Interleukin-10 receptor-1 expression in monocyte-derived antigen-presenting cell populations: dendritic cells partially escape from IL-10's inhibitory mechanisms.

Interleukin (IL)-10 is an important immunoregulatory cytokine that mediates its effects via a transmembrane receptor complex consisting of two different chains, IL-10R1 and IL-10R2. While IL-10R2 is ubiquitously expressed and does not bind IL-10 primarily, the expression of IL-10R1 determines cellular responsiveness. However, the current knowledge about the expression and regulation of IL-10R1 is still limited. Here we analyzed the expression of IL-10R1 on monocytic cells and demonstrated that human blood monocytes carried about 720 IL-10-binding sites on their surface. Compared with lymphocytes and various tissue cells and tissues, blood monocytes expressed the highest IL-10R1 levels. The in vitro differentiation of these cells into macrophages provoked a further increase of IL-10R1 surface expression. In contrast, their differentiation into myeloid dendritic cells (mDCs) resulted in reduced surface IL-10R1 levels. The different IL-10R1 levels expressed by monocyte-derived antigen-presenting cell populations were reflected in their different responsiveness toward IL-10. Importantly, also in vivo developed immature macrophages and mDCs showed different IL-10 sensitivity. These data suggest that, compared with monocytes and macrophages, mDCs partially escape from IL-10's inhibitory mechanisms by downregulating IL-10R1.

[Tympanotomy and sealing of the round window membrane in sudden sensorineural hearing loss: a retrospective analysis]. / Tympanoskopie mit Abschottung der Rundfenstermembran beim idiopathischen Hörsturz: Eine retrospektive Analyse.

BACKGROUND: The pathogenesis of idiopathic sudden sensorineural hearing loss (ISSHL) still remains unclear. This retrospective study was performed to evaluate the effectiveness of tympanotomy and sealing of the round window membrane after unilateral idiopathic sudden sensorineural hearing loss. METHODS: A total of 74 patients with idiopathic sudden sensorineural hearing loss were treated with antiphlogistic-rheologic infusion therapy according to Stennert (steroids and pentoxyphylline). In addition, all patients underwent exploratory tympanotomy and sealing of the round window membrane. Pure tone audiometry was performed pre- and postinterventionally. RESULTS: The average hearing loss (four pure tone average) of all patients was 58.9 dB pre-, and 46 dB postinterventionally, which is an average improvement of 12.9 dB. Patients with hearing loss of more than 60% improved significantly compared to patients with hearing loss less than 60% (33.9% vs. +3.3%). Sealing of the round window membrane was found to be more effective when performed within 8 days after ISSHL. A membrane rupture did not lead to better therapy results. No significant correlation was found between therapy outcome and other clinical symptoms. CONCLUSION: Sealing of the window membrane shows equal results to conservative methods. If patients with hearing loss of more than 60% have more benefit in tympanotomy with sealing of the window membrane than patients with less hearing loss--as shown as in this study--has to be proved in randomized examinations. Intraoperatively found ruptures of the round window membrane had no significant effect on the therapy outcome.

[Chronic obstructive eustachian tube dysfunction in adults: long-term results of balloon eustachian tuboplasty]. / Chronisch obstruktive Tubenfunktionsstörung des Erwachsenen: Langzeitergebnisse der Ballondilatation der Tuba Eustachii.

BACKGROUND: Sufficient diagnostic tools and effective therapies for chronic obstructive eustachian tube dysfunction are lacking. PATIENTS AND METHODS: A total of 120 patients (209 ears) with chronic obstructive eustachian tube dysfunction were treated over a 2-year period using transnasal endoscopic balloon dilatation of the cartilaginous part of the eustachian tube (balloon eustachian tuboplasty, BET). A balloon catheter is inserted into the eustachian tube via the pharyngeal opening and dilated with a pressure of 10 bar for 2 min. RESULTS: The first 12 patients (20 dilatations) had a pretreatment average tube score of 1.25 (± 1.83 SD), and 1 year after treatment, the score improved to 6.2 (± 2.61 SD). Furthermore, the pretreatment and 2-month posttreatment data of 66 additional patients (115 dilatations) were analyzed. In these patients, the tube score improved significantly from 2.21 (± 2.02 SD) to 5.4 (± 2.53 SD). CONCLUSION: The initial long-term results suggest that BET is feasible and safe for the treatment of chronic obstructive eustachian tube dysfunction.

