Edith van der Linden

Expression of integrins E-cadherin in cells from menstrual effluent, endometrium, peritoneal fluid, peritoneum, endometriosis

Objective To detect the expression of integrins and E-cadherin in cells from peritoneal fluid (PF), endometrium, menstrual effluent, peritoneum, and endometriotic lesions during the early follicular phase of the menstrual cycle. Design An immunohistochemical study. Setting Tertiary care university medical center. Patients Sixteen patients undergoing a diagnostic laparoscopy as part of a subfertility work-up. All patients had regular and ovulatory cycles. Interventions A laparoscopy was performed in the early follicular phase (days 2 to 5). Simultaneously, samples were taken from endometrium, menstrual effluent, and PF, and a representative biopsy of an endometriotic lesion was obtained. If endometriosis was not noted, a peritoneal biopsy was obtained instead. Main Outcome Measures The expression of cell adhesion molecules, including the integrin α 2 β 1, α 3 β 1, α 4 β 1, α 5 β 1, and α 6 β 1 and E-cadherin, as determined by immunohistochemistry on frozen sections. Results All integrins tested could be detected in the endometrium samples and in endometriotic lesions. In menstrual effluent samples, positive staining for the integrins α 2 β 1 and α 3 β 1 was found in epithelial cells in 13 of 16 cases. Integrin α 5 β 1 was detected in 11 of 16 samples, and integrins α 4 β 1 and α 6 β 1 were detected in 5 of 16 samples. In PF, integrin α 3 β 1 was found in epithelial cells in 12 of 16 samples, integrin α 5 β 1 in 5 of 16, and integrins α 4 β 1 and α 6 β 1 in 2 of 16. The antibody for E-cadherin showed positive staining of epithelial cells in 6 of 16 menstrual effluent samples. All endometrial tissue samples showed positive staining for E-cadherin. In PF, E-cadherin was detected in the epithelial cells of one sample. One peritoneum biopsy revealed positive staining for E-cadherin. Conclusion Integrins α 2 β 1, α 3 β 1, α 4 β 1, α 5 β 1, and α 6 β 1, and E-cadherin, important cell adhesion molecules, are expressed in endometriotic lesions and in cells and tissues that are potentially involved in the development of endometriosis. These cell adhesion molecules could be involved in the shedding of endometrial tissue during menstruation and the attachment of endometrial tissue fragments to the peritoneum.

Isolation of endothelial cells from fresh tissues

Here, we present a protocol for the isolation of endothelial cells (ECs) from tissues. ECs make up a minor population of cells in a tissue, but play a major role in tissue homeostasis, as well as in diverse pathologies. To understand the biology of ECs, characterization of this cell population is highly desirable, but requires the availability of purified cells. For this purpose, tissues are mechanically minced and subsequently digested enzymatically with collagenase and dispase. ECs in the resulting single-cell suspension are labeled with Abs against EC surface antigens and separated from the remainder of the cells and debris by capture with magnetic beads or by high-speed cell sorting. Purified ECs are viable and suitable for characterization of diverse cellular properties. This protocol is optimized for human tissues but can also be adapted for use with other species. Depending on the tissue, the procedure can be completed in ∼6 h.

Human specific anti-type IV collagen monoclonal antibodies, characterization and immunohistochemical application

SummaryThis paper describes two new monoclonal antibodies reactive with human specific type IV collagen epitopes in frozen as well as routinely fixed and processed tissue sections. The antibodies (1042 and 1043) were raised against human placental type IV collagen and were shown by immunoblotting and ELISA tests to react exclusively with type IV collagen determinants. Extensive immunohistochemical survey studies on panels of tissues from various species, using unfixed cryosat sections, demonstrated that antibody 1042 reacted only with human type IV collagen whereas antibody 1043 in addition reacted with rabbit type IV collagen. All tissues showed homogeneous staining of the basement membrane, indicating that the detected epitopes did not show organ-specific distribution.Tissue processing protocols for using these monoclonal antibodies on routinely processed paraffin embedded tissues were developed. It was found that whereas polyclonal antitype IV collagen antisera required pepsin digestion, our monoclonal antibodies required pronase or papain digestion to restore type IV collagen immunoreactivity in paraffin sections.It is concluded that these monoclonal anti-type IV collagen antibodies detect species specific epitopes which can be detected in routinely processed paraffin embedded tissues after appropriate enzyme pretreatment.

