Cell-autonomous Hedgehog signaling controls Th17 polarization and pathogenicity.

Th17 cells are key drivers of autoimmune disease. However, the signaling pathways regulating Th17 polarization are poorly understood. Hedgehog signaling regulates cell fate decisions during embryogenesis and adult tissue patterning. Here we find that cell-autonomous Hedgehog signaling, independent of exogenous ligands, selectively drives the polarization of Th17 cells but not other T helper cell subsets. We show that endogenous Hedgehog ligand, Ihh, signals to activate both canonical and non-canonical Hedgehog pathways through Gli3 and AMPK. We demonstrate that Hedgehog pathway inhibition with either the clinically-approved small molecule inhibitor vismodegib or genetic ablation of Ihh in CD4+ T cells greatly diminishes disease severity in two mouse models of intestinal inflammation. We confirm that Hedgehog pathway expression is upregulated in tissue from human ulcerative colitis patients and correlates with Th17 marker expression. This work implicates Hedgehog signaling in Th17 polarization and intestinal immunopathology and indicates the potential therapeutic use of Hedgehog inhibitors in the treatment of inflammatory bowel disease.

Blocking the proteolytic activity of zymogen matriptase with antibody-based inhibitors.

Matriptase is a member of the type-II transmembrane serine protease (TTSP) family and plays a crucial role in the development and maintenance of epithelial tissues. As all chymotrypsin-like serine proteases, matriptase is synthesized as a zymogen (proform), requiring a cleavage event for full activity. Recent studies suggest that the zymogen of matriptase possesses enough catalytic activity to not only facilitate autoactivation, but also carry out its in vivo functions, which include activating several proteolytic and signaling cascades. Inhibition of zymogen matriptase may therefore be a highly effective approach for limiting matriptase activity. To this end, here we sought to characterize the catalytic activity of human zymogen matriptase and to develop mAb inhibitors against this enzyme form. Using a mutated variant of matriptase in which the serine protease domain is locked in the zymogen conformation, we confirmed that the zymogen form of human matriptase has catalytic activity. Moreover, the crystal structure of the catalytic domain of zymogen matriptase was solved to 2.5 Å resolution to characterize specific antibody-based matriptase inhibitors and to further structure-based studies. Finally, we describe the first antibody-based competitive inhibitors that target both the zymogen and activated forms of matriptase. We propose that these antibodies provide a more efficient way to regulate matriptase activity by targeting the protease both before and after its activation and may be of value for both research and preclinical applications.

CL-L1 and CL-K1 Exhibit Widespread Tissue Distribution With High and Co-Localized Expression in Secretory Epithelia and Mucosa.

Collectin liver 1 (CL-L1, alias collectin 10) and collectin kidney 1 (CL-K1, alias collectin 11) are oligomeric pattern recognition molecules associated with the complement system, and mutations in either of their genes may lead to deficiency and developmental defects. The two collectins are reportedly localized and synthesized in the liver, kidneys, and adrenals, and can be found in the circulation as heteromeric complexes (CL-LK), which upon binding to microbial high mannose-like glycoconjugates activates the complement system via the lectin activation pathway. The tissue distribution of homo- vs. heteromeric CL-L1 and -K1 complexes, the mechanism of heteromeric complex formation and in which tissues this occurs, is hitherto incompletely described. We have by immunohistochemistry using monoclonal antibodies addressed the precise cellular localization of the two collectins in the main human tissues. We find that the two collectins have widespread and almost identical tissue distribution with a high expression in epithelial cells in endo-/exocrine secretory tissues and mucosa. There is also accordance between localization of mRNA transcripts and detection of proteins, showing that local synthesis likely is responsible for peripheral localization and eventual formation of the CL-LK complexes. The functional implications of the high expression in endo-/exocrine secretory tissue and mucosa is unknown but might be associated with the activity of MASP-3, which has a similar pattern of expression and is known to potentiate the activity of the alternative complement activation pathway.

