Galectin-8 expression decreases in cancer compared with normal and dysplastic human colon tissue and acts significantly on human colon cancer cell migration as a suppressor.

BACKGROUND AND AIMS: Galectins are beta-galactoside binding proteins. This ability may have a bearing on cell adhesion and migration/proliferation in human colon cancer cells. In addition to galectins-1 and -3 studied to date, other members of this family not investigated in detail may contribute to modulation of tumour cell features. This evident gap has prompted us to extend galectin analysis beyond the two prototypes. The present study deals with the quantitative determination of immunohistochemical expression of galectin-8 in normal, benign, and malignant human colon tissue samples and in four human colon cancer models (HCT-15, LoVo, CoLo201, and DLD-1) maintained both in vitro as permanent cell lines and in vivo as nude mice xenografts. The role of galectin-8 (and its neutralising antibody) in cell migration was investigated in HCT-15, LoVo, CoLo201, and DLD-1 cell lines. METHODS: Immunohistochemical expression of galectin-8 and its overall ability to bind to sugar ligands (revealed glycohistochemically by means of biotinylated histochemically inert carrier bovine serum albumin with alpha- and beta-D-galactose, alpha-D-glucose, and lactose derivatives as ligands) were quantitatively determined using computer assisted microscopy. The presence of galectin-8 mRNA in the four human colon cancer cell lines was examined by reverse transcriptase-polymerase chain reaction. In vitro, cellular localisation of exogenously added galectin-8 in the culture media of these colon cancer cells was visualised by fluorescence microscopy. In vitro galectin-8 mediated effects (and the influence of its neutralising antibody) on migration levels of living HCT-15, LoVo, CoLo201, and DLD-1 cells were quantitatively determined by computer assisted phase contrast microscopy. RESULTS: A marked decrease in immunohistochemical expression of galectin-8 occurred with malignancy development in human colon tissue. Malignant colon tissue exhibited a significantly lower galectin-8 level than normal or benign tissue colon cancers; those with extensive invasion capacities (T3-4/N+/M+) harboured significantly less galectin-8 than colon cancers with localised invasion capacities (T1-2/N0/M0). The four experimental models (HCT-15, LoVo, CoLo201, and DLD-1) had more intense galectin-8 dependent staining in vitro than in vivo. Grafting the four experimental human colon cancer models onto nude mice enabled us to show that the immunohistochemical expression of galectin-8 was inversely related to tumour growth rate. In vitro, galectin-8 reduced the migration rate of only those human experimental models (HCT-15 and CoLo201) that exhibited the lowest growth rate in vivo. CONCLUSIONS: Expression of galectin-8 correlated with malignancy development, with suppressor activity, as shown by analysis of clinical samples and xenografts. In vitro, only the two models with low growth rates were sensitive to the inhibitory potential of this galectin. Future investigations in this field should involve fingerprinting of these newly detected galectins, transcending the common focus on galectins-1 and -3.

Immunohistochemical profile of galectin-8 expression in benign and malignant tumors of epithelial, mesenchymatous and adipous origins, and of the nervous system.

This study aims to investigate whether the immunohistochemical expression of galectin-8 could be used as a diagnostic marker in tumor tissues of various histogenetic origins including specimens from epithelial (n=145), mesenchymatous (n=16), adipous (n=10) and central and peripheral nervous system (n=25) tissue, and 4 mesotheliomas. Immunohistochemical reactions were carried out with a polyclonal anti-galectin-8 antibody and histological slides from tissues derived from the files of the Laboratory of Anatomopathology of University Erasmus Hospital, Brussels. Formalin-fixed paraffin-embedded tissues of 45 normal cases as well as 41 benign and 114 malignant tumors were studied. Marked decreases in immunohistochemical galectin-8 expression were observed in colon (p=0.001), pancreas (p=0.007), liver (p=0.0008), skin (p=0.002) and larynx (p=0.02) tissue when comparing malignant tissue to normal tissue and/or benign tumors. The reverse relationship was observed for breast tissue (p=0.007). No statistically significant differences (p>0.05) were detected when comparing normal tissue and/or benign to malignant tumors in lung, bladder, kidney, prostate and stomach tissue. Significant galectin-8 expression was also measured in non-epithelial tissue including tumors of the central and peripheral nervous system as well as in skeletal muscle and mesotheliomas. Immunohistochemical monitoring of galectin-8 thus reveals an organ-type-dependent regulation of expression upon malignant transformation of various tissue types of epithelial origin. This observation will prompt further studies to delineate any relationship with prognosis.

