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Biomedical subjects

S A Stacker

Publications and source records attributed to S A Stacker.

At least 55 records · Page 3Linked to original sources
PubMed1991

Characterization of ICAM-2 and evidence for a third counter-receptor for LFA-1.

In an endeavor to further characterize human intercellular adhesion molecule-2 (ICAM-2), two murine monoclonal antibodies (mAb) were generated to ICAM-2 transfected COS cells, and designated CBR-IC2/1 and CBR-IC2/2. Immunoprecipitated, reduced ICAM-2 migrated as a broad band of Mr 60,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Treatment with N-glycanase revealed a peptide backbone of Mr 31,000, consistent with the size predicted from the cDNA. ICAM-2 had a broad distribution on hematopoietic cell lines and little expression on other cell lines, the sole exception being cultured endothelial cells which possess high levels of ICAM-2. Resting lymphocytes and monocytes expressed ICAM-2, while neutrophils did not. Staining of tissue sections with anti-ICAM-2 mAb confirmed their strong reactivity to vascular endothelium, but demonstrated a lack of ICAM-2 expression on other tissues. Small clusters of ICAM-2 positive cells were, however, seen in germinal centers. In contrast to ICAM-1 there was little or no induction of ICAM-2 expression on lymphocytes or cultured endothelium upon stimulation with inflammatory mediators. One of the two mAb, CBR-IC2/2, was found to totally inhibit binding of ICAM-2+ COS cells to purified lymphocyte function-associated antigen-1 (LFA-1). Using this mAb, LFA-1-dependent binding to both stimulated and unstimulated endothelium was found to be totally accounted for by ICAM-1 and ICAM-2. Homotypic aggregation of an Epstein-Barr virus-transformed B cell line, JY, was found to be solely ICAM-1 and ICAM-2-dependent, while in the case of the T cell lymphoma cell line, SKW3, anti- ICAM-2 mAb in conjunction with anti-ICAM-1 mAb could not inhibit the LFA-1-dependent aggregation. This suggests an additional LFA-1 ligand exists. Using a cell binding assay to purified LFA-1 in conjunction with anti-ICAM-1 and anti-ICAM-2 mAb, we have demonstrated that this putative third ligand for LFA-1 exists on SKW3 and other cell lines.

Animals↗
PubMed1991

Regulation of adhesion of ICAM-1 by the cytoplasmic domain of LFA-1 integrin beta subunit.

Interactions between cytotoxic lymphocytes and their targets require the T cell antigen receptor (TCR) and the integrin lymphocyte function-associated molecule-1 (LFA-1, CD11a/CD18). LFA-1 is not constitutively avid for its counter-receptors, intercellular adhesion molecules (ICAMs)-1 and -2. Cross-linking of the TCR transiently converts LFA-1 to a high avidity state and thus provides a mechanism for regulating cellular adhesion and de-adhesion in an antigen-specific manner. Truncation of the cytoplasmic domain of the beta, but not the alpha, subunit of LFA-1 eliminated binding to ICAM-1 and sensitivity to phorbol esters. Thus, LFA-1 binding to ICAM-1 was found to be regulated by the cytoplasmic domain of the beta subunit of LFA-1.

Amino Acid Sequence↗
PubMed1991

Leukocyte integrin P150,95 (CD11c/CD18) functions as an adhesion molecule binding to a counter-receptor on stimulated endothelium.

