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Biomedical subjects

I F McKenzie

Publications and source records attributed to I F McKenzie.

At least 19 recordsLinked to original sources
PubMed1999

Expression of mucin 1 (MUC1) in esophageal squamous-cell carcinoma: its relationship with prognosis.

Using 2 anti-mucin 1 (MUC1) monoclonal antibodies (MAbs), DF3 and BCP8, we examined MUC1 expression immunohistochemically in 192 esophageal squamous-cell carcinomas (SCCs). In normal squamous epithelium of the esophagus, DF3 was not expressed, but BCP8 was expressed on the cell membrane, mainly in the surface layer. In esophageal SCCs, DF3 and BCP8 were expressed mainly on the cell membrane of SCC cells, but also in the cytoplasm in several cases. To analyze the correlation of MUC1 expression and the prognosis of the patients, the 192 cases were divided into 2 groups: high-expression group (HEG, > 50% of the neoplastic cells stained) and low-expression group (LEG, < 50% of neoplastic cells stained). DF3-HEG (24 patients) showed a significantly poorer survival rate than DF3-LEG (168 patients), whereas there was no significant difference in survival between BCP8-HEG (43 patients) and BCP8-LEG (149 patients). Also, in the analysis of 162 patients with advanced stage (submucosal or deeper invasion) to exclude the influence of low expression of DF3 and BCP8 in 30 patients with early stage (up to the level of muscularis mucosae), DF3-HEG (24 patients) showed significantly poorer survival than DF3-LEG (138 patients), whereas there was no significant difference in survival between BCP8-HEG (42 patients) and BCP8-LEG (120 patients). The results of our study on esophageal SCC suggest that the expression of sialyl oligosaccharides detected by DF3 is related to poor prognosis.

Carcinoma, Squamous Cell↗
PubMed1999

Induction of humoral and cellular responses in cynomolgus monkeys immunised with mannan-human MUC1 conjugates.

Mice immunised with oxidised mannan conjugated to the human mucin 1 (MUC1), produce MHC Class 1 restricted CD8+ cytotoxic T-cells which eradicate MUC1 + tumours, indicating potential for the immunotherapy of MUC1 + cancers in humans. We now describe preclinical studies performed in cynomolgus monkeys immunised with human or murine MUC1 conjugated to oxidised mannan, where immune responses and toxicity were examined. High titred antibodies specific for MUC1 were produced, MUC1 specific CD4+ and CD8+ T-cell proliferative responses and specific cytotoxic precursor cells (CTLp) were found, but not MUC1 specific cytotoxic T-cells (CTL). There was no toxicity and monkeys can be immunised against human MUC1 with mannan-MUC1 conjugates, but a humoral response (Th2 type) predominates. The results contrast with those obtained in mice when a CTL response (Th1 type) predominates.

Amino Acid Sequence↗
PubMed1999

Fine structure analysis of interaction of FcepsilonRI with IgE.

The high affinity receptor for IgE (FcepsilonRI) plays an integral role in triggering IgE-mediated hypersensitivity reactions. The IgE-interactive site of human FcepsilonRI has previously been broadly mapped to several large regions in the second extracellular domain (D2) of the alpha-subunit (FcepsilonRIalpha). In this study, the IgE binding site of human FcepsilonRIalpha has been further localized to subregions of D2, and key residues putatively involved in the interaction with IgE have been identified. Chimeric receptors generated between FcepsilonRIalpha and the functionally distinct but structurally homologous low affinity receptor for IgG (FcgammaRIIa) have been used to localize two IgE binding regions of FcepsilonRIalpha to amino acid segments Tyr129-His134 and Lys154-Glu161. Both regions were capable of independently binding IgE upon placement into FcgammaRIIa. Molecular modeling of the three-dimensional structure of FcepsilonRIalpha-D2 has suggested that these binding regions correspond to the "exposed" C'-E and F-G loop regions at the membrane distal portion of the domain. A systematic site-directed mutagenesis strategy, whereby each residue in the Tyr129-His134 and Lys154-Glu161 regions of FcepsilonRIalpha was replaced with alanine, has identified key residues putatively involved in the interaction with IgE. Substitution of Tyr131, Glu132, Val155, and Asp159 decreased the binding of IgE, whereas substitution of Trp130, Trp156, Tyr160, and Glu161 increased binding. In addition, mutagenesis of residues Trp113, Val115, and Tyr116 in the B-C loop region, which lies adjacent to the C'-E and F-G loops, has suggested Trp113 also contributes to IgE binding, since the substitution of this residue with alanine dramatically reduces binding. This information should prove valuable in the design of strategies to intervene in the FcepsilonRIalpha-IgE interaction for the possible treatment of IgE-mediated allergic disease.

