Expression of human beta-defensins-1-4 in thyroid cancer cells and new insight on biologic activity of hBD-2 in vitro.

UNLABELLED: The study was aimed on analysis of human beta-defensin-1-4 (hBDs) mRNA expression in cultured thyroid cancer cells and evaluation of effects of recombinant hBD-2 (rec-hBD-2) on growth patterns, migration properties and expression of E-cadherin and vimentin in these cells. METHODS: The study was performed on cultured follicular thyroid cancer WRO cells, papillary thyroid cancer TPC1 cells, and anaplastic thyroid cancer KTC-2 cells. For analysis of hBD-1-4 mRNA expression in thyroid cancer cells, semiquantitative RT-PCR was used. Effects of rec-hBD-2 on cell proliferation, viability, and migration were analyzed using direct cell counting, MTT test, and scratch assay respectively. Expression of vimentin and E-cadherin was evaluated by quantitative PCR (qPCR). RESULTS: By the data of RT-PCR, all three studied thyroid cancer cell lines express hBD-1 and -4 mRNA, but not hBD-2 mRNA, while hBD-3 expression was detected in WRO and KTC-2 cells. The treatment of TPC-1, WRO, and KTC-2 cells with 100-1000 nM rec-hBD-2 resulted in significant concentration-dependent suppression of cell proliferation, viability, and migratory property. By the data of qPCR, significant up-regulation of vimentin expression was registered in KTC-2 and WRO cells treated with 500 nM rec-hBD-2. Significant down-regulation of E-cadherin expression (p < 0.05) was detected only in KTC-2 cells treated with the defensin. Also, it has been shown that TPC-1 cells treated with 500 nM rec-hBD-2 acquired more elongated morphology. CONCLUSION: The data demonstrate that hBD-2 in concentrations higher than 100 nM exerts significant concentration-dependent suppression of thyroid cancer cell growth and migration, and affects vimentin and E-cadherin expression dependent on histologic type of thyroid cancer cells.

Involvement of human beta-defensin-2 in regulation of malignant potential of cultured human melanoma cells.

BACKGROUND AND AIM: Human beta-defensin-2 (hBD-2) is an antimicrobial cationic peptide capable to control human carcinoma cell growth via cell cycle regulation. The present study was aimed on determination of hBD-2 influence on the growth patterns and malignant potential of cultured human melanoma cells. METHODS: The study was performed on cultured human melanoma cells of mel Z and mel Is lines treated with recombinant hBD-2 (rec-hBD-2); cell viability, proliferation, cell cycle distribution, and anchorage-independent growth were analyzed using MTT test, direct cell counting, flow cytometry, and colony forming assay respectively. Expression and/or phosphorylation levels of proteins involved in cell cycle control were evaluated by Western blotting. RESULTS: The treatment of mel Z and mel Is cells with rec-hBD-2 in a concentration range of 100-1000 nM resulted in a concentration-dependent suppression of cell proliferation, viability, and colony forming activity. It has been shown that rec-hBD-2 exerts its growth suppression effects via significant downregulation of B-Raf expression, activation of pRB and upregulation of p21(WAF1) expression, downregulation of cyclin D1 and cyclin E resulting in cell cycle arrest at G1/S checkpoint. CONCLUSION: According to obtained results, hBD-2 exerts its growth suppression effect toward human melanoma cells via downregulation of B-Raf, cyclin D1 and cyclin E expression, upregulation of p21(WAF1) expression and activation of pRB.

Biologic activities of recombinant human-beta-defensin-4 toward cultured human cancer cells.