The levels of expression of galectin-3, but not of galectin-1 and galectin-8, correlate with apoptosis in human cholesteatomas.

OBJECTIVES: To investigate whether galectins 1, 3, and 8 are expressed in human cholesteatomas and whether any such expression does correlate with the level of apoptosis, which is, as we have previously shown, predictive of recurrence.7 STUDY DESIGN: The analysis of 52 cholesteatomas resected by the same surgeon by means of canal wall up and canal wall down procedures. METHODS: The immunohistochemical levels of expression of galectins 1, 3, and 8 were quantitatively determined (using computer-assisted microscopy) on conventional histological slides by means of specific anti-galectin-1, anti-galectin-3, and anti-galectin-8 antibodies. The level of apoptosis in each cholesteatoma under study had already been determined 7 by means of the in situ labeling of nuclear DNA fragmentation (Tolt-mediated dUTP nick end labeling [TUNEL] staining). RESULTS: Galectin-1 was expressed markedly in both the epithelial and the connective tissue areas of all the cholesteatomas under study. The levels of expression of galectin-3 and galectin-8 were considerably lower than that of galectin-1. The level of expression of galectin-3 correlated both highly and positively with the level of apoptosis. CONCLUSIONS: An upregulation of galectin-3 (known to have an antiapoptotic and antianoikis effect in certain model systems) expression, which is associated with pronounced apoptotic activity, could have a physiologically protective effect against the characteristically substantial apoptotic features occurring in recurrent cholesteatomas.

The levels of retinoid RARbeta receptors correlate with galectin-1, -3 and -8 expression in human cholesteatomas.

Cholesteatoma is a benign disease characterized by the presence of an unrestrained growth and the accumulation of keratin debris in the middle ear cavity. This often recurs, even when surgical resection is thought to be complete. In a previous study we showed that cholesteatomas with the highest apoptotic indices recurred more rapidly and also exhibited a high level of p53 immunopositive cells. In view of their relevance to the characterization of the cell differentiation status, the present study focuses on the expression of retinoid acid receptors (RARs) and galectins in human cholesteatomas. Retinoids control the differentiation processes in keratinocytes while galectins play strikingly modulatory roles at apoptosis and cell adhesion levels in a wide variety of tissue (embryonic, normal and neoplastic). To clarify the expression of these two protein families in human cholesteatomas we examined and quantified the levels of immunohistochemical expression of RARalpha, beta and gamma, and also galectin-1, -3 and -8 in a series of 70 human cholesteatomas. Our data show clearly that predominantly RARbeta and galectin-1 were expressed. The RARgamma concentration was significantly lower than that of the RARalpha; this was also observed for the galectin-8 concentration in comparison with the galectin-3 one. Furthermore, the level of RARbeta expression correlated highly (P=0.00001) with the level of galectin-8 expression, which also correlated significantly with the level of RARalpha and RARgamma expression. In addition, this parameter also correlated with the level of galectin-1 and galectin-3 expression. These data suggest that cholesteatomas may originate in an undifferentiated population of keratinocytes, and that a relation may exist between retinoid activity and galectins.

Evidence for stimulation of tumor proliferation in cell lines and histotypic cultures by clinically relevant low doses of the galactoside-binding mistletoe lectin, a component of proprietary extracts.

