Hans-Georg Wisniewski

Agarose and polyacrylamide gel electrophoresis methods for molecular mass analysis of 5-to 500-kDa hyaluronan
Bhilocha, Shardul; Amin, Ripal; Pandya, Monika; Yuan, Han; Tank, Mihir; LoBello, Jaclyn; Shytuhina, Anastasia; Wang, Wenlan; Wisniewski, Hans-Georg; de la Motte, Carol; Cowman, Mary K.
2011 OCT 1 ;417(1):41-49, Analytical biochemistry
Agarose and polyacrylamide gel electrophoresis systems for the molecular mass-dependent separation of hyaluronan (HA) in the size range of approximately 5-500 kDa were investigated. For agarose-based systems, the suitability of different agarose types, agarose concentrations, and buffer systems was determined. Using chemoenzymatically synthesized HA standards of low polydispersity, the molecular mass range was determined for each gel composition over which the relationship between HA mobility and logarithm of the molecular mass was linear. Excellent linear calibration was obtained for HA molecular mass as low as approximately 9 kDa in agarose gels. For higher resolution separation, and for extension to molecular masses as low as approximately 5 kDa, gradient polyacrylamide gels were superior. Densitoinetric scanning of stained gels allowed analysis of the range of molecular masses present in a sample as well as calculation of weight-average and number-average values. The methods were validated for polydisperse HA samples with viscosity-average molecular masses of 112, 59, 37, and 22 kDa at sample loads of 0.5 mu g (for polyacrylarnide) to 2.5 mu g (for agarose). Use of the methods for electrophoretic mobility shift assays was demonstrated for binding of the HA-binding region of aggrecan (recombinant human aggrecan G1-IGD-G2 domains) to a 150-kDa HA standard. (C) 2011 Elsevier Inc. All rights reserved
— id: 136613, year: 2011, vol: 417, page: 41, stat: Journal Article,

Evolutionary conservation of heavy chain protein transfer between glycosaminoglycans
Sanggaard, Kristian W; Hansen, Lone; Scavenius, Carsten; Wisniewski, Hans-Georg; Kristensen, Torsten; Thogersen, Ida B; Enghild, Jan J
2010 Apr;1804(4):1011-1019, Biochimica & biophysica acta
The bikunin proteins are composed of heavy chains (HCs) covalently linked to a chondroitin sulfate chain originating from Ser-10 of bikunin. Tumor necrosis factor stimulated gene-6 protein (TSG-6)/heavy chain 2 (HC2) cleaves this unique cross-link and transfers the HCs to hyaluronan and other glycosaminoglycans via a covalent HC*TSG-6 intermediate. In the present study, we have investigated if this reaction is evolutionary conserved based on the hypothesis that it is of fundamental importance. The results revealed that plasma/serum samples from mammal, bird, and reptile were able to form TSG-6 complexes suggesting the presence of proteins with the same function as the human bikunin proteins. To substantiate this, the complex forming protein from Gallus gallus (Gg) plasma was purified and identified as a Gg homolog of human HC2*bikunin. In addition, Gg pre-alpha-inhibitor and smaller amount of high molecular weight forms composed of bikunin and two HCs were purified. Like the human bikunin proteins, the purified Gg proteins were all stabilized by a protein-glycosaminoglycan-protein cross-link, i.e. the HCs were covalently attached to a chondroitin sulfate originating from bikunin. Furthermore, the complex formed between Gg HC2*bikunin and human TSG-6 appeared to be identical to that of the human proteins. Akin to human, Gg HC2 was further transferred to hyaluronan when present, and when incubated in vitro, Gg pre-alpha-inhibitor and TSG-6, failed to form the intermediate covalent complex, essential for HC transfer. Significantly, Gg HC2, analogous to human HC2, promoted complex formation between human HC3 and human TSG-6, substantiating the evolutionary conservation of these interactions. The present study demonstrates that the unique interactions between bikunin proteins, glycosaminoglycans, and TSG-6 are evolutionary conserved, emphasizing the physiological importance of the TSG-6/HC2-mediated HC-transfer reaction. In addition, the data show that the evolution of HC transfer is likely to predate the role of HC.HA complexes in female fertility and thus has evolved in the context of inflammation rather than fertility
— id: 133468, year: 2010, vol: 1804, page: 1011, stat: Journal Article,