[Therapy of chronic obstructive eustachian tube dysfunction: evolution of applied therapies]. / Therapie chronisch obstruktiver Funktionsstörungen der Tuba Eustachii: Konzepte im Wandel der Zeit.

This paper reviews the past and present developments in the treatment of chronic obstructive eustachian tube dysfunction. Alongside tube catheterization and bougie insertion, modern approaches such as laser eustachian tuboplasty and balloon eustachian tuboplasty (BET) are described. In BET, transnasal endoscopic insertion via the pharyngeal ostium places a balloon catheter in the cartilaginous portion of the eustachian tube. This is then dilated to a pressure of 10 bar for 2 min. Up until January 2013, 351 chronic obstructive eustachian tube dysfunction patients had been treated in our department using BET. The average preoperative eustachian tube score was 2.1 (± 1.8 standard deviation, SD); 12 months postoperatively it was 6.1 (± 2.6 SD). Of these patients, 87% expressed satisfaction with the improvement in chronic obstructive dysfunction. These results demonstrate that BET is a safe and effective treatment for improving eustachian tube function and ear ventilation.

[Intratumoral Onyx embolisation in the management of juvenile nasopharyngeal angiofibroma]. / Intratumorale Onyx-Embolisation in der Therapie von juvenilen Nasenrachenangiofibromen.

Preoperative embolization for the treatment of juvenile nasopharyngeal angiofibroma was successfully accomplished with Onyx by intratumoral puncture for the first time. Extratumoral migration of Onyx particles was not observed, precluding the necessity to inflate the shield balloon. Postinterventional angiography showed complete occlusion of all supporting blood vessels. Transnasal surgery on the following day achieved complete resection of the angiofibroma without complications. Direct intratumoral embolization of juvenile nasopharyngeal angiofibromas appears to be a safe and effective preoperative method without complications. It could represent a new strategy for the treatment of JNA, as is already the case with other highly vascularized head and neck tumors. Moreover, it increases the likelihood of achieving complete resection.

[Acquired postinflammatory stenosis of the external auditory canal]. / Postinflammatorische Gehörgangsstenose.

There are only few publications about acquired postinflammatory stenosis of the auditory canal, its etiology, its progress and an adequate therapy. Chronic inflammatory processes of the ear canal, the tympanic membrane or the middle ear could lead to a replacement of the origin epithelium by fibrotic tissue and ends in a total obliteration of the ear canal. Lateral parts of the canal remain open and have the form of a blind-ending sac. Patients can suffer from severe conductive hearing loss. Treatment implies local therapy in the first stadium of the disease, then in the second one surgical removement of the fibrotic tissue is indicated before ear drum and bony canal are covered with skin graft. This technique results in stable, wide ears with good hearing.

[Superelastic nitinol stapes prostheses]. / Superelastische Nitinol Stapes Prothesen.

UNLABELLED: SUPERELASTIC NITINOL STAPES PROSTHESESINTRODUCTION: Many different prostheses are available for stapes surgery. The newly designed Nitinol piston band-shaped stapes prosthesis could minimize postoperative complications due to its new superelastic characteristic and design. PATIENTS AND METHODS: A stapedectomy and implantation of superelastic Nitinol piston stapes prosthesis was performed in 22 patients with a mean age of 45.2 years. Examination and audiometry was performed pre- and postoperatively. RESULTS: The average observed air-bone-gap in all frequencies was 12.8 dB postoperatively, whereas in the frequencies 0.5-4 kHz ABG was under 10 dB in 68.2% of the patients, between 10 and 15 dB in 18.2% and between 15 and 20 dB in 9.1% of the patients. No patient showed air-bone-gap of more than 20 dB. One patient missed follow-up examination. CONCLUSION: In stapes surgery the step of fixation of the prosthesis onto the incus is critical. Implantation of the new Nitinol piston stapes prosthesis is facilitated due to the superelasticity and the design of the prosthesis. Crimping is not needed anymore. Postoperative hearing results are very good comparable to other Nitinol (shape memory) or titanium prostheses. Long-term results in a larger number of patients will be studied in the future.