Fast form alpha-2-macroglobulin - A marker for protease activation in plasma exposed to artificial surfaces.

OBJECTIVES: Investigation of the blood compatibility requires a number of sensitive assays to quantify the activation of the blood protein cascades and cells induced by biomaterials. A global assay measuring the blood compatibility of biomaterials could be a valuable tool in such regard. In this study, we investigated whether an enzyme-linked immunosorbent assay (ELISA), that specifically measures the electrophoretic "fast form" of α2-macroglobulin (F-α2M), could be a sensitive and global marker for activation of calcium dependent and in-dependent proteases in plasma exposed to biomaterials in vitro. METHODS: A F-α2M specific monoclonal antibody was generated and applied in an ELISA setup. Using the F-α2M ELISA, we investigated activation of calcium dependent and in-dependent proteases by polyvinylchloride (n=10), polytetrafluoroethylene (n=10) and silicone (n=10) tubings as well as glass tubes (n=10). RESULTS: We found that F-α2M is a sensitive marker for activation of both calcium dependent and in-dependent proteases. A significant difference between F-α2M concentrations in the control sample and plasma exposed to the artificial surfaces was found (p>0.001). This was observed both in the presence and absence of calcium. Furthermore, the highest F-α2M concentration was in both cases found in plasma incubated with glass. CONCLUSIONS: Our findings demonstrate that F-α2M is a sensitive marker for detection of protease activation in plasma by artificial surfaces. Potentially, levels of F-α2M could be a global marker of the blood compatibility of biomaterials.

A novel antihuman C3d monoclonal antibody with specificity to the C3d complement split product.

The complement component C3 and the cleavage products of C3b/iC3b, C3c and C3d are used as biomarkers in clinical diagnostics. Currently, no specific antibodies are able to differentiate C3d from other fragments, although such a distinction could be very valuable considering that they may reflect different pathophysiological mechanisms. We have developed a rat antihuman C3d monoclonal antibody with specificity to the end sequence of the N-terminal region of C3d. The antibody can therefore only bind to C3d when it manifests itself as the final end product of cleaved C3. We believe that this specificity is it first of its kind, and predicts that it can be used as a detection tool in several immunological methods with great value in diagnostics.

Calcium-sensitive immunoaffinity chromatography: Gentle and highly specific retrieval of a scarce plasma antigen, collectin-LK (CL-LK).

Immunoaffinity chromatography is a powerful fractionation technique that has become indispensable for protein purification and characterization. However, it is difficult to retrieve bound proteins without using harsh or denaturing elution conditions, and the purification of scarce antigens to homogeneity may be impossible due to contamination with abundant antigens. In this study, we purified the scarce, complement-associated plasma protein complex, collectin LK (CL-LK, complex of collectin liver 1 and kidney 1), by immunoaffinity chromatography using a calcium-sensitive anti-collectin-kidney-1 mAb. This antibody was characterized by binding to CL-LK at hypo- and physiological calcium concentrations and dissociated from CK-LK at hyperphysiological concentrations of calcium. We purified CL-LK from plasma to a purity of 41% and a yield of 38%, resulting in a purification factor of more than 88,000 in a single step. To evaluate the efficiency of this new purification scheme, we purified CL-LK using the same calcium-sensitive mAb in combination with acidic elution buffer and by using calcium-dependent anti-CL-K1 mAbs in combination with EDTA elution buffer. We found that calcium-sensitive immunoaffinity chromatography was superior to the traditional immunoaffinity chromatographies and resulted in a nine-fold improvement of the purification factor. The technique is applicable for the purification of proteins in complex mixtures by single-step fractionation without the denaturation of eluted antigens, and it allows for the purification of scarce proteins that would have otherwise been impossible to purify and, hence, to characterize. This technique may also potentially be applied for the purification of proteins that only interact with calcium ions at hyperphysiological concentrations.

Three monoclonal antibodies against the serpin protease nexin-1 prevent protease translocation.

