Immunohistochemical detection of lymph node-DTCs in patients with node-negative HNSCC.

This study was performed to systematically assess the prevalence, topography and prognostic impact of disseminated tumor cells (DTCs) in lymph nodes (LN) of patients with primary, regional and distant metastasis-free head and neck squamous cell carcinoma (HNSCC) who underwent resection with elective neck dissection. From the routinely processed resection specimen, we could prospectively analyze a total of 1.137 exactly mapped LNs of 50 pN0-HNSCC patients, classified as tumor free by routine histopathology. Three immunohistochemistry (IHC) assays using antibodies directed against CK5/14, a broad spectrum of CKs (1-8, 10, 14-16 and 19), and CD44v6, respectively, were applied on 4.190 LN sections to detect DTCs. The IHC results were correlated with clinicopathologic parameters and clinical follow-up data. We detected seven micrometastases (MM) in five patients and 31 DTCs in 12 patients. Overall, 15 (30%) patients were positive for DTCs or MMs. Strikingly, the anatomical distribution of LN affected with DTCs was not random, but was dependent on the lateralization of the primary tumor and clustered significantly most proximal to the primary tumor. None of the investigated patients developed loco-regional lymphatic or distant metastasis during the mean follow-up period of 71 months. Our results reveal clinically occult tumor cell dissemination as an early and frequent event in HNSCC. Considering that higher rates of recurrences in therapeutic LN dissection concepts have been reported than in elective neck dissection strategies, our DTC-data support to perform elective neck dissections, since they appear to be effective in preventing loco-regional lymphatic recurrence from LN DTCs or MMs.

Expression and purification of RHC-EGFP fusion protein and its application in hyaluronic acid assay.

Hyaluronan is a widely distributed glycosaminoglycan which has multiple functions. Hyaluronic acid (HA) accumulation has been reported in many human diseases. Understanding the role of hyaluronan and its binding proteins in the pathobiology of disease will facilitate the development of novel therapeutics for many critical diseases. Current techniques described for the analysis of HA are mainly for HA quantification in solutions, not for the direct detection of HA in tissues or on cell surfaces. In our study, a fusion protein, named C-terminal domain of RHAMM-enhanced green fluorescence protein (RHC-EGFP), combined the HA-binding domain, C-terminal of receptor for hyaluronan-mediated motility, with EGFP, a widely used enhanced green fluorescence protein, was expressed and purified from Escherichia coli with high purity. Based on the sensitivity and convenience of fluorescence detection, methods for direct assay of HA in solutions, on cell surface or in tissues were established using RHC-EGFP. The binding specificity was also confirmed by competitive binding experiment and hyaluronidase degradation experiment. Our results provide an alternative choice for the specific and convenient assay of HA in various samples, and maybe helpful for further understanding of the fundamental and comprehensive functions of HA.

Characterization of a novel recombinant hyaluronan binding protein for tissue hyaluronan detection.

Tumor necrosis factor-Stimulated Gene 6 protein (TSG-6) is a hyaluronan (HA)-binding glycoprotein containing an HA-binding Link module. Because of its well-defined structure, HA binding properties and small size, TSG-6 is an excellent candidate as an alternative to animal-derived HA-binding protein (HABP) for the detection of HA. The present work describes the generation and characterization of a novel recombinant HA-binding probe obtained by fusion of a modified TSG-6 Link module with mutationally inactivated heparin-binding sequence and the Fc portion of human IgG1 (TSG-6-ΔHep-Fc) for tissue HA detection in histological samples. Direct binding assays indicated strong binding of TSG-6-ΔHep-Fc to HA, with little residual binding to heparin. Histolocalization of HA in formalin-fixed, paraffin-embedded tissue sections using biotin-TSG-6-ΔHep-Fc resulted in hyaluronidase-sensitive staining patterns similar to those obtained with biotin-HABP, but with improved sensitivity. HA was detected in many human tissues, and was most abundant in soft connective tissues such as the skin dermis and the stroma of various glands. Digital image analysis revealed a linear correlation between biotin-HABP and biotin-TSG-6-ΔHep-Fc staining intensity in a subset of normal and malignant human tissues. These results demonstrate that TSG-6-ΔHep-Fc is a sensitive and specific probe for the detection of HA by histological methods.