p150,95 is a member of the beta 2 family of integrins, which includes both LFA-1 and Mac-1. These molecules are known to play a role in the adhesion of lymphocytes, granulocytes, and monocytes to various cell types including vascular endothelium. p150,95 is presumed to have an adhesive function because of its structural relationship to the other beta 2 integrins and the ability of anti-p150,95 mAb to inhibit some myeloid cell interactions with tumor cells, endothelial cells, and other substrates. In an endeavor to demonstrate directly that p150,95 can act as an adhesion molecule, we raised a mAb (CBRp150/4G1) to the alpha subunit of p150,95, which allows for the purification of functional intact p150,95 heterodimers. The antibody was selected by using a high pH elution ELISA. The assay was designed to select for antibodies directed to the alpha-chain of p150,95, which could be readily dissociated from p150,95 under conditions of high pH and 2 mM MgCl2. p150,95 purified under these conditions with CBRp150/4G1-Sepharose could be immunoprecipitated by using antibodies to the alpha- and beta-chains of p150,95 indicating that the structural integrity of the heterodimer was preserved during purification and elution. Elution in the absence of divalent cations yielded primarily dissociated alpha and beta subunits. Other antibodies previously made to p150,95 alpha-chain such as SHCL3 were greatly reduced in their efficiency of yielding intact heterodimer under these conditions. Mapping of the epitopes by using chimeric molecules of p150,95/Mac-1 revealed that antibodies that react with the divalent cation sites of p150,95 are inferior for the purification of intact p150,95. The adhesive capacity of p150,95 was demonstrated by the specific binding of 18-h rIL-1 beta or LPS-stimulated endothelial cells to purified p150,95 absorbed to plastic microtiter plates. These results indicate that p150,95 can function independently as an adhesion molecule and that it can interact with a counter-receptor on stimulated endothelium.

Animals↗
PubMed1991

The prognostic value of immunoperoxidase staining with monoclonal antibodies NCRC-11 and 3E1.2 in breast cancer.

The variation in survival of women with clinically similar breast cancers may lead to difficulty in clinical management so it is important to recognise factors which indicate the prognosis. Immunoperoxidase staining patterns of primary breast tumours using monoclonal antibody NCRC-11 have been shown to relate to overall survival (Ellis et al., 1985) but the results have not been reproducible in other centres. In this study paraffin sections of 483 primary breast cancers were stained with NCRC-11 and 3E1.2 using an immunoperoxidase system. The tumour staining patterns were compared with overall survival using life tables and tested for relative prognostic significance by Cox's multivariate analysis. NCRC-11 related to survival in all 483 cases (chi 2 5.8, P = 0.02) but both antibodies achieved maximum prognostic significance in lymph node negative patients (chi 2 9.4, P less than 0.002 and chi 2 10.7, P less than 0.001) in whom no other factor was more significant. Immunoperoxidase staining patterns produced by monoclonal antibodies NCRC-11 and 3E1.2 are important prognostic factors in breast cancer.

Antibodies, Monoclonal↗
PubMed1990

Distinct mutations in two patients with leukocyte adhesion deficiency and their functional correlates.

Two patients with leukocyte adhesion deficiency (LAD), one with a moderate phenotype (patient 14) and one with a severe phenotype (patient 2) who had been shown to have a normal sized beta subunit protein precursor, were analyzed in an attempt to determine the molecular basis for their disease. RNase mapping located possible mutations to two distinct but adjacent regions of the beta subunit cDNA. Sequencing of patient-derived cDNA clones in this region revealed a C for T difference at amino acid 149 in patient 14 which resulted in the substitution of a leucine for a proline, and an A for G substitution at amino acid 169 in patient 2 which mutated a glycine to an arginine. The mutated amino acids are in a region of the cDNA that is highly conserved between the beta subunits of the integrin family and are identical in all known integrin beta subunits. Co-transfection of the beta subunit cDNA containing the patient 2 mutation with the wild-type alpha subunit of LFA-1 in a mammalian expression system resulted in no expression of LFA-1. In the case of the mutation in patient 14 there was markedly diminished expression of LFA-1 with loss of function and loss of the epitope for a number of anti-beta mAbs. Normal half-life of the mutant beta subunits, and previous demonstration of a lack of alpha/beta complex formation during biosynthesis in patient cells, suggest a defect in association with the alpha subunit. Association with beta is required for expression of the alpha subunit of LFA-1. Loss of functional expression with both of these beta subunit mutations suggests that they lie in a site critical for association with the alpha subunit.

Amino Acid Sequence↗
PubMed1990

ICAM-1 (CD54): a counter-receptor for Mac-1 (CD11b/CD18).