The toxic galactoside-specific lectin from mistletoe, a component of proprietary extracts with unproven efficacy in oncology, exhibits capacity to trigger enhanced secretion of proinflammatory cytokines at low doses (ng/ml or ng/kg body weight) and reductions of cell viability with increasing concentrations. To infer any tumor selectivity of this activity, cytofluorimetric and cell growth assays with a variety of established human tumor cell lines were performed. Only quantitative changes were apparent, and the toxicity against tumor cells was within the range of that of the tested fibroblast preparations from 5 donors. No indication for any tumor selectivity was observed. In kinetic studies with 8 sarcoma and 4 melanoma lines, this evidence for quantitative variability of the response in interindividual comparison was further underscored. At 50 pg lectin/ml x 10(5) cells, even a growth-stimulatory impact was noted in 5 of 12 tested cases. To mimic in vivo conditions with presence of cytokine-secreting inflammatory and stromal cells, exposure to the lectin was extended to histotypic cultures established from 30 cases of surgically removed tumor. As salient result, 5 specimens from 4 of the 8 tested tumor classes responded with a significant increase of [3H]-thymidine incorporation relative to controls during the culture period of 72 hours, when the lectin was present at a concentration in the described immunomodulatory range (1 ng/ml). A relation of this activity to the extent of the actual proliferative status of the reactive samples could not be delineated. Therefore, a non-negligible percentage of the established tumor cell lines (e.g., 3 from 8 sarcoma lines) can be markedly stimulated by the lectin at a very low dose and with dependence on the cell type. Furthermore, the feasibility to elicit a significant growth enhancement is likewise documented for human tumor explants in 16.6% of the examined cases. In view of the uncontrolled application of lectin-containing extracts in alternative/complementary medicine, the presented results on unquestionably adverse lectin-dependent effects in two culture systems call for rigorous examination of the clinical safety of this unconventional, scientifically entirely experimental treatment modality.

Galectin-1 is highly expressed in human gliomas with relevance for modulation of invasion of tumor astrocytes into the brain parenchyma.

Protein (lectin)-carbohydrate interaction is supposed to be relevant for tumor cell behavior. The aims of the present work are to investigate whether galectin-1 modulates migration/invasion features in human gliomas in vitro, whether it can be detected in human gliomas immunohistochemically, and whether its expression is attributable to certain glioma subgroups with respect to invasion and prognosis. For this purpose, we quantitatively determined (by computer-assisted microscopy) the immunohistochemical expression of galectin-1 in 220 gliomas, including 151 astrocytic, 38 oligodendroglial, and 31 ependymal tumors obtained from surgical resection. We also xenografted three human glioblastoma cell lines (the H4, U87, and U373 models) into the brains of nude mice in order to characterize the in vivo galectin-1 expression pattern in relation to tumor invasion of the normal brain parenchyma. In addition, we characterized the role in vitro of galectin-1 in U373 tumor astrocyte migration and kinetics. Our data reveal expression of galectin-1 in all human glioma types with no striking differences between astrocytic, oligodendroglial, and ependymal tumors. The level of galectin-1 expression correlated with the grade in the group of astrocytic tumors only. Furthermore, immunopositivity of high-grade astrocytic tumors from patients with short-term survival periods was stronger than that of tumors from patients with long-term survivals. In human glioblastoma xenografts, galectin-1 was preferentially expressed in the more invasive parts of these xenografts. In vitro experiments revealed that galectin-1 stimulates migration of U373 astrocytes.

Distinct differences in binding capacity to saccharide epitopes in supratentorial pilocytic astrocytomas, astrocytomas, anaplastic astrocytomas, and glioblastomas.

We monitored the expression of glycan-binding sites on a panel of 10 biotinylated neoglycoconjugates by means of quantitative computer-assisted microscopy to further study the molecular mechanisms in the extensive infiltration of the surrounding brain parenchyma by most astrocytic tumors. Three distinct histological compartments were analyzed for each of the 108 astrocytic tumors (15 pilocytic astrocytomas (WHO grade I), 25 astrocytomas (WHO grade II), 30 anaplastic astrocytomas (WHO grade III), and 38 glioblastomas (WHO grade IV) included in our series. These compartments were tumors (nonperivascular tumor astrocytes), perivascular tumor astrocytes, and blood vessel walls. Clear differences were observed between the pilocytic and the diffuse astrocytic tumors. Furthermore, malignant progression in the latter category was paralleled by a decrease in cells' ability to bind distinct sugar epitopes, especially the D-GalNAc(alpha1-3)-D-GalNAc-beta1-R determinant of the Forssman pentasaccharide in tumors, the alpha-L-fucose in perivascular tumor areas, and the beta-D-glucose in tumor vessel walls. Markedly, the level of binding site expression for alpha-D-mannose decreased in the tumors, the perivascular tumor areas, and the vessel walls. These glycohistochemical results imply the functional relevance of protein-carbohydrate interactions in this tumor system.

Mucins and secreted factors.