AIM: The aim of the study was in vitro analysis of biological activity of recombinant human beta-defensin-4 (rec-hBD-4). METHODS: hBD-4 cDNA was cloned into pGEX-2T vector, and recombinant plasmid was transformed into E. coli BL21(DE3) cells. To purify soluble fusion GST-hBD-4 protein, affinity chromatography was applied. Rec-hBD-4 was cleaved from the fusion protein with thrombin, and purified by reverse phase chromatography on Sep-Pack C18. Effects of rec-hBD-4 on proliferation, viability, cell cycle distribution, substrate-independent growth, and mobility of cultured human cancer cells of A431, A549, and TPC-1 lines were analyzed by direct cell counting technique, MTT assay, flow cytofluorometry, colony forming assay in semi-soft medium, and wound healing assay. RESULTS: Rec-hBD-4 was expressed in bacterial cells as GST-hBD-4 fusion protein, and purified by routine 3-step procedure (affine chromatography on glutathione-agarose, cleavage of fusion protein by thrombin, and reverse phase chromatography). Analysis of in vitro activity of rec-hBD-4 toward three human cancer cell lines has demonstrated that the defensin is capable to affect cell behaviour in concentration-dependent manner. In 1-100 nM concentrations rec-hBD-4 significantly stimulates cancer cell proliferation and viability, and promotes cell cycle progression through G2/M checkpoint, greatly enhances colony-forming activity and mobility of the cells. Treatment of the cells with 500 nM of rec-hBD-4 resulted in opposite effects: significant suppression of cell proliferation and viability, blockage of cell cycle in G1/S checkpoint, significant inhibition of cell migration and colony forming activity. CONCLUSION: Recombinant human beta-defensin-4 is biologically active peptide capable to cause oppositely directed effects toward biologic features of cancer cells in vitro dependent on its concentration.

Human beta-defensin-2 controls cell cycle in malignant epithelial cells: in vitro study.

AIM: In the present research we analyze the mechanism of human beta-defensin-2 (hBD-2) influence on cultured malignant epithelial cell growth. MATERIALS AND METHODS: The analysis of a concentration-dependent effect of recombinant hBD-2 (rec-hBD-2) on cell growth patterns and cell cycle distribution has been performed in vitro with 2 cell lines (human lung adenocarcinoma A549 cells and human epidermoid carcinoma A431 cells) using MTT test, flow cytometry and direct cell counting. To study intracellular localization of hBD-2 immunocytofluorescent and immunocytochemical analyses were applied, and effect of hBD-2 on signal cascades involved in cell cycle regulation has been studied by Western blotting. RESULTS: According to our data, rec-hBD-2 exerts a concentration-dependent effect on the viability of cultured A549 and A431 cells. It causes proproliferative effect at concentrations below 1 nM, significant suppression of cell proliferation at concentration range from 10 nM to 1 µM (p<0.05), and cell death at higher concentrations. Using flow cytometry we have demonstrated that hBD-2 dependent growth suppression is realized via cell cycle arrest at G1/S phase (p<0.05). Also, we have registered significant activation of pRB and decreased expression of Cyclin D1 in cells treated with the defensin compared to untreated control cells, while the expression of p53 remains unaffected. The study of intracellular localization of hBD-2 in these cells has revealed that exogeneously added defensin molecules enter the cells, are distributed throughout the cytoplasm and could be detected in cell nuclei. The model study using A549 cells treated with 1,25-(OH)(2)D(3) has shown similar cell growth suppression effect of native endogenously produced hBD-2. CONCLUSION: The results of our study suggest that in malignant epithelial cells hBD-2 may control cell growth via arrest of G1/S transition and activation of pRB.

Administration of vitamin D3 improves antimetastatic efficacy of cancer vaccine therapy of Lewis lung carcinoma.

AIM: To analyze antitumor efficacy of experimental cancer vaccine therapy combined with introduction of vitamin D3 (VD3) for treatment of Lewis lung carcinoma (3LL). MATERIALS AND METHODS: Cancer vaccines composed from recombinant murine beta-defensin-2 (mBD-2) and 3LL cell lysate, or DNA, coding for mBD-2-Muc1 fusion construct cloned in pcDNA3+ vector, were prepared and used for intradermal vaccination. Experimental cancer vaccines introduced i. d. at therapeutic and prophylactic regimens to 3LL-bearing C57Bl mice, were applied alone or in combination with VD3 (administered per os) and/or low-dose cyclophosphamide (CP, administered intraperitoneal). Efficacy of treatments was analyzed by primary tumor growth dynamics indexes and by metastasis rate in vaccinated animals. RESULTS: As it has been shown, administration of the protein-based vaccine composed from mBD-2 and 3LL cell lysate in combination with VD3 and CP, but not in VD3 free setting, led to significant suppression of primary tumor growth (p < 0.005) and had significant antimetastatic effect. Introduction of VD3 with or without CP in the scheme of treatment with mBD- 2-Muc1-DNA vaccine at therapeutic regimen has led to significant suppression of primary tumor (p < 0.05) and metastasis volumes (p < 0.005), while in the groups of animals treated with DNA-vaccine + VD3 with or without CP at prophylactic regimen, significant antimetastatic effect (p < 0.05) and elevation of average life-span (p < 0.05) have been registered. CONCLUSION: The results of this pilot study have shown promising clinical effects of VD3 administration in combination with cancer vaccinotherapy in vivo.