Soni-removal of nucleic acids from inclusion bodies.

Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs.

Bioactive hyaluronan fragment (hexasaccharide) detects specific hexa-binding proteins in human breast and stomach cancer: possible role in tumorogenesis.

Hyaluronan (HA) is a component of extracellular matrix that influences cell-proliferation, migration, development, regeneration, normal tissue remodeling, tissues undergoing malignancy and tumor cell interaction. The widespread occurrence of HA binding proteins, their involvement in tissue organization and the control of cellular behavior are well documented. The low molecular mass HA fragments can also induce a variety of biological events, including chemokine gene expression, transcription factor expression and angiogenesis. It is believed that these fragments are more potent in cellular activities than high molecular mass HA. In this study, we isolated the various fragments by gel permeation chromatography of hyaluronidase digested HA and characterized by fluoro assisted carbohydrate electrophoresis (FACE) and matrix assisted laser desorption ionization analysis (MALDI). Detection and distribution of cellular receptors in invasive tumor tissues for HA polymer and HA fragments were determined both by Western blot and histochemistry. The study demonstrated the overexpression of HA-hexa binding protein in human tumors of breast and stomach and its involvement in tumorogenesis.

Biological characteristics of a cell subpopulation in tongue squamous cell carcinoma.

OBJECTIVES: To isolate the CD133+CD44+ cells from human tongue squamous cell carcinoma (TSCC) Tca8113 cell line and investigate biological characteristics of them. MATERIALS AND METHODS: Immunomagnetic microbeads were applied to sort the CD133+CD44+ cells. Flow cytometry was used to detect isolation purity. The proliferation, clone-formation efficiencies, invasion and migration, gene expressions, and tumor-formation abilities were analyzed among CD133+CD44+, CD133-CD44-, and total population of cells. RESULTS: The average purities of CD133+ and CD44+ cells reached 97.3% and 98.7%, respectively. The proliferation of CD133+CD44+ cells was significantly higher than the other two groups. The clone-forming efficiency of three groups was 70%, 8%, and 14%, respectively. The average invaded and migrated cell numbers of CD133+CD44+ and total population cells were 132 and 36.2, 311.6, and 156.2, respectively. The expressions of Bcl-2 and Sox2 in CD133+CD44+ cells were significantly higher than those in total population cells. A total of 10(4) CD133+CD44+ cells could form secondary tumors in nude mice, while the total population group needed 10(6) cells. CONCLUSIONS: The CD133+CD44+ subpopulation cells possess stem-like characteristics. They appear to be the potential targets for future biology therapy of human TSCC.

Inhibitors of autoactivation of plasma hyaluronan-binding protein (factor VII activating protease).

Plasma hyaluronan-binding protein (PHBP), a serine protease that can activate coagulation factor VII and prourokinase, circulates in a single-chain form (pro-PHBP) and autoproteolytically converts to an active two-chain form with the aid of an effector such as spermidine and heparin. It has been postulated that PHBP plays roles in regulating inflammation, vascular function, fibrosis and atherosclerosis. From the comprehensive screening of natural sources for inhibitors of spermidine-induced pro-PHBP autoactivation, we identified several compounds with a polyphenol feature. Of these inhibitors, tannic acid (IC(50)=0.020 µM), delphinidin (IC(50)=0.079 µM), hamamelitannin (IC(50)=0.19 µM), (-)-epicatechin gallate (IC(50)=0.24 µM), and 3,5-di-O-caffeoylquinic acid (IC(50)=1.0 µM) were potent and selective, and did not inhibit heparin-induced pro-PHBP autoactivation and the active form of PHBP at concentrations 100 times higher than the respective IC(50) values. From evaluation of the activities of related compounds, it has been suggested that a compound with multiple aromatic rings with plural phenolic hydroxyl substituents exhibits potent activity. The inhibitory actions of delphinidin, hamamelitannin, (-)-epicatechin gallate and 3,5-di-O-caffeoylquinic acid were attenuated by catechol, a minimum polyphenol unit. Thus, it is likely that pro-PHBP binds these potent inhibitors through its site(s) that recognize a catechol-like structure. Our results would facilitate understanding of the molecular mechanism of pro-PHBP autoactivation and rational design of a compound for suppressing unregulated pro-PHBP activation.