Mucin is a large glycoprotein (M up to 4-6.10(6)) with a high content of serine, threonine, and proline residues and numerous O-linked saccharides, often occurring in clusters on the polypeptide. Nine mucin genes exist in humans that encode an apomucin highly modified by O-glycans in forming epithelial mucins. These are variably expressed by epithelial cells of the gastrointestinal, tracheobronchial and reproductive tracts. They may be found either membrane-associated or secreted. As might be imagined from their ubiquitous and complex nature, the biological roles of glycans are quite varied. Protection against infection is an important biological role. Alterations in their carbohydrate moiety have been reported in airway and salivary mucins secreted by patients suffering of cystic fibrosis (CF). Moreover, changes in expression of glycans are also often reported in the setting of transformation and progression to malignancy. Due to the key roles played by glycans of glycoconjugates in both physiological and pathological events, glycobiology and carbohydrate chemistry have become of increasing importance in modern biotechnology.

Labeled neoglycoproteins and human lectins as diagnostic and potential functional markers in salivary glands of patients with Sjögren's syndrome.

OBJECTIVE: The profile of glycans and their recognition by endogenous receptors (lectins) are increasingly attributed to disease process. Monitoring this can provide information on the pathogenesis of Sjögren's syndrome (SS). Commonly, plant lectins are employed for phenomenological glycan mapping. To go beyond this approach restricted to binding of exogenous probes, new markers measure ligand properties of glycans to human (not plant) lectins and the presence of sugar receptors completing a protein-carbohydrate recognition system. Carrier-immobilized sugar epitopes (neoglycoproteins) and purified human lectins establish this innovative panel. METHODS: The host defence molecules mannan binding lectin, serum amyloid P component, and the macrophage migration inhibitory factor-binding sarcolectin, selected for their involvement in cell destructive mechanisms, were purified and labeled. The plant lectins SNA and MAA were employed to monitor regulation of potential ligand sites for I-type lectins and galectins. Asialofetuin was tested as a "pan-galectin selective" probe. The specific binding characteristics were determined by quantitative morphometry and statistical analysis. RESULTS: Diagnostic information emerged from this analysis. The percentage of stained tissue area was significantly different between SS and control specimens after processing with GlcNAc and Man-bearing neoglycoproteins and the 2 tested serum lectins. For separation of cases of primary and secondary SS, the staining intensity with the asialoglycoprotein, sarcolectin, and the exogenous alpha2,6-sialylated glycan-binding lectin SNA was statistically significant. CONCLUSION: Saccharide-presenting probes to measure the cellular capacity to bind glycan epitopes and human lectins as sensors for endogenous binding sites have proven to be useful as diagnostic tools. We suggest the differences we observed reflect aberrations from the normal cellular homeostasis with relevance for the pathogenesis of SS and its manifestation as a primary or secondary syndrome.

Correlation of galectin-3/galectin-3-binding sites with low differentiation status in head and neck squamous cell carcinomas.

The accurate determination of levels of differentiation is of prognostic value in human head and neck squamous cell carcinomas (HNSCCs). Because the deliberate selection of biochemical determinants accompanying certain stages of differentiation can refine the predictive power of histochemical assessments, the application of the quantitative evaluation of staining distribution and intensity by computer-assisted microscopy is one prerequisite to potential improvements. We used 2 innovative approaches with peanut agglutinin based on encouraging results with respect to common lectin-histochemistry. First, we used a custom-made neoglycoprotein to monitor the presence of Thomsen-Friedenreich (T) antigen-binding sites. Second, we measured the presence of 2 galectins immunohistochemically and, at the same time, measured lectin-histochemically the presence of accessible ligands for the endogenous lectins. We also monitored the presence of calcyclin, a protein with relevance to cell cycle progression or exocytosis. With 61 cases of HNSCC as their basis, including 31 oral, 20 laryngeal, and 10 hypopharyngeal lesions, the data show that the main modifications observed in connection with a loss of differentiation are related to a modification in the levels of both galectin-3/galectin-3-binding site and T-antigen/T-antigen-binding site expressions. The data obtained also suggest that galectin-3 could act as an acceptor site for the T antigen. Because the level of differentiation is known to be indicative of the recurrence rate in HNSCCs and our data clearly indicate that galectin-3 and the T antigen (and their respective binding sites) are involved in dedifferentiation processes, further investigation is warranted into the roles of galectins in HNSCC tumor progression and recurrence analysis.

