Are differences in the expression of carbohydrate-binding sites (endogenous lectins) responsible for recurrences of benign tumours of the parotid gland?

The expression of endogenous lectins in pleomorphic adenomas and papillary cystadenoma lymphomatosum (Warthin tumour) as well as in the tissue of the normal parotid gland was investigated with the aim to determine the capacity of the different cells to specifically bind carbohydrate ligand structures. The loss of sugar-binding sites of pleomorphic adenomas - lactose (diazonium derivative; diaz.), beta-N-acetylgalactosamine (diaz.), sialic acid [(2,3-epoxypropane)-4-oxybutyric acid linked; epichl.], heparin, mannose-6-phosphate (diaz.), maltose (diaz.), xylose (diaz.), melibiose (diaz.), N-acetylgalactosamine (epichl.) - was detected in the investigated tumours, leading to the hypothesis that altered carbohydrate-protein interactions have been involved in the adhesion of lost pleomorphic adenoma cells after damaging the tumour capsule. The papillary cystadenoma lymphomatosum in its cytoplasmic areas expressed significantly fewer sugar-binding sites for alpha-N-acetylgalactosamine (diaz.) and melibiose (diaz.).

The role of ABO blood groups in infections induced by Staphylococcus saprophyticus and Pseudomonas aeruginosa.

Recently, considerable evidence has been accumulated showing that carbohydrate-containing blood group substances represent prime candidates for the specific interaction with microbial surface lectins in infectious diseases. Accordingly, clinical studies have proved that urinary tract infections by Staphylococcus saprophyticus and outer ear canal infections by Pseudomonas aeruginosa can be positively correlated with the patients blood group. Apparently, the blood group antigens (terminal carbohydrates) represent receptors recognized by S. saprophyticus and P. aeruginosa surface lectins.

Are ABH antigenic determinants on human outer ear canal epithelium responsible for Pseudomonas aeruginosa infections?

The outer ear canal expression of ABH human blood group antigens has been analyzed with a standardized routine histological procedure by monoclonal antibodies in the case of blood groups A and B, and a corresponding lectin in the case of blood group 0, respectively. In all 20 cases the blood groups were histochemically confirmed. Furthermore, Pseudomonas aeruginosa-specific inhibition experiments were performed with different sugar solutions as well as A-like substance incubating outer ear canal tissue sections with P. aeruginosa strain (No. 60) presenting lectin specificity for N-acetyl-galactosamine (GalNAc). P. aeruginosa lectins with GalNAc specificity apparently adhere to GalNAc as terminal blood group A determinant and indicate that patients presenting with blood group A may have a genetic predisposition to this form of otitis externa.

Blood group phenotype determines lectin-mediated adhesion of Pseudomonas aeruginosa to human outer ear canal epithelium.

Pseudomonas aeruginosa is the most frequent bacterial pathogen causing acute diffuse otitis externa. In a recent prospective phase II study we demonstrated that lectin-mediated bacterial adhesion can be blocked by receptor-analogue carbohydrates in patients suffering from Pseudomonas aeruginosa-induced acute otitis externa. In this investigation, human ABO blood group antigens were analysed on outer ear canal epithelial cells with standard routine histological procedures by monoclonal antibodies for the blood groups A and B, and with Ulex europaeus I lectin for the blood group O, respectively. In all cases (n = 20) the blood groups could be shown immunohistologically. P. aeruginosa-specific adhesion and inhibition assays were performed in the presence of N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), D-mannose and A-like substance. Outer ear canal tissue sections were incubated with P. aeruginosa (strain PA 60), presenting lectin-specificity for GalNAc. Sections from patients presenting with blood group A were closely settled with bacteria in the presence of non-specific GlcNAc, D-mannose and PBS however, GalNAc and A-like substance inhibited the microbial adhesion. Amongst others, P. aeruginosa present adhesion molecules (lectins) with specificity for GalNAc. Thus, the correlation between blood group A phenotype and P. aeruginosa-induced acute diffuse otitis externa was investigated. Statistical evaluation proved a highly significant association. These data support the hypothesis that P. aeruginosa lectins with GalNAc specificity apparently adhere to GalNAc moieties, representing the terminal blood group A-determinant and further indicate that patients presenting with blood group A may have a genetic disposition for this form of otitis externa.

Will neoadjuvant chemotherapy affect the capacity of head and neck squamous cell carcinoma to bind neoglycoproteins?