While the leukocyte integrin lymphocyte function-associated antigen (LFA)-1 has been demonstrated to bind intercellular adhesion molecule (ICAM)-1, results with the related Mac-1 molecule have been controversial. We have used multiple cell binding assays, purified Mac-1 and ICAM-1, and cell lines transfected with Mac-1 and ICAM-1 cDNAs to examine the interaction of ICAM-1 with Mac-1. Stimulated human umbilical vein endothelial cells (HUVECs), which express a high surface density of ICAM-1, bind to immunoaffinity-purified Mac-1 adsorbed to artificial substrates in a manner that is inhibited by mAbs to Mac-1 and ICAM-1. Transfected murine L cells or monkey COS cells expressing human ICAM-1 bind to purified Mac-1 in a specific and dose-dependent manner; the attachment to Mac-1 is more temperature sensitive, lower in avidity, and blocked by a different series of ICAM-1 mAbs when compared to LFA-1. In a reciprocal assay, COS cells cotransfected with the alpha and beta chain cDNAs of Mac-1 or LFA-1 attach to immunoaffinity-purified ICAM-1 substrates; this adhesion is blocked by mAbs to ICAM-1 and Mac-1 or LFA-1. Two color fluorescence cell conjugate experiments show that neutrophils stimulated with fMLP bind to HUVEC stimulated with lipopolysaccharide for 24 h in an ICAM-1-, Mac-1-, and LFA-1-dependent fashion. Because cellular and purified Mac-1 interact with cellular and purified ICAM-1, we conclude that ICAM-1 is a counter receptor for Mac-1 and that this receptor pair is responsible, in part, for the adhesion between stimulated neutrophils and stimulated endothelial cells.

Antibodies, Monoclonal↗
PubMed1990

Transfection of cells from patients with leukocyte adhesion deficiency with an integrin beta subunit (CD18) restores lymphocyte function-associated antigen-1 expression and function.

Leukocyte adhesion deficiency (LAD) is an inherited immunodeficiency disease that is characterized by the deficient expression of the leukocyte adhesion glycoproteins lymphocyte function-associated antigen-1 (LFA-1), Mac-1, and p150,95. This loss of expression is attributed to heterogeneous defects in the common beta subunit shared by these glycoproteins. Here we demonstrate that expression of the LFA-1 alpha beta heterodimer in EBV-transformed B lymphoblastoid cells from LAD patients can be recovered after transfection with the beta subunit cDNA contained in an EBV-based vector. Four patients with differing severities of LAD comprising three distinct classes of mutations were studied. Flow cytometry analysis of stably transfected patient cells revealed near normal levels of expression of both the alpha and beta chains of LFA-1, and immunoprecipitation studies confirmed that fully processed alpha and beta chains were being expressed at the cell surface. In addition, Northern analysis of mRNA expression also demonstrated that the transfected LAD patient cells were expressing high quantities of exogenous beta subunit mRNA. Functional studies such as homotypic adhesion and adhesion to a purified counterreceptor for LFA-1, intracellular adhesion molecule-1, demonstrated that LFA-1 function had been restored in the stably transfected LAD patient cell lines. These studies unequivocally show that the defect in cells from patients with LAD is in the leukocyte integrin beta subunit.

Animals↗
PubMed1989

Purification and biochemical characterisation of a novel breast carcinoma associated mucin-like glycoprotein defined by antibody 3E1.2.

A member of the high molecular weight glycoproteins of human milk and breast cancer was isolated from the sera, ascites and breast carcinoma tissue of patients with breast cancer using monoclonal antibody 3E1.2. The 3E1.2 defined antigen, termed mammary serum antigen (MSA) was obtained by immunoaffinity chromatography and a solid phase immuno-precipitation technique (SPIT) from serum of patients with metastatic breast cancer. MSA was found to be a high molecular weight glycoprotein with a Mr greater than 300,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and a native Mr approximately 1 x 10(6) by gel filtration chromatography; in accord with the published Mr of other high molecular weight glycoproteins obtained from human milk and breast cancer. A high degree of glycosylation of MSA molecule was shown by its poor staining with Coomassie blue but good staining in a PAS-silver stain. In addition, MSA contained N-acetyl neuraminic acid and N-acetyl glucosamine as indicated by its binding to wheat-germ agglutinin. The epitope defined by antibody 3E1.2 is sensitive to treatment by sodium periodate and neuraminidase, implying that both carbohydrate and sialic acid are required for binding of antibody 3E1.2. Sandwich immunoassays demonstrated that MSA+ molecules are likely to express repeated 3E1.2 defined epitopes. Furthermore, MSA was susceptible to degradation by pronase, subtilisin and proteinase K and gave a different peptide profile from that of the PAS-O glycoprotein of human milk. MSA+ molecules were found to carry epitopes for a number of other monoclonal antibodies which were reactive with the PAS-O glycoprotein. It is suggested that MSA has the same core protein as is recognised by antibody DF3 which has been used to clone the same cDNA as was cloned with antibodies HMFG-1, HMFG-2 and SM-3. However, the epitope detected by the 3E1.2 antibody is either absent or weakly expressed on human milk, human milk-fat globule membrane (HMFGM) or deglycosylated HMFGM--all of which react strongly with various anti-HMFG antibodies. The antibody 3E1.2 thus recognises a unique epitope of the high molecular weight glycoproteins of human milk and breast cancer, being found in cancer tissue, serum and ascitic fluid of patients with breast cancer but weakly expressed or absent in human milk.