The TSG-6/HC2-mediated Transfer Is a Dynamic Process Shuffling Heavy Chains between Glycosaminoglycans
Sanggaard, KW; Scavenius, C; Rasmussen, AJ; Wisniewski, HG; Thgersen, IB; Enghild, JJ
2010 JUL 16 ;285(29):21988-21993, Journal of biological chemistry
The heavy chain (HC) subunits of the bikunin proteins are covalently attached to a single chondroitin sulfate (CS) chain originating from bikunin and can be transferred to different hyaluronan (HA) molecules by TSG-6/HC2. In the present study, we demonstrate that HCs transferred to HA may function as HC donors in subsequent transfer reactions, and we show that the CS of bikunin may serve as an HC acceptor, analogous to HA. Our data suggest that TSG-6/HC2 link HCs randomly on the CS chain of bikunin, in contrast to the ordered attachment observed during the biosynthesis. Moreover, the results show that the transfer activity is indifferent to the new HC position, and the relocated HCs are thus prone to further TSG-6/HC2 induced transfer reactions. The data suggest that HCs may be transferred directly from HA to HA without the involvement of the bikunin CS chain. The results demonstrate reversibility of the interactions between HCs and glycosaminoglycans and suggest that a dynamic shuffling of the HCs occur in vivo
— id: 111335, year: 2010, vol: 285, page: 21988, stat: Journal Article,

Transfer of inter-alpha-inhibitor heavy chains to hyaluronan by surface-linked hyaluronan-TSG-6 complexes
Colon, Elisa; Shytuhina, Anastasia; Cowman, Mary K; Band, Philip A; Sanggaard, Kristian W; Enghild, Jan J; Wisniewski, Hans-Georg
2009 Jan 23;284(4):2320-2331, Journal of biological chemistry
Inter-alpha-inhibitor, TSG-6, and hyaluronan have important functions in fertility and inflammation. Two subunits of inter-alpha-inhibitor, the heavy chains, form covalent bonds with TSG-6 or hyaluronan in vitro. TSG-6-heavy chain complexes serve as intermediates in the transfer of heavy chains from inter-alpha-inhibitor to hyaluronan. In vivo, in addition to these complexes, stable ternary complexes of hyaluronan with both TSG-6 and heavy chains have been demonstrated in the ovulatory cumulus oophorus. In our ongoing efforts to characterize the multiple interactions between hyaluronan, TSG-6 and inter-alpha-inhibitor, we recently characterized the formation of highly stable complexes of TSG-6 with hyaluronan that had been tethered to a solid surface. Here we show that these hyaluronan-TSG-6 complexes are functionally active and transfer heavy chain subunits from inter-alpha-inhibitor to either free or surface-bound hyaluronan. Transitional hyaluronan-TSG-6-heavy chain complexes do not accumulate in vitro. Our data show the capability for heavy chain transfer by both free TSG-6 and preformed hyaluronan-TSG-6 complexes, suggesting that both might contribute to hyaluronan modification in vivo. Transfer of heavy chains to surface-tethered hyaluronan by either free TSG-6 or surface-tethered hyaluronan-TSG-6 complexes did not affect the CD 44-mediated binding of BW 5147 cells in vitro. We show how TSG-6 and hyaluronan together can deplete inter-alpha-inhibitor and generate bikunin, as has been observed in sepsis, and discuss the role of TSG-6 in the generation of hyaluronan-heavy chain complexes associated with ovulation, arthritis, and sepsis
— id: 94330, year: 2009, vol: 284, page: 2320, stat: Journal Article,

TSG-6 transfers proteins between glycosaminoglycans via a Ser28-mediated covalent catalytic mechanism
Sanggaard, Kristian W; Sonne-Schmidt, Carsten S; Krogager, Toke P; Kristensen, Torsten; Wisniewski, Hans-Georg; Thogersen, Ida B; Enghild, Jan J
2008 Dec 5;283(49):33919-33926, Journal of biological chemistry
Studies of the interaction between Bikunin proteins, tumor necrosis factor-stimulated gene-6 protein (TSG-6), and glycosaminoglycans have revealed a unique catalytic activity where TSG-6/heavy chain 2 transfer heavy chain subunits between glycosaminoglycan chains. The activity is mediated by TSG-6/heavy chain 2 and involves a transient SDS stable interaction between TSG-6 and the heavy chain to be transferred. The focus of this study was to characterize the molecular structure of this cross-link to gain further insight into the catalytic mechanism. The result showed that the C-terminal Asp residue of the heavy chains forms an ester bond to Ser(28) beta-carbon of TSG-6 suggesting that this residue plays a role during catalysis
— id: 94331, year: 2008, vol: 283, page: 33919, stat: Journal Article,