Glycohistochemical characteristics of nasal polyps from patients with and without cystic fibrosis.

OBJECTIVE: To investigate whether cystic fibrosis (CF)-related nasal polyps exhibit significantly distinct glycohistochemical characteristics when compared with single vs massive nasal polyps obtained from patients without CF. DESIGN: Glycohistochemical characteristics were identified by means of 8 histochemical probes, including 5 plant lectins (peanut, gorse seed, wheat germ, Maackia amurensis, and Sambucus nigra agglutinins), 2 animal lectins (14- and 16-kd galectins), and 1 neoglycoprotein (exposing the Thomsen-Friedenreich antigen). The binding of the 8 glycohistochemical markers was determined by means of computer-assisted microscopy. For each probe, 3 quantitative parameters were computed: the labeling index, which describes the percentage of tissue area specifically stained by a given marker; the mean optical density, which reflects the staining intensity; and the concentrational heterogeneity, which characterizes the level of heterogeneity of the staining intensity. SUBJECTS: A series of 61 nasal mucosa specimens was analyzed, including 6 normal cases, 23 single and 18 massive polyposis cases without CF, and 14 nasal polyps associated with CF. RESULTS: Normal and polyposal nasal mucosa differed in terms of the amounts and linkage types of sialic acids (revealed by the wheat germ, M amurensis, and S nigra agglutinins) rather than the characteristics of galactoside expression (monitored with the peanut agglutinin and 2 animal galectins). In contrast, nasal polyps markedly differed between patients with and without CF with respect to galactoside expression (revealed by the peanut agglutinin and the 14-kd galectin) and the display of binding site(s) for the neoglycoprotein. CONCLUSION: Normal and polyposal nasal mucosa differ essentially in sialic acid presentation, while nasal polyps from patients with CF have a higher level of various lectin-reactive galactoside residues than nasal polyps from those without CF.

Characterization of ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also of the expression of calcyclin in thyroid lesions.

The purpose of this study was to characterize ligands for galectins, natural galactoside-binding immunoglobulin G subfractions and sarcolectin and also the expression of calcyclin in various benign and malignant thyroid lesions. The extent of the binding of eight glycochemical probes was quantitatively assessed using computer-assisted microscopy on 76 thyroid lesions including 10 not-otherwise-specified multinodular goiters (S_MNG), 11 multinodular goiters with adenomatous hyperplasia (AH_MNG), 8 normomacrovesicular (NM_ADE) and 12 microvesicular (MIC_ADE) adenomas, and 9 papillary (P_CAR), 10 follicular variants of papillary (FvarP_CAR), 7 follicular (F_CAR) and 9 anaplastic (A_CAR) carcinomas. The 8 histochemical probes included 5 animal lectins (including galectins and sarcolectin), 1 polyclonal antibody (raised against calcyclin) and 2 immunoglobulin G subfractions from human serum with selectivity to alpha- and beta-galactosyl residues. The results show that multinodular goiters with adenomatous hyperplasia exhibited histochemical characteristics intermediate to those of normal multinodular goiters and microvesicular adenomas. Normomacrovesicular adenomas behaved very distinctly from microvesicular ones. Microvesicular adenomas were more closely related to differentiated thyroid carcinomas than any other type of benign thyroid lesions of epithelial origin. Papillary and follicular carcinomas seemed to represent the two extremes of the same biological entity with the follicular variant of the papillary carcinoma serving as a biological link between these two extremes. Anaplastic carcinomas behaved in a significantly different manner when compared to the differentiated forms of thyroid carcinomas. The results suggest that the patterns of expression of the glycoconjugates investigated in the present study may constitute useful tools for characterizing lesions in the human thyroid.

Improving accuracy in the grading of renal cell carcinoma by combining the quantitative description of chromatin pattern with the quantitative determination of cell kinetic parameters.