NCI-H716 cells as a model for endocrine differentiation in colorectal cancer

SummaryIn colonic neoplasms, endocrine differentiation is encountered not only in carcinoid tumors but also in adenocarcinomas, where endocrine cells may represent a distinct line of differentiation in the tumor. The significance of endocrine differentiation in colorectal cancer is not well established, partly because of the paucity of tumor cell lines which can serve as a model for studying endocrine differentiation. In this report we describe the properties of NCI-H716 cells, a cell line derived from a poorly differentiated adenocarcinoma of the caecum, under various in vitro conditions and as xenografts in athymic mice. Phenotypical properties were immunohistochemically assessed using a panel of differentiation related antibodies, and also by Northern blot analysis and by electron microscopy. Receptors for biogenic amines and peptide hormones were analyzed by ligand binding assay. These studies show that:1.NCI-H716 cells can be undifferentiated, or show endocrine, mucin-producing or “amphicrine” properties.2.Endocrine differentiation of NCI-H716 cells preferentially occurs in xenografts in athymic mice, which suggests that mesenchymal elements induce endocrine differentiation.3.NCI-H716 cells express large amounts of high affinity receptors for gastrin, serotonin and somatostatin and these substances can regulate growth. Thus, NCI-H716 cells form a suitable model for the study of endocrine differentiation in intestinal epithelium and of auto- or paracrine growth regulation in intestinal neoplasia.

Patterns of α- and β-catenin and E-cadherin expression in colorectal adenomas and carcinomas

Previous in vitro and in vivo model studies have shown that when E‐cadherin expression in carcinoma cells is reduced, invasive behaviour ensues. The situation in human cancer in vivo, however, appears to be more complex, as immunohistochemically determined E‐cadherin expression in various carcinomas, including colorectal cancer, does not always correlate with invasive growth. Loss of cell adhesion during invasion in spite of E‐cadherin expression might be associated with a defective cadherin–catenin complex. The expression of α‐ and β‐catenin in comparison with E‐cadherin was therefore examined in colorectal adenomas and carcinomas and in lymph node and liver metastases. In normal colonic mucosa, α‐ and β‐catenin immunoreactivity occurred along the lateral plasma membranes of the epithelial cells, in a pattern identical to E‐cadherin staining. A similar pattern was found in colorectal adenomas and in most malignancies. In general, in neoplastic epithelia, the majority of the cancer cells displayed a normal (matching) pattern of E‐cadherin and catenin expression. It is concluded that the patterns of expression of E‐cadherin and α‐ and β‐catenin are very similar in colorectal neoplasms. This observation indicates that invasion in colorectal cancer is not paralleled by consistent loss of expression of the components of the cadherin–catenin complex.

A Human Immunoglobulin G1 Antibody Originating from an in Vitro-Selected Fab Phage Antibody Binds Avidly to Tumor-Associated MUC1 and Is Efficiently Internalized

We describe the engineering and characterization of a whole human antibody directed toward the tumor-associated protein core of human MUC1. The antibody PH1 originated from the in vitro selection on MUC1 of a nonimmune human Fab phage library. The PH1 variable genes were reformatted for expression as a fully human IgG1. The resulting PH1-IgG1 human antibody displays a 160-fold improved apparent kd (8.7 nmol/L) compared to the kd of the parental Fab (1.4 micromol/L). In cell-binding studies with flow cytometry and immunohistochemistry, PH1-IgG1 exhibits staining patterns typical for antibodies recognizing the tumor-associated tandem repeat region on MUC1, eg, it binds the tumor-associated glycoforms of MUC1 in breast and ovarian cancer cell lines, but not the heavily glycosylated form of MUC1 on colon carcinoma cell lines. In many tumors PH1-IgG1 binds to membranous and cytoplasmic MUC1, with often intense staining of the whole-cell membrane (eg, in adenocarcinoma). In normal tissues staining is either absent or less intense, in which case it is found mostly at the apical side of the cells. Finally, fluorescein isothiocyanate-labeled PH1-IgG1 internalizes quickly after binding to human OVCAR-3 cells, and to a lesser extent to MUC1 gene-transfected 3T3 mouse fibroblasts. The tumor-associated binding characteristics of this antibody, its efficient internalization, and its human nature, make PH1-IgG1 a valuable candidate for further studies as a cancer-targeting immunotherapeutic.

Epithelial cells in peritoneal fluid--of endometrial origin?