[Hamartoma : a rare nasopharyngeal pathology as a cause of nasal obstruction]. / Das Hamartom : Eine seltene nasopharyngeale Pathologie als Ursache einer Nasenatmungsbehinderung.

We present a case of a 44-year-old man with complaints of double-sided nasal obstruction and a foreign body sensation. A polypoid lesion was seen in the nasopharynx and was later completely excised. Histologic examination showed a respiratory epithelial adenomatoid hamartoma. The clinical and pathological features of this rare tumor as a cause of nasal obstruction are discussed.

[Direct percutaneous embolization of a carotid body tumor with Onyx]. / Perkutane Onyx(R)-Embolisation zur Therapie von Glomus-caroticum-Tumoren.

Carotid body tumors are highly vascularized lesions that require successful preoperative embolization to achieve favorable clinical results in terms of morbidity and complete tumor resection. The procedure of percutaneous embolization was performed using ethylene-vinyl alcohol copolymer (Onyx) in addition to balloon-catheter protection to prevent particle displacement into the internal carotid artery. The procedure resulted in nearly complete tumor embolization and facilitated the uneventful complete surgical resection. Percutaneous angiographic embolization of carotid body tumors in the head and neck was found to be safe and effective. This technique is likely to result in improved surgical outcomes and tumor resectability.

A synthetic mimic of a discontinuous binding site on interleukin-10.

We synthetically reconstructed a discontinuous binding site on interleukin-10 (IL-10) that recognizes the neutralizing anti-IL-10 antibody CB/RS/1. To design the 32-mer IL-10 mimic, a discontinuous interaction site on IL-10 was mapped, and binding studies with epitope-derived peptides led to specific replacement of several amino acids. Both parts of the interaction site were combined by addition of a linker molecule. Systematic analoging of the combined molecule then led to introduction of several additional substitutions in both regions and the linker. All possible disulfide bridge-containing variants of the 32-mer were tested by binding studies. Parallel syntheses were performed on continuous cellulose membranes by spot synthesis. As a result, a conformationally stabilized IL-10-derived molecule was obtained that both binds to and neutralizes the biological activity of CB/RS/1 in the low nanomolar range. This synthetic approach is a powerful alternative to phage display methods for the design of protein mimics.

Antigen sequence- and library-based mapping of linear and discontinuous protein-protein-interaction sites by spot synthesis.

The knowledge (antigen-derived peptide scans)- and library (de novo)-based mapping of linear and discontinuous antibody epitopes as well as protein-protein contact sites in general by spot synthesis now is a well established technique. Due to its automation, this technique also promises great potential for applications in functional genomics. It should help to elucidate the complex network of interacting protein molecules involved in signal transduction events (Adam-klages et al. 1996; Hoffmüller et al. 1999). Although little chemistry is involved in the preparation of peptide scans or libraries and the synthesis procedure is relatively simple, the laboratories of immunologists or molecular biologists are often not equipped to perform spot synthesis. In this case scans or libraries can be purchased from commercial suppliers.

Applications of peptide arrays prepared by the SPOT-technology.

The growing range of applications for peptide arrays synthesized on coherent membranes by the SPOT-synthesis method proves they have emerged as a powerful proteomics technique to study molecular recognition events and identify biologically active peptides. Several developments, such as the introduction of novel polymeric surfaces, linkers, synthesis/cleavage strategies and detection methods, are facilitating an increasing spectrum of accessible compounds and applications in biological or pharmaceutical research.

Mapping of the interleukin-10/interleukin-10 receptor combining site.

