Inter-α-trypsin Inhibitor Research Articles

Serum interα-trypsin inhibitor heavy chain H4 may be an anti-inflammatory marker reflecting disease risk, activity and treatment outcome of ankylosing spondylitis

Interα-trypsin inhibitor heavy chain H4 (ITIH4) modulates inflammation and immunity, which take part in the pathogenesis of ankylosing spondylitis (AS). The current research intended to discover the clinical value of serum ITIH4 quantification for AS management. Serum ITIH4 among 80 AS patients before current treatment initiation (baseline) at weeks (W) 4, 8 and 12 after treatment was detected by ELISA. Serum ITIH4 from 20 disease controls (DCs) and 20 healthy controls (HCs) was detected. ITIH4 expression was lower in AS patients than in DCs (p = 0.002) and HCs (p < 0.001). Among AS patients, ITIH4 was negatively associated with C-reactive protein (CRP) (r = −0.311, p = 0.005), bath AS disease activity index (BASDAI) (r = −0.223, p = 0.047), total pack pain (r = −0.273, p = 0.014) and AS disease activity score (ASDAS) (CRP) (r = −0.265, p = 0.018). Meanwhile, ITIH4 was negatively related to tumor necrosis factor (TNF)-α (r = −0.364, p = 0.001), interleukin (IL)-1β (r = −0.251, p = 0.025), IL-6 (r = −0.292, p = 0.009) and IL-17A (r = −0.254, p = 0.023). After treatment, the assessment of the spondylitis arthritis international society 40 response rate was 28.7% at W4, 46.3% at W8 and 55.0% at W12; ITIH4 showed an increasing trend from baseline to W12 (p < 0.001). Furthermore, ITIH4 at W8 (p = 0.020) and W12 (p = 0.035), but not at baseline or W4 (both p > 0.05), was enhanced in response patients vs. nonresponse patients. Additionally, ITIH4 at W12 was increased in AS patients receiving TNF inhibitors vs. those receiving nonsteroidal anti-inflammatory drugs (NSAIDs) (p = 0.024). Serum ITIH4 increases after treatment, and its augmentation is correlated with lower disease activity, decreased inflammation and enhanced treatment response in AS patients.

Amplification of Inflammation by Lubricin Deficiency Implicated in Incident, Erosive Gout Independent of Hyperuricemia.

In gout, hyperuricemia promotes urate crystal deposition, which stimulates the NLRP3 inflammasome and interleukin-1β (IL-1β)-mediated arthritis. Incident gout without background hyperuricemia is rarely reported. To identify hyperuricemia-independent mechanisms driving gout incidence and progression, we characterized erosive urate crystalline inflammatory arthritis in a young female patient with normouricemia diagnosed as having sufficient and weighted classification criteria for gout according to the American College of Rheumatology (ACR)/EULAR gout classification criteria (the proband). We conducted whole-genome sequencing, quantitative proteomics, whole-blood RNA-sequencing analysis using serum samples from the proband. We used a mouse model of IL-1β-induced knee synovitis to characterize proband candidate genes, biomarkers, and pathogenic mechanisms of gout. Lubricin level was attenuated in human proband serum and associated with elevated acute-phase reactants and inflammatory whole-blood transcripts and transcriptional pathways. The proband had predicted damaging gene variants of NLRP3 and of inter-α trypsin inhibitor heavy chain 3, an inhibitor of lubricin-degrading cathepsin G. Changes in the proband's serum protein interactome network supported enhanced lubricin degradation, with cathepsin G activity increased relative to its inhibitors, SERPINB6 and thrombospondin 1. Activation of Toll-like receptor 2 (TLR-2) suppressed levels of lubricin mRNA and lubricin release in cultured human synovial fibroblasts (P < 0.01). Lubricin blunted urate crystal precipitation and IL-1β induction of xanthine oxidase and urate in cultured macrophages (P < 0.001). In lubricin-deficient mice, injection of IL-1β in knees increased xanthine oxidase-positive synovial resident M1 macrophages (P < 0.05). Our findings linked normouricemic erosive gout to attenuated lubricin, with impaired control of cathepsin G activity, compounded by deleterious NLRP3 variants. Lubricin suppressed monosodium urate crystallization and blunted IL-1β-induced increases in xanthine oxidase and urate in macrophages. The collective activities of articular lubricin that could limit incident and erosive gouty arthritis independently of hyperuricemia are subject to disruption by inflammation, activated cathepsin G, and synovial fibroblast TLR-2 signaling.