The transfer of heavy chains from bikunin proteins to hyaluronan requires both TSG-6 and HC2
Sanggaard, Kristian W; Sonne-Schmidt, Carsten S; Krogager, Toke P; Lorentzen, Karen A; Wisniewski, Hans-Georg; Thogersen, Ida B; Enghild, Jan J
2008 Jul 4;283(27):18530-18537, Journal of biological chemistry
Tumor necrosis factor-stimulated gene-6 protein (TSG-6) is involved in the transfer of heavy chains (HCs) from inter-alpha-inhibitor (IalphaI), pre-alpha-inhibitor, and as shown here HC2.bikunin to hyaluronan through the formation of covalent HC.TSG-6 intermediates. In contrast to IalphaI and HC2.bikunin, pre-alpha-inhibitor does not form a covalent complex in vitro using purified proteins but needs the presence of another factor (Rugg, M. S., Willis, A. C., Mukhopadhyay, D., Hascall, V. C., Fries, E., Fulop, C., Milner, C. M., and Day, A. J. (2005) J. Biol. Chem. 280, 25674-25686). In the present study we purified the required component from human plasma and identified it as HC2. Proteins containing HC2 including IalphaI, HC2.bikunin, and free HC2 promoted the formation of HC3.TSG-6 and subsequently HC3.hyaluronan complexes. HC1 or HC3 did not possess this activity. The presented data reveal that both HC2 and TSG-6 are required for the transesterification reactions to occur
— id: 94332, year: 2008, vol: 283, page: 18530, stat: Journal Article,

2-Methoxyestradiol inhibits prostate tumor development in transgenic adenocarcinoma of mouse prostate: role of tumor necrosis factor-alpha-stimulated gene 6
Garcia, Gretchen E; Wisniewski, Hans-Georg; Lucia, M Scott; Arevalo, Nicole; Slaga, Thomas J; Kraft, Susan L; Strange, Robert; Kumar, Addanki P
2006 Feb 1;12(3 Pt 1):980-988, Clinical cancer research
PURPOSE: 2-Methoxyestradiol, an estrogenic metabolite, is in clinical trials for the treatment of hormone-refractory prostate cancer. However, neither the chemopreventive role nor the mechanism of 2-methoxyestradiol-induced biological activities is fully understood. EXPERIMENTAL DESIGN: Eight- and 24-week-old transgenic adenocarcinoma of mouse prostate (TRAMP) mice were fed a diet containing 50 mg 2-methoxyestradiol/kg body weight for 16 and 8 weeks, respectively. Chemopreventive efficacy was evaluated by magnetic resonance imaging, determining the prostate-seminal vesicle complex volume and histologic analysis of prostate tumor or tissue. Tumor invasion assays were used to show the role of tumor necrosis factor-alpha-stimulated gene (TSG-6), a 2-methoxyestradiol-up-regulated gene identified by DNA array analysis. Expression of TSG-6 was analyzed in a human tissue array containing different grades of prostate tumors. RESULTS: Dietary administration of 2-methoxyestradiol prevented the development of preneoplastic lesions independent of progression stage. TSG-6 was low or undetectable in prostate cancer cells (LNCaP, PC-3, and DU145) and TRAMP tumors but up-regulated in response to 2-methoxyestradiol. Immunohistochemistry of the human prostate tumor array showed a decrease in TSG-6-positive cells with increasing grade relative to normal prostate (P = 0.0001). Although overexpression of TSG-6 inhibited invasion of androgen-independent cells (P = 0.007), antisense TSG-6 reversed this effect. CONCLUSIONS: To the best of our knowledge, this is the first report showing the potential of 2-methoxyestradiol as a chemopreventive agent. We have also identified TSG-6 as a potential marker that could be used for early diagnosis and prognosis of cancerous or precancerous lesions
— id: 94334, year: 2006, vol: 12, page: 980, stat: Journal Article,

Evidence for a two-step mechanism involved in the formation of covalent HC x TSG-6 complexes
Sanggaard, Kristian W; Sonne-Schmidt, Carsten S; Jacobsen, Christian; Thogersen, Ida B; Valnickova, Zuzana; Wisniewski, Hans-Georg; Enghild, Jan J
2006 Jun 20;45(24):7661-7668, Biochemistry
IalphaI and TSG-6 interact to form a covalent bond between the C-terminal Asp alpha-carbon of an IalphaI heavy chain (HC) and an unknown component of TSG-6. This event disrupts the protein-glycosaminoglycan-protein (PGP) cross-link and dissociates IalphaI. In simple terms the interaction involves 5 components: (i) the IalphaI HCs, (ii) bikunin, (iii) chondroitin sulfate chain, (iv) TSG-6, and (v) divalent cations. To understand the molecular mechanism of complex formation, the effect of these were separately examined. The data show that although the mature covalent cross-link between the HCs and TSG-6 only involves the C-terminal Asp residue, the native fold of both IalphaI and TSG-6 was essential for the reaction to occur. Similarly, complex formation was prevented if the chondroitin sulfate chain was cleaved, releasing bikunin but maintaining the HC1 and HC2 PGP cross-links. In contrast, releasing the majority of the bikunin protein moiety by limited proteolysis did not prevent complex formation. An analysis of the divalent-cation requirements revealed two distinct interactions between IalphaI and TSG-6: (i) a noncovalent manganese, magnesium, or calcium-independent interaction between TSG-6 and the chondroitin sulfate chain (Kd 180 nM) and (ii) a covalent manganese, magnesium, or calcium-dependent interaction generating HC1 x TSG-6, HC2 x TSG-6, and high molecular weight (HMW) IalphaI. Significantly, both free TSG-6 and HC x TSG-6 complexes were able to bind the chondroitin sulfate chain suggesting that the sites on TSG-6 were distinct. On the basis of these findings, we propose a two-step reaction mechanism involving two putative binding sites. Initially, a cation-independent interaction between TSG-6 and the chondroitin sulfate chain is formed at site 1. Subsequently, a cation-dependent transesterification occurs, generating the covalent HC x TSG-6 cross-link at another site, site 2
— id: 94333, year: 2006, vol: 45, page: 7661, stat: Journal Article,

