Human Milk Fat Globule Research Articles

A useful low temperature method for post-embedding electron immunocytochemistry in routine histopathology.

Using a low temperature resin, we have developed a reliable technique for post-embedding electron immunocytochemistry which is rapid enough to be used in a routine histopathology laboratory. Glutaraldehyde fixed human tissues were dehydrated and embedded at -25 degrees C in a new acrylic resin called LR-Gold. Using the immunogold technique, ultra-thin sections of the tissues were labelled with monoclonal antibodies to cytokeratin, human milk fat globule and HLA-D region antigen; and with polyclonal antisera to lysozyme, kappa and lambda light chains and immunoglobulin M. The resin was easy to section and the preservation of fine structure was excellent. Immunolabelling procedures gave clean and consistent results, and electron micrographs of examples of this are included. It was felt that the preservation of ultrastructure and antigenicity compared well with the results of other workers using low temperature resins such as Lowicryl K4M, but LR-Gold was superior to Lowicryl K4M because sectioning was considerably easier and the sections were more stable in the electron beam.

Epithelial membrane antigen as a marker of human salivary gland acinar and ductal cell function

Monoclonal antibody to epithelial membrane antigen (EMA) from human milk fat globule membrane was used with the immunoperoxidase method to examine the distribution of EMA in normal human salivary glands and in their obstructive lesions. EMA staining in the normal salivary gland was limited to luminal and lateral borders of acinar cells. The antigen was also present in high concentration on the luminal side of ductal cells. In obstructive lesions or sialoadenitis, lateral border and luminal positive staining in atrophic acinar cells was reduced or absent, and the EMA staining in duct-like structures was also markedly decreased on even absent in chronic stage. The degree of reduction in EMA staining was dependent on the extent of degeneration. Expression of immunohistochemically detectable EMA in paraffin sections suggests the antigen to be useful as a new marker of salivary gland function, of either acinar compartments or ductal segments.

Immunohistochemical expression of monoclonal antibody against epithelial membrane antigen in salivary gland tumors.

A total of 154 cases of salivary gland tumors were studied by an indirect method with the use of monoclonal antibody to epithelial membrane antigen (EMA) from human milk fat globule membrane. Normal salivary glands displayed positive reaction of EMA staining in the luminal and lateral borders of serous acinar cells, and in the luminal border of ducts. Immunohistochemical expression of EMA in salivary gland tumors was chiefly classified into two types: one is the luminal surface-positive type and the other is the whole cellpositive type. Pleomorphic adenoma was usually positive for EMA staining in luminal borders of tubular, duct-like and cystic structures, and occasionally positive in the cytoplasm of the cells comprising these structures. Warthin's tumor was positive for EMA staining on the luminal surface of eosinophilic tumor epithelial cells, or EMA-positive reaction was evident in various areas scattered throughout the epithelial structures. Acinic cell tumor, carcinoma in pleomorphic adenoma (carcinoma cell), and mucoepidermoid tumor showed positive reaction of EMA staining throughout their cytoplasm depending on histological malignancy. No positive reaction to EMA staining was given by either normal myoepithelial cells, and transformed or neoplastic myoepithelial cells.

Immunohistological staining of reactive mesothelium, mesothelioma, and lung carcinoma with a panel of monoclonal antibodies.

A panel of seven monoclonal antiepithelial antibodies of different specificities, including anticytokeratin, human milk fat globule membrane, C, and carcinoembryonic antigen (CEA) were used with the alkaline phosphatase-antialkaline phosphatase (APAAP) immunostaining technique to determine their value in the differentiation between benign and malignant mesothelial cells and lung carcinoma in histological preparations. The anticytokeratin antibody reacted strongly with all cases of reactive mesothelium, mesothelioma, and lung carcinoma. Antibodies to human milk fat globule membrane and the Ca antigen stained mesothelioma and carcinoma and 43% of cases of reactive mesothelium. Staining for carcinoembryonic antigen was not detected in reactive mesothelium or mesothelioma, but was present in most of the lung carcinomas. CEA seemed to be the single most useful marker in distinguishing carcinoma from mesothelioma in that a positive reaction for CEA would indicate carcinoma rather than mesothelioma.