A Retrospective Analysis of the Cartilage Kunitz Protease Inhibitory Proteins Identifies These as Members of the Inter-α-Trypsin Inhibitor Superfamily with Potential Roles in the Protection of the Articulatory Surface.

Aim: The aim of this study was to assess if the ovine articular cartilage serine proteinase inhibitors (SPIs) were related to the Kunitz inter-α-trypsin inhibitor (ITI) family. Methods: Ovine articular cartilage was finely diced and extracted in 6 M urea and SPIs isolated by sequential anion exchange, HA affinity and Sephadex G100 gel permeation chromatography. Selected samples were also subjected to chymotrypsin and concanavalin-A affinity chromatography. Eluant fractions from these isolation steps were monitored for protein and trypsin inhibitory activity. Inhibitory fractions were assessed by affinity blotting using biotinylated trypsin to detect SPIs and by Western blotting using antibodies to α1-microglobulin, bikunin, TSG-6 and 2-B-6 (+) CS epitope generated by chondroitinase-ABC digestion. Results: 2-B-6 (+) positive 250, 220,120, 58 and 36 kDa SPIs were detected. The 58 kDa SPI contained α1-microglobulin, bikunin and chondroitin-4-sulfate stub epitope consistent with an identity of α1-microglobulin-bikunin (AMBP) precursor and was also isolated by concanavalin-A lectin affinity chromatography indicating it had N-glycosylation. Kunitz protease inhibitor (KPI) species of 36, 26, 12 and 6 kDa were autolytically generated by prolonged storage of the 120 and 58 kDa SPIs; chymotrypsin affinity chromatography generated the 6 kDa SPI. KPI domain 1 and 2 SPIs were separated by concanavalin lectin affinity chromatography, domain 1 displayed affinity for this lectin indicating it had N-glycosylation. KPI 1 and 2 displayed potent inhibitory activity against trypsin, chymotrypsin, kallikrein, leucocyte elastase and cathepsin G. Localisation of versican, lubricin and hyaluronan (HA) in the surface regions of articular cartilage represented probable binding sites for the ITI serine proteinase inhibitors (SPIs) which may preserve articulatory properties and joint function. Discussion/Conclusions: The Kunitz SPI proteins synthesised by articular chondrocytes are members of the ITI superfamily. By analogy with other tissues in which these proteins occur we deduce that the cartilage Kunitz SPIs may be multifunctional proteins. Binding of the cartilage Kunitz SPIs to HA may protect this polymer from depolymerisation by free radical damage and may also protect other components in the cartilage surface from proteolytic degradation preserving joint function.

Potential of Protein-based Anti-metastatic Therapy with Serpins and Inter α-Trypsin Inhibitors.

In this review we summarize the principles of anti-metastatic therapy with selected serpin family proteins, such as pigment epithelial-derived factor (PEDF) and maspin, as well as inter α-trypsin inhibitor (IαIs) light chains (bikunin) and heavy chains (ITIHs). Case-by-case, antimetastatic activity may be dependent or independent of the protease-inhibitory activity of the corresponding proteins. We discuss the incidence of target deregulation in different tumor entities, mechanisms of deregulation, context-dependent functional issues as well as in vitro and in vivo target validation studies with transfected tumor cells or recombinant protein as anti-metastatic agents. Finally, we comment on possible clinical evaluation of these proteins in adjuvant therapy.

Identification of novel serum peptide biomarkers for high-altitude adaptation: a comparative approach.

We aimed to identify serum biomarkers for screening individuals who could adapt to high-altitude hypoxia at sea level. HHA (high-altitude hypoxia acclimated; n = 48) and HHI (high-altitude hypoxia illness; n = 48) groups were distinguished at high altitude, routine blood tests were performed for both groups at high altitude and at sea level. Serum biomarkers were identified by comparing serum peptidome profiling between HHI and HHA groups collected at sea level. Routine blood tests revealed the concentration of hemoglobin and red blood cells were significantly higher in HHI than in HHA at high altitude. Serum peptidome profiling showed that ten significantly differentially expressed peaks between HHA and HHI at sea level. Three potential serum peptide peaks (m/z values: 1061.91, 1088.33, 4057.63) were further sequence identified as regions of the inter-α trypsin inhibitor heavy chain H4 fragment (ITIH4 347–356), regions of the inter-α trypsin inhibitor heavy chain H1 fragment (ITIH1 205–214), and isoform 1 of fibrinogen α chain precursor (FGA 588–624). Expression of their full proteins was also tested by ELISA in HHA and HHI samples collected at sea level. Our study provided a novel approach for identifying potential biomarkers for screening people at sea level who can adapt to high altitudes.