Animals↗
PubMed1999

Target cell susceptibility to lysis by human natural killer cells is augmented by alpha(1,3)-galactosyltransferase and reduced by alpha(1, 2)-fucosyltransferase.

Susceptibility of porcine endothelial cells to human natural killer (NK) cell lysis was found to reflect surface expression of ligands containing Gal alpha(1,3)Gal beta(1,4)GlcNAc [corrected], the principal antigen on porcine endothelium recognized by xenoreactive human antibodies. Genetically modifying expression of this epitope on porcine endothelium by transfection with the alpha(1,2)-fucosyltransferase gene reduced susceptibility to human NK lysis. These results indicate that surface carbohydrate remodeling profoundly affects target cell susceptibility to NK lysis, and suggest that successful transgenic strategies to limit xenograft rejection by NK cells and xenoreactive antibodies will need to incorporate carbohydrate remodeling.

Animals↗
PubMed1999

Definition and characterization of chicken Gal alpha(1,3)Gal antibodies.

BACKGROUND: The Gal alpha(1,3)Gal epitope is of interest as, in pig-to-primate xenotransplantation, it is the major target of naturally occurring human IgM and IgG antibodies, leading to hyperacute rejection. Human and Old World monkeys make anti-Gal alpha(1,3)Gal antibodies as they lack a functional gene and do not express Gal alpha(1,3)Gal. Interestingly, the cultured fibroblasts of some other species, such as chickens, have been reported also not to express Gal alpha(1,3)Gal--if this is true for other tissues, and chickens do not express Gal alpha(1,3)Gal antigen, then they would have anti-Gal antibodies--which could have diagnostic and therapeutic value, particularly as chicken antibodies do not fix mammalian complement. METHODS: Standard serological methods were used to characterize the antibodies. Several baboons received pig kidney xenografts that had been perfused with hyperimmune chicken anti-Gal antibodies. RESULTS AND CONCLUSIONS: We now demonstrate that chickens do not express Gal alpha(1,3)Gal on their red cells, leukocytes, or tissues, and that their serum contains large amounts of anti-Gal alpha(1,3)Gal antibodies. In addition, chickens could be immunized to produce high-titer, high-avidity antibodies (9.5x10(9) M(-1))--an avidity considerably greater than that of the Gal alpha(1,3)Gal binding lectin IB4 (2.9x10(8) M(-1)) or Gal antibodies in human serum (2.2x10(5) M(-1)). Chicken antibodies, obtained from both normal and immunized chickens, could block the in vitro cytolysis of pig endothelial cells or lymphocytes by human or baboon antibodies. However, such antibodies tested in vivo in pig-to-baboon xenotransplantation failed to block hyperacute rejection and, indeed, may have accelerated this.

Animals↗
PubMed1999

High-level porcine endothelial cell expression of alpha(1,2)-fucosyltransferase reduces human monocyte adhesion and activation.