The discontinuous interleukin-10(IL-10)/interleukin-10 receptor (IL-10R) combining site was mapped using sets of overlapping peptides derived from both binding partners bound to continuous cellulose membranes. Low affinity binding of single regions of the discontinuous contact sites on IL-10 and IL-10R could be identified due to (1) high peptide density on the membrane support, (2) incubation with high protein concentrations, (3) indirect immunodetection of the ligates after electrotransfer onto polyvinylene difluoride membranes, and (4) use of highly overlapping peptide scans of different length (6-mers and 15-mers). The single binding regions identified for each protein species are separated in the protein sequences, but form continuous areas on the surface of IL-10 (X-ray structure) and IL-10R (computer model). Furthermore, four epitopes of neutralizing anti-IL-10 and anti-IL-10R antibodies were mapped and overlap with these binding regions. Soluble peptides (15- to 19-mers) each spanning one of the three identified IL-10-derived receptor binding regions displayed no significant affinity to IL-10R as expected, whereas a peptide (35-mer) comprising two of these regions had considerably higher binding activity. The data are consistent with a previously published computer model of the IL-10/IL-10R complex. This approach should be generally applicable for the mapping of non-linear protein-protein contact sites.

The antigen binding domain of non-idiotypic human anti-F(ab')2 autoantibodies: study of their interaction with IgG hinge region epitopes.

In previous studies we described a natural human IgG-anti-F(ab')2 autoantibody family with immunoregulatory properties. Genes coding for the variable regions of the heavy and light chains of the Abs were isolated from a natural Ig gene library and scFv Abs were expressed in E. coli. The scFv Abs bound to F(ab')2 but not to Fab fragments. This points to an epitope located in the hinge region since Fab fragments are lacking most of the hinge. In order to verify our hypothesis, double chain peptides comprising the lower-, middle-, and part of the upper hinge subregion of IgG1-IgG4 were synthesized on cellulose membranes and tested for binding to the Abs. The results show binding of Abs to IgG1 and IgG4 hinge region peptides. In order to identify the key residues of the discontinuous epitopes we carried out complete substitutional analyses in which each amino acid of the wt peptides was substituted by all other amino acids except cysteine. The exchange of proline in the IgG1 or IgG4 middle hinge region abrogated the binding, revealing the importance of this subregion for epitope expression. No binding to the IgG2 or IgG3 hinge was detected. These results indicate that scFv anti-F(ab')2 Abs recognize the hinge region of IgG1 and IgG4 and that the expression of the epitope depends on an intact middle hinge subregion.

Is there an interaction between interleukin-10 and interleukin-22?

Interleukin(IL)-10 and IL-22 are structurally related cytokines. Their heterodimeric receptors consist of the cytokine-specific chains IL-10R1 and IL-22R1, respectively, and the common chain IL-10R2. This study focused on the question of whether IL-10 modulates IL-22 effects and vice versa. This question is important because IL-10 and IL-22 exert anti- and proinflammatory effects, respectively, and, as we show here, are simultaneously present in both systemic and local inflammation. The revealed lacking concomitance of IL-10R1 and IL-22R1 on identical cells excluded any possible interaction between IL-10 and IL-22 apart from the competition for IL-10R2. To study this competition, monocytes and hepatocytes were chosen. The dependence of the cytokine action on IL-10R2 was verified. Interestingly, no influence of IL-22 on IL-10 effects was observed. The same was true when IL-22 was used in complex with IL-22-binding protein. Similarly, no influence of IL-10 was found on IL-22 action. This missing competition seemed to be due to a lack of binding between IL-10R2 and the native cytokines in the absence of their corresponding R1 chain. However, IL-10R2 interacted with defined IL-10- and IL-22-derived peptides supporting the hypothesis that cytokine binding to its corresponding R1 chain creates a binding site on this cytokine for IL-10R2.

Spatially addressed synthesis of amino- and amino-oxy-substituted 1, 3,5-triazine arrays on polymeric membranes.

Effective spatially addressed parallel assembly of trisamino- and amino-oxy-1,3,5-triazines was achieved by applying the SPOT-synthesis technique on cellulose and polypropylene membranes. In addition to developing a suitable linker strategy and employing amines and phenolate ions as building blocks, a highly effective microwave-assisted nucleophilic substitution procedure at membrane-bound monochlorotriazines was developed. The 1,3, 5-triazines obtained could be cleaved in parallel from the solid support by TFA vapor to give compounds adsorbed on the membrane surface in a conserved spatially addressed format for analysis and screening. The reaction conditions developed were employed for the synthesis of 8000 cellulose-bound 1,3,5-triazines which were probed in parallel for binding to the anti-transforming growth factor-alpha monoclonal antibody Tab2 in order to identify epitope mimics.