The concentration, gene expression, and spatial distribution of aggrecan in canine articular cartilage, meniscus, and anterior and posterior cruciate ligaments: a new molecular distinction between hyaline cartilage and fibrocartilage in the knee joint.

The concentration, spatial distribution, and gene expression of aggrecan in meniscus, articular cartilage, and the anterior and posterior cruciate ligaments (ACL and PCL) was determined in the knee joints of five mature dogs. An anti-serum against peptide sequences specific to the G1 domain of aggrecan was employed in competitive-inhibition ELISA of guanidine HCl extracts and immunofluorescence microscopy. Gene expression was determined by Taqman real-time PCR. The concentration of aggrecan in articular cartilage (240.1 +/- 32 nMol/g dry weight) was higher than that in meniscus (medial meniscus: 33.4 +/- 4.3 nMol/g) and ligaments (ACL: 6.8 +/- 0.9 nMol/g). Aggrecan was more concentrated in the inner than the outer zone of the meniscus. Aggrecan in meniscus showed an organized, spatial network, in contrast to its diffuse distribution in articular cartilage. Thus, differences in the concentration, gene expression, and spatial distribution of aggrecan constitute another molecular distinction between hyaline cartilage and fibrocartilage of the knee.

Soluble CD44 is cytotoxic to trabecular meshwork and retinal ganglion cells in vitro.

PURPOSE: Current glaucoma research targets neuroprotective therapies for retinal ganglion cells (RGCs) in primary open-angle glaucoma (POAG). The purpose of this study was to determine whether the 32-kDa ectodomain fragment of CD44-soluble CD44 (sCD44)-which is increased in the aqueous of patients with POAG, affects RGC and trabecular meshwork (TM) cell survival in vitro. METHODS: sCD44 was isolated from human or fetal calf serum (FCS) by urea solubilization and immunoprecipitation. A transformed rat RGC-like cell line (RGC-5), human and bovine TM cells, and control cells were grown in Dulbecco's modified Eagle's medium containing 10% FCS until confluent and then were incubated in medium containing 0.1% FCS and treated with various doses of purified sCD44 and 17-alpha-methyl testosterone (17-alpha-MT). The cytotoxicity of sCD44 was verified by heat-inactivation, pretreatment with a pan-caspase inhibitor, and coadministration of anti-CD44 neutralizing antibody or hyaluronic acid (HA). Cell viability was assessed by trypan blue staining, cell counting, and phase-contrast microscopy. RESULTS: There was a statistically significant dose- and time-dependent decrease in the number of cells and viability in the RGC-5 and TM cells treated with sCD44. Within 12 hours of sCD44 treatment, RGC-5 and TM cells displayed cell rounding, detachment, and swelling. sCD44-induced cell death was cell specific. Smooth muscle cells were resistant to sCD44, whereas human cortical neuronal-like cells were susceptible to sCD44 after 24 hours, but recovered. The cytotoxicity of sCD44 was blocked by heat-inactivation, pretreatment with a pan-caspase inhibitor, or coadministration of anti-CD44 antibody or HA. 17-alpha-MT prevented sCD44 cytotoxicity in both RGC-5 and TM cells. CONCLUSIONS: The results indicate that exogenous sCD44 adversely affects RGC-5 and TM cell survival in vitro by activating proapoptotic pathways.

Constitutive and induced CD44 shedding by ADAM-like proteases and membrane-type 1 matrix metalloproteinase.