An assay for bacterial and eukaryotic chondroitinases using a chondroitin sulfate-binding protein
Wisniewski, Hans-Georg; Sweet, Moshe H; Stern, Robert
2005 Dec 1;347(1):42-48, Analytical biochemistry
A simple, rapid, and reproducible microtiter-based chondroitinase (CSase) assay is reported here, based on the competition of chondroitin sulfate (CS) with immobilized hyaluronan (HA) for the binding of TSG-6 protein, the product of TNF-inducible gene 6. Although the catabolic reaction of bacterial and other prokaryotic CSase enzymes, often referred to as the chondroitin lyases, can be followed by tracking the generation of unsaturated bonds by the spectrophotometrical determination of the absorbance at 232 nm, no rapid, sensitive, and simple assay has been devised to date for measuring the activity of the vertebrate enzymes that cleave their substrate exclusively by hydrolysis. We provide data demonstrating that the CSase assay described here is suitable for the determination of the activities of both classes of enzymes. For the bacterial enzyme CSase ABC, both the determination of the absorbance at 232 nm and the assay based on TSG-6 binding are suitable using the same range of enzyme activities. However, for testicular hyaluronidase, considerably higher enzyme activities were needed to cleave CS than to cleave HA. Using the HA-binding domain of aggrecan for a comparison, we determined that the interaction between TSG-6 and chondroitin sulfate is uniquely suited for this CSase assay
— id: 94335, year: 2005, vol: 347, page: 42, stat: Journal Article,

Protective effect of TSG-6 against collagen-induced arthritis in DBA/1J mice expressing the TSG-6 transgene
Mindrescu, C; Dias, A; Olszewki, R; Klein, M; Reis, L; Wisniewski, HG
2002 Jun;103(3):8-79, Clinical immunology
— id: 32370, year: 2002, vol: 103, page: 8, stat: Journal Article,

Alzheimer's disease presenilin-1 expression modulates the assembly of neurofilaments
Dowjat WK; Wisniewski H; Wisniewski T
2001 ;103(1):1-8, Neuroscience
Mutations in presenilin-1 gene are responsible for the majority of early-onset familial Alzheimer's disease cases. The function of this protein and the mechanism underlying the pathogenicity of its mutations are still unclear. To elucidate the role of presenilin-1 in the Alzheimer's disease pathology, we tested two such mutations (P117L and M146L) for their effect in stably transfected mouse neuroblastoma cell lines. Over-expression of the wild-type presenilin-1 gene induced formation of a well-extended, orderly organized network consisting of neurofilaments assembled from the L and H subunits, while in cells with the mutant gene this network was markedly reduced to short filaments concentrated in structures resembling cups. Cells expressing the mutant gene displayed altered processing of the transgene protein and neurofilament-H, suggesting that presenilin-1 is the mediator of changes targeted at neurofilaments. The two different mutations produced similar alterations, implying that this is a common pathogenic mechanism. Presenilin-1, neurofilament-H and tau proteins showed co-localization as evidenced by confocal microscopy, suggesting a possible physiological connection between these three proteins. Presenilin-1 appears to influence assembly of the H subunit into neurofilaments and the subsequent formation of new neurites. Mutations impair this function of presenilin-1, resulting in inhibition of neurite outgrowth. That presenilin-1 influences the assembly of neurofilaments may represent a novel pathway through which presenilin-1 mutations are involved in Alzheimer's disease pathology. In this hypothesis, presenilin-1 mutations will be associated with aberrant sprouting leading to synaptic loss, a key neuropathological feature of Alzheimer's disease
— id: 23494, year: 2001, vol: 103, page: 1, stat: Journal Article,