Characterization of the Polyproline Region of the Hepatitis E Virus in Immunocompromised Patients

Little is known about virus adaptation in immunocompromised patients with chronic genotype 3 hepatitis E virus (HEV3) infections. Virus-host recombinant strains have been isolated recently from chronically infected patients. The nature and incidence of such recombinant events occurring during infections of solid-organ transplant (SOT) recipients are essentially unknown. The polyproline region (PPR) of strains isolated from SOT patients was sequenced during the acute-infection phase (n = 59) and during follow-up of patients whose infections became chronic (n = 27). These 27 HEV strains included 3 (11%) that showed recombinant events 12, 34, 48, or 88 months after infection. In one strain, parts of the PPR and the RNA-dependent RNA polymerase were concomitantly inserted. In the second, a fragment of a human tyrosine aminotransferase (TAT) gene was inserted first, followed by a fragment of PPR. A fragment of the human inter-α-trypsin inhibitor (ITI) gene was inserted in the third. All the inserted sequences were rich in aliphatic and basic amino acids. In vitro growth experiments suggest that the ITI insertion promoted more vigorous virus growth. In silico studies showed that the inserted sequences could provide potential acetylation, ubiquitination, and phosphorylation sites. We found that recombinant events had occurred in the HEV PPR in approximately 11% of the strains isolated from chronically infected transplant patients followed up in Toulouse University Hospital. These inserted fragments came from the HEV genome or a human gene and could enhance virus replication. Importance: Hepatitis E virus (HEV) can cause chronic infections in immunocompromised patients, including solid-organ transplant (SOT) recipients. Two strains that had undergone recombination with human ribosomal genes were described recently. The strains with inserted sequences replicated better in vitro. Little is known about the frequency of such recombinant events or how such an insertion enhances replication. We therefore investigated 59 SOT patients infected with HEV and found 3 strains with 4 recombinant events in 27 of these patients whose infection became chronic. The 4 inserted sequences were of different origins (human gene or HEV genome), but all were enriched in aliphatic and basic amino acids and provided potential regulation sites. Our data indicate that recombinant events occur in approximately 11% of strains isolated from chronically infected patients. The structures of the inserted sequences provide new clues as to how the inserted sequences could foster virus replication.

Effects of RU486 and indomethacin on meiotic maturation, formation of extracellular matrix, and progesterone production by porcine oocyte-cumulus complexes

This study was designed to determine whether inhibition of either cyclooxygenase-2 (COX-2) by indomethacin or progesterone receptor (PR) by PR antagonist, RU486, affects oocyte maturation, progesterone production, and covalent binding between hyaluronan (HA) and heavy chains of inter-α trypsin inhibitor, as well as expression of cumulus expansion-associated proteins (HA-binding protein, tumor necrosis factor α-induced protein 6, pentraxin 3) in oocyte-cumulus complexes (OCCs). The experiments were based on freshly isolated porcine OCC cultures in which the consequences of PR and COX-2 inhibition on the final processes of oocyte maturation were determined. Granulosa cells (GCs) and OCCs were cultured in medium supplemented with FSH/LH (both 100 ng/mL) in the presence/absence of RU486 or indomethacin. Western blot analysis, 3H-glucosamine hydrochloride assay, immunofluorescence, and radioimmunoassay were performed. Only treatment with RU486 (25 μM) caused a decrease in the number of oocytes that reached germinal vesicle breakdown and metaphase II stage compared with indomethacin (100 μM) or FSH/LH treatment alone after 44 h. All treated OCCs synthesized an almost equal amount of HA. Heavy chains (of inter-α trypsin inhibitor)-HA covalent complexes were formed during in vitro FSH/LH-stimulated expansion in RU486- or indomethacin-treated OCCs. Follicle-stimulating hormone/LH-induced progesterone production by OCCs was increased in the presence of RU486 after 44 h. In contrast, a decrease of FSH/LH-stimulated progesterone production by GCs was detected in the presence of either RU486 or indomethacin after 72 h. We suggest that the PR-dependent pathway may be involved in the regulation of oocyte maturation. Both PR and COX-2 regulate FSH/LH-stimulated progesterone production by OCCs and GCs.

