Factors influencing long-term survival after cytoreductive surgery and hyperthermic intraperitoneal chemotherapy for pseudomyxoma peritonei originating from appendiceal neoplasms.

Background: Pseudomyxoma peritonei (PMP) is a rare disease, most commonly of appendiceal origin. Treatment consists of cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (CRS-HIPEC). The aim of this study was to identify prognostic factors for recurrence and survival. Methods: This was an observational study using a prospectively designed database containing consecutive patients with PMP originating from the appendix, undergoing CRS-HIPEC at a tertiary referral centre between 1996 and 2015. Histopathological slides were reassessed. Cox regression was used for multivariable analyses. Results: Of 225 patients identified, 36 (16·0 per cent) were diagnosed with acellular mucin, 149 (66·2 per cent) had disseminated peritoneal adenomucinosis (DPAM) and 40 (17·8 per cent) had peritoneal mucinous carcinomatosis (PMCA). The 5-year overall survival (OS) rates were 93, 69·8 and 55 per cent respectively. Recurrence was observed in 120 patients (53·3 per cent), 39 of whom (17·3 per cent) were treated with a second CRS-HIPEC procedure. Factors independently associated with poor disease-free survival were six or seven affected regions (hazard ratio (HR) 6·01, 95 per cent c.i. 2·04 to 17·73), incomplete cytoreduction (R2a resection: HR 1·67, 1·05 to 2·65; R2b resection: HR 2·00, 1·07 to 3·73), and more than threefold raised carcinoembryonic antigen (CEA) and/or carbohydrate antigen (CA) 19-9 level (HR 2·31, 1·30 to 4·11). Factors independently associated with poorer OS were male sex (HR 1·74, 1·09 to 2·77), incomplete cytoreduction (R2a resection: HR 1·87, 1·14 to 3·08; R2b resection: HR 2·28, 1·19 to 4·34), and more than threefold raised CEA and/or CA19-9 level (HR 2·89, 1·36 to 6·16). Conclusion: CEA and CA19-9 levels raised more than threefold above the upper limit identify patients with PMP of appendiceal origin and poorer survival.

Plant Lectins Targeting O-Glycans at the Cell Surface as Tools for Cancer Diagnosis, Prognosis and Therapy.

Aberrant O-glycans expressed at the surface of cancer cells consist of membrane-tethered glycoproteins (T and Tn antigens) and glycolipids (Lewis a, Lewis x and Forssman antigens). All of these O-glycans have been identified as glyco-markers of interest for the diagnosis and the prognosis of cancer diseases. These epitopes are specifically detected using T/Tn-specific lectins isolated from various plants such as jacalin from Artocarpus integrifola, and fungi such as the Agaricus bisporus lectin. These lectins accommodate T/Tn antigens at the monosaccharide-binding site; residues located in the surrounding extended binding-site of the lectins often participate in the binding of more extended epitopes. Depending on the shape and size of the extended carbohydrate-binding site, their fine sugar-binding specificity towards complex O-glycans readily differs from one lectin to another, resulting in a great diversity in their sugar-recognition capacity. T/Tn-specific lectins have been extensively used for the histochemical detection of cancer cells in biopsies and for the follow up of the cancer progression and evolution. T/Tn-specific lectins also induce a caspase-dependent apoptosis in cancer cells, often associated with a more or less severe inhibition of proliferation. Moreover, they provide another potential source of molecules adapted to the building of photosensitizer-conjugates allowing a specific targeting to cancer cells, for the photodynamic treatment of tumors.

Structural analysis and unique molecular recognition properties of a Bauhinia forficata lectin that inhibits cancer cell growth.