Immunohistochemical analysis of beta-defensin-2 expression in human lung tumors.

AIM: The present research was directed on analysis of the expression patterns of human beta-defensin-2 (hBD-2) in human lung tumors. MATERIALS AND METHODS: Specimens of surgically resected human lung tumors (n = 31) of different histological type (1 case of small cell lung cancer, and 30 cases of non-small cell lung cancer (1 case of clear cell carcinoma, 9 cases of squamous cell carcinoma (SCC), and 20 cases of adenocarcinoma (AC)) were analyzed for expression of hBD-2 with the use of immunohistochemical analysis. RESULTS: Immunohistochemical analysis has revealed that all lung tumor samples independently on their histological type express hBD-2 peptide, however at different levels (from < 5% to 100% cells). According to our observations, low-differentiated AC differs from moderately differentiated AC by significantly lower hBD-2 expression levels (p < 0.05). No correlation between hBD-2 expression patterns and PCNA or Bcl-2 expression has been found. CONCLUSION: Human beta-defensin-2 expression levels may depend on differentiation grade of lung adenocarcinoma.

Expression patterns of murine beta-defensin-2 mRNA in Lewis lung carcinoma cells in vitro and in vivo.

AIM: To evaluate the anti-tumor activity of murine beta-defensin-2 (mBD-2) expression in vitro and in vivo. MATERIALS AND METHODS: Based on pcDNA3 vector, constructs containing mBD-2 cDNA coding mature defensin molecule (pcDNA3-mBD2), and Igk-mBD-2 insertion, coding secretory sequence plus mature defensin molecule (pcDNA3-Igk-mBD-2) were generated. Lewis lung carcinoma (3LL) cells were transfected in vitro with these plasmids and with blank pcDNA3 vector, and the proliferative rate and clonogenic ability of obtained cell lines cultivated in vitro were analyzed using (3)H-incorporation technique and colony formation in semi-soft medium, respectively. Expression of mBD-2 mRNA was studied by semiquantative RT-PCR analysis. Also, transfected cells were transplanted to C57B mice, and the patterns of tumor growth in vivo were analyzed by routine techniques. RESULTS: We have found out that in the 3LL cells transfected with pcDNA3-mBD-2 and pcDNA3-Igk-mBD-2, the expression of mBD-2 mRNA is significantly down regulated compared to wild-type cells and 3LL cells transfected with blank vector. The cells with suppressed mBD-2 expression differed from parental cells and cells transfected with blank vector by higher proliferation rate (p < 0.001) and higher clonogenic ability. The 3LL-mBD-2 and 3LL-Igk-mBD-2 cells that are transplanted to C57B mice gave rise to more aggressive tumors that possessed significantly higher growth rate (p < 0.01) than those that arise from wild-type 3LL cells. CONCLUSION: The obtained results indicate the relation between mBD-2 expession in 3LL cells and their proliferation rate and malignant phenotype, and also allow to hypothesize the possibility of regulation of mBD-2 mRNA expression in these cells by a feedback mechanism.

Expression of human beta-defensins-1, 2 and 4 mRNA in human lung tumor tissue: a pilot study.

AIM: To analyze the patterns of human beta-defensin-1, 2, 4 (hBDs) expression in human lung tumors. MATERIALS AND METHODS: Tissue samples of surgically resected human lung tumors (squamous cell carcinoma (SCC), n=10; adenocarcinoma (AC), n=10) paired with conditionally normal tissue samples were analyzed for expression of hBD-1, 2, 4 mRNA by semiquantitative RT-PCR. RESULTS: In a number of studied lung cancer tissue samples, overexpression of defensin mRNA was registered: hBD-1 mRNA (50% of SCC and 60% AC), hBD-2 mRNA (60% of SCC and 50% of AC) or hBD-4 (40% of SCC and 20% AC). No correlation was detected between the levels of hBD-1, hBD-2 and hBD-4 mRNA and histological type, differentiation grade of the tumor, and the stage of the disease, as well as the content of hBD-2 peptide in blood serum of lung cancer patients. CONCLUSION: Human beta-defensins-1 and -2 are often up-regulated in human lung tumors.