Antibodies, Monoclonal↗
PubMed1989

The detection of axillary lymph node metastases from breast cancer by radiolabelled monoclonal antibodies: a prospective study.

In a prospective study to assess the accuracy of monoclonal immunoscintigraphy for the detection of axillary lymph node metastases in breast cancer, two murine monoclonal antibodies that react with human breast cancer (3E1.2 and RCC-1) were labelled with 131iodine, and the radiolabelled antibody was injected subcutaneously into the interdigital spaces of both hands of 40 patients, 36 of whom had breast cancer and the remaining four of whom had fibroadenoma (the normal, contralateral axilla was used as a control). Of the patients with breast cancer, the findings from the scintigraphy images were correlated with histopathology or cytology of the axillary lymph nodes; images were regarded as positive and hence indicative of lymph node metastases if the amount of background-subtracted radioactive count in axilla on the side of breast cancer exceeded the contralateral normal side by a ratio greater than or equal to 1.5:1.0 as assessed by computer analysis. Using this method, immunoscintigraphy had an overall sensitivity of 33% (23% with 131I-3E1.2 and 50% with 131I-RCC-1) for the detection of lymph node metastases and a specificity of 63% (67% with 131I-3E1.2 and 60% with 131I-RCC-1) with problems of non-specific uptake by presumably normal lymph nodes. The results of immunoscintigraphy obtained with 131I-RCC-1 (IgG) were superior to 131I-3E1.2 (IgM) although the accuracy of immunoscintigraphy using 131I-RCC-1 (56%) was not much better than preoperative clinical assessment (50%). However, there were cases when immunoscintigraphy using radiolabelled antibody (IgM or IgG) detected axillary lymph node metastases not suspected by clinical examination. Thus it appears that while immunoscintigraphy may be a useful adjunct to preoperative clinical assessment and is simple and safe, a major improvement in its accuracy is needed before it can replace axillary dissection and histological examination in the accurate staging of axilla in breast cancer.

Antibodies, Monoclonal↗
PubMed1989

Monoclonal antibodies reactive with mucin expressed in breast cancer.

Three murine monoclonal antibodies (BC1, BC2 and BC3) were developed against human milk fat globule membrane (HMFGM). By immunoperoxidase staining, it was found that the antigenic determinants had a predominant distribution in breast cancer tissue. In addition, the antibodies reacted preferentially with mucin derived from human milk rather than that derived from the breast cancer cell line ZR75; they also recognized polymorphic high molecular weight components (MW greater than or equal to 230,000) in serum and in human milk fat globule membrane. Thus the antibodies appear to react with a component of the family of mucins found in breast cancer and human milk and it appears likely that at least part of each epitope is protein in nature. Antibodies BC1, BC2 and BC3 recognized related but not identical epitopes, and they appear to be co-expressed on the same molecules as 3E1.2-defined antigen (mammary serum antigen, MSA) which is also a member of the family of breast cancer-related mucin. However, the 3E1.2 epitope is distinct and non-cross-reactive with those described for BC1, BC2 and BC3. The BC2 and BC3 defined epitopes were examined for their value in serum assays. Immunoassay was developed with a combination of two antibodies, using antibody BC3 for antigen capture and antibody BC2 or 3E1.2 for antigen detection and gave reasonable sensitivity (approximately 85%) and specificity (approximately 95%) in such serum tests for breast cancer. In a limited study, these tests appeared to complement the MSA test in the detection of breast cancer.