The determination of grade and stage in renal cell carcinomas (RCCs) often fails to predict the actual clinical outcome for individual patients. The aim of the present work was to investigate whether it is possible to significantly improve the prognostic accuracy of the grading system by using the combination of two independent computer-assisted microscopy techniques. The first technique relates to the quantitative description of morphonuclear and nuclear DNA content features by means of the image analysis of Feulgen-stained cell nuclei, and the second quantitatively characterizes tumor growth by means of different cell kinetic parameters. These parameters consist of a duplication of a time-related parameter determined by means of the technique of using silver-stained proteins in interphase nucleolar organizer regions (AgNOR), a proliferation index determined by means of MIB-1 immunohistochemistry, and an apoptotic index determined by means of the terminal dUTP nick end labeling technique. The prognostic value of these quantitative features was investigated in a series of 60 RCCs. The quantitative analysis of Feulgen-stained nuclei made it possible to identify subgroups of patients with significantly different prognoses in both grade II and grade III RCCs. We labeled the RCCs associated with the most favorable prognoses as grade II- and III- and those with the least favorable ones as grade II+ and III+. The two most important kinetic variables to identify patients with different clinical outcomes were the MIB-1 index and the mean AgNOR area in the MIB-1-positive cells. Three significantly different survival curves were obtained for the 53 grade II and III RCC patients. Our results show that conventional RCC grading can be significantly improved by the quantitative analysis of Feulgen-stained nuclei, by cell kinetic parameter determination, and, more importantly, by combining the proliferation index with the mean AgNOR area parameter.

The levels of expression of galectin-1, galectin-3, and the Thomsen-Friedenreich antigen and their binding sites decrease as clinical aggressiveness increases in head and neck cancers.

BACKGROUND: The aim of this study was to investigate whether an increase in malignancy level is accompanied by significant modifications of the expression of galectin-1, galectin-3, and Thomsen-Friedenreich antigen (T antigen) as well as the expression of binding sites for these three markers in head and neck squamous cell carcinomas (HNSCCs). METHODS: Immunohistochemical and glycohistochemical staining reactions were carried out with antibodies, labeled lectins, and a custom-made neoglycoprotein on the basis of histologic slides from a retrospective series of 40 normal and 75 HNSCC formalin fixed, paraffin embedded tissues, and were quantitatively described with the aid of computer-assisted microscopy. RESULTS: Whatever the histologic type, the epithelial tissues in HNSCC exhibited very significantly (P < 0.01 to P < 0. 0001) lower amounts of galectin-1, galectin-3, and T antigen and their respective binding sites than their corresponding normal counterparts. The tumors of the larynx differed very significantly (P < 0.0001 to P < 0.000001) from all the other tumor types. A loss of differentiation in the HNSCCs is accompanied first by the loss of expression of galectin-3 and galectin-3-reactive sites and then by that of the T antigen and its binding site(s). The opposite feature was observed when the parameters associated with the TNM classification were taken into account. The negative lymph node HNSCCs could be distinguished (P = 0.02) from the positive lymph node HNSCCs on the basis of a loss of galectin-3 expression. The modifications occurring in the extent of expression of galectin-1 and galectin-1-reactive sites were relatively marginal in comparison with those observed for galectin-3-dependent and T- antigen-dependent staining. CONCLUSIONS: The decrease in the extent of expression of galectin-3 and galectin-3-reactive sites, T antigen and T antigen-binding sites, and, to a lesser extent, galectin-1 and galectin-1-reactive sites correlates significantly with an increasing level of clinically detectable HNSCC aggressiveness.

Sialic acid residues in the labial salivary glands from Sjögren's syndrome patients.

OBJECTIVE: To investigate the composition and expression of sialic acid in the labial salivary glands (LSG) in Sjögren's syndrome (SS). METHODS: LSG of 19 patients with primary SS (n = 11) or secondary SS (n = 8) were studied. Specimens from 7 healthy women served as controls. Computer-assisted microscopy was employed to quantitatively determine the percentage of positive structures, the staining intensity and the heterogeneity for the 4 biotinylated plant lectins Tritricum vulgaris L. (WGA), Maackia amurensis (MAA), Sambucus nigra (SNA) and Canavalia ensiformis L. (Con A). RESULTS: In the acini there was a significant decrease in the staining heterogeneity of WGA in SS compared to controls; the same was observed with respect to MAA staining in the connective tissue and extralobular ducts. In the intralobular ducts, primary SS differed from normal and secondary SS mainly in terms of a decrease in the percentage of positively labeled MAA tissue. In addition, Con A stained acinar cells were significantly more numerous in secondary SS compared with primary SS. CONCLUSION: Differences in the degree of glycoconjugate sialylation were found in SS labial salivary glands, and may play a role in the disease process.