Amelioration of collagen-induced arthritis in DBA/1J mice by recombinant TSG-6, a tumor necrosis factor/interleukin-1-inducible protein.[In Process Citation]
Mindrescu C; Thorbecke GJ; Klein MJ; Vilcek J; Wisniewski HG
2000 Dec;43(12):2668-2677, Arthritis & rheumatism
OBJECTIVE: To examine the effect of recombinant TSG-6 on collagen-induced arthritis (CIA) in DBA/1J mice. TSG-6 is a tumor necrosis factor (TNF)/ interleukin-1 (IL-1)-inducible hyaluronan-binding protein produced by synovial cells and chondrocytes that is present in synovial fluids of patients with rheumatoid arthritis. METHODS: To determine the effect of TSG-6 on chronic inflammatory joint disease, we induced CIA in DBA/1J mice by immunization with bovine type II collagen. Animals were treated with 12 intraperitoneal doses of 200 microg of recombinant TSG-6, beginning 3 days before the expected onset of disease symptoms. Progression of arthritis was monitored by determining the disease incidence, arthritis index, and footpad swelling. Levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen and serum concentrations of IL-6 were determined at various time points. Histologic examination of affected joints was performed approximately 20 days after the onset of arthritis. RESULTS: Treatment with recombinant TSG-6 protein had a potent ameliorative effect, manifested by decreases in the disease incidence, arthritis index, and footpad swelling. Histologic examination of affected joints in TSG-6-treated animals revealed little pannus formation and cartilage erosion, features which were conspicuous in control mice. Animals treated with recombinant TSG-6 developed significantly reduced levels of IgG1, IgG2a, and IgG2b antibodies against bovine and murine type II collagen. CONCLUSION: The antiinflammatory effect of the TNF/IL-1-inducible TSG-6 protein in murine CIA suggests a role for this protein as an endogenous regulator of the inflammatory process
— id: 15527, year: 2000, vol: 43, page: 2668, stat: Journal Article,

TSG-6: an IL-1/TNF-inducible protein with anti-inflammatory activity
Wisniewski HG; Vilcek J
1997 Jun;8(2):143-156, Cytokine & growth factor reviews
The pro-inflammatory cytokines IL-1 and TNF-alpha are primary mediators of the acute phase response, the complex reaction of the mammalian organism to infection and injury. Among the genes activated by TNF-alpha and IL-1 in a variety of cells is TNF-stimulated gene 6 (TSG-6). The TSG-6 cDNA encodes a secreted 35 kDa glycoprotein which is abundant in synovial fluids of patients with various forms of arthritis and detectable in serum of patients with different inflammatory or autoimmune disorders. TSG-6 protein consists of two structural domains: a hyaluronan-binding link module, the characteristic domain of the hyaladherin family of proteins, and a C-terminal CUB domain, present in a variety of diverse proteins. TSG-6 forms a stable complex with components of the plasma protein inter-alpha-inhibitor (I[alpha]I), a Kunitz-type serine protease inhibitor. TSG-6 and I(alpha)I synergize to inhibit plasmin, a serine protease involved in the activation of matrix metalloproteinases which are part of the proteolytic cascade associated with inflammation. Recombinant human TSG-6 protein exerts a potent anti-inflammatory effect in a murine model of acute inflammation. Modulation of the proteolytic network associated with inflammatory processes may be a mechanism whereby TSG-6, in cooperation with I(alpha)I, inhibits inflammation. Activation of the TSG-6 gene by pro-inflammatory cytokines, presence of TSG-6 protein in inflammatory lesions and its anti-inflammatory effect suggest a role for TSG-6 in a negative feed-back control of the inflammatory response
— id: 7281, year: 1997, vol: 8, page: 143, stat: Journal Article,

In vivo structural studies of the hippocampus in normal aging and in incipient Alzheimer's disease
de Leon MJ; Convit A; George AE; Golomb J; de Santi S; Tarshish C; Rusinek H; Bobinski M; Ince C; Miller D; Wisniewski H
1996 Jan 17;777:1-13, Annals of the New York Academy of Sciences
Population trends indicate that in the near future the size of the elderly population will increase. This will result in a large increment in the numbers of persons suffering mild to severe levels of cognitive impairment. While considerable efforts continue to be made to explain brain changes associated with Alzheimer disease (AD), little is known of the brain changes in aging without dementia or so-called normal aging. Pathologic studies suggest that the medial temporal lobe is informative in the examination of the early brain changes related to AD. However, pathologic studies only offer a single observation and considerable uncertainty exists regarding the likelihood of progression of disease and the development of dementia. Several structural neuroimaging studies have recently investigated this anatomy and recent reports are encouraging for a medial temporal lobe based diagnosis for age-related cognitive impairments. We will present our findings on the MRI anatomy of the hippocampal formation as well as data bearing on the use of hippocampal formation imaging in the diagnosis of AD and as a predictive marker for future dementia. Our findings suggest an anatomically specific relationship between hippocampal volume and secondary memory performance. Because these observations apply to nondemented and normal elderly subjects, we are encouraged that the anatomy of age-related cognitive impairments can be reliably recognized and possibly put to use in therapeutic studies
— id: 6989, year: 1996, vol: 777, page: 1, stat: Journal Article,