Antibodies, Monoclonal↗
PubMed1988

Detection of mammary serum antigen in sera from breast cancer patients using monoclonal antibody 3E1.2.

Murine monoclonal antibody 3E1.2, made against human breast cancer cells, detects a glycoprotein (Mr greater than 300,000) called mammary serum antigen (MSA) which is elevated in the serum of patients with breast cancer. An enzyme immunoassay was developed to detect MSA in human serum and used to detect MSA in subjects with breast cancer and other diseases. Raised levels of MSA (greater than 300 inhibition units) were found in the serum of 1.9% of 2406 blood donors, in 18% of sera from 40 subjects with benign breast disease, and in 16% of sera from 222 subjects with non-breast cancers. However, in patients with a diagnosis of breast cancer, 76% (84 of 110) of Stage I and II, and 86% (142 of 166) of Stage III and IV had levels of greater than 300 inhibition units. Nineteen % of patients, classified clinically disease free, had raised MSA levels. In 34 of 37 (92%) patients followed over 2 to 11 mo the level of MSA correlated with the clinical course of disease. Changes in MSA levels not only corresponded to changes in the clinical course of disease, but also preceded the clinical detection of progressive disease. Immunoblotting has detected a heterogeneous molecule of Mr greater than 300,000 and been used to confirm the elevation of MSA in breast cancer patients. Determination of MSA level may be useful for the detection of breast cancer and for monitoring progress of disease and response to therapy.

Antibodies, Monoclonal↗
PubMed1988

Application of mammary serum antigen assay in the management of breast cancer: a preliminary report.

A monoclonal antibody (3E1.2) based serum test using an enzyme immunoassay has been used to determine circulating levels of the breast cancer associated antigen--mammary serum antigen (MSA). Of 157 patients with early breast cancer (stage I and II) and 199 patients with advanced breast cancer (stage III and IV), 73 per cent and 87 per cent respectively had elevated MSA levels (i.e. greater than 300 inhibition units (IU). Furthermore, 40 of 44 patients (91 per cent) had a significant fall of MSA levels with reduction in tumour load by mastectomy. In addition, there was a correlation of MSA levels with the clinical course: changes in MSA levels correlated with changes in disease status (progressive disease, stable disease, disease regression) in 54 of 61 patients and antedated disease progression or recurrence by up to 8 months in some patients; and in 32 of 36 patients (89 per cent) with no clinical evidence of recurrence MSA levels did not vary by more than 25 per cent of the original MSA value over a period of 2-15 months. MSA is therefore a useful tumour marker in the diagnosis and staging of breast cancer. There is also evidence that serial estimations of MSA levels may be used to detect subclinical recurrence and the fluctuations in MSA levels might be useful in assessing response to therapy. Furthermore, it was also noted that surgical procedures such as fine needle aspiration biopsy or incisional biopsy could lead to a rise in MSA levels.

Adult↗
PubMed1988

Evaluation of MSA as a serum marker in breast cancer: a comparison with CEA.

In a blind study, 518 serum samples were assayed for serum levels of mammary serum antigen (MSA) by an enzyme immunoassay (EIA) using the 3E1.2 monoclonal antibody. Using 300 IU as the arbitrary cut off to distinguish normal from abnormal individuals, 75% of patients with primary Stage I carcinoma of the breast (n = 12), 89% of those with Stage II (n = 9) and 93% of those with Stage IV (n = 57) had elevated levels of MSA. A relationship was observed between the level of MSA and stage of disease, and therefore with the extent of tumour burden. Levels of MSA were also determined in a series of 19 patients undergoing chemotherapy for breast cancer. Over a 2-24 month period, the change of MSA levels corresponded with the clinical course of the disease in 17 (89%) cases. MSA levels were also raised in some patients with ovarian, colon, lung and kidney cancer, but the average level was lower than in patients with breast cancer. A comparison of CEA and MSA levels in these patients revealed that MSA was a substantially better marker for breast cancer than CEA. The results of this study demonstrate that MSA levels are elevated in patients with breast cancer and may provide a useful means of following the clinical course of patients with this disease.