Protease nexin-1 (PN-1) belongs to the serpin family and is an inhibitor of thrombin, plasmin, urokinase-type plasminogen activator, and matriptase. Recent studies have suggested PN-1 to play important roles in vascular-, neuro-, and tumour-biology. The serpin inhibitory mechanism consists of the serpin presenting its so-called reactive centre loop as a substrate to its target protease, resulting in a covalent complex with the inactivated enzyme. Previously, three mechanisms have been proposed for the inactivation of serpins by monoclonal antibodies: steric blockage of protease recognition, conversion to an inactive conformation or induction of serpin substrate behaviour. Until now, no inhibitory antibodies against PN-1 have been thoroughly characterised. Here we report the development of three monoclonal antibodies binding specifically and with high affinity to human PN-1. The antibodies all abolish the protease inhibitory activity of PN-1. In the presence of the antibodies, PN-1 does not form a complex with its target proteases, but is recovered in a reactive centre cleaved form. Using site-directed mutagenesis, we mapped the three overlapping epitopes to an area spanning the gap between the loop connecting α-helix F with ß-strand 3A and the loop connecting α-helix A with ß-strand 1B. We conclude that antibody binding causes a direct blockage of the final critical step of protease translocation, resulting in abortive inhibition and premature release of reactive centre cleaved PN-1. These new antibodies will provide a powerful tool to study the in vivo role of PN-1's protease inhibitory activity.

Complement defects in patients with chronic rhinosinusitis.

The complement system is an important part of our immune system, and complement defects lead generally to increased susceptibility to infections and autoimmune diseases. We have studied the role of complement activity in relation with chronic rhinosinusitis (CRS), and more specifically studied whether complement defects collectively predispose individuals for CRS or affect CRS severity. The participants comprised 87 CRS patients randomly selected from the general population, and a control group of 150 healthy blood donors. The CRS patients were diagnosed according to the European Position Paper on Rhinosinusitis and nasal Polyps criteria, and severity was evaluated by the Sino-nasal Outcome Test-22. Serum samples were analysed by ELISA for activity of the respective pathways of complement, and subsequently for serum levels of relevant components. We found that the frequency of complement defects was significantly higher among CRS patients than among healthy control subjects. A majority of Mannan-binding lectin deficient CRS patients was observed. The presence of complement defects had no influence on the severity of subjective symptoms. Our studies show that defects in the complement system collectively may play an immunological role related to the development of CRS. However, an association between severity of symptoms and presence of complement defects could not be demonstrated.

Cellular and humoral factors involved in the response of rainbow trout gills to Ichthyophthirius multifiliis infections: molecular and immunohistochemical studies.

The parasitic ciliate Ichthyophthirius multifiliis infecting skin, fins and gills of fish induces a protective immune response in rainbow trout (Oncorhynchus mykiss) surviving the infection and a similar protection can be conferred by i.p. injection of live theronts. A combined molecular and immunohistochemical approach has been used in this work for pinpointing cellular and humoral immune factors in gill tissue involved in the response and indicating interactions between the systemic and local responses. Fish were immunized by intra-peritoneal injection of live I. multifiliis theronts, control fish were injected with PBS and subgroups were treated with the immuno-suppressant hydrocortisone before fish were challenged with live theronts. Significant up-regulations of genes encoding IgM, IgT, C3, SAA, IL-8, IL-22 and IFN-γ were induced by immunization and challenge. Hydrocortisone treatment had a significant down-regulating effect on genes incoding IgT, IgM, CD4, CD8, IFN-γ, IL-8 and IL-22 in all groups. Immunohistochemistry, using monoclonal antibodies to detect cellular markers, demonstrated active involvement of CD8, MHC II, IgT and IgM positive cells in gill tissue. Putative T-cells (CD8 positive cells) were detected in the intraepithelial lymphoid tissue located at the base of gill filaments and in hyperplastic gill tissue but following infection a clear efflux of these cells was detected. MHC II positive cells were distributed across the gill filaments and accumulated in hyperplastic tissue but hydrocortisone treatment affected their density negatively in both immunized and non-immunized fish. IgT positive cells were present in the epithelial lining of the gill lamellae (suggesting a primary role of this protein in the mucosal defence against the ciliate) whereas IgM positive cells were found only in gill arterioles and the lamellar capillaries. The present work indicates an intensive activity and specialized function of immune cells (B-cells, T-cells and macrophages) and humoral elements such as immunoglobulins IgT and IgM which are orchestrated by cytokines in gill tissue reacting against I. multifiliis.