Carotid atherosclerotic plaques: Proteomics study after a low‐abundance protein enrichment step

Atherosclerosis is one of the most important causes of cardiovascular and cerebrovascular events. Although phenotypic differentiation between stable and unstable plaques is currently possible, proteomic analysis of the atherosclerotic plaque could offer a global view of the atherosclerosis pathology. With the objective to highlight the detection of low-abundance proteins, we reduced the dynamic range of proteins by combinatorial peptide ligand library treatment of human carotid artery atherosclerotic plaques. After enrichment step, abundance of major proteins was decreased, revealing different protein profiles as assessed by both SDS-polyacrylamide gel electrophoresis and two-dimensional electrophoresis comparative analyses. Identification of proteins that were contained in a spot allowed finding large differences between noncomplicated and complicated plaques from carotid atherosclerotic lesions. Novel low-abundance proteins were detected correlating very well with biological alterations related to atherosclerosis (heat shock protein 27 (HSP27) isoforms, aldehyde dehydrogenase, moesin, Protein kinase C delta-binding protein, and inter-α trypsin inhibitor family heavy chain-related protein (ITIH4)). At the same time, the differential expression of known proteins of interest such as hemoglobin β-chain and heat shock protein 27 between noncomplicated and hemorrhagic complicated plaques was maintained after enrichment step. The detection of different isoforms of a low-abundance protein such as heat shock protein 27 species was actually improved after enrichment of tissue protein extracts. All of these findings clearly support further investigations in view to confirm the role of these proteins as possible biomarkers.

Isolation and identification of a snake venom metalloproteinase inhibitor from California ground squirrel (Spermophilus beecheyi) blood sera

California ground squirrels (Spermophilus beecheyi) show blood-based defenses to a variety of toxins in the venom of the Northern Pacific rattlesnake (Crotalus oreganus oreganus). In this study we demonstrate the presence of an effective snake venom metalloproteinase inhibitor (SVMPI) in S. beecheyi. The blood sera of California ground squirrels were effective at reducing the metalloproteinase activity of Northern Pacific (C. o. oreganus) and prairie rattlesnake (Crotalus viridis viridis) venoms by over 75%, significantly more than its ability to reduce the activity of western diamondback rattlesnake venom. We used anion exchange and affinity chromatography to isolate this protein from the blood sera of S. beecheyi. This SVMPI had a molecular mass of 108.3 kDa and a pI of 5.1. The IC50 of this inhibitor against whole venom from C. o. oreganus was determined to be 3.14 × 10−8 M. Subsequent LC MS/MS analysis of a CNBr/tryptic digest of the inhibitor yielded multiple internal peptide sequences. These sequences showed homology to three other known mammalian plasma proteins: inter-α trypsin inhibitor, and two hibernation-associated proteins, HP25 and HP27. The presence of SVMPI in S. beecheyi blood sera is consistent with the resistance of these animals to venom-induced hemorrhage and tissue damage, and consistent with the protective factors conferring venom resistance in other mammals. However, the variety of SVMPI identified to date from mammalian taxa suggests that different species have converged on neutralization of venom metalloproteinase activity as a key step in venom neutralization.

Sporadic intracranial aneurysms in patients with relatively proteomics research

Objective To find out the differences on the protein expressions between human intracranial aneurysm and normal adult by way of proteomics. Methods The blood serums were obtained from sporadic aneurysms and normal adults,and then separated by two-dimensional gel electrophoresis(2-DE). And the differential protein spots were identified by comparing the aneurysms and normals. And then to analyze the differential protein spots by matrix-assisted laser desorption ionization-time of flight-mass spectrometry(MALDI-TOF-MS) and The Protein Structure Database. The results were further validated by Western blot on the two selected proteins. Result Fourteen differential proteins were successfully identified, which were expressed significantly and differently in aneurysms and in controls, 11 peptide mass fingerprinting were received, and seven kinds of proteins were recognized. Haptoglobin, C-reactive protein, B factors, serum amyloid protein A and apolipoprotein E precursor group in the expression of aneurysms increased, while Apolipoprotein A-Ⅳ precursors and inter-α trypsin inhibitor heavy chain H4 precursor expression declined. The results of Western blotting were consistent with those of 2D-PAGE.Conclusion Seven proteins were identified with cerebral aneurysms, intracranial aneurysms in order to further clarify the pathophysiological mechanism of the formation of a new idea. Key words: Intracranial aneurysm; Proteomics; 2-dimensional Gel Electrophoresis; MALDI-TOF-MS