Involvement of human beta-defensin-2 in intracellular signaling: in vitro study.

AIM: To analyze involvement of human beta-defensin-2 (hBD-2) in intracellular signaling in vitro. MATERIALS AND METHODS: A431cells were cultured in the presence of 1 microg/ml of recombinant hBD-2 and/or 10 ng/ml EGF. For evaluation of expression of mRNAs for p70S6 kinase, isoforms alpha and beta, RT-PCR analysis was applied. Expression and activity of p70S6K, phosphorylation of PDK1, ERK, JNK, p38 kinases and EGF receptor (EGFR) was evaluated using Western blot analysis. RESULTS: 30 min incubation of A431 cells with 1 mug/ml of hBD-2 didn't influence autophosphorylation level of EGFR, but resulted in activation of p70S6K, 12 h treatment - in prominently increased level of mRNA for alpha and beta-isoforms of p70S6 kinase, whilst 24 h treatment - in elevation of p70S6K synthesis on protein level. Up-stream kinase phosphorylating p70S6K, PDK1, is also phosporylated upon influence of exogenous hBD-2 in vitro. CONCLUSION: Our data point on the involvement of PDK1-p70S6K pathway in mediation of action of hBD-2 in A431 cells.

Regulated expression of human beta-defensin-2 leads to altered phenotype and growth patterns of cultured human embryonal kidney cells.

AIM: To create cell line with regulated expression of human beta-defensin-2 (hBD-2) and evaluate the influence of expressed peptide on its phenotypic and growth patterns. MATERIALS AND METHODS: Using cloning techniques, on the base of human embryonic kidney cells of HEK293T line, stable T-rex HEK-hBD2-m cell subline expressing mature biologically active hBD-2 molecule upon the presence of tetracycline in culture medium was generated. The morphological patterns, growth characteristics and colony forming activity of these cells were studied using routine techniques. RESULTS: T-rex HEK-HBD2-m cell subline was shown to express both mRNA and hBD-2m protein upon the presence of 1 mug/ml tetracycline in culture medium as it was demonstrated by RT-PCR and immunocytochemical approach. Upon prolonged expression of hBD-2, the cells acquired special features: they lost ability to grow in monolayer in vitro and to form colonies in soft agar, characteristic to parental HEK293T cells, but possess higher growth rate and longer survival in FBS-free medium than wild type cells. CONCLUSION: Expression of hBD-2 in T-rex HEK-HBD2-m cell subline results in specific biological consequences that favor cell survival.

Involvement of human beta-defensin-2 in proliferation of transformed cells of human cervix.

AIM: To evaluate the influence of human beta-defensin-2 (hBD-2) on viability and proliferation of cultured human epithelial cells and the patterns of hBD-2 expression in normal tissues and early-stage human cervical neoplasia in the relation to proliferative state of these cells. MATERIALS AND METHODS: The influence of recombinant hBD-2 on viability and proliferation of cultured cells of A431 and M-HeLa lines in vitro was performed by MTT-test, 3H-thymidine incorporation and cell counting techniques. Immunohistochemical analysis of expression of hBD-2 and PCNA in tissue samples (10 normal cases (control), 30 carcinomas of the cervix uteri: 15 - squamous cell carcinoma in situ (Stage 0), and 15 squamous cell carcinoma (Stage Ia)) was performed with the use of anti-hBD-2 and anti-PCNA-mAbs, respectively. RESULTS: We have revealed that hBD-2 significantly stimulated proliferation of A431 and M-HeLa cells in a concentration-dependent manner in the range of 0.1-2 microg/ml, whilst at higher concentrations (> 3-5 microg/ml) it negatively influenced cell viability. The results of immunohistochemical study have shown that malignant transformation of human cervical epithelium is accompanied by the increase of expression of hBD-2 and PCNA. However, the correlative analysis of the expression of the mentioned markers has revealed no relation between them. CONCLUSION: The effect of hBD-2 on viability and proliferation of cultured epithelial cells possesses a concentration-dependent character. Expression of hBD-2 is increased in early-stage cervical carcinoma.

Epidermoid carcinoma-derived antimicrobial peptide (ECAP) inhibits phosphorylation by protein kinases in vitro.