Sodium polyanethole sulfonate as an inhibitor of activation of complement function in blood culture systems.

Sodium polyanethole sulfonate (SPS; trade name, Liquoid) is a constituent in culture media used to grow bacteria from blood samples from patients suspected of bacteremia. SPS prevents the killing of bacteria by innate cellular and humoral factors. We analyzed the effect of SPS on the three complement activation pathways: the classical, alternative, and lectin pathways, respectively. Inhibition of complement activity by SPS is caused by a blocking of complement activation and is not a result of complement consumption. The classical pathway is inhibited at SPS concentrations greater than 0.1 mg/ml, and complete inhibition is seen at 0.4 mg/ml. An SPS concentration of 0.5 mg/ml completely inhibits the binding of C1q and subsequent incorporation of C3, C4, and C9. The same was observed for the alternative pathway with an inhibition at SPS concentrations from 0.1 mg/ml and a complete inhibition from 0.4 mg/ml. Here, properdin binding was completely absent, and no incorporation of C3 and C9 was observed. In contrast, the lectin complement pathway remains unaffected at these SPS concentrations, and inhibition is first observed from 0.7 mg/ml. A complete inhibition required concentrations greater than 1 mg/ml. SPS is used in growth media (e.g., BACTEC and BacT/Alert) at concentrations from 0.3 to 0.5 mg/ml. The well-known finding that certain bacteria are growth inhibited by blood factors could therefore be a consequence of the lectin pathway, which is not inhibited at these concentrations. In addition, our findings also open up the possibility of a new assay for the assessment of the functional capacity of the lectin complement pathway.

Upregulation of estrogen receptor alpha and vitellogenin in eelpout (Zoarces viviparus) by waterborne exposure to 4-tert-octylphenol and 17beta-estradiol.

The mechanisms of action of an estrogenic chemical have been examined in a viviparous fish the eelpout (Zoarces viviparus), by identification of an upregulated estrogenic pathway--the induction of hepatic estrogen receptor mRNA, hepatic estrogen binding activity and plasma vitellogenin. A relative quantitative RT-PCR assay has been established to measure hepatic estrogen receptor alpha (ER) mRNA levels in eelpout. Assay conditions were optimised using control and induced samples to ascertain its applicability in the actual working range of ER mRNA concentrations. beta-Actin was co-amplified and used as an internal standard. Time-course effects of water exposure to 0.5 microg/L 17beta-estradiol (E(2)) and 25 microg/L of the xeno-estrogen 4-tert-octylphenol (4-tert-OP) on ER mRNA levels in the male eelpout was examined. After 48 h of exposure, ER transcripts were induced 15-fold and 6-fold in the E(2)- and OP-treated fish, respectively. This difference, however, was not apparent after 1 week of exposure, when similar high levels of ER mRNA were present in both groups (20-fold induction). This indicates that the estrogenic capacity of 4-tert-OP increases with exposure time. The effect of treatment was also evaluated by examining the induction of specific E(2) binding capacity in hepatic cytosolic extracts and by measuring vitellogenin in plasma. Both parameters were also induced by the treatments, but later in the time course. The measurement of ER mRNA by the RT-PCR assay showed to be the most sensitive method for the detection of estrogenic responses in eelpout.

Molecular cloning, characterisation, and tissue distribution of oestrogen receptor alpha in eelpout (Zoarces viviparus).