Lectins have been used at length for basic research and clinical applications. New insights into the molecular recognition properties enhance our basic understanding of carbohydrate-protein interactions and aid in the design/development of new lectins. In this study, we used a combination of cell-based assays, glycan microarrays, and X-ray crystallography to evaluate the structure and function of the recombinant Bauhinia forficata lectin (BfL). The lectin was shown to be cytostatic for several cancer cell lines included in the NCI-60 panel; in particular, it inhibited growth of melanoma cancer cells (LOX IMVI) by over 95%. BfL is dimeric in solution and highly specific for binding of oligosaccharides and glycopeptides with terminal N-acetylgalactosamine (GalNAc). BfL was found to have especially strong binding (apparent Kd  = 0.5-1.0 nm) to the tumor-associated Tn antigen. High-resolution crystal structures were determined for the ligand-free lectin, as well as for its complexes with three Tn glycopeptides, globotetraose, and the blood group A antigen. Extensive analysis of the eight crystal structures and comparison to structures of related lectins revealed several unique features of GalNAc recognition. Of special note, the carboxylate group of Glu126, lining the glycan-binding pocket, forms H-bonds with both the N-acetyl of GalNAc and the peptide amido group of Tn antigens. Stabilization provided by Glu126 is described here for the first time for any GalNAc-specific lectin. Taken together, the results provide new insights into the molecular recognition of carbohydrates and provide a structural understanding that will enable rational engineering of BfL for a variety of applications. DATABASE: Structural data are available in the PDB under the accession numbers 5T50, 5T52, 5T55, 5T54, 5T5L, 5T5J, 5T5P, and 5T5O.

Using CRISPR/Cas9-Mediated GLA Gene Knockout as an In Vitro Drug Screening Model for Fabry Disease.

The CRISPR/Cas9 Genome-editing system has revealed promising potential for generating gene mutation, deletion, and correction in human cells. Application of this powerful tool in Fabry disease (FD), however, still needs to be explored. Enzyme replacement therapy (ERT), a regular administration of recombinant human α Gal A (rhα-GLA), is a currently available and effective treatment to clear the accumulated Gb3 in FD patients. However, the short half-life of rhα-GLA in human body limits its application. Moreover, lack of an appropriate in vitro disease model restricted the high-throughput screening of drugs for improving ERT efficacy. Therefore, it is worth establishing a large-expanded in vitro FD model for screening potential candidates, which can enhance and prolong ERT potency. Using CRISPR/Cas9-mediated gene knockout of GLA in HEK-293T cells, we generated GLA-null cells to investigate rhα-GLA cellular pharmacokinetics. The half-life of administrated rhα-GLA was around 24 h in GLA-null cells; co-administration of proteasome inhibitor MG132 and rhα-GLA significantly restored the GLA enzyme activity by two-fold compared with rhα-GLA alone. Furthermore, co-treatment of rhα-GLA/MG132 in patient-derived fibroblasts increased Gb3 clearance by 30%, compared with rhα-GLA treatment alone. Collectively, the CRISPR/Cas9-mediated GLA-knockout HEK-293T cells provide an in vitro FD model for evaluating the intracellular pharmacokinetics of the rhα-GLA as well as for screening candidates to prolong rhα-GLA potency. Using this model, we demonstrated that MG132 prolongs rhα-GLA half-life and enhanced Gb3 clearance, shedding light on the direction of enhancing ERT efficacy in FD treatment.

O-glycosylation of α-1-acid glycoprotein of human milk is lactation stage related.

