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Biomedical subjects

A M Lew

Publications and source records attributed to A M Lew.

108 records · Page 6Linked to original sources
PubMed1986

Secretion of a malarial histidine-rich protein (Pf HRP II) from Plasmodium falciparum-infected erythrocytes.

Plasmodium falciparum-infected erythrocytes (IRBCs) synthesize several histidine-rich proteins (HRPs) that accumulate high levels of [3H]histidine but very low levels of amino acids such as [3H]isoleucine or [35S]methionine. We prepared a monoclonal antibody which reacts specifically with one of these HRPs (Pf HRP II) and studied the location and synthesis of this protein during the parasite's intracellular growth. With the knob-positive Malayan Camp strain of P. falciparum, the monoclonal antibody identified a multiplet of protein bands with major species at Mr 72,000 and 69,000. Pf HRP II synthesis began with immature parasites (rings) and continued through the trophozoite stage. The Mr 72,000 band of Pf HRP II, but not the faster moving bands of the multiplet, was recovered as a water-soluble protein from the culture supernatant of intact IRBCs. Approximately 50% of the total [3H]histidine radioactivity incorporated into the Mr 72,000 band was extracellular between 2 and 24 h of culture. Immunofluorescence and cryothin-section immunoelectron microscopy localized Pf HRP II to several cell compartments including the parasite cytoplasm, as concentrated "packets" in the host erythrocyte cytoplasm and at the IRBC membrane. Our results provide evidence for an intracellular route of transport for a secreted malarial protein from the parasite through several membranes and the host cell cytoplasm.

Animals↗
PubMed1986

Characteristics of the expression of the murine soluble class I molecule (Q10).

Q10 is a class I molecule previously proven to be secreted rather than membrane bound. To measure the amount of Q10 in various mouse sera, a quantitative Western blot assay was developed. Q10 was the only class I molecule detectable in mouse sera. It occurs as a high m.w. complex of 200,000 to 300,000. The amount of Q10 in serum varies among different mouse strains and is controlled by a region telomeric to H-2S. Mice of the f haplotype do not express Q10, but all other mice examined (20 strains) with inbred or wild-derived H-2 haplotypes do. The H-2 haplotypes rank according to their levels of Q10 as follows: z, s greater than k, b greater than d, q greater than f; and the actual values range from to 60 micrograms/ml to undetectable levels in serum. In some strains the levels are higher in males than in females. The levels increase with age and decrease during pregnancy but not during lactation. There is a dramatic decrease after the injection of irritants or syngeneic tumor transplantation, but allostimulation has no effect on Q10 levels. The possible significance of this soluble class I molecule is discussed in the light of our findings.

Animals↗
PubMed1985

Secretion of a soluble class I molecule encoded by the Q10 gene of the C57BL/10 mouse.

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.

Animals↗
PubMed1985

Five groups of antigenic determinants on DNA identified by monoclonal antibodies from (NZB X NZW)F1 and MRL/Mp-lpr/lpr mice.

A library of monoclonal antibodies (MCA) reactive with DNA was derived from mice with lupus-like disease. The combining reactions of the antibodies was determined by ELISA, precipitation assay and indirect immunofluorescence assay on cells. On the basis of their reactions in these assays, the MCA have been classified into five taxonomic groups. MCA in Group I react with conformational determinants on double-stranded DNA (dsDNA); those in Group II with conformational backbone-dependent sugar-phosphate determinants on dsDNA and single-stranded DNA (ssDNA); those in Group III with determinants predominantly expressed on ssDNA; those in Group IV with base-dependent determinants on ssDNA, and those in Group V with determinants on both DNA and RNA. It is concluded that antibodies which react with DNA are collectively of limited heterogeneity with regard to their specificity. The study illustrates how, upon traditional interpretation, different assay systems may give discordant results in the assignment of specificity to antibodies reactive with DNA.

Animals↗
PubMed1984

Influence of antibody affinity on the performance of different antibody assays.

The effect of antibody affinity on the performance of 5 commonly used assays was studied. The assays used were measurement of antigen binding capacity in a Farr type assay, haemagglutination, solid-phase radioimmunoassay (SP-RIA), solid-phase ELISA and precipitation. The first 4 assays were all more sensitive for high affinity antibodies. Precipitation was not related to affinity, suggesting that factors secondary to antigen-antibody binding may be more important in determining the level of precipitate formation. The effect of epitope density of the antigen was also investigated in the SP-RIA and ELISA. Affinity dependence was more marked when antigen of low epitope density was used and this dependence could be reduced in the ELISA by choosing a low OD endpoint. Thus, the most reliable way to estimate antibody content by the ELISA may be to determine a low OD endpoint titre against antigen of high epitope density. When epitope density per molecule cannot be increased, an alternative approach to the problem is to increase epitope density by covalent coupling of antigen to the solid-phase rather than by adsorption.