BACKGROUND: Monocyte binding to and activation by human endothelium requires a number of interactions, including those involving sialylated endothelial cell ligands. As porcine endothelial cell transfection with alpha(1,2)-fucosyltransferase has been shown to reduce terminal sialylation, we investigated whether high-level expression of alpha(1,2)-fucosyltransferase by porcine endothelium would reduce human monocyte adhesion and functional activation. METHOD: Purified human monocytes were labeled with 51Cr, and measured for adherence to human or porcine endothelial cell monolayers in the presence of either medium or monoclonal antibodies against monocyte lectins or sialylated endothelial cell ligands. Monocyte production of prostaglandin E2 (PGE2) and interleukin-1beta (IL-1beta) was measured by enzyme-linked immunosorbent assay, using supernatants collected from cultures performed between human monocytes and human or porcine endothelial cell monolayers. Finally, monocyte adhesion and activation were measured after culture with a porcine endothelial cell line transfected with alpha(1,2)-fucosyltransferase, expressing reduced surface expression of terminal Gal alpha(1,3)-Gal and sialic acid residues. RESULTS: Human monocytes adhered by 50% higher levels to porcine endothelium than to human endothelium. This increased level of adherence was associated with augmented monocyte activation, as defined by 3.3-fold higher levels of PGE2 production and 7.3-fold higher levels of IL-1beta production. Monoclonal antibodies against CD62L (L-selectin) on monocytes or CD15s (sialylated Lewis X) on porcine endothelium reduced monocyte adhesion by 38% and 52%, respectively. Porcine endothelial cell transfection with alpha(1,2)-fucosyltransferase reduced terminal sialic acid expression by 65%, monocyte adherence by 50%, and the production of PGE2 and IL-1beta by 67% and 38%, respectively. CONCLUSIONS: Together, these results demonstrate that human monocytes use surface lectins to bind to sialylated carbohydrate structures on porcine endothelium, and indicate that reduction in porcine endothelial cell surface expression of terminally sialylated structures by high-level alpha(1,2)-fucosyltransferase activity reduces monocyte adherence and activation.

Animals↗
PubMed1999

Carbohydrate/peptide mimics: effect on MUC1 cancer immunotherapy.

Recent clinical studies with mannan mucin immunotherapeutic agents indicate that patients produce predominantly antibody responses while mice produce a high cytotoxic T lymphocyte response. In studying the reason for the 'immune deviation' occurring in mice to humans from cellular to antibody responses, it has been found that natural anti-Galalpha(1,3)Gal antibodies, present in all humans, react with the mucin component of the agent, providing an example of a carbohydrate-peptide mimic. The immune deviation can be overcome by in vitro sensitization of antigen-presenting cells in the absence of anti-Gal antibodies - at least in mice. The review examines the background of these observations and discusses other peptide carbohydrate mimics and immune deviation

Animals↗
PubMed1999

Crystal structure of the human leukocyte Fc receptor, Fc gammaRIIa.

Fc gamma receptors bind IgG to initiate cellular responses against pathogens and soluble antigens. We have determined the three-dimensional structure of the extracellular portion of human Fc gammaRIIa to 2.0 A resolution providing a structural basis for the unique functions of the leukocyte FcR family. The receptor is composed of two immunoglobulin domains and arranged to expose the ligand-binding site at one end of domain 2. Using alanine mutants we find that the binding sites for IgG1 and 2 are similar but the relative importance of specific regions on the receptor varies. In crystals, Fc gammaRIIa molecules associate to resemble V(L)V(H) dimers, suggesting that two Fc gammaRIIa molecules could cooperate to bind IgG in an asymmetric manner.

Amino Acid Sequence↗
PubMed1999

Molecular cloning and characterization of the pig secretor type alpha 1,2fucosyltransferase (FUT2).

The existence of at least two distinct alpha 1,2fucosyltransferases has been postulated for many years, and recently confirmed in humans with the cloning of the human and rabbit secretor type alpha 1,2fucosyltransferase. We now describe the cloning and analysis of PFUT2, the pig secretor type alpha 1,2fucosyltransferase, which shows a high level of amino acid identity with previously cloned alpha 1,2fucosyltransferases, but more so with human and rabbit FUT2. Expression of PFUT2 in COS cells showed cell surface staining for H substance with UEAI lectin and anti-H monoclonal antibody, but not for A blood group substance. Kinetic studies were consistent with PFUT2 having a preference for type 1 and type 3 acceptors, as do the human and rabbit homologues, in contrast to PFUT1 which shows a preference for type 2 substrates. Like HuFUT1 and PFUT1, PFUT2 was able to dominate over the pig alpha 1,3galactosyltransferase in co-expression studies in COS cells and give preferential expression of H substance and reduced expression of Gal alpha (1,3)Gal. Cotransfection studies demonstrate that a combination of FUT1 and FUT2 cDNAs has an additive effect in suppressing expression of Gal alpha (1,3)Gal.