Evidence is growing that the carbohydrate portion of glycoconjugates is not merely an inert structural addition to the protein or lipid background, but is involved in normal and altered physiological processes. Conjugation of the potentially bioactive carbohydrate ligand to an inert labeled carrier generates the tools to histochemically monitor the presence of sugar receptors like lectins in tissue sections. In head and neck squamous cell carcinoma endogenous lectins have been systematically characterized by a selected panel of conjugates of such synthetic probes. In this investigation we provide evidence that the antineoplastic agents carboplatin and 5-fluorouracil influence the expression of carbohydrate-binding receptors. In cases with a statistically significant alteration, an increasing loss of binding capacity from treatment cycle to cycle was observed in cytoplasmic areas. This means that the extent of binding of beta-N-acetyl-galactosamine, cellobiose, galactose, and sialic acid was affected. The pattern of diminished binding capacity in nuclear structures encompassed beta-N-acetyl-galactosamine, beta-N-acetyl-glucosamine, sialic acid as carbohydrate part of the neoglycoprotein. Thus, exposure to certain chemotherapeutic agents clearly influences the capacity of tumor cells to mediate protein-carbohydrate interactions. Concomitant with the elucidation of the functions of such an interplay the significance of this down-regulation will be apparent.

Characterization of sugar receptor expression by neoglycoproteins in oral and oropharyngeal squamous cell carcinomas.

Recognition of the carbohydrate part of cellular glycoconjugates by sugar receptors like lectins may contribute to biosignaling and interactions between normal and transformed cells. Such recognitions may be essential for establishing phenotypic characteristics in neoplastic cells, including metastasis-associated properties. To evaluate various glycoconjugates in tumor diagnosis and clinical therapy, a panel of 18 biotinylated neoglycoproteins was prepared. This included conjugates of a histochemically inert carrier protein and crucial sugar moieties such as D-glucuronic acid, alpha- and beta-N-acetyl-galactosamine, beta-N-acetyl-glucosamine, melibiose, lactose, maltose, cellobiose, mannose, mannose-6-phosphate, fucose, rhamnose, and xylose. In so doing the diazo derivative of the respective p-aminophenyl glycosides was coupled with galactose, beta-N-acetyl-galactosamine or beta-N-acetyl-glucosamine via an epoxy group-containing aliphatic spacer. Other glycoconjugates used were the proteoglycan heparin and the sulfated fucan fucoidan. Labeling was effected with cyanogen bromide activation and aminoalkylation for specific detection of endogeneous sugar receptors, especially lectins. Tissues studied were paraformaldehyde-fixed, paraffin-embedded surgical biopsies from patients with different stages of squamous cell carcinomas (SCCs) of the oral cavity (n = 16) and oropharynx (n = 17), including three lymph node metastases from oropharyngeal primary tumors. Semiquantitative binding differences of probes to tumor stages were evaluated statistically by the Mann-Whitney U-Wilcoxon rank sum W test. Specific binding of a probe to cytoplasmic and nuclear structures was detected with apparent quantitative differences. Overall, the cytoplasmic compartment revealed a higher intensity of histochemical reaction than did nuclear structures, indicating a comparatively higher density of specific carbohydrate receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

[Immunoactive action of mistletoe lectin-1 in relation to dose]. / Immunaktive Wirkung von Mistellektin-1 in Abhängigkeit von der Dosierung.

Galactoside-specific mistletoe lectin-1 (ML-1) was isolated by affinity chromatography from proprietary mistletoe extract and checked in BALB/c-mice for its immunoactive potency. To investigate the optimal immunomodulating dosage, ML-1 (0.5, 1.0, 2.5, 5.0 ng/kg body weight, b.w.) was subcutaneously administered for three subsequent days followed by another injection 48 h later. These studies proved that injections of 1 ng ML-1/kg b.w. induced optimal immunomodulation, since thymocyte proliferation, maturation and emigration were significantly enhanced in this murine model as compared to non-treated control mice. Further on, counts of peripheral blood lymphocytes and monocytes as well as expression of relevant activation markers on these cells revealed significant increases after ML-1 (1 ng/kg b.w.) administration. However, increase of cell counts and activity of peritoneal macrophages were less pronounced but still statistically significant for this ML-1 concentration. Determination of immune responses after low dose ML-1 treatment (0.5 ng/kg b.w.) presented relevant (partly statistically significant) increases, too. However, high dose ML-1 treatment (2.5, 5.0 ng/kg b.w.) did not enhance (but suppress) relevant immune functions. For future clinical/therapeutical treatment strategies, ML-1 dosages ranging from 0.5-1.0 ng/kg b.w. may be supposed to be optimal.

Immunoprotective activity of the galactoside-specific mistletoe lectin in cortisone-treated BALB/c-mice.