OBJECTIVE Our purpose was to examine the immunohistochemical properties of epithelial cells in peritoneal fluid and to compare the staining characteristics with cells of endometrium, menstrual effluent, peritoneum, and endometriotic lesions. STUDY DESIGN Samples of menstrual effluent, endometrium, and peritoneal fluid and biopsy specimens of endometriotic lesions and peritoneum from 16 patients were examined. Monoclonal antibodies against vimentin, cytokeratin 18 and 19, and the monoclonal antibody BW495/36, staining an epithelial marker present in endometrium and absent in peritoneal epithelium, were used. RESULTS All but one sample of menstrual effluent and peritoneal fluid cells stained positively with antibodies against vimentin and cytokeratin 18 and 19. BW495/36 stained 14 of 16 menstrual effluent samples and nine of 16 peritoneal fluid cell samples. Endometriotic specimens showed staining with all markers. No major differences in staining properties were observed in menstrual effluent, endometrium, and peritoneal fluid cells between patients with or without endometriosis. CONCLUSION These results support the contention of transport of menstrual detritus to the peritoneal cavity in women with patent fallopian tubes.

Evidence for a bias toward intracellular antigens in the local humoral anti-tumor immune response of a colorectal cancer patient revealed by phage display

Many patients with colorectal carcinoma (CRC) mount a cellular as well as a humoral immune response to the tumor. To investigate the nature and specificity of the humoral immune response in a CRC patient, lymphocytes infiltrating the primary colorectal tumor and lymph nodes draining the tumor were used as antibody variable (V)‐gene pools for the construction of phage antibody repertoires. These libraries were first validated by selection on the antigen tetanus toxoid and shown to contain antibodies that were probably derived from both naive and memory B cells. The repertoires were then screened for the presence of antibodies directed to CRC by selection on the cell line CaCo2. For comparison, the same selections were performed with a phage antibody repertoire made from B cells of healthy donors. Striking differences were observed in the panel of specificities selected from these different repertoires: although a large panel of antibodies reactive with patient‐derived primary tumors was obtained from the immune repertoires, none of these discriminated between normal colonic epithelium and colon cancer and none were reactive with cell‐surface antigens. However, selections using the non‐immune library did result in numerous antibodies that recognized cell surface markers on CaCo2. These data suggest a bias in the local humoral immune response in this CRC patient, directed primarily toward intracellular epithelial‐cell specific target antigens.

In vitro characterisation of a monovalent and bivalent form of a fully human anti Ep-CAM phage antibody

Abstract Antibodies to tumour-associated antigens are increasingly being used as targeting vehicles for the visualisation and for therapy of human solid tumours. The epithelial cell adhesion molecule (Ep-CAM) is an antigen that is overexpressed on a variety of human solid tumours and constitutes an attractive target for immunotargeting. We set out to obtain fully human antibodies to this antigen by selecting from a large antibody repertoire displayed on bacteriophages. Two single-chain variable antibody fragments (scFv) were identified that specifically bound recombinant antigen in vitro. One of the selected antibodies (VEL-1) cross-reacted with extracellular matrix components in immunohistochemistry of colon carcinoma, whereas the other scFv (VEL-2) specifically recognised colon cancer cells. The latter antibody was further characterised with respect to epitope specificity and kinetics of antigen-binding. It showed no competition with the well-characterised anti Ep-CAM MOC-31 monoclonal antibody and had an off-rate of 5 × 10−2 s−1. To obtain an antibody format more suitable for in vivo tumour targeting and to increase the apparent affinity through avidity, the genes of scFv VEL-2 were re-formatted by fusion to a human (γ1) hinge region and CH3 domain. This “minibody” was expressed in Escherichia coli, specifically bound the Ep-CAM antigen and showed a 20-fold reduced off-rate in surface plasmon resonance analysis. These results show that phage antibody selection, combined with antibody engineering, may result in fully human antibody molecules with promising characteristics for in vivo use in tumour targeting.

Solid-phase adsorption of antigens for efficient production of antibodies reactive with native and fixed tissue antigens☆

A study has been made of the efficacy of different immunization protocols using low antigen levels for the generation of monoclonal antibodies capable of detecting antigens (ADCP) in processed tissues. Protocols using unprocessed native antigen immobilized on nitrocellulose were more efficient than soluble antigen in generating serum antibodies reactive with both native antigen and processed tissues. The derived monoclonal antibodies reacted with native but not processed antigen. The use of antigen immobilized on polyvinylidene (PVDF) and subsequently processed as for histochemistry was successful in generating monoclonal antibodies reactive with processed antigen.