Intraperitoneal administration of an I 125 labelled monoclonal antibody for localization of human colorectal cancer in the nude mouse

The monoclonal antibody (MoAb) HMGF-1 was evaluated in the radioimmunodetection of human colonic cancer transplanted intraperitoneally (i.p.) into athymic nude mice. This antibody reacts with a component of the human milkfat globule, as well as a wide range of epithelial cells and adeonocarcinomas of various origins. Purified MoAb was iodinated with 125I and administered i.p. into nu/nu mice bearing (i.p.) xenografts of human colonic adenocarcinoma (X56). Differential tissue counts of radioactivity demonstrated preferential localization of the antibody in i.p. and subcutaneous (s.c.) tumor tissue as compared to normal tissues. Maximum per cent dose per g of tumor (25.17±1.37), maximum tumor: blood ratio (4.45±0.14) and maximum tumor: tissue ratios (34.2±0.12) were obtained at the optimal labelling time of 5 days after antibody injection. Selective localization to tumor was confirmed with a control anti-hepatitis virus MoAb of the same isotype and by localization studies in non-tumor bearing athymic mice. Half lives of the persistence of the iodine 125 in the tumor bearing and non tumor bearing mice were 5 and 7 days, respectively, indicating approximate antibody half lives. Whole body scans showed distinct tumor images without the use of subtraction techniques. This pilot experimental study demonstrates the feasibility of i.p. administration of labelled antitumor MoAb in the imaging of i.p. tumors in an athymic mouse system. Whether or not these observations are applicable to the human situation remains to be carefully established.

The prognostic significance of the monoclonal antibody III D 5 to human milk fat globule antigen in breast cancer.

The monoclonal antibody III D 5 identifies an antigen in human milk fat globule membrane, found also on breast epithelial cells. Immunohistochemistry with the antibody was used in the staining of formalin-fixed, paraffin-embedded sections of 80 breast carcinomas. The duration of follow-up of patients was from seven to twelve years. The presence or absence of metastases in regional lymph nodes at the time of primary operation, histological grade and necrosis of tumours correlated highly significantly to the survival of patients. The patients whose tumours exhibited positive staining with the antibody III D 5 had an improved survival-rate compared with those with III D 5 negative tumours. In the patients without metastases at the time of primary operation, the histological grade of primary tumour had only a weak correlation with survival, while the same patients with III D 5 positive tumours had significantly better prognosis compared to the III D 5 negative cases. Inclusion of the III D 5 immunoreactivity with other factors in a prognostic index could permit a more accurate estimation of prognosis in patients with mammary carcinoma.

Detection of surface antigens defined by monoclonal antibodies in primary mucinous breast carcinomas. Relation to prognostic factors and recurrence-free survival.

Three monoclonal antibodies, 67D11, 115H10 and 115C2, raised against human milk fat globule membranes, have been applied to 207 primary mucinous breast carcinomas. The tumours reacted positively in 18% (67D11), 54% (115H10), and 20% (115C2) of the cases. The detected epitopes (MAM-3a (67D11), MAM-3b (115H10), and MAM-3c (115C2)) have formerly been shown to be markers of differentiation in infiltrating ductal carcinomas. In the present group of mucinous breast carcinomas, statistically significant correlation to high risk factors, such as occurrence of primary lymph node metastases, large tumour size, and local invasion of the tumour into overlying skin or deep fascie, were found. Furthermore, the antigen expression was less marked in pure mucinous carcinomas as compared to carcinomas also presenting with non-mucinous tumour areas. Thus, especially the antigen MAM-3b, is more frequently present in mixed tumours, in large tumours, in tumours with local invasion, and in tumours with primary lymph node metastases. However, no association could be demonstrated between expression of MAM-3b and recurrence-free survival. Mucinous carcinomas of the breast apparently differ from other carcinomas not only with respect to morphology, but also in their pattern of antigenic expression in relation to other prognostic factors.

Immunohistochemical reactivity of monoclonal antibodies to human milk fat globule with breast carcinoma and with other normal and neoplastic tissues.

The immunohistochemical reactivity of three monoclonal antibodies, generated against human milk fat globule membranes (HMFG), were studied using peroxidase conjugated secondary antibody or avidin-biotin-peroxidase complex method. Two of the antibodies, III E 8 and III H 2 showed rather broad reactivity, staining all mammary, ovarian, endometrial, cervical and bronchial adenocarcinomas. They also stained all pancreatic adenocarcinomas but only 30% of the carcinomas of the colon. Reaction with prostatic and renal adenocarcinomas varied. No reaction was seen with squamous or transitional cell carcinomas. Antibody III D 5, which previously has been shown to recognize an estrogen receptor activation associated antigen in mammary carcinomas, stained 60% of mammary or gynecological adenocarcinomas. This antibody had a more limited reactivity with other tissues as well, as all intestinal carcinomas were negative. It stained all bronchial adenocarcinomas, however, and 50% of the prostatic carcinomas. The three monoclonal antibodies, although not specific, show a more restricted reactivity than the so far described HMFG antibodies and they may therefore be of value in histopathological diagnosis.