Amino Acid Sequence↗
PubMed1998

Pig islet xenografts are susceptible to "anti-pig" but not Gal alpha(1,3)Gal antibody plus complement in Gal o/o mice.

Hyperacute rejection due to Galalpha(1,3)Gal (Gal) Ab plus complement (C') is a major problem in xenografting vascularized organs from pigs to primates, but the fate of neovascularized xeno islets is unclear. Nonendocrine islet cells are Gal+, and there is a large rise in Gal Abs after transplantation, but graft remnants persist for some days in monkeys and humans. To define the role of alphaGal Ab plus C' in porcine islet graft rejection, cultured porcine fetal islets were grafted to mice lacking the alpha(1,3)galactosyltransferase gene. Anti-Gal Ab plus C' did not cause islet damage or rejection in mice lacking the alpha(1,3)galactosyltransferase gene, even when additional Ab plus C' was given; in addition, hyperimmune mice (titer >1/ 20,000) did not reject pig islets, showing that islets are resistant to Gal Ab plus C'. However, islets can be destroyed by polyclonal mouse anti-pig Abs. Thus, the focus of islet xenografting should not be on Gal Ab plus C'.

Animals↗
PubMed1998

A murine model of antibody-mediated hyperacute rejection by galactose-alpha(1,3)galactose antibodies in Gal o/o mice.

BACKGROUND: In pig-to-primate/human xenografts, hyperacute rejection of primarily vascularized organs usually occurs in 10-60 min and is due to the reaction of the recipients' natural antibodies with antigens expressed on the donor endothelium, the fixation of complement, and ultimately vascular stasis and hemorrhage. Surprisingly, the major target of the natural antibodies is the disaccharide galactose-alpha(1,3)galactose (Gal alpha(1,3)Gal), which is found on many different molecules in pig tissues and reacts with naturally occurring human anti-pig IgM and IgG antibodies. There are a number of strategies to remove/block/alter Gal alpha(1,3)Gal expression in pig tissues, all of which involve the expression of transgenes in pigs. To overcome the difficulty of preclinical studies using primates, we describe a model of hyperacute rejection of heart transplants to Gal o/o mice, which are similar to humans in that they have anti-Gal alpha(1,3)Gal antibodies. METHODS: Gal o/o mice received skin or heart grafts from Gal+ mice or rats, and additional antibody and complement were provided; hyperacute rejection was monitored by observation and histology. RESULTS: Gal alpha(1,3)Gal+ mouse tissues (skin or heart) are not rejected by Gal o/o mice. This was not unexpected, as mice do not utilize alloantibody/complement systems satisfactorily in experimental transplantation studies. However, with the addition of anti-Gal alpha(1,3)Gal antibody and complement, hyperacute rejection of hearts can occur in 10-20 min; it is mediated by IgM, not IgG, antibodies and leads predominantly to tissue hemorrhage. CONCLUSION: Gal alpha(1,3)Gal antigen modification by expression of the H transferase cDNA leads to "indefinite" survival (>120 min) and no hyperacute rejection, which shows that this model is suitable for the study of antibody-mediated rejection of relevance to pig-to-human xenografts.

Acute Disease↗
PubMed1998

Anti-MUC1 antibodies react directly with MUC1 peptides presented by class I H2 and HLA molecules.

Peptides bound in the groove of MHC class I molecules and detected by CTLs are not normally accessible to Ab. We now report that MUC1 peptides that are bound within the groove of MHC class I molecules (H2 and HLA) and that can be detected by CTLs can also be detected by anti-MUC1 Abs. mAbs to the middle and C-terminal regions of the class I-associated peptides but not to the N terminus were able to react with MUC1 peptides bound to H2Kb and HLA-A*0201, and only to the mid-region for H2Db, by flow cytometry and also to block CTL activity. Molecular modeling showed that the N terminus is buried (and not accessible), whereas the midpeptide residues form a loop and the C terminus is free, making these two regions accessible to Ab. The findings demonstrate for the first time that peptides associated with class I molecules can be detected by anti-peptide Abs.