BACKGROUND: Human milk provides a multitude of glycoproteins, including highly glycosylated α-1-acid glycoprotein (AGP), which elicits anti-inflammatory and immunomodulatory properties. The milk AGP glycoforms may provide the breastfed infant with a wide range of biological benefits. Here, we analyzed the reactivity of O-linked sugar-specific lectins with human milk AGP over the process of lactation and compared the results with those of the lactating mother's plasma. MATERIALS AND METHODS: Relative amounts of human skim milk AGP O-glycans were analyzed in early colostrum, colostrum, and transitional and mature milk samples of 127 healthy mothers by lectin-AGP enzyme-linked immunosorbent assay using sialyl T (sialyl-α2,3/α2,6 Galß1,3GalNAc-), asialyl T (Galß1,3GalNAc-), and Tn (GalNAc-) antigen-specific biotinylated Artocarpus integrifolia (Jacalin), Arachis hypogaea (PNA), and Vicia villosa (VVA) lectins, respectively. RESULTS: Milk AGP elicited high expression of Jacalin- and PNA-reactive glycotopes and low expression of VVA-reactive glycotopes, which were absent on plasma AGP of lactating mothers and healthy individuals. The expression of sialyl, asialyl T, and Tn glycotopes of human milk AGP was lactation stage related. The relative amount of Jacalin-reactive AGP glycotope was highest in the colostrum samples and then decreased starting from Day 8 of lactation. In contrast, an increase of the relative amount of PNA-reactive glycotope with milk maturation was observed. The relative amount of VVA-reactive glycotope remained almost constant over the development of lactation. CONCLUSIONS: Milk AGP differs from mother's plasma AGP by the presence of O-linked sialylated and asialylated T as well as Tn antigens. The variation of the expression of sialylated and asialylated T and Tn antigens on AGP is associated with milk maturation.

A Tn antigen binding lectin from Myrsine coriacea displays toxicity in human cancer cell lines.

The Tn antigen (GalNAc-O-Ser/Thr) is one of the most specific human cancer-associated structures. In the present study we characterize the biochemical and functional properties of the Myrsine coriacea lectin (McL). We show that McL is an unusual high molecular weight highly glycosylated protein, which displays a strong Tn binding activity. The lectin exhibits in vitro inhibition of proliferation in the six cancer cell lines evaluated, in a dose-dependent manner (the strongest activity being against HT-29 and HeLa cells), whereas it does not exhibit toxicity against normal lymphocytes. McL could be exploited in the design of potential new tools for the diagnosis or treatment of cancer.

Isolation and characterisation of a Salvia bogotensis seed lectin specific for the Tn antigen.

A lectin was isolated and characterised from Salvia bogotensis seeds. Removal of the abundant pigments and polysaccharides, which are present in seeds, was an essential step in its purification. Several procedures were assayed and the best suited, including Pectinex treatment, DEAE-cellulose and affinity chromatography, led to a protein being obtained amounting to 18-20mg/100g seeds having high specific agglutination activity (SAA). The lectin specifically agglutinated human Tn erythrocytes and was inhibited by 37mM GalNAc, 0.019mM ovine submaxillary mucin (OSM) or 0.008mM asialo bovine submaxillary mucin (aBSM). Enzyme-linked lectinosorbent assay (ELLSA) revealed strong binding to aOSM and aBSM, corroborating Tn specificity, whereas no binding to fetuin or asialo fetuin was observed. The lectin's monomer MW (38,702Da), amino acid composition, pI, carbohydrate content, deglycosylated form MW, thermal stability and Ca(2+) and Mn(2+) requirements were determined. Evidence of the existence of two glycoforms was obtained. The lectin's specificity and high affinity for the Tn antigen, commonly found in tumour cells, makes this protein a useful tool for immunohistochemical and cellular studies.

Lectinochemical studies on the glyco-recognition factors of a Tn (GalNAcalpha1-->Ser/Thr) specific lectin isolated from the seeds of Salvia sclarea.