Lectin binding affinities of human milk fat globule (HMFG) membrane antigens

Six biotinylated lectins with differing specificities and two monoclonal antibodies (III D 5 and III H 2) were used to characterize the sugar-residues in human milk fat globule (HMFG) membrane antigens. Immunoblotting analysis revealed that most of the antigens contain several sugars. However, the molecules exclusively reacting with anti-HMFG III D 5, a monoclonal antibody previously shown to detect antigen(s) positively correlating with the expression of estrogen receptors in mammary and gynaecological carcinomas, could only be stained with peanut agglutinin and Ricinus communis-leclins. One of these antigens, a 42–57 kDa molecule, was shown to have a complexed quaternary structure with galactose determining the antigenic specificity. It is suggested that the production of this glycoprotein in estrogen sensitive tissues results from activation of galactosyl-transferase-enzyme at the same time as the expression of estrogen receptors.

Recognition with a monoclonal antibody of a cytoplasmic mammary carcinoma antigen, correlated to the estrogen receptor status.

Immunohistochemical reactivity of mammary carcinomas with monoclonal antibodies (MAbs) to human milk fat globule (HMFG) membrane antigens was compared with the estrogen (ER) and progesterone receptor (PR) status of the tumors. Antibody III D 5 stained 55 of the 74 tumors studied, the reaction being of borderline intensity in 19 cases and unequivocally positive in 36 cases. The staining was always cytoplasmic; in addition occasional extracellular III D 5-positive secretory material was observed. Positive reactivity of the tumor with antibody III D 5 was significantly correlated with ER and/or PR content of the tumors. The presence of extracellular, III D 5-positive secretory material correlated to the ER but not to the PR status of the tumors.

Reactivity of a monoclonal antibody recognizing an estrogen receptor regulated glycoprotein in relation to lectin histochemistry in breast cancer.

We have raised monoclonal antibodies against human milk fat globule membrane antigens and previously shown that one of them, called III D 5, recognises a glycoprotein associated with estrogen receptor activity of breast cancer. In immunoblotting it was shown that the molecule in human milk exclusively stained with III D 5 also binds peanut agglutinin (PNA) and Ricinus communis. In this study we correlate the staining of III D 5 and binding of lectins to tissue sections fixed in formalin and embedded in paraffin. Similar reactions were seen only with III D 5 and PNA. Our results suggest that III D 5 and PNA detect overlapping antigenic epitopes in mammary carcinoma. This is in keeping with previous results that PNA or III D 5 reactivity is correlated with estrogen receptor status of breast cancer.

A review on antigens of milkfat globule membranes and their value for diagnosis and prognosis of breast cancer and other carcinomas.

A short review is presented on the usefulness of monoclonal antibodies against human milkfat globule membranes in pathology and clinical chemistry for the Cancer Clinic, with particular reference to antibodies reacting with the so-called MAM-3 and MAM-6 groups, as defined by a series of antibodies generated in the Netherlands Cancer Institute.

Hormonally regulated expression of an apocrine epithelial antigen in the cell membranes of endometrial glands.

We have raised a rabbit antiserum against formalin-treated membrane glycoproteins isolated by lectin affinity chromatography from human milk fat globules. In immunofluorescence staining the antiserum reacts strongly and exclusively with the apical membrane of the glandular epithelial cells in normal endometrium during the proliferative phase. No membrane-bound antigen is seen during the secretory phase but some positively staining material is found in the glandular lumina and a weak cytoplasmic staining is also seen. The antigen is absent in postmenopausal endometrium but is found in abundance in the cell membrane of the glandular epithelium in endometria from postmenopausal women receiving estrogen treatment. The glandular epithelium of hyperestrogenism-induced endometrial hyperplasia is very strongly decorated by the antiserum. Molecular characterization of the antigen by immunoblotting under reducing conditions reveals two polypeptides of 110,000 daltons and 93,000 daltons from normal endometrium during the proliferative phase. A single band of 75,000 daltons, probably representing shed fragment, is seen in secretory endometrium. This hormonally regulated and strictly located protein differs from many other endometrial proteins in that it is estrogen-induced, while others are dependent on progesterone effect, and it constitutes an interesting histochemical marker of endometrial glands.