CD44 is a receptor for hyaluronan and mediates signaling that regulates complex cell behavior including cancer cell migration and invasion. Shedding of the extracellular portion of CD44 is the last step in the regulation of the molecule-releasing interaction between the ligand and cell. However, highly glycosylated forms of CD44 have hampered the identification of the exact cleavage sites for shedding and the responsible proteases. In this study, we found that expression of membrane-type 1 matrix metalloproteinase (MT1-MMP) increased shedding of the 65-70 kDa CD44H (standard form) fragments and generated two additional smaller fragments. We purified the shed fragments and identified the cleaved sites by mass spectrometry. Specific antibodies that recognize the newly exposed COOH terminus by cleavage were prepared and used to analyze shedding at each site. Shedding of the 65-70 kDa fragments was inhibited by tissue inhibitor of metalloproteinase 3 (TIMP-3) but not by TIMP-1 and TIMP-2, suggesting involvement of a disintegrin and metalloproteinase (ADAM)-like proteases, although shedding is affected by MT1-MMP. Conversely, shedding of the two smaller fragments was inhibited by TIMP-2 and TIMP-3 but not TIMP-1, suggesting involvement of MT1-MMP itself. Shed fragments cleaved at these sites were also detected in human tumor tissues. Increased shedding at one of the MT1-MMP-sensitive sites was observed in the tumor compared with the surrounding normal tissue. However, no significant difference was observed with shedding by ADAM-like proteases. Thus, the cleavage sites for the shedding of CD44H were identified for the first time, and the results provide a basis for exploring the unknown biologic roles of shedding at different sites.

Purification and molecular identification of the human hyaluronan receptor for endocytosis.

The clearance of hyaluronan (HA) and chondroitin sulfates from the circulating blood and lymph in the body is mediated by the membrane-bound HA receptor for endocytosis (HARE). Previously, we found that two HARE species of approximately 175 kDa and approximately 300 kDa are abundant in the sinusoidal endothelial cells in rat liver, spleen, and lymph nodes (Zhou et al. [2000], J. Biol. Chem., 275, 37733-37741). In the present study, immunocytochemical analysis of human tissues showed a similar pattern with abundant expression of HARE in the sinusoidal endothelial cells of human liver, spleen, and lymph nodes. The two human HARE proteins were immunoaffinity-purified from human spleen. Each protein was recognized in western blots using several anti-rat HARE monoclonal antibodies and was able to bind 125I-HA specifically. In nonreducing SDS-PAGE, these two human HARE species migrated at approximately 190 kDa and approximately 315 kDa; both proteins are approximately 15 kDa larger than the corresponding rat HAREs, although the de-N-glycosylated core proteins are essentially the same mass. After reduction, the human 190-kDa HARE gave a single 196-kDa species, which was not seen in the approximately 315-kDa HARE after reduction. The reduced approximately 315-kDa HARE yielded two major proteins at approximately 250 kDa and approximately 220 kDa. We determined the sequence of the human 190-kDa HARE cDNA based on analysis of internal tryptic peptides, as well as RT-PCR and 5' RACE analyses using human spleen and lymph node cDNA libraries. The human gene that encodes HARE is on chromosome 12.

Sequence, protein expression and extracellular-regulated kinase association of the hyaladherin RHAMM (receptor for hyaluronan mediated motility) in PC12 cells.

Differentiation of PC12 cells by nerve growth factor (NGF) or fibroblast growth factor-2 (FGF2) is dependent on signaling mediated by extracellular regulated kinase (ERK). We investigated the involvement of receptor for hyaluronan mediated motility (RHAMM) in this signaling pathway. A single RHAMM 3.2 kb transcript was detected in PC12 RNA. Reverse transcriptase-polymerase chain reaction generated a 2141 bp cDNA that had identical sequence to rat brain RHAMM and showed no evidence of alternate splicing. Several RHAMM species were identified by Western blotting. Immunohistochemistry showed RHAMM localization to the cytoskeleton, neurites and growth cones. Following stimulation of PC12 cells with NGF or FGF2 RHAMM was co-immunoprecipitated by phosphorylation-specific anti-ERK antibodies, indicating a role for RHAMM in ERK signaling in PC12 cells.

Proteolytic activation and inactivation of the serine protease activity of plasma hyaluronan binding protein.