A cDNA encoding the eelpout (Zoarces viviparus) oestrogen receptor alpha (eERalpha) has been isolated from eelpout liver, cloned and sequenced. The cDNA contains a complete open reading frame encoding 570 amino acid residues (mw: 63.0 kDa). The amino acid sequence of eERalpha showed a high degree of identity to ERalpha of other teleost species. The tissue distribution of eERalpha mRNA was examined using Northern blotting, RT-PCR and in situ hybridisation (ISH). All three methods identified a pronounced expression of eERalpha in liver, pituitary, testis and ovary. In the brain ISH experiments showed that ERalpha mRNA was highly expressed in distinct regions of the preoptic area and the mediobasal hypothalamus. We have provided evidence that the receptor is auto-regulated by 17beta-oestradiol (E(2)) not only in liver but also in the testis, indicating an important role for E(2) during spermatogenesis in male eelpout. RT-PCR analysis showed a broader expression pattern including significant expression in the brain, kidney, heart, and gut of adult eelpout. In eelpout embryos eERalpha expression has also been identified, indicating a possible role for the receptor in early development. This study contributes to the accumulating evidence that in fish E(2) is not only involved in the regulation of liver specific proteins, but has a much broader range of targets.

Genomic and molecular characterization of CL-43 and its proximal promoter.

Collectins are part of the innate immune system as they bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination or opsonization of the invaders. Conglutinin and CL-43 are serum proteins that have only been found and characterized in Bovidae. We have studied molecular and genomic characteristics of CL-43 to identify polymorphisms that might be associated with disease-susceptible phenotypes or other traits in cattle, and to elucidate how the Bovidae may benefit from possessing additional collectins. Screening a bovine cDNA library resulted in the isolation of two plasmid clones that encoded the entire translated sequence of CL-43. The 5'-untranslated end and start point of transcription were identified by 5'-RACE and showed that the mRNA transcript comprises either 1326 or 1241 nucleotides because of alternative splicing. Both transcripts encode a protein of 321 amino acids including a signal peptide of 20 residues. Characterization of two overlapping genomic lambda phage clones showed that the gene comprised seven exons spanning 8.5 kbp. The CL-43 gene, like the conglutinin gene, was mapped to Bos taurus chromosome 28 at q1.8. The CL-43 promoter has 96% identity with the conglutinin promoter recently described by us, and the assignment of potential cis-regulatory elements shows that several hepatic transcription factors may regulate transcription in the acute phase response and in response to metabolic changes.

CL-46, a novel collectin highly expressed in bovine thymus and liver.

Collectins are oligomeric molecules with C-type lectin domains attached to collagen-like regions via alpha-helical neck regions. They bind nonself glycoconjugates on the surface of microorganisms and inhibit infection by direct neutralization, agglutination, or opsonization. During the characterization of the gene encoding bovine CL-43 (43-kDa collectin), we identified a novel collectin-gene. We report the cloning and partial characterization of the novel collectin CL-46. The mRNA comprises 1188 nucleotides encoding a protein of 371 aa with an included leader peptide of 20 residues. CL-46 has two cysteine residues in the N-terminal segment, a potential N-glycosylation site in the collagen region, and an extended hydrophilic loop close to the binding site of the carbohydrate recognition domain. It is expressed in the thymus, liver, mammary gland, and tissues of the digestive system. Recombinant CL-46 corresponding to the alpha-helical neck region and the C-type lectin domain binds preferential N-acetyl-D-glucoseamine and N-acetyl-D-mannoseamine. The gene encoding CL-46 spans approximately 10 kb and consists of eight exons, with high structural resemblance to the gene encoding human surfactant protein D. It is located on the bovine chromosome 28 at position q1.8 together with the gene encoding conglutinin and CL-43. Several potential thymus-related cis-regulatory elements were identified in the 5'-upstream sequence, indicating that the expression in thymus may be modulated by signals involved in T cell development.