Monoclonal antibodies, electrophoretically transferred from polyacrylamide gels, retain their ability to bind specific antigens

Antibodies subjected to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose paper, have been found to retain the ability to bind specific antigen. This has been demonstrated for two groups of antibodies, directed to a) a large molecular weight glycoprotein of the human milk fat globule and b) human interferon α2. Immunoreactive antibody fragments produced by protease digestion could also be identified in this way on Western blots, thus permitting the development of optimal conditions for digestion, without the need for extensive purification procedures.

Epithelial membrane antigen—A diagnostic discriminant in surgical pathology: Immunohistochemical profile in epithelial, mesenchymal, and hematopoietic neoplasms using paraffin sections and monoclonal antibodies

Glycoproteins isolated from human milk fat globule membranes, designated epithelial membrane antigen (EMA), have been detected immunohistochemically in most nonneoplastic epithelia and are potentially a highly effective marker for establishing the epithelial nature of neoplastic cells. With commercially available monoclonal antibodies and an indirect immunoperoxidase technique, EMA localization was evaluated in paraffin-embedded tissues from a wide variety of neoplasms (320 specimens). Adenocarcinomas from various primary sites (breast, lung, colon, stomach, pancreas, gallbladder, prostate, endocrine glands, ovary, kidney, thyroid) were immunoreactive for EMA in 88 of 97 cases (91 per cent). Cytoplasmic and apical luminal membrane staining were the most common patterns of immunoreactivity, with peripheral membrane staining or other patterns also seen in some neoplasms. Squamous cell (13 of 13 cases) and transitional cell (12 of 12 cases) carcinomas, small cell anaplastic carcinomas (12 of 12 cases), and mesotheliomas (six of six cases) were also uniformly EMA-positive. Malignant lymphomas of the Hodgkin's (15 cases) and non-Hodgkin's types (74 cases), except for the true histiocytic lymphomas and occasional T-cell lymphomas, were nonreactive for EMA. Neoplastic and nonneoplastic plasma cells showed variable EMA positivity. Endocrine neoplasms (17 cases), including carcinoid tumors, medullary carcinoma of thyroid, adrenocortical carcinomas and pheochromocytomas, and germ cell tumors (eight cases, embryonal carcinoma and seminoma), and a wide variety of soft tissue tumors (27 cases) generally lacked immunoreactivity for EMA; the exceptions to this finding were synovial sarcomas and an epithelioid sarcoma. Malignant melanomas (eight cases) were typically nonreactive. Based on the observations in this large series of neoplasms, EMA is an excellent marker of epithelial differentiation, appears to be highly reliable for discriminating between poorly differentiated carcinomas and malignant lymphomas, and is especially helpful in characterizing small cell anaplastic carcinomas. Epithelial membrane antigen immunoreactivity is well preserved in paraffin sections of routinely processed tissues, facilitating application of this technique in diagnostic surgical pathology.

Novel antigenic specificity involving the blood group antigen, Lea, in combination with onco-developmental antigen, SSEA-1, recognized by two monoclonal antibodies to human milk-fat globule membranes

Two monoclonal antibodies to human milk-fat globule membranes, which recognize an epithelial antigen designated MAM-3c, were found to bind strongly to epithelial glycoproteins derived from non-secretors. Further investigations, using purified glycoproteins and structurally defined ollgosaccharides, established that the optimal antigenic structure for both antibodies involves the Type 1 based blood group antigen. Lea. In combination with the Type 2 based onco-developmental antigen. SSEA-1. ▪ as in lacto-N-difucohexaose II. The antibodies may also react with the corresponding monofucosyl structures lacking the 3- or 4- linked fucose residues and to a lesser extent with the afucosyl tetrasaccharide sequence as in lacto-N-tetraose. The Lea and SSEA-1 antigens are known to occur on human epithelial glycoproteins. However, this is the first report of an antigenic specificity involving a combination of the Type 1 and Type 2 based fuco-oligosaccharides and occurring on epithelial glycoproteins.

Monoclonal antibodies recognizing epitopes carried on both glycolipids and glycoproteins of the human milk fat globule membrane.