Identification of disease- and therapy-associated proteome changes in the sera of patients with myelodysplastic syndromes and del(5q)

Using ProteinChip array technology, which is based on the surface-enhanced laser desorption/ionization time-of-flight mass spectrometry, we performed proteomic analyses on sera from myelodysplastic syndromes (MDSs) patients with an interstitial deletion of the long arm of chromosome 5 (del(5q)) and those from control individuals. One analysis with 80 samples from 29 patients and 51 control subjects resulted in the detection of 61 peak differences. Another analysis with 36 paired-samples from 18 patients collected before and after the treatment with lenalidomide (Revlimid) identified 19 differential peak features. We also observed differential profiles between the pre-treatment samples from the responders and those from the non-responders reflected by eight peak differences. On the basis of these data we developed two classification models that could distinguish between the diseased and the control subjects or between the responders and the non-responders. Efforts were made to purify and identify a range of differential peak proteins. We conclude that inter-α trypsin inhibitor, heavy chain H4 (fragments), serum transferrin, transthyretin (variants), haemoglobin and a protein peak at m/z 2791 could be potential disease-associated markers for del(5q) MDS. Platelet factor 4 (PF-4) and a peak at m/z 8559 may serve as therapy-associated markers and be potentially useful for monitoring and predicting the response to therapy.

Endometrial gene expression of acute phase extracellular matrix components following estrogen disruption of pregnancy in pigs

In pigs, administration of estrogen to gilts on Days 9 and 10 of pregnancy causes conceptus fragmentation and death between Days 15 and 18 of gestation. Conceptus degeneration is associated with breakdown of the microvilli surface glycocalyx on the lumenal epithelium (LE). We previously identified endometrial expression of inter-α-trypsin inhibitor (ITI) and hyaluronic acid (HA), which are key components of extracellular matrix (ECM), during the period of conceptus attachment to the uterine surface in the pig. Tumor necrosis factor-α-inducible protein-6 (TNFAIP6) serves as a linker for ECM expansion and is stimulated by prostaglandin E (PGE). We hypothesized that early estrogen administration alters the normal ECM components forming glycocalyx on the LE. Bred gilts (4 gilts/trt/day) were treated with either 5 mg estradiol cypionate (E) or corn oil (CO) on Days 9 and 10 of gestation. The uterus was surgically removed on either Days 10, 12, 13, 15 and 17 of gestation and endometrial tissue snap frozen in liquid nitrogen. Endometrial tumor necrosis factor-α (TNF), TNFAIP6, interleukin 6 (IL6), and inter-α-trypsin inhibitor heavy chains (ITIH) were detected during early pregnancy thereby indicating all components for maintenance of the extracellular glycocalyx are present in the endometrium of pigs. However, only gene expression of ITIH2 was suppressed by E-treatment. TNFAIP6 protein was detected across all days of gestation but was not affected by E-treatment. The present study demonstrates that while the pig endometrium expresses key components of ECM only ITIH2 gene expression was altered by E-treatment. A decrease in ITIH2 could lead to the possible loss of the uterine glycocalyx leading to conceptus degeneration; however, other factors may be involved with the loss of glycocalyx during implantation in the pig following E-treatment.