Simultaneous synthesis of peptide libraries on single resin and continuous cellulose membrane supports: examples for the identification of protein, metal and DNA binding peptide mixtures.

Peptide libraries were simultaneously synthesized on single supports by double coupling 0.8 equivalents of an equimolar acylating amino acid mixture consisting of 19 amino acids (cysteine omitted) at randomized sites, thus compensating for the different coupling rates of the amino acids. Peptide epitope mixtures, as well as very complex mixtures such as a completely randomized hexapeptide, were prepared and analyzed by HPLC and amino acid analysis. The results obtained indicate that this method can be applied to the synthesis of peptide libraries. Parts of a simultaneously synthesized solution phase combinatorial library XXB1B2XX were successfully used for the detection of the linear epitope HFND of transforming growth factor-alpha (TGF alpha) recognized by the monoclonal antibody Tab2. Furthermore, novel combinatorial peptide libraries XXB1B2XX were prepared on continuous cellulose membrane supports, also allowing the identification of TGF alpha epitope sequences. In addition, peptide mixtures that bound to a double-stranded DNA (15mer) and silver were identified. These preliminary results indicate that cellulose-bound combinatorial peptide libraries can be used for the rapid and inexpensive screening of millions of peptides to identify single molecules that bind any given ligand such as proteins, nucleic acids and metals.

Coherent membrane supports for parallel microsynthesis and screening of bioactive peptides.

Since its invention the SPOT-synthesis methodology has become one of the most efficient strategies for the miniaturized assembly of large numbers of peptides. The combination of a facile synthetic method with high throughput solid- and solution-phase screening assays qualifies the SPOT-technique as a valuable tool in biomedical research. Recent developments such as the introduction of novel polymeric surfaces, new linker and cleavage strategies as well as automated robot systems extended the scope of practical chemical reactions that can be accommodated as well as the numbers of compounds obtainable by this technique. Thus, highly complex spatially addressed compound arrays have become accessible. Together with the introduction of novel screening assays, the method is excellently suited to elucidate recognition events on the molecular level.

Mutations of the core promoter and response to interferon treatment in chronic replicative hepatitis B.

In chronic replicative hepatitis B the significance of mutations in the basic core promoter (BCP), core upstream regulatory sequences (CURS) and negative regulatory element (NRE) for response to interferon (IFN) is unknown. A sequence analysis of the NRE, CURS, BCP, and precore region was performed from sera of 96 patients with chronic replicative hepatitis B (64 hepatitis B e antigen [HBeAg]-positive patients and 32 HBeAg-negative patients) treated with alfa-IFN (IFN-alpha). The overall sustained response (SR) rate to IFN was 30% with no significant difference between HBeAg-positive and HBeAg-negative patients. IFN responsiveness correlated to hepatitis B virus (HBV)-DNA levels, hepatitis B surface antigen (HBsAg) levels, the number of mutations in the complete BCP, especially nucleotide (nt) region 1753 to 1766 and mutations at nt 1762 and 1764. In HBeAg-positive hepatitis, SR to IFN was associated with a high number of mutations in the BCP (P <.04) and nucleotide region 1753 to 1766 (P <.015) as well as mutations at nucleotide 1764 (P <.007). In HBeAg-negative hepatitis, SR to IFN correlated with a low number of mutations in the BCP (P <.04) and nucleotide region 1753 to 1766 (P <.02) and a wild-type sequence at nt 1764 (P <.003). Prediction of IFN response was possible on the basis of nt 1764 in 77% of HBeAg-positive patients and 78% of HBeAg-negative patients. IFN response did not correlate with the occurrence of the 1896 mutation, mutations in the CURS or NRE, disease duration, ethnic origin of the patient, alanine transaminase (ALT) levels and HBV genotype. Our data suggest that HBV genome mutations located within the BCP are determinants of a response to IFN therapy.