The lectin extracted from the seeds of Salvia sclarea (SSL) recognizes the Tn antigen (GalNAc alpha1-->Ser/Thr) expressed in certain human carcinomas. In previous studies, knowledge of the binding properties of SSL was restricted to GalNAcalpha1--> related oligosaccharides and glycopeptides. Thus, the requirements of functional groups in monosaccharide and high-density polyvalent carbohydrate structural units for SSL binding and an updated affinity profile were further evaluated by enzyme-linked lectinosorbent (ELLSA) and inhibition assays. Among the glycoproteins (gps) tested for interaction, a high density of exposed Tn-containing glycoproteins such as in the armadillo salivary Tn glycoprotein and asialo ovine salivary glycoprotein reacted best with SSL. When the gps were tested for inhibition of SSL binding, which was expressed as 50% nanogram inhibition, the high density polyvalent Tn present in macromolecules was the most potent inhibitor. Among the monosaccharide and carbohydrate structural units studied, which were expressed as nanomole inhibition, GalNAc alpha1-->3GalNAc beta1-->3Gal alpha1-->4Gal beta1-->4Glc (Fp), GalNAc alpha1-->3Gal beta1-->4Glc (A(L)), GalNAc alpha1-->3GalNAc beta1-->Me (F beta), GalNAc alpha1-->3GalNAc alpha1-->Me (F alpha) and GalNAc alpha1--> Ser/Thr (Tn) were the most active ligands, being 2.5-5.0 x 10(3) and 1.25-2.5 times more active than Gal and GalNAc, respectively. From the results, it is suggested that the combining site of SSL is a shallow groove type, recognizing the monosaccharide of GalNAc as the major binding site or Tn up to the Forssman pentasaccharide (Fp). It can be concluded that the three critical factors for SSL binding are the -NH CH(3)CO at carbon-2 in Gal, the configuration of carbon-3 in GalNAc, and the polyvalent Tn (GalNAc alpha1-->Ser/Thr) present in macromolecules. These results should assist in understanding the glyco-recognition factors involved in carbohydrate-lectin interactions in biological processes. The effect of the polyvalent F alpha, F beta and GalNAc beta1-->3Gal alpha1--> (P alpha) glycotopes on binding should be examined. However, this is hampered by the lack of availability of suitable reagents.

Tumor-associated CD75s gangliosides and CD75s-bearing glycoproteins with Neu5Acalpha2-6Galbeta1-4GlcNAc-residues are receptors for the anticancer drug rViscumin.

The anticancer drug rViscumin, currently under clinical development, has been shown in previous studies to be a sialic acid specific ribosome inactivating protein (RIP). Comparative binding assays with the CD75s-specific monoclonal antibodies HB6 and J3-89 revealed rViscumin to be a CD75s-specific RIP due to identical binding characteristics toward CD75s gangliosides. The receptor gangliosides are IV6nLc4Cer, VI6nLc6Cer, and the newly characterized ganglioside VIII6nLc8Cer, all three carrying the Neu5Acalpha2-6Galbeta1-4GlcNAc motif. To elucidate the clinical potential of the rViscumin targets, CD75s gangliosides were determined in several randomly collected gastrointestinal tumors. The majority of the tumors showed an enhanced expression of CD75s gangliosides compared with the unaffected tissues. The rViscumin binding specificity was further investigated with reference glycoproteins carrying sialylated and desialylated type II N-glycans. Comparative Western blots of rViscumin and ricin, an rViscumin homologous but galactoside-specific RIP, revealed specific recognition of type II N-glycans with CD75s determinants by rViscumin, whereas ricin failed to react with terminally sialylated oligosaccharides such as CD75s motifs and others. This strict binding specificity of rViscumin and the increased expression of CD75s gangliosides in various tumors suggest this anticancer drug as a promising candidate for an individualised adjuvant therapy of human tumors.

Salivary CA130 with and without unilateral autonomic parotid denervation of rats fed different diets.

BACKGROUND: Tumor markers such as CA130 can be determined in human whole saliva. Saliva represents an attractive body fluid for longitudinal studies. MATERIALS AND METHODS: CA130 was determined in parotid saliva from 8 rats fed different diets, with or without autonomic denervation. RESULTS: CA130 could be determined in parotid saliva of rats, irrespective of diet and/or autonomic denervation. Whether the numerical decrease in CA130 observed after autonomic denervation is statistically significant requires further work. CONCLUSIONS: Since salivary CA130 has been shown to decrease following treatment with anti-cancer drugs in humans, the ability to determine this tumor marker in rat saliva opens new opportunities for optimizing cancer chronotherapy in the experimental laboratory.

