 of 15 shoulders. Transtendon repair (mean +/- SEM, 0.8 +/- 0.1 MPa) and double-row repair (1 +/- 0.09 MPa) showed 3-fold (P < .001) greater footprint contact pressures than tension-band single-row repair (0.3 +/- 0.03 MPa) and no repair (0.3 +/- 0.02 MPa). The ultimate load to failure for transtendon repair (544 +/- 22 N) was more than 3 times greater than that for the double-row repair (157 +/- 23 N) (P < .001) and the single-row repair (116 +/- 11 N) (P < .001). CONCLUSIONS: Transtendon repair of partial-thickness tears by use of specifically designed anchors biomechanically outperformed tear completion and repair in an ovine model. Transtendon repair showed the best combination of high footprint contact pressure and high ultimate failure load. However, the high insertion failure rate of these transtendon anchors is of concern. CLINICAL RELEVANCE: On the basis of the biomechanical data, transtendon repair of partial-thickness rotator cuff tears may be used as an alternative to tear completion and repair, but the specific transtendon anchors used in this study need further evaluation before their clinical use can be recommended.

Midterm results of cementless total hip replacement in rapidly destructive arthropathy and a review of the literature.

BACKGROUND AND PURPOSE: Rapidly destructive arthropathy (RDA) of the hip is a disease of unknown etiology characterized by a rapid destruction of the acetabular and femoral aspects of the hip joint. The purpose of this study was to assess the outcome of cementless total hip replacement in this category of patients. METHODS: A prospective study was performed of all cases of rapidly destructive arthropathy treated by cementless total hip replacement between 1998 and 2005. There were 6 female patients (8 hips) meeting the criteria of RDA. Median age at surgery was 74 years (range 64-83). Using the Paprosky classification of acetabular defects, five hips had a type 2B acetabular defect and three a type 3A acetabular defect. In all cases a cementless prosthesis was used. In two cases a shelf plasty of the acetabulum was added. Radiographic and clinical follow-up was performed up to 9 years postoperatively (mean follow-up 69 months, range 24-104 months). RESULTS: At radiographic follow-up, no signs of prosthetic loosening or migration were seen. Harris Hip Score improved from 25.8 (SD 7.3, range 11-34) preoperatively to 88.3 (SD 9.7, range 71-98) at latest follow-up. CONCLUSION: Cementless total hip replacement in patients with rapidly destructive arthropathy led to a good result in a series of eight cases at midterm follow-up.

Multimerization of peptide mimotopes for blocking of factor VIII neutralizing antibodies.

About 30 % of patients with severe hemophilia A develop neutralizing antibodies (inhibitors) to coagulation factor VIII (FVIII) upon treatment with exogenous factor preparations. Two peptides, C6 (NPVENMMDRDSQ) and H10 (QSPWQTWFTRAL), that mimic putative inhibitor epitopes (mimotopes), were previously selected by phage display screening of plasma samples from patients with inhibitors. Synthetic peptide mimotopes inhibited IgG binding to FVIII (IC(50): 30-50 microM). This effect was increased by an equimolar combination of both mimotopes. Mimotopes were fused to the C-terminal multimerization domain of the C4bp alpha-chain and expressed as multimers in 293T cells. Multimerized mimotopes showed improved binding to anti-FVIII IgG and prolonged in vitro half-life relative to synthetic peptides. The two mimotopes were combined in heteromultimers by co-transfection of 293T cells with respective vectors, resulting in bi-specific molecules that almost completely blocked polyclonal antibody binding to FVIII (IC(50): 2-3 microM). This strategy is capable of functionally improving synthetic peptides by multimerization and could provide a basis for novel therapeutic approaches for patients with hemophilia A and inhibitors.