Hydrocortisone-acetate (HA)-treatment of BALB/c-mice induced a profound suppression of the lymphatic immune system with statistically significant decreases of thymocyte proliferation and maturation rates as well as peripheral blood lymphocyte (PBL) counts. To check its putative immunoprotective/immunorestoring activity, the optimal immunomodulating dosage of commercially available mistletoe extract standardized for the galactoside-specific mistletoe lectin (ML-1) was regularly administered (1 ng ML-1 per kg body weight; subcutaneously). As compared to counts of thymic lymphatic subsets of HA-treated mice, a considerable up regulation of mature cells expressing helper/inducer (L3T4+) as well as cytotoxic/suppressor (Lyt-2+) phenotypes and immature cells expressing both antigens (L3T4+/Lyt-2+) could be found after ML-1 co-administration. Counts of BALB/c-mouse peripheral blood mononuclear cells also revealed statistically significant increases after ML-1 co-administration, as compared to HA-treated animals. Accordingly, ML-1 treatment may be supposed to restore the lymphatic system after immunosuppressive corticoid treatment and thus this treatment may be of great benefit to patients.

An estimate on the frequency of duplicated haplotypes and silent alleles of human C4 protein polymorphism. II. Investigations in healthy Negro families.

The first investigation of complete MHC marker data in South African Negroes by segregation analysis in 11 families with up to three generations is presented, including quantitative evaluation of C4 allotype patterns and C4 beta chain determinations according to Steuer et al. (1). The frequency of homo- and heteroduplicated, hybrid, and non-expressed C4 alleles was determined from C4 protein phenotyping, including C4 alpha and beta chains, quantitative estimates of the relative electrophoretic C4 banding patterns by scanning densitometry, and from the other classical MHC markers by submitting all results to the family analysis program (FAP). From unrelated non-diseased individuals (n = 105) in these families with 62 haplotypes, the following frequencies were observed for non-expressed alleles: C4A*Q0 0.1189, C4B*Q0 0.2552, and for the total of heteroduplicated alleles: C4A 0.0645, C4B 0.0608. Applying additionally quantitative determinations of C4 banding patterns, homoduplications such as C4A*3 A*3, C4B*1 B*1, C4B*3 B*3, and the heteroduplication C4A*3 A*2 were assumed. In the investigated individuals the heteroduplications of C4A*12 and C4A*3 with the A*91 allele and of C4B*2 with C4B*92 were observed. It was concluded that not only allele frequencies but also the frequency of heteroduplications seems to be of specific ethnic character. Furthermore, the prior hypothesis that deletion or non-expression at one C4 locus is accompanied by duplication at the other was only confirmed for non-expressed B-alleles with C4A*3 A*91 or C4A*12 A*91.(ABSTRACT TRUNCATED AT 250 WORDS)

Thymocyte proliferation and maturation in response to galactoside-specific mistletoe lectin-1.

Commercially available mistletoe extract standardized for the galactoside-specific mistletoe lectin-1 (ML-1) could be shown to induce thymocyte proliferation and maturation in BALB/c-mice after regular subcutaneous administration of the optimal immunomodulating dosage (1ng lectin/kg body weight). The increase in thymocyte numbers per mg organ weight was statistically significant. Determinations of thymic lymphatic subsets revealed considerable up-regulation of mature cells expressing helper/inducer (L3T4) or cytotoxic/suppressor (Lyt-2) phenotype and immature cells presenting both (L3T4/Lyt-2) antigens. Thus, administration of ML-1 accelerated murine thymocyte proliferation and maturation. Counts of BALB/c-mouse peripheral blood lymphocytes (PBL) revealed evident (but statistically non-significant) increases after ML-1 administration. The determination of activated PBL expressing interleukin (Il)-2 receptors proved, however, that ML-1 induced a potent immunostimulation since these cells were significantly enhanced after ML-1 administration.

[Effect of mistletoe lectin therapy on serum level of defined serum proteins (acute phase proteins) in tumor patients]. / Einfluss der Mistellektintherapie auf den Serumspiegel definierter Serumproteine (Akutphaseproteine) bei Tumorpatienten.

The influence of galactoside-specific mistletoe lectin (ML-1) administration on defined acute phase reactants in the serum of cancer patients (mammary carcinoma, n = 4; larynx carcinoma, n = 11; TNM-stages II to IV; after appropriate surgery, chemotherapy, radiation) was studied. Regular subcutaneous injections of the optimal doses of ML-1 (1 mg/kg body weight, twice a week) yielded statistically significant increases of certain acute phase reactants (C-reactive protein, haptoglobin, coeruloplasmin, C3-complement, albumin, immunoglobulin IgM) after four weeks of treatment. However, serum concentrations of transferrin, C4-complement and the immunoglobulins IgG and IgA were found within the biological range (means +/- 2 s). The increase of acute phase reactants after administration of ML-1 correlates positively with the activity of lymphatic cells (e.g. expression of IL-2 and HLA-DR receptors) in FACS (fluorescence-activated cell sorter) staining experiments and indicates the immunoreactive potency of this substance which may be speculated to be cytokine-induced.