Determination of the levels of expression of sarcolectin and calcyclin and of the percentages of apoptotic but not proliferating cells to enable distinction between recurrent and nonrecurrent cholesteatomas.

OBJECTIVES: To investigate in a series of cholesteatomas 1. whether subgroups of cholesteatomas with specific proliferative/apoptotic features exhibit distinct differentiation markers and 2. whether these different subgroups identified at the biological level relate to specific groups of clinically identified cholesteatomas. STUDY DESIGN: Analysis of 55 cholesteatomas resected by the same surgeon, by means of canal wall up and canal wall down surgical procedures. METHODS: Two differentiation markers were used: biotinylated sarcolectin (to identify sarcolectin-binding sites) and a monoclonal antibody directed against calcyclin (which is the S100A6 protein). The growth pattern in cholesteatomas was characterized at three distinct levels: 1. the cell proliferation level determined by means of the MIB-1 antibody, which enables the Ki-67 cell-cycle-related antigen to be identified on archival material; 2. the apoptosis level determined by means of the in situ labeling of nuclear DNA fragmentation (TUNEL staining); and 3. the p53 tumor suppressor gene-related product determined by means of p53 immunohistochemistry. RESULTS: The cholesteatomas that exhibited the highest proportion of apoptotic cells were those which exhibited the highest level of sarcolectin-binding sites (i.e., sialic acids). In contrast, the cholesteatomas exhibiting the lowest level of both proliferation and apoptosis showed the highest level of calcyclin. Recurrent cholesteatomas can be identified from nonrecurrent ones on the basis of three features, namely, the level of apoptotic cells, the way in which the apoptotic cells are distributed (i.e., homogeneously vs. heterogeneously), and the percentage of calcyclin-positive cells. CONCLUSIONS: The present data emphasize the existence of distinct subgroups of cholesteatomas identifiable at both cell kinetic and differentiation levels. Some of the biological variables used here to identify distinct biological subgroups of cholesteatomas in turn enabled some biological variables to be identified, so making it possible to classify the cholesteatomas in terms of recurrence versus nonrecurrence.

Grading dysplasia in colorectal adenomas by means of the quantitative binding pattern determination of Arachis hypogaea, Dolichos biflorus, Amaranthus caudatus, Maackia amurensis, and Sambucus nigra agglutinins.

The current study deals with the setting up of a new tool that enables the benign versus the malignant nature of colorectal adenomas to be determined accurately. The 2 objectives are to determine (1) whether adenomas should, or should not, be included in a 2- or a 3-tier grading system, and (2) whether severe dysplasias and carcinomas in situ share common or different biological characteristics. The levels of expression of different types of glycoconjugates were characterized in a series of 166 colorectal specimens, including 14 normal, 90 dysplastic, and 62 cancerous cases. The glycoconjugate expressions were demonstrated for 5 lectins, namely, Arachis hypogaea (PNA), Dolichos biflorus (DBA), Amaranthus caudatus (ACA), Maackia amurensis (MAA) and Sambucus nigra (SNA). The glycoconjugates demonstrated by these 5 lectins belong to the family of the Thomsen-Friedenreich antigens. The binding patterns of the 5 lectins were quantitatively determined by means of computer-assisted microscopy. The quantitative data were submitted to discriminant analyses. Our results show that the specific glycochemical staining patterns could be identified unambiguously and without misclassification between benign (normal and low dysplasia) and malignant (ie, either as moderate/severe dysplasia, carcinoma in situ, or cancer) cases. The data also strongly suggested that (1) dysplasias seem to be distinguishable in 2 instead of 3 groups, that is, low versus moderate/severe (high); and (2) moderate/severe dysplasias are biologically distinct from carcinomas in situ. The methodology developed can be applied directly in routine diagnosis to identify moderate/severe dysplasia specimens already exhibiting features common to carcinomas, and which therefore should be treated consistently in view of the fact that our data strongly suggest that most moderate/severe dysplasias are still benign, whereas carcinomas in situ are real carcinomatous lesions.

Galectin-3 and galectin-3-binding site expression in human adult astrocytic tumours and related angiogenesis.