Animals↗
PubMed1984

Localization of covalent immune complexes on the epithelial side of the glomerular basement membrane in mice.

The site of immune complex localization in human glomerulonephritis is important in determining the kind of disease that may develop. Immune complex deposits found on the subepithelial side of the glomerular basement membrane are characteristic of membranous nephropathy, a common and often severe form of human renal disease. The pathogenesis of this disease remains controversial and arguments have recently been put forward that the subepithelial deposits arise from an in situ mechanism. This mechanism involves the initial localization of antigen allowing the antibody to subsequently combine, i.e. the immune complex is formed locally. This mechanism is thus in contrast to that in which immune complexes formed in the circulation are localized in the glomeruli. Covalent immune complexes do not dissociate and hence are ideal tools for studies to investigate whether complexes are able to cross the glomerular basement membrane. Covalent immune complexes of defined size and antigen-antibody ratio were prepared with a monoclonal antibody and photoaffinity labelling antigen, 4-azido-2-nitro-phenylated bovine serum albumin. When these complexes were injected into mice, complexes of 550,000 MW molecular weight (but not those of 800,000 MW) were shown by electron microscopic autoradiography to localize on the subepithelial side of the glomerular basement membrane. It is thus proposed that small circulating immune complexes may be important in the pathogenesis of subepithelial deposits and there is a need for devising reliable tests for measuring small immune complexes.

Animals↗
PubMed1984

Glomerulonephritis: the use of grafted hybridomas to investigate the role of epitope density, antibody affinity and antibody isotype in active serum sickness.

Two experimental models were used to investigate the role of antigen epitope density, antibody isotype and antibody affinity in immune-complex glomerulonephritis. In both models, two antigens were used: dinitrophenylated bovine serum albumin of high and low substitution ( DNP19BSA and DNP4BSA , respectively). In the first, acute actively induced model, a single intravenous injection of either of the two antigens was given to mice bearing hybridomas that secreted one of five monoclonal antibodies--four IgGl with varying affinities for the dinitrophenol hapten, and an IgM antibody with a similar affinity to that of the lowest affinity IgGl. The second, chronic actively induced model was similar, except that mice were immunosuppressed to reduce interference by the hosts' antibody responses and that 15 daily intraperitoneal injections of antigen were given. The immunofluorescent findings in both models were similar. Glomerular deposition of antibody and antigen in mice with high affinity IgG or low affinity IgM antibodies was mesangial when DNP4BSA or DNP19BSA was injected. However in mice with the lowest affinity IgG antibodies, deposition was extensive and predominantly capillary when DNP19BSA was injected but was minimal when DNP4BSA was injected. The relevance of these findings to the pathogenesis of glomerulonephritis is discussed.

Acute Disease↗
PubMed1984

Glomerulonephritis induced by pre-formed immune complexes containing monoclonal antibodies of defined affinity and isotype.

The work presented here represents the first report of the induction of experimental immune complex (IC) disease in mice using monoclonal antibodies (MoAb) derived from somatic cell hybridization. IC were formed using two antigens of either high (DNP19BSA) or low (DNP4BSA) epitope density and five MoAb (four IgGl with varying affinities for the dinitrophenol hapten and one IgM with a similar affinity to that of the lowest affinity IgGl). Circulating levels and sizes of IC were dependent on the affinity of the antibody component of the complex. When antigen of high epitope density was used, the glomerular localization of injected IC was diffuse mesangial for the IgM antibody, focal mesangial for the highest affinity IgG and diffuse, and predominantly capillary for the low affinity IgG antibodies. Subepithelial electron dense deposits were observed only with IC made with the low affinity IgG antibodies. When IC containing antigen of a lower epitope density were injected, localization was only observed with IC made near equivalence. Deposition of these IC was less prominent than that found when IC containing antigen of higher epitope density were injected. The relevance of these findings to the pathogenesis of glomerulonephritis is discussed.

Animals↗
PubMed1983

The measurement of antibody affinity: a comparison of five techniques utilizing a panel of monoclonal anti-DNP antibodies and the effect of high affinity antibody