Animal peptide antibiotics are thought to mediate their cytotoxic and growth inhibitory action on bacteria, fungi, and cancer cells through a membrane-targeted mechanism. Although the membrane interactions of the peptide antibiotics and their penetration through the membranes have been studied in several models, the precise chain of events leading to cell death or growth arrest is not established yet. In this study we used in vitro kinase assays followed by imaging analyses to examine the effect of human cationic antimicrobial peptide ECAP on the activity of the protein kinases. We report that HPLC-grade ECAP is responsible for inhibition of EGFR autophosphorylation in plasma membrane fractions obtained from A-431 cells. The activity of ECAP is concentration dependent with a half-inhibitory concentration in the range of 0.1-0.2 microM. Marked decrease in autophosphorylation of immunoprecipitated non-receptor protein kinases belonging to different families, namely PKCmu, Lyn and Syk, is observed in the presence of as little as 0.2 microM of the peptide. Among the examined non-receptor protein kinases PKCmu was the most sensitive to the inhibitory action of ECAP, whereas Syk was inhibited least of all. ECAP exerted no detectable cytotoxicity on non-nucleate animal cells at concentrations up to 3 microM. The capability of ECAP to inhibit protein kinases at concentrations, that are at least 10 fold lower than antibacterial and cytotoxic ones, suggests that the protein kinases are possible intracellular targets for antimicrobial peptides. We suppose that inhibition of the protein kinases may provide a mechanism for the action of cationic antimicrobial peptides on host cells including tumour cells.

A protein kinase inhibitor from A431 subline overexpressing TGF alpha possesses antimicrobial activity.

During the last decade the key role of antimicrobial peptides in innate immunity has been argued. They were found in plants and in different phylogenic groups of animals (insects, amphibia, and even in mammals). We report the production of a human peptide antibiotic that was previously characterized as an EGF receptor tyrosine kinase inhibitor in epidermoid carcinoma A431/1522 cell subline overexpressing TGF alpha. It is a 3 kDa hydrophobic cationic peptide cytotoxic for different species of Gr+ and Gr- bacteria in micromolar concentration range, and demonstrating slight fungicidal activity.

[Antigenic bacterial polysaccharides. Structure of O-specific polysaccharides from Pseudomonas cepacia serogroups C, I, O1, and O4]. / Antigennye polisakharidy bakterii. Stroenie O-spetsificheskikh polisakharidov Pseudomonas cepacia serogrupp C, I, O1 i O4.

Like some Pseudomonas cepacia serogroups studied earlier, serogroups C, I (Nakamura), O1 and O4 (Heidt) are characterized by the presence of at least two structurally different O-antigenic polysaccharide chains in cell-wall lipopolysaccharides. On the basis of acid hydrolysis, methylation, 1H- and 13C-NMR spectroscopy, including computer-assisted 13C-NMR-based analysis, the complete structures of the predominant polysaccharides of serogroups I (I), C and O4 (III) and the minor polysaccharides of serogroups I (II) and O1 (V) were established, and the structure of the predominant polysaccharide of serogroup O1 (IV) established earlier (Cox A. D., Wilkinson S. G.@Carbohydr. Res. 1990. V. 195. No 2. P. 295-301) was confirmed.

[Antigenic polysaccharides of bacteria. 34. Structure of O-specific polysaccharide chains of lipopolysaccharides from Pseudomonas cepacia strains IMV 4207 (Serotype A) and IMV 598/2]. / Antigennye polisakharidy bakterii. 34.Struktura O-spetsificheskikh polisakharidnykh tsepei lipopolisakharidov Pseudomonas cepacia IMV 4207 (serotip A) i IMV 598/2.

On the basis of non-destructive analysis by means of 1H and 13C NMR spectroscopy and calculation of specific optical rotation, it was concluded that O-specific polysaccharide of Pseudomonas cepacia strain IMV 4207 (serotype A) has the structure (I): (formula; see text) Two structurally different polysaccharides were found in the ratio of approximately 2.5:1 in P. cepacia strain IMV 598/2 which is serologically related to serotype A in Nakamura classification and serotype 2 in Heidt classification. The minor polysaccharide has the structure (I) whereas the major one possesses the structure (II) which is characteristic of the formerly studied O-specific polysaccharide of P. cepacia strain IMV 4137 belonging to serotype 2: ----4)-beta-D-Galp-(1----2)-alpha-L-Rhap-(1----.