Long pentraxins: an emerging group of proteins with diverse functions
Goodman AR; Cardozo T; Abagyan R; Altmeyer A; Wisniewski HG; Vilcek J
1996 Aug;7(2):191-202, Cytokine & growth factor reviews
The earliest described pentraxins, C reactive protein (CRP) and serum amyloid P component (SAP), are cytokine-inducible acute phase proteins implicated in innate immunity whose concentrations in the blood increase dramatically upon infection or trauma. The highly conserved family of pentraxins was thought to consist solely of approximately 25 kDa proteins. Recently, several distinct larger proteins have been identified in which only the C-terminal halves show characteristic features of the pentraxin family. One of the recently described 'long' pentraxins (TSG-14/PTX3) is inducible by TNF or IL-1 and is produced during the acute phase response. Other newly identified long pentraxins are constitutively expressed proteins associated with sperm-egg fusion (apexin/p50), may function at the neuronal synapse (neuronal pentraxin I, NPI), or may serve yet other, unknown functions (NPII and XL-PXN1). Evidence obtained by molecular modeling and by direct physicochemical analysis suggests that TSG-14 protein retains some characteristic structural features of the pentraxins, including the formation of pentameric complexes
— id: 12559, year: 1996, vol: 7, page: 191, stat: Journal Article,

TSG-6 expression in human articular chondrocytes. Possible implications in joint inflammation and cartilage degradation
Maier R; Wisniewski HG; Vilcek J; Lotz M
1996 Apr;39(4):552-559, Arthritis & rheumatism
OBJECTIVE: The hyaluronan-binding protein TSG-6 (tumor necrosis factor-stimulated gene 6) forms a stable complex with the serine protease inhibitor, inter-alpha-inhibitor, potentiates the inhibition of plasmin activity, and has antiinflammatory effects in vivo. This study examines the expression of TSG-6 in human articular chondrocytes and cartilage. METHODS: Human articular chondrocytes and cartilage explants were stimulated with cytokines, growth factors, and other agents. TSG-6 expression was analyzed by imaging-assisted Northern and Western blotting. RESULT: TSG-6 messenger RNA (mRNA) expression was upregulated by cytokines and growth factors, predominantly interleukin-1 beta (IL-1 beta), tumor necrosis factor alpha (TNF alpha), platelet-derived growth factor AA (PDGF-AA), and transforming growth factor beta 1 (TGF beta 1). TSG-6 mRNA induction by TGF beta 1 was delayed as compared with IL-1beta. Treatment of the cells with the glucocorticoid dexamethasone neither induced TSG-6 mRNA nor did it affect IL-1 beta-induced transcript levels. TSG-6 mRNA induction may involve several signal transduction pathways. The strong transcriptional stimulation by phorbol myristate acetate suggests protein kinase C (PKC)-mediated signaling. In contrast, PKA- and Ca- dependent signals are only marginally involved as messengers leading to increased TSG-6 levels after IL-1beta and TNF alpha treatment. In chondrocyte and cartilage organ cultures, both free TSG-6 (35 kd) and the complex with inter-alpha-inhibitor (120 kd) were present and upregulated by IL-1 beta, TNF alpha, or TGF beta 1. CONCLUSION: Chondrocytes are a source of TSG-6 which may play a role in cartilage remodeling and joint inflammation
— id: 15531, year: 1996, vol: 39, page: 552, stat: Journal Article,

TNF/IL-1-inducible protein TSG-6 potentiates plasmin inhibition by inter-alpha-inhibitor and exerts a strong anti-inflammatory effect in vivo
Wisniewski HG; Hua JC; Poppers DM; Naime D; Vilcek J; Cronstein BN
1996 Feb 15;156(4):1609-1615, Journal of immunology
TNF-stimulated gene 6 (tsg6), encoding a 35-kDa secretory glycoprotein (TSG-6), is induced in fibroblasts, chondrocytes, synovial cells, and mononuclear cells by the proinflammatory cytokines TNF-alpha and IL-1, or by LPS. Large amounts of TSG-6 protein were found in synovial fluids of patients with rheumatoid arthritis. TSG-6 protein forms a stable complex with components of the serine protease inhibitor, inter-alpha-inhibitor (I alpha I). In this work, we show that TSG-6 potentiates the inhibitory effect of l alpha l on the protease activity of plasmin. The plasmin/plasminogen activator system is important in the protease network associated with inflammation. To test the hypothesis that through their cooperative inhibitory effect on plasmin TSG-6 and l alpha l can modulate the protease network and thus inhibit inflammation, we examined the effect of TSG-6 on experimentally induced inflammation. Human recombinant TSG-6 protein showed a potent anti-inflammatory activity in the murine air pouch model of carrageenan- or IL-1-induced acute inflammation. The inhibitory effect of locally administered TSG-6 on the IL-1-induced cellular infiltration was comparable with that of systemic dexamethasone treatment. Two mutant TSG-6 proteins with single amino acid substitutions close to the N terminus showed a complete or partial loss of anti-inflammatory activity. The anti-inflammatory effect of the TNF/IL-1-inducible TSG-6 protein, along with its ability to inhibit protease action through interaction with l alpha l, suggests that TSG-6 production during inflammation is part of a negative feedback loop operating through the protease network
— id: 6973, year: 1996, vol: 156, page: 1609, stat: Journal Article,