Using computer-assisted microscopy, the present work aimed to quantitatively characterize the level of the histochemically detectable expression of galectin-3 and galectin-3-binding sites in sections of a series of 84 astrocytic tumours (including 22 grade II, 21 grade III and 41 grade IV specimens) and seven non-tumoural specimens used as controls. The presence of galectin-3 and reactive sites for this lectin were monitored by means of a specific polyclonal anti-galectin-3 antibody (aGal3) and biotinylated galectin-3 (Gal3), respectively. The pattern of expression of galectin-3-binding sites is compared to the pattern of expression of laminin (a potential galectin-3 ligand) revealed using a biotinylated anti-laminin antibody (aLam). Three variables quantitatively characterizing histochemical staining reactions were evaluated by means of computer-assisted microscopy for each of the 3 probes under study (aGal3, Gal3 and aLam). The labelling index (LI) is the percentage of tissue area specifically stained by a histochemical probe. The mean optical density (MOD) denotes staining intensity. The concentration heterogeneity (CH) feature expresses the concentrational spread of individual fields. The data obtained in the present study show that: (i) white matter of a non-tumoural brain expresses galectin-3 (and also galectin-3-binding sites); (ii) the level of galectin-3 expression significantly decreases in the majority of tumour astrocytes from low to high grade astrocytic tumours; while (iii) some tumour cell clones expressing high amounts of galectin-3 emerged with increasing levels of malignancy; and (iv) the level of accessible galectin-3-binding sites was apparently not heavily modified in the course of malignancy progression. In conclusion, the results obtained in the present study show that human astrocytic tumours are very heterogenous in their galectin-3 levels of expression. If high levels of galectin-3 determine the invasiveness potential of a tumour cell, then within a heterogenous tumour the presence of even a small, but actively proliferating number of tumour cell clones expressing high levels of galectin-3 can be expected to lead to tumour invasiveness.

Galectin-1 and galectin-3 binding pattern expression in renal cell carcinomas.

We studied 2 families of molecules whose role remains uncharacterized or obscure in the progress of renal cell carcinoma (RCC): galectins, a major class of glycoproteins, and the Thomsen-Friedenreich (T) antigen. We characterized the level of expression of galectin-1 and galectin-3 and their respective binding sites in a series of 74 RCCs. We also characterized the level of expression of laminin, a natural ligand for galectins. Finally, we characterized the level of T antigen expression and the T antigen binding sites. All levels of expression were quantitatively determined by using computer-assisted microscopy on immunohistochemically or glycohistochemically stained slides. A small concentration of galectin-1 binding sites or a large concentration heterogeneity of galectin-3 can be associated with unfavorable prognoses for patients with grade II or III RCCs. In contrast, T antigen and T antigen binding sites revealed no change across the 2 RCC groups that exhibited different clinical outcomes. We established discriminant scores that permitted a clear distinction between the 2 RCC groups analyzed. Modifications to the expression of galectin-1 and galectin-3, but not of T antigen, parallel an increase in RCC aggressiveness. Galectins represent a family of molecules with a meaningful role in RCC progression.

Quantitative glycohistochemical characterization of normal nasal mucosa, and of single as opposed to massive nasal polyps.

A series of 41 nasal polyps (23 single and 18 massive) and 6 normal nasal mucosa specimens was glycohistochemically investigated. Five plant lectins were used, i.e., the peanut agglutinin (PNA), the wheat germ agglutinin (WGA), the gorse seed agglutinin (UEA-I), the Maackia amurensis agglutinin (MAA), and the elderberry bark agglutinin (SNA). A neoglycoconjugate and 2 animal lectins (CL-14 and CL-16) were also used. Three quantitative features were calculated by means of computer-assisted microscopy: the percentage of tissue area specifically stained by the histochemical probe, the staining intensity, and the heterogeneity level of the staining distribution. The results show that with respect to sialic acid-glycoprotein binding characteristics as determined by SNA, MAA, and WGA probes, the normal nasal mucosa differed markedly (p<.00001) from the polyposal one. The single nasal polyps exhibited glycohistochemical characteristics that differed markedly (p = .0004) from those exhibited by the massive ones. These differences related mainly to the UEA-I, PNA, and the Thomsen-Friedenreich antigen-exposing neoglycoprotein binding characteristics. In conclusion, the present study shows unambiguously that polyposal mucosa, whether of the single or the massive type, exhibits markedly distinct glycohistochemical characteristics when compared to normal nasal mucosa, and that single nasal polyps also differ markedly from massive ones.