ProteinChip Array Profiling for Identification of Disease- and Chemotherapy-Associated Biomarkers of Nasopharyngeal Carcinoma

We previously used ProteinChip array profiling analysis to discover a serum biomarker associated with nasopharyngeal carcinoma (NPC). In this study, we used the same method to examine other biomarkers associated with NPC and response to chemotherapy (CT) in NPC patients. We performed ProteinChip array analysis in 209 serum samples from 66 relapsed patients before and after salvage CT with gemcitabine and cisplatin or etoposide and cisplatin combinations, 11 patients in remission, and 35 healthy individuals. Intensities of the biomarker peaks were correlated with CT response of the patients and other clinical parameters. We discovered 13 candidate biomarkers associated with different clinical parameters. Two biomarkers (2803 and 3953 Da) were significantly increased in patients compared with controls at all stages of disease. Analysis of pre- and post-CT paired serum samples revealed 7 biomarkers correlated with impact of CT. Of these 7 biomarkers, 2 (2509 and 2756 Da) were significantly increased and 5 (7588, 7659, 7765, 7843, and 8372 Da) were significantly decreased post-CT in either 1 or both CT cohorts. Four biomarkers from pre-CT sera were correlated with CT response, with 3 (2950, 13 510, and 14 855 Da) being significantly decreased and 1 (6701 Da) significantly increased in patients who did not respond to CT. Tandem mass spectrometric sequencing and/or immunoaffinity capture assay identified the 3953 Da biomarker as a fragment of interalpha-trypsin inhibitor precursor and 7765 Da biomarker as platelet factor-4. Treatment-associated serum biomarkers found might serve to triage NPC patients for appropriate CT treatment.

Isolation of a Natural Inhibitor of Human Malignant Glial Cell Invasion: Inter α-Trypsin Inhibitor Heavy Chain 2

Malignant central nervous system (CNS) tumors, such as glioblastoma multiforme, invade the brain and disrupt normal tissue architecture, making complete surgical removal virtually impossible. Here, we have developed and optimized a purification strategy to isolate and identify natural inhibitors of glioma cell invasion in a three-dimensional collagen type I matrix. Inter alpha-trypsin inhibitor heavy chain 2 (ITI H2) was identified from the most inhibitory fractions and its presence was confirmed both as a single protein and in a bikunin-bound form. Stable overexpression in U251 glioma cells validated ITI H2's strong inhibition of human glioma cell invasion together with significant inhibition of cell proliferation and promotion of cell-cell adhesion. Analysis of primary human brain tumors showed significantly higher levels of ITI H2 in normal brain and low-grade tumors compared with high-grade gliomas, indicating an inverse correlation with malignancy. The phosphatidylinositol 3-kinase/Akt signaling cascade seemed to be one of the pathways involved in the effect of ITI H2 on U251 cells. These findings suggest that reduction of ITI H2 expression correlates with brain tumor progression and that targeting factors responsible for its loss or restoring the ITI supply exogenously may serve as potential therapeutic strategies for a variety of CNS tumors.

ITIH5, a novel member of the inter-α-trypsin inhibitor heavy chain family is downregulated in breast cancer

The inter-α-trypsin inhibitor (ITI) family constitutes a group of proteins built up from one light chain and a variable set of heavy chains. Originally identified as plasma protease inhibitors, recent data indicate that ITI plays a role in extracellular matrix (ECM) stabilization and in prevention of tumor metastasis. Here we describe cloning as well as phylogenetic and expression analysis of a novel member of the heavy chain gene family, ITIH5. ITIH5 contains the two domains conserved in all known ITIHs, the vault protein inter-alpha-trypsin (VIT) domain and a von Willebrand type A (vWA) domain. However, ITIH5 diverged early from a common ancestor of the other subfamilies. We found strong downregulation of ITIH5 expression in breast tumors by real-time PCR and RNA in situ hybridization. While normal breast epithelial cells clearly express ITIH5, expression is consistantly lost or strongly downregulated in invasive ductal carcinoma. ITIH5 mRNA was neither detectable in cancerous nor benign breast cell lines. We propose that loss of ITIH5 expression may be involved in breast cancer development.

TSG-6 and calcium ions are essential for the coupling of inter-alpha-trypsin inhibitor to hyaluronan in human synovial fluid

Objective: To investigate the role of tumor necrosis factor stimulated gene 6 (TSG-6) and metal ions in the coupling of inter-α-trypsin inhibitor (ITI) to hyaluronan in human synovial fluid. Design: The concentration of ITI heavy chains bound to hyaluronan was determined by a two-step electrophoretic technique. Synovial fluid, TSG-6 depleted synovial fluid and metal chelated synovial fluid were tested for their ability to support the coupling of ITI heavy chains to hyaluronan. Results: When synovial fluid was mixed with an ITI-source (serum or purified ITI), coupling of ITI heavy chains to hyaluronan took place. TSG-6 immunodepleated synovial fluid lost the coupling activity, but addition of recombinant TSG-6 restored the activity. EDTA inhibited the coupling activity, but combinations of the metal-ion chelators Mg-EGTA and Ca-EGTA demonstrated, that Ca ++is essential for the coupling of ITI heavy chains to hyaluronan. Conclusions: Tumor necrosis factor stimulated gene 6 (TSG-6) and calcium ions are both essential for the coupling of inter-α-trypsin inhibitor to hyaluronan in human synovial fluid.