We prepared anti-plasma hyaluronan binding protein (PHBP) mouse monoclonal antibodies and studied the fragmentation profile of PHBP with them. PHBP is present in human plasma as a single polypeptide chain (70 kDa). During the purification, PHBP partially fragmentated into the 50-kDa N-terminal fragment and the 27-kDa C-terminal fragment. After the incubation of the purified PHBP, the 70-kDa precursor form was completely cleaved to the 50- and 27-kDa fragments, followed by the 50-kDa to the 26-kDa, and the 27-kDa to the 17-kDa plus the 8-kDa fragments, respectively. Because the purified PHBP contained no other detectable proteins and PHBP has a typical serine protease domain, we concluded that the fragmentation of PHBP was caused by own serine protease activity. PHBP cleaved the C-terminal side of Arg in the peptide effectively and that of Lys weakly. The results of the pre-incubation experiments of PHBP suggested that the single-chain form of PHBP is a precursor, the two-subunit structure is an active form and the three- or four-chain structure is an inactive form of a serine protease.

Regulation mechanism of the serine protease activity of plasma hyaluronan binding protein.

The inhibitor for the serine protease activity of plasma hyaluronan binding protein (PHBP) was purified from human plasma by polyethylene glycol (PEG) fractionation, diethylaminoethyl (DEAE)-Sephacel ion-exchange chromatography, Phenyl Toyopearl 650M hydrophobic chromatography, Bio Gel A-0.5 m gel-filtration and hydroxyapatite chromatography. The serine protease activity of PHBP was measured with Boc-Phe-Ser-Arg-methylcoumarine amide (MCA) as the synthetic substrate of PHBP. The results of the amino acid sequence analyses of the purified PHBP inhibitor indicated that it was C1 inhibitor of the serpin family. C1 inhibitor formed a complex with PHBP, suggesting that it is the actual inhibitor of PHBP in human plasma. On the other hand, dextran sulfate and phosphatidylethanolamine enhanced the auto-fragmentation and the serine protease activity of pro-PHBP, but kaolin did not. These results suggested that the serine protease activity of PHBP was regulated in a similar manner to that of factor XII of the coagulation system.

Identification of the substrates for plasma hyaluronan binding protein.

Plasma hyaluronan biding protein (PHBP) is a novel serine protease, which has an amino acid sequence homology to that of hepatocyte growth factor activator (HGFA), and has a similar domain structure to that of urinary plasminogen activator (u-PA), found in human plasma. We searched the PHBP substrate in human plasma by measuring the digested protein bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that fibrinogen and fibronectin were the major substrates of PHBP. PHBP cleaved the alpha-chain at multiple sites and the beta-chain between lysine53 and lysine54 but not the gamma-chain of fibrinogen. Therefore, PHBP did not initiate the formation of the fibrin clot and did not cause the fibrinolysis directly. PHBP did not cleave (activate) prothrombin and plasminogen, but it converted the inactive single chain urinary plasminogen activator to the active two chain form.

Epitope mapping of four novel CD44 monoclonal antibodies using surface plasmon resonance and soluble CD44.

CD44 is a ubiquitous multistructural and multifunctional cell surface adhesion molecule. The molecular diversity of this glycoprotein is generated by both post-translational modification and the differential use of alternatively spliced exons which play a critical role in determining the exact conformation of the molecule. CD44 isoforms are found in many tissues and in soluble form in plasma. Soluble CD44 was purified by a two-step purification combining ion exchange and immuno-affinity chromatography. Our aim was to check that all the known antigenic epitopes are present on sCD44, which could thus be used for the mapping of new anti-CD44 antibodies. Competitive binding assays using reference antibodies and novel anti-CD44 monoclonal antibodies were performed by real-time biospecific interaction analysis. Reference mAbs identified on soluble CD44 the three distinct epitopes previously defined using red blood cell membrane CD44. From the four novel CD44 mAbs, two mAbs (NaM198-6B5, NaM198-10B4) mapped to epitope group 1, whereas the others (NaM10-8F4, NaM77-9D6) mapped to epitope group 2. Immunopurified sCD44 obtained from the plasma of healthy donors appears to be a usable tool for the mapping of anti-CD44 mAbs.

Protection against virulent Mycobacterium avium infection following DNA vaccination with the 35-kilodalton antigen is accompanied by induction of gamma interferon-secreting CD4(+) T cells.