Antigens, Neoplasm↗
PubMed1987

Comparison of mammary serum antigen (MSA) and CA15-3 levels in the serum of patients with breast cancer.

Serum levels of mammary serum antigen (MSA) and CA15-3 were evaluated in 135 individuals in order to determine their single and combined value in the diagnosis and monitoring of breast cancer. Raised MSA levels (greater than 300 IU) were found in 68% of patients with Stage I and II breast cancer compared to only 3% having raised CA15-3 levels (greater than 40 U ml-1). Of 38 patients with Stage IV breast cancer, 95% had raised levels of MSA and CA15-3 combined with each test individually detecting 82% of those with Stage IV disease. No correlation was found between MSA and CA15-3 levels. Four patients being treated for breast cancer were followed over a 5-17 week period; MSA levels correlated with disease course in 3 and CA-15 in 2. The overall sensitivity, specificity and accuracy in detecting breast cancer were 76%, 91% and 96% for MSA; and 47%, 95% and 97% for CA15-3 respectively. When both tests were used together combined evaluation gave the highest sensitivity (84%) and specificity (100%). MSA seems to be superior to CA15-3 for early breast cancer diagnosis and a combination of the two tests gave the best results for metastatic disease.

Antigens, Neoplasm↗
PubMed1987

Mammary serum antigen (MSA) levels as a marker for breast cancer: a survey of blood donors and patients with cancer.

A murine monoclonal antibody (3E1.2) to human breast carcinoma cells was made. The antibody, which was selected for its ability to react with formalin fixed sections of the immunizing tissue, detects an antigen in the serum of breast cancer patients which we have called Mammary Serum Antigen. Immunoperoxidase staining has shown that there is an increased expression of this antigen on malignant breast epithelium compared to normal breast epithelium and other normal tissues. An assay (a serum inhibition enzyme immunoassay) using this antibody has been established to determine levels of MSA in patients' sera. Two independent studies involving greater than 2,000 blood donors and greater than 400 patients with breast cancer have shown that MSA is elevated in patients with carcinoma of the breast. MSA levels were found to be raised in approx. 2% of blood donors, approx. 55% of patients with localised breast cancer (Stage I and II) and in approx. 85% of patients with advanced breast cancer (Stage III and IV). Raised levels were also found in some patients with benign breast disease and other tumors, however these were generally much lower than those found in breast cancer patients. MSA levels may therefore be of some use for the monitoring of breast cancer patients, and as a diagnostic aid to screen populations for breast cancer.

Antigens, Neoplasm↗
PubMed1985

A new breast carcinoma antigen defined by a monoclonal antibody.

A new monoclonal antibody (MoAb), 3E1-2, to human breast carcinoma cells was made. With the use of the immunoperoxidase technique, 3E1-2 was tested on Formalin-fixed and fresh sections of 27 normal and 81 neoplastic tissues, including 37 carcinomas of the breast, 15 lung tumors, 5 colon tumors, and other tumors. Strong uniform staining of the cytoplasm and membrane occurred with the breast carcinoma, whereas with normal breast tissue less intense staining of the luminal membrane was seen; not all cells were reactive with the MoAb. Most other human tumors (with the exception of some lung, kidney, and uterine carcinomas) were nonreactive, and few normal tissues were reactive. The unique features of this new MoAb are: a) reaction with Formalin-fixed as well as fresh tissue; b) lack of a reaction with the cell surface of 43 established cell lines, including 10 lines derived from breast carcinoma cultures; c) variable staining patterns in different breast carcinomas, varying from all cells staining to dense cytoplasmic staining to minimal membrane staining of a few cells; d) a great differential in staining patterns between normal and neoplastic tissue (nonetheless, some normal tissues were 3E-1.2+). The antibody does not detect a tumor-specific antigen, but has a high carcinoma-to-normal breast ratio of staining. In addition, preliminary studies on the sera of 20 patients with carcinoma of the breast have shown that the antigen detected by 3E1-2 is elevated in their serum; 3E1-2 thus has the potential to be used for diagnosis of this disease.

Animals↗