TSG-6, a glycoprotein associated with arthritis, and its ligand hyaluronan exert opposite effects in a murine model of inflammation
Wisniewski HG; Naime D; Hua JC; Vilcek J; Cronstein BN
1996 ;431(6 Suppl 2):R225-R226, Pflugers archiv = European journal of physiology
TSG-6 is an arthritis-associated hyaluronan binding protein whose production in synovial cells, chondrocytes, fibroblasts and mononuclear cells is stimulated by TNF-alpha and IL-1. The purpose of this study was to gain insights into the role of TSG-6 and its functional interactions with hyaluronan in inflammation. In the murine air pouch model of carrageenan/IL-1-induced inflammation TSG-6 showed a dramatic inhibitory effect on the cellular infiltration of the inflammatory site by neutrophilic PMN, while hyaluronan enhanced cellular infiltration. There was no indication of a neutralizing or cooperative effect when TSG-6 and hyaluronan were injected together. The potent antiinflammatory effect of TSG-6 along with its induction by proinflammatory cytokines suggests that TSG-6 is part of a negative feedback loop in the control of the inflammatory response
— id: 9819, year: 1996, vol: 431, page: R225, stat: Journal Article,

TSG-6, an arthritis-associated hyaluronan binding protein, forms a stable complex with the serum protein inter-alpha-inhibitor
Wisniewski HG; Burgess WH; Oppenheim JD; Vilcek J
1994 Jun 14;33(23):7423-7429, Biochemistry
TSG-6 is a secreted 35-kDa glycoprotein, inducible by TNF and IL-1. The N-terminal portion of TSG-6 shows sequence homology to members of the cartilage link protein family of hyaluronan binding proteins. The C-terminal half of TSG-6 contains a so-called CUB domain, characteristic for developmentally regulated proteins. High levels of TSG-6 protein are found in the synovial fluid of patients with rheumatoid arthritis and some other arthritic diseases. Here we show that TSG-6 readily formed a complex with a protein present in human, bovine, rabbit, and mouse serum. This complex was stable during SDS-PAGE under reducing conditions, and in the presence of 8 M urea. The protein that binds TSG-6 was purified from human serum and identified as inter-alpha-inhibitor (I alpha I) by N-terminal microsequencing. Microsequencing of the complex itself revealed the presence of TSG-6 and two of the three polypeptide chains of I alpha I (bikunin and HC2). Experiments with recombinant TSG-6 and I alpha I purified from human serum showed that the TSG-6/I alpha I complex is rapidly formed even in the apparent absence of other proteins at 37 degrees C, but not at 4 degrees C. The TSG-6/I alpha I complex was cleaved by chondroitin sulfate ABC lyase, suggesting that cross-linking by chondroitin sulfate is required for the stability of the complex.(ABSTRACT TRUNCATED AT 250 WORDS)
— id: 6555, year: 1994, vol: 33, page: 7423, stat: Journal Article,

Hippocampal atrophy in early Alzheimer's disease: anatomic specificity and validation
Convit A; de Leon MJ; Golomb J; George AE; Tarshish CY; Bobinski M; Tsui W; De Santi S; Wegiel J; Wisniewski H
1993 Winter;64(4):371-387, Psychiatric quarterly
We evaluated three groups of elderly individuals who were carefully screened to rule out clinically significant diseases that could affect cognition. They were matched for age and education. The groups included normals (N = 18), Alzheimer's Disease (AD) patients (N = 15), and minimally impaired individuals with memory complaints and impairments but who did not fulfill criteria for AD (N = 17). Volumetric measurements of different regions of the temporal lobe on the coronal scan as well as ratings of the perihippocampal cerebrospinal fluid (CSF) accumulation (HCSF) on the negative angle axial MR were carried out. Volume reductions were found in AD relative to the normals for both medial and lateral temporal lobe volumes. Only hippocampal volume reductions were found in the minimal group. The minimally impaired individuals had equivalent hippocampal volume reductions and significantly larger parahippocampal and lateral temporal lobe gyri than the AD group. The axial HCSF was validated using the coronal volumes. The combination of coronal hippocampal and perihippocampal CSF was the best predictor of the axial HCSF rating. The parahippocampal volume did not add to the predictive ability of the hippocampal-perihippocampal CSF combination. Future work should validate these findings with longitudinal designs as well as assess the issue of normal aging of these structures and their relationship to cognitive function
— id: 6340, year: 1993, vol: 64, page: 371, stat: Journal Article,