Decreased Expression of Tumor Necrosis Factor-α-Stimulated Gene 6 in Cumulus Cells of the Cyclooxygenase-2 and EP2 Null Mice

Ovulation, the release of fertilizable oocytes from mature follicles, involves tissue remodeling and increased prostaglandin (PG) signaling. Cyclooxygenase (COX)-2 is the rate-limiting enzyme during PG synthesis. Female mice null for either COX-2 or the PGE(2) receptor EP2 are infertile, show decreased ovulation, and exhibit abnormal cumulus expansion. Cumulus expansion is the production of a complex extracellular matrix surrounding the cumulus-oocyte complex (COC). Matrix components consist of hyaluronan, proteoglycans, and proteins with hyaluronan binding domains. One such hyaluronan binding protein is TNFalpha-stimulated gene 6 (TSG-6). By various methods, we show induction of TSG-6 and hyaluronan synthase-2 mRNA in ovaries of mice treated with pregnant mare serum gonadotropin and human chorionic gonadotropin. By in situ hybridization, we show that both genes are expressed in periantral mural granulosa cells and cumulus cells of the mouse ovary. Notably, RT-PCR and in situ hybridization show that TSG-6 mRNA but not hyaluronan synthase-2 mRNA expression is selectively reduced in cumulus cells of COX-2 and EP2 null mice. Western analysis further confirms that TSG-6 protein is reduced in isolated COCs but remains covalently associated with inter alpha-trypsin inhibitor in COX-2 null mice. These observations identify TSG-6 as a target of PG action and show that its production in ovulatory follicles is associated with proper formation of the cumulus-derived extracellular matrix.

Glycosaminoglycan-bound and free inter-alpha-trypsin inhibitor components of follicular fluid.

The proteinase inhibitor inter-alpha trypsin inhibitor (ITI) is a blood-derived protein necessary for normal female fertility. Absence of ITI leads to ovulation of naked oocytes that cannot fertilise. ITI consists of two heavy chains (ITI-HC) and bikunin linked by a chrondroitin sulphate. By binding to hyaluronate, ITI-HC stabilises the extracellular matrix, but ITI-HC also binds to proteoglycans in follicular fluid. In vivo concentrations of ITI components in preovulatory follicular fluid, free as well as bound to hyaluronate or proteoglycan, are unknown. In order to quantify these components, 58 follicular fluids and 13 blood samples were collected in connection with in vitro fertilisation and embryo transfer treatment of 13 women. Quantitation of glycosaminoglycan-bound ITI-HC was performed after separation from free ITI in agarose gel. ITI components were determined by immunoelectrophoresis and hyaluronate by an ELISA method. The follicular fluid concentration of ITI was on average 70% of that in plasma and the concentration of hyaluronate remained low despite follicular production, suggesting that the production of hyaluronate is the rate-limiting step in the formation of the extracellular matrix of the oocyte-cumulus complex. In follicular fluid, the concentration of free ITI-HC was higher than that of glycosaminoglycan-bound ITI-HC. Addition of exogeneous hyaluronate doubled the amount of hyaluronate-bound ITI-HC, further supporting the notion that ITI in follicular fluid is not rate-limiting for cumulus expansion in vivo.

Assignment1 of the porcine inter-α trypsin inhibitor heavy chain 4 (ITIH4) gene to SSC13q2.1→q2.2 by fluorescence in situ hybridization and radiation hybrid mapping

Assignment<footref rid="foot01">1</footref> of the porcine inter-α trypsin inhibitor heavy chain 4 (ITIH4) gene to SSC13q2.1→q2.2 by fluorescence in situ hybridization and radiation hybrid mapping

Immunohistochemical Distribution of Inter-α-trypsin Inhibitor Chains in Normal and Malignant Human Lung Tissue

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)