Amino Acid Sequence↗
PubMed1998

Mouse mucin 1 (MUC1) defined by monoclonal antibodies.

Mucins are highly expressed in many different human cancers and numerous murine monoclonal antibodies (MAbs) to human mucins, particularly Mucin 1 (MUC1), have been produced. However, no such antibodies to murine mucin 1 (muc1) have been described and we now describe 6 different antibodies produced to murine muc1 and to human MUC1 cytoplasmic tail, either by immunising rats, or muc1 o/o mice with synthetic peptides or a fusion protein composed of glutathione-s-transferase (GST) linked to the tandem repeat region of muc1. The antibodies to both the extracellular tandem repeat region and to the cytoplasmic tail were found to react with mucin-containing murine tissues such as breast, stomach, colon, ovary, kidney and pancreas, and the staining patterns were similar to those found in humans. The reagents reacted specifically with muc1 peptides and tissues; however, some cross reactivity with other mucin-derived peptides was noted, particularly those containing the amino acid sequence TSS. Three different epitopes (TSS, TAVLSGTS and LSGTSSP) of the M30, M70 and MFP25 MAbs were detected. Of interest was the finding that some of the antibodies reacted with murine lymphocytes; it was not clear whether these reactions were due to mucin 1 on mouse lymphocytes (MUC1 was considered to be absent from human lymphocyte), or due to cross reaction with a sialic adhesion molecule on lymphocytes. The antibodies should prove valuable reagents when studying differentiation and expression in murine glandular tissues and the ontogeny of mucin-secreting tumours.

Animals↗
PubMed1998

Oxidised mannan antigen conjugates preferentially stimulate T1 type immune responses.

It is desirable to be able to produce either T1 or T2 responses and we have found that, in mice, mannose--coupled antigens stimulated T2 type responses antibodies and CTLs, whereas if oxidized, mannose--coupled antigens stimulated T1 responses little antibody and a potent CTL response. In addition, the cytokine profiles support the T1rT2 differentiation with these immunizations, in that oxidized mannan antigen gives IFNg, IL-2 and IL-12 production, whereas in the absence of oxidization, IL-4 and not the other cytokines is produced. A number of antigens have been examined--particularly Mucin 1 and the delivery method using mannose may be applicable to the other antigens.

Animals↗
PubMed1998

Parameters for using mannan-MUC1 fusion protein to induce cellular immunity.

We have previously reported preclinical studies in mice of the human mucin 1 (MUC1) antigen covalently linked to the yeast cell-wall mannan polysaccharide (MFP), and shown strong cellular responses of the T1 type using mice. We now describe the optimum parameters for administration of MFP to obtain cellular immunity [as measured by the cytotoxic T cell precursor (CTLp) frequency]. In dose/response studies, in which 1 microg-150 microg was given by the i.p. route, it was clear that doses of 1-7 microg led to cellular and not humoral immunity; at doses above 7 microg humoral immunity prevailed with little cellular immunity increasing doses giving greater amounts of antibody. The most favoured routes of administration were intraperitoneal or intradermal immunisation, which were substantially better than i.m., i.v.; s.c. administration was the worst. Three immunisations were necessary for a maximum cellular response, further immunisation decreasing the CTLp frequency. Six different adjuvants were used with MFP [complete and incomplete Freund's adjuvant (CFA, IFA) Alum, Adjuprime, muramyl dipeptide (MDP) and glutaminyl-muramyl dipeptide (GMDP)]; Alum, GMDP, MDP and IFA moderately increased the CTLp frequency, IFA being the best. Even though preclinical studies of the immunogen in mice may not necessarily mirror the behaviour of the immunogen in humans, these studies demonstrate the factors to be taken into account for phase I/II clinical trials.

Animals↗