[Antigenic polysaccharides of bacteria. 35. Establishment of the structure of polysaccharide chains of lipopolysaccharides of Pseudomonas cepacia IMV 4176 and IMV 4202 (Serotype 3)]. / Antigennye polisakharidy bakterii. 35. Ustanovlenie struktury polisakharidnykh tsepei lipopolisakkharidov Pseudomonas cepacia IMV 4176 i IMV 4202 (serotip 3).

On mild acid degradation of the Pseudomonas cepacia strain IMV 4176 lipopolysaccharide, two polysaccharides were obtained, one of which is a homopolymer of N-acetyl-D-galactosamine and the other is composed of equal amounts of N-acetyl-D-galactosamine and D-ribose. Partial hydrolysis with aqueous oxalic acid caused depolymerization of the heteropolysaccharide, and the homopolysaccharide was isolated in the individual state. On the basis of methylation and 13C NMR analysis, it was concluded that both polysaccharides are built up of disaccharide repeating units having the following structures: ----4)-alpha-D-GalpNAc-(1----4)-beta-D-GalpNAc-(1---- and ----4)-alpha-D-GalpNAc-(1----2)-beta-D-Ribf-(1----. The heteropolysaccharide from P. cepacia strain 4176 is identical by the structure of the repeating unit to the O-specific polysaccharide of P. cepacia strain IMV 4202 (serotype 3), Pseudomonas aeruginosa O12 and Serratia marcescens O14.

[Antigenic polysaccharides of bacteria. 33. The structure of O-specific polysaccharide chain of lipopolysaccharide from Pseudomonas cepacia serotype 6]. / Antigennye polisakharidy bakterii. 33. Struktura O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Pseudomonas cepacia, serotip 6.

On mild acid degradation of the Pseudomonas cepacia serotype 6 lipopolysaccharide, the O-specific polysaccharide was obtained, which contains D-mannose and D-galactose residues in the ratio approximately 1:1, as well as O-acetyl groups. On the basis of 1H and 13C NMR analysis, calculation of specific optical rotation, and methylation, it was concluded that the polysaccharide possesses the following structure: (formula; see text) Regularities in glycosidation effects in 13C NMR spectra of 1,3-linked disaccharides containing furanoside residues are discussed.

[Antigenic polysaccharides of bacteria. 32. The structure of O-specific polysaccharide chains of Pseudomonas cepacia serotype B and E lipopolysaccharides containing D-fucose]. / Antigennye polisakharidy bakterii. 32. Struktura O-spetsificheskikh polisakharidnykh tsepei lipopolisakharidov Pseudomonas cepacia serotipov B i E, soderzhashchikh D-fukozu.

On the basis of acid hydrolysis, methylation, 1H and 13C NMR analysis, and calculation of specific optical rotation, the following structures were established for O-specific polysaccharides of Pseudomonas cepacia serotypes B and E: ----3)-beta-D-Galf-(1----3)-alpha-D-Fucp-(1----serotype B ----3)-beta-D-GlcpNAc-(1----3)-alpha-D-Fucp-(1----serotype E A characteristic feature of the polysaccharides is the presence of D-fucose, rather rare for bacterial antigens.

[Antigenic polysaccharides of bacteria. 25. Structure of the O-specific polysaccharide chain of Pseudomonas cepacia 673/2 lipopolysaccharide containing L-glycero-D-manno-heptose]. / Antigennye polisakharidy bakterii. 25. Struktura O-spetsificheskoi polisakharidnoi tsepi lipopolisakharida Pseudomonas cepacia 673/2, soderzhashchei L-glitsero-D-manno-geptozu.

O-Specific polysaccharide, consisting of D-rhamnose and L-glycero-D-manno-heptose (LD-Hep) in a 2 : 1 ratio, was obtained on the mild acid degradation of the Pseudomonas cepacia IMV 673/2 lipopolysaccharide; monosaccharide LD-Hep has not previously been found in O-specific chains of lipopolysaccharides. On the basis of methylation and 13C-NMR data, it was concluded that the polysaccharide is composed of trisaccharide repeating units having the following structure: ----3)-alpha-D-Rha-(1----3)-alpha-D-Rha-(1----2)-alpha-LD-Hep-(1----