The molecules of the human milk fat globule membrane (MFGM) which bind four murine monoclonal antibodies (LICR LON M3, M8, M18 and M24) raised against the human MFGM have been identified. By using 'Western' blotting [Burnette (1981) Anal. Biochem. 112, 195-203] it was shown that each antibody reacted with a different set of proteins. M3 and M24 were similar in their pattern of reaction with the membrane proteins, but were quite distinct from M8 and M18, which also differed from each other. Glycopeptides prepared from the MFGM by exhaustive Pronase digestion were able to inhibit partially the binding of M3 and M24, and prevent totally the binding of M8 and M18, to the MFGM in an enzyme-linked immunoabsorbent assay. Oligosaccharides obtained by the deproteination of human milk also completely inhibited the binding of M3, M18 and M24 to the MFGM. However, the binding of M8 was not inhibited by these saccharides, and therefore M8 may not be recognizing a simple carbohydrate determinant. By using an enzyme-linked assay, M8 and M18 were shown not to bind to MFGM glycolipid, whereas M3 and M24 did, and this was confirmed by overlaying thin layer chromatograms of MFGM lipids with these antibodies. Both M3 and M24 showed a similar complex pattern of reaction, binding to more than one glycolipid moiety. By these means all four antibodies have been shown to react with antigens which involve carbohydrate side chains carried on different proteins, and two were also shown to react with such determinants on glycolipids.

Glycoprotein PAS-0 from the milk fat globule membrane carries antigenic determinants for epithelial membrane antigen

Epithelial membrane antigen (EMA) can be found in human tissues using antisera raised against defatted human cream. PAS-0 is a glycoprotein which has been extracted from human milk fat globule membranes. Using a polyclonal antiserum and a series of monoclonal antibodies, we have shown that the major antigenic determinant for EMA is carried by PAS-0. A more detailed comparison of the two glycoproteins has been made by establishing a set of radioimmunossays using the different antibodies.

Characterization and partial purification of human milk fat globule membrane antigens by polyacrylamide gel electrophoresis and immunoblotting using monoclonal antibodies.

Polyclonal and monoclonal antibodies were used to characterize human milk fat globule (HMFG) membrane antigens, using gel filtration, ion exchange chromatography and western blotting. Although originally generated against HMFG, monoclonal antibodies also reacted with skim milk. In western blotting, several antigen molecules were seen, of which some were detected by all available antibodies, and one only with monoclonal antibody III D5, previously shown to react with mammary and ovarian carcinomas bearing estrogen receptors. This or these antigens, with a molecular weight of about 53 kd, were isolated by fractionation in SDS-PAGE. A low polypeptide content was demonstrated but the antigenic structure could be stained with periodic-acid-Schiff and biotinylated peanut agglutinin, indicating the presence of galactose or N-acetylgalactosamine residues. While this 53-kd molecule is the only determinant exclusively stained by III D 5 antibody, it is suggested that it carries epitopes related to estrogen receptor activation.

Epithelial markers for paraffin-embedded human tissues. Immunohistochemistry with monoclonal antibodies against milk fat globule antigens.

About 200 human tumours and corresponding normal tissue samples were investigated by immunoperoxidase tests for the expression of MAM-3, MAM-5 and MAM-6 antigens, which had previously been defined by monoclonal antibodies to human milk fat globule membranes. All tissue specimens had been treated for routine histopathology, i.e. fixed in formalin and embedded in paraffin. One of the antigens, MAM-6, appeared to be an important epithelial marker, present in all normal and neoplastic breast tissue samples, in about 80% of non-mammary normal tissues and in more than 90% of non-mammary epithelial tumours. It could never be detected in normal and neoplastic mesenchymal and neuroectodermal structures. Direct comparison with the distribution of Carcinoembryonic Antigen (CEA), Tissue Polypeptide Antigen (TPA) and keratin provided clear evidence that MAM-6 is different from these well known epithelial markers. MAM-3 proved to be an additional important marker exhibiting a characteristic distribution pattern in those epithelial tissues investigated. In contrast to MAM-6, it could never be detected in renal cell cancers and carcinomas of the prostate gland, thus allowing differential diagnosis on the basis of immunohistochemistry. MAM-5, known to be associated with lactoferrin, was mainly detectable in secretory organs and their tumours. In the group of breast tumours, its expression was mainly seen in lobular cancers. These findings suggest a use for these new markers for routine histopathology.