Developmental expression of a unique carbohydrate antigen, Tn antigen, in mouse central nervous tissues.

Using an anti-Tn monoclonal antibody, the Tn antigen was detected immunohistochemically in prenatal and early postnatal central nervous tissues. On embryonic day 9 (E9), the antigen was distributed throughout the single neuroepithelial layer in the neocortex and then became more prominent in the preplate than in the ventricular zone along with formation of the preplate. Following division of the preplate and concomitant formation of the cortical plate, distinct labeling of the neocortex occurred in the marginal, subplate and intermediate zones, whereas in the cortical plate and ventricular zone were virtually not immunostained. It is notable that thalamocortical afferent fibers were also immunostained specifically on E14. After birth, the localization of the antigen became less noticeable and by 3 weeks after birth, the antigen had substantially disappeared. In the developing cerebellum, prominent labeling was also observed in the molecular layer and outskirts of the cerebellar nuclei on early postnatal days. To characterize the glycoprotein bearing the Tn antigen biochemically, immunoblot analysis was performed. The glycoprotein, most of which was extracted with a salt solution, migrated as a broad smeared band corresponding to a molecular weight of about 250 kDa on SDS-PAGE. Among the various tissues examined, this glycoprotein was only detected in the brain and its amount increased until an early postnatal stage with a peak on postnatal day 3 (P3), and then decreased gradually with age. This spatially and developmentally regulated expression of the Tn antigen suggests that this antigen plays a significant role in brain development.

Secretory immunoglobulin A heavy chain presents Galbeta1-3GalNAc binding structures for Actinomyces naeslundii genospecies 1.

Adherence of Actinomyces naeslundii ATCC 12104 to hydroxyapatite beads coated with protein fractions of parotid saliva, obtained by gel filtration on S-200 HR columns, showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to high-molecular-weight proteins (Strömberg et al., 1992). The present study investigates the nature of these high-molecular-weight binding proteins and determines their specific ability to mediate adherence to representative strains of Actinomyces species. Strain ATCC 12104 bound specifically in a lactose-inhibitable manner to the heavy chain of secretory immunoglobulin A (S-IgA), contained within a high-molecular-weight parotid protein fraction separated on SDS-PAGE and transferred to a solid membrane support. Lactose-inhibitable binding to the heavy chain of S-IgA from human colostrum was also demonstrated. Peanut agglutinin bound to the heavy chain of parotid and colostrum S-IgAs contained on solid support membranes, confirming the presence of Galbeta1-3GalNAc residues on these molecules. Both salivary and colostrum S-IgA aggregated with strain ATCC 12104 in a GalNAcbeta1-3Galalpha-O-ethyl-inhibitable fashion. Further separation of high-molecular-weight salivary proteins on S-500 HR columns showed GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to both mucin- and S-IgA-containing fractions. The presence of S-IgA in salivary pellicles formed in vivo on teeth was demonstrated by Western blot analysis of pellicle extracts with anti-IgA antibodies. Among strains representing A. naeslundii genospecies 1 and 2 and A. odontolyticus, only those of genospecies 1 with a particular adherence profile showed efficient GalNAcbeta1-3Galalpha-O-ethyl-inhibitable binding to S-IgA. Thus, oligosaccharides on S-IgA may promote bacterial aggregation (or adherence) and provide a mechanism by which S-IgA can interact with bacteria without prior immunological challenge.

Down-regulation of human sialyltransferase gene expression during in vitro human keratinocyte cell line differentiation.