[Intralesional therapy with natural interferon-beta in refractory squamous epithelial cancers of the ENT area]. / Intraläsionale Therapie mit natürlichem Interferon-beta bei ausbehandelten Patienten mit Plattenepithelkarzinomen im HNO-Bereich.

It has been the aim of the present investigation to study the effect of intratumorally applied human fibroblast interferon (nIFN-beta; Fiblaferon 5 for the first two weeks, and Fiblaferon 3 three times a week) in a phase-II clinical trial of thirteen patients with advanced head and neck squamous cell carcinomas. All of the patients had failed established therapeutic modalities before and could not be treated by conventional procedures. nIFN-beta was injected intratumorally and its effect on tumour size was assessed by an independent, second observer as well as via CT and MR imaging. All assessments were done prior to treatment, 8 weeks after beginning treatment and at 16 weeks. Three female and ten male patients with primary tumours of the hypopharynx (n = 5), the larynx (n = 4), the oropharynx (n = 1), the glandula submandibularis (n = 1), the oral cavity (n = 1) and the oesophagus (n = 1) have undergone outpatient treatment three times a week. Tumour size showed no change in six patients while progressive disease occurred in seven cases after eight weeks of treatment. Radiological findings did not change in the nine patients continuing treatment while five showed progressive disease. There were no serious local or systemic side effects due to the intratumoral nIFN-beta treatment. The survival time was 9.73 months after the onset of nIFN-beta treatment.

[Histochemical identification of endogenous lectins using labelled neoglycoproteins in human head-and-neck squamous cell carcinoma]. / Histochemische Identifizierung endogener Lektine durch markierte Neoglykoproteine bei humanen Kopf-Hals-Plattenepithelkarzinomen.

According to the "Population-based cancer register" of the Federal Republic of Germany only malignant neoplasms of the buccal cavity, the pharynx and larynx as well as cancers of the respiratory tract show an increasing rate of incidence and mortality. The molecular mechanisms and etiological factors causing this phenomenon are still little understood despite intensive research work. Recognition between receptors on a cellular level may be mediated by specific amino acid sequences on the level of protein-protein recognition. Additionally, the interactions between cell sugars and the corresponding protein receptor may play a decisive role in development, regeneration and organisation of cells and tissue. The high specificity of the binding of biotinylated neoglycoproteins in tissue sections enables to detect glycohistochemically binding sites for the carbohydrate ligands of the glycosylated carrier protein. The evidence of lectins in squamous cell cancer of the oral cavity, oropharynx, larynx, and hypopharynx has not been established so far. Squamous cell cancer tissue samples of twelve patients with different tumour locations were investigated by incubation of sections of paraffin-embedded samples and application of an avidin-biotin-peroxidase complex for visualisation with synthetic biotinylated neoglycoproteins. Altogether 168 stained sections were evaluated including controls. Pronounced cytoplasmatic staining was seen with the following neoglycoproteins: sialic acid-bovine serum albumin (BSA), glucuronic acid-BSA, N-acetylglucosamine (glcNAc)-BSA, N-acetylgalactosamine (beta-galNAc)-BSA, lactose-BSA, maltose-BSA, mannose-BSA, mannose-6-phosphate-BSA. No corresponding lectins seems to exist for the following investigated sugars: fucoidan, heparin, and the alpha-anomeric form of N-acetylgalactosamine, because no specific staining was seen.(ABSTRACT TRUNCATED AT 250 WORDS)

Early onset sensorineural hearing loss: association studies with major histocompatibility class III (complement) markers.

In 39 families with at least one child suffering from moderate or severe bilateral sensorineural hearing loss (SNHL), major histocompatibility complex (MHC) class III complement phenotypes were retrospectively determined by standard methods; MHC class I segregation data was also available. The families were treated in the Hospital for Communication Disorders and selected for the HLA-B16 and B18 specificities, respectively. Haplotype and allele frequencies were derived from segregation analysis in the families. From 31 unrelated children with random and familiar forms of SNHL significant deviations in the distribution were seen for the following MHC class III alleles using as a control population 60 German healthy individuals: duplicated C4A alleles (C4"DA") p = 0.009, silent C4A alleles (C4A*Q0) p = 0.006, duplicated heavy C4 beta-chain alleles (C4 beta"DHH") p = 0.0003, and silent C4 beta-chain alleles (C4 beta*Q0) p = 0.0075. In serum samples from patients with an assumed genetic disposition according to clinical criteria indications for an association were found for C4"DA" (p = 0.03), C4A*Q0 (p = 0.003), C4B*3 (p = 0.046), C4 beta"DHH" (p = 0.004), and C4 beta*Q0 (p = 0.02). The underrepresentation of C4A*Q0 may be an indicator for aberrant or duplicated C4 alleles on the same haplotype or exhibit a protection mechanism for acquiring the inheritable forms of early onset SNHL.