TSG-6: a TNF-, IL-1-, and LPS-inducible secreted glycoprotein associated with arthritis
Wisniewski HG; Maier R; Lotz M; Lee S; Klampfer L; Lee TH; Vilcek J
1993 Dec 1;151(11):6593-6601, Journal of immunology
TSG-6 (TNF-stimulated gene 6) was originally discovered by differential screening of a cDNA library prepared from TNF-stimulated human diploid FS-4 fibroblasts. We show that the 35-kDa protein encoded by TSG-6 was undetectable in the medium of untreated FS-4 cultures, whereas its production reached approximately 1400 and 700 ng/10(6) cells after 24-h treatment with IL-1 or TNF, respectively. Stimulation of TSG-6 protein and mRNA levels was also demonstrated in normal human mononuclear cells by treatment with TNF and, especially, by LPS. In view of the inducibility of TSG-6 by inflammatory cytokines and its earlier demonstrated affinity for hyaluronan, we examined the presence of TSG-6 protein in the synovial fluids from patients with various forms of arthritis. TSG-6 protein was undetectable in the joint fluids of persons with no known history of arthritis, but high levels of TSG-6 oere demonstrated in the synovial fluids of a majority of arthritis patients. TSG-6 protein was also detected in the sera of some of the arthritis patients, albeit at concentrations that were less than in the joint fluids. To investigate the source of TSG-6 in the synovial fluids, we examined the production of TSG-6 protein in cultures of synovial cells. Synoviocytes from rheumatoid arthritis patients produced TSG-6 protein constitutively, and this production was increased by treatment with TNF or IL-1, but not with TGF-beta. Steady-state levels of TSG-6 mRNA were also increased in synoviocytes after treatment with TNF or IL-1. The presence of high levels of TSG-6 protein in the synovial fluids of arthritis patients and its inducibility by inflammatory cytokines in fibroblasts, mononuclear cells, synoviocytes, and chondrocytes suggest a role for TSG-6 in arthritis and inflammation
— id: 6553, year: 1993, vol: 151, page: 6593, stat: Journal Article,

A novel secretory tumor necrosis factor-inducible protein (TSG-6) is a member of the family of hyaluronate binding proteins, closely related to the adhesion receptor CD44
Lee TH; Wisniewski HG; Vilcek J
1992 Jan;116(2):545-557, Journal of cell biology
TSG-6 cDNA was isolated by differential screening of a lambda cDNA library prepared from tumor necrosis factor (TNF)-treated human diploid FS-4 fibroblasts. We show that TSG-6 mRNA was not detectable in untreated cells, but became readily induced by TNF in normal human fibroblast lines and in peripheral blood mononuclear cells. In contrast, TSG-6 mRNA was undetectable in either control or TNF-treated human vascular endothelial cells and a variety of tumor-derived or virus-transformed cell lines. The sequence of full-length TSG-6 cDNA revealed one major open reading frame predicting a polypeptide of 277 amino acids, including a typical cleavable signal peptide. The NH2-terminal half of the predicted TSG-6 protein sequence shows a significant homology with a region implicated in hyaluronate binding, present in cartilage link protein, proteoglycan core proteins, and the adhesion receptor CD44. The most extensive sequence homology exists between the predicted TSG-6 protein and CD44. Western blot analysis with an antiserum raised against a TSG-6 fusion protein detected a 39-kD glycoprotein in the supernatants of TNF-treated FS-4 cells and of cells transfected with TSG-6 cDNA. Binding of the TSG-6 protein to hyaluronate was demonstrated by coprecipitation. Our data indicate that the inflammatory cytokine (TNF or IL-1)-inducible, secretory TSG-6 protein is a novel member of the family of hyaluronate binding proteins, possibly involved in cell-cell and cell-matrix interactions during inflammation and tumorigenesis
— id: 13722, year: 1992, vol: 116, page: 545, stat: Journal Article,

CRITICAL ROLE OF THE C-TERMINUS IN THE BIOLOGICAL-ACTIVITIES OF HUMAN TUMOR-NECROSIS-FACTOR-ALPHA
Gase, K; Korobko, VG; Wisniewski, HG; Le, J; Dobrynin, VN; Filippov, SA; Gutsche, W; Maksimova, YN; Schlott, B; Shingarova, LN; Vilcek, J; Behnke, D
1990 Nov;71(3):368-371, Immunology
— id: 31829, year: 1990, vol: 71, page: 368, stat: Journal Article,