Sialic acids play important roles in biological processes, such as cell-cell communication and cell-matrix interaction. Histochemical analysis using PNA and LFA lectin has shown that the expression of alpha 2,3-sialic acid linked to Gal beta 1,3GalNAc is high in basal cells and decreases following further keratinocyte differentiation. In the present study, we used an in vitro keratinocyte cell line differentiation model to study expression of alpha 2,3-sialic acid linked to Gal beta 1,3 GalNAc. Treatment of the human papillomavirus type 16-immortalized human keratinocyte (PHK16) cell line with high concentrations (1.0 mM) of Ca2+ resulted in PHK16 cell differentiation and redistribution of PNA binding glycoproteins. The synthesis of alpha 2,3-sialic acid linked to Gal beta 1,3GalNAc is mediated by three beta-galactoside alpha 2,3-sialytransferases, which are the gene products of hST30, hST30/N and hST3 Gal II. Ca2+ treatment of PHK16 cells decreased the mRNA expression of hST30/N, whereas the mRNA of hST30 and hST3Gal II was not detected by Northern blot analysis, suggesting that the hST30/N gene is responsible for sialic acid down regulation during keratinocyte differentiation. In order to examine transcriptional regulation of the hST30/N gene, we first determined the transcriptional starting sites of the hST30/N gene in PHK 16 using 5'-RACE analysis. Two kinds of type B isoforms, types B3 and BX, were identified. Type BX is a novel isoform related to the type B form, but which differs upstream of the B3 exon. The results of Northern blot analysis using a type BX-specific probe suggest that the B3 promoter may be regulated by Ca2+. Using a luciferase assay, we identified a functional DNA portion within hST30/N genomic DNA that confers negative transcriptional regulation on the hST30/N B3 promoter during Ca2+ stimulated human keratinocyte differentiation. This element contains some putative transcriptional factor binding sequence motifs such as AP2.

Amino acid sequence and three-dimensional structure of the Tn-specific isolectin B4 from Vicia villosa.

The partial amino acid sequence of the tetrameric isolectin B4 from Vicia villosa seeds has been determined by peptide analysis, and its three-dimensional structure solved by molecular replacement techniques and refined at 2.9 A resolution to a crystallographic R-factor of 21%. Each subunit displays the thirteen-stranded beta-barrel topology characteristic of legume lectins. The amino acid residues involved in metal- and sugar-binding are similar to those of other GalNAc-specific lectins, indicating that residues outside the carbohydrate-binding pocket modulate the affinity for the Tn glycopeptide. Isolectin B4 displays an unusual quaternary structure, probably due to protein glycosylation.

Distribution of CA 125 in embryonic tissues and adult derivatives of the fetal periderm.

New murine monoclonal antibodies to a partially purified CA 125 antigen were developed and identified as M 2 and M 11. With immunohistochemical techniques, these new antibodies and OC 125 antibody were used to search for CA 125 in embryonic tissues and adult apocrine sweat glands and mammary glands. The embryonic skin, the periderm, expressed CA 125 antigen and its adult derivatives, the mammary glands and apocrine sweat glands, expressed CA 125 while in the active state of secretion. In a 6-week-old formalin-fixed and paraffin-embedded ectopic embryo specimen, antibodies M 2 and M 11 recognized CA 125 in the periderm, the notochord, the myocardium, the pericardium, the gastroenteric tract, enteric duct remnants in the umbilical cord (vitelline and allantoic ducts), the mesonephric duct, and the amnion. OC 125 staining of these formalin-fixed specimens was either very faint or absent. In a formalin-fixed and paraffin-embedded specimen of apocrine sweat glands from the axilla, antibodies M 2 and M 11 detected CA 125 antigen intracellularly in the secretory cells. Again no staining was observed with OC 125 antibody. In a frozen and acetone-fixed specimen of lactating mammary glands, antibodies M 2 and OC 125 detected CA 125 antigen intraductally. Colostrum and milk collected from 25 mothers at various stages post partum had mean CA 125 levels of 34,213 U/ml in colostrum, 1469 U/ml at 3 to 7 days, and 105 U/ml at 5 to 26 weeks.