Mycobacterium avium is an opportunistic pathogen that primarily infects immunocompromised individuals, although the frequency of M. avium infection is also increasing in the immunocompetent population. The antigen repertoire of M. avium varies from that of Mycobacterium tuberculosis, with the immunodominant 35-kDa protein being present in M. avium and Mycobacterium leprae but not in members of the M. tuberculosis complex. Here we show that a DNA vector encoding this M. avium 35-kDa antigen (DNA-35) induces protective immunity against virulent M. avium infection, and this protective effect persists over 14 weeks of infection. In C57BL/6 mice, DNA vaccines expressing the 35-kDa protein as a cytoplasmic or secreted protein, both induced strong T-cell gamma interferon (IFN-gamma) and humoral immune responses. Furthermore, the antibody response was to conformational determinants, confirming that the vector-encoded protein had adopted the native conformation. DNA-35 immunization resulted in an increased activated/memory CD4(+) T-cell response, with an accumulation of CD4(+) CD44(hi) CD45RB(lo) T cells and an increase in antigen-specific IFN-gamma production. The protective effect of the DNA-35 vectors against M. avium infection was comparable to that of vaccination with Mycobacterium bovis BCG and significantly greater than that for previous treated infection with M. avium. These results illustrate the importance of the 35-kDa protein in the protective response to M. avium infection and indicate that DNA vaccination successfully promotes a sustained level of protection during chronic M. avium infection.

Identification and characterization of murine cytotoxic T cells that kill Mycobacterium tuberculosis.

As we seek to develop and evaluate new vaccines against tuberculosis, it is desirable that we understand the mechanisms of protective immunity in our models. Adoptive transfer of protection with hsp65-specific T-cell clones from infected or vaccinated mice into naïve mice had indicated that cytotoxic T cells can make a major contribution to protection. We characterized 28 CD4(+) CD8(-) and 28 CD4(-) CD8(+) hsp65-specific T-cell clones derived from infected or vaccinated mice. Half of the CD4(+) CD8(-) and 64% of the CD4(-) CD8(+) clones were cytotoxic. Cytotoxicity was associated with high expression of CD44 and gamma interferon production. Most (86%) of the cytotoxic CD4(+) CD8(-) clones lysed target cells via the Fas-FasL pathway, and most (83%) of the cytotoxic CD4(-) CD8(+) clones lysed target cells via cytotoxic granules. Only the clones using the granule-mediated pathway caused substantial loss of viability of virulent Mycobacterium tuberculosis during lysis of infected macrophages, and the degree of killing closely correlated with the availability of granule marker enzyme activity. Granule-mediated cytotoxicity thus may have a key role in protection against tuberculosis by delivering mycobactericidal granule contents.

Purification and subunit characterization of the rat liver endocytic hyaluronan receptor.

The endocytic hyaluronan (HA) receptor of liver sinusoidal endothelial cells (LECs) is responsible for the clearance of HA and other glycosaminoglycans from the circulation in mammals. We report here for the first time the purification of this liver HA receptor. Using lectin and immuno-affinity chromatography, two HA receptor species were purified from detergent-solubilized membranes prepared from purified rat LECs. In nonreducing SDS-polyacrylamide gel electrophoresis (PAGE), these two proteins migrated at 175- and approximately 300 kDa corresponding to the two species previously identified by photoaffinity labeling of live cells as the HA receptor (Yannariello-Brown, J., Frost, S. J., and Weigel, P. H. (1992) J. Biol. Chem. 267, 20451-20456). These two proteins co-purify in a molar ratio of 2:1 (175:300), and both proteins are active, able to bind HA after SDS-PAGE, electrotransfer, and renaturation. After reduction, the 175-kDa protein migrates as a approximately 185-kDa protein and is not able to bind HA. The 300-kDa HA receptor is a complex of three disulfide-bonded subunits that migrate in reducing SDS-PAGE at approximately 260, 230, and 97 kDa. These proteins designated, respectively, the alpha, beta, and gamma subunits are present in a molar ratio of 1:1:1 and are also unable to bind HA when reduced. The 175-kDa protein and all three subunits of the 300-kDa species contain N-linked oligosaccharides, as indicated by increased migration in SDS-PAGE after treatment with N-glycosidase F. Both of the deglycosylated, nonreduced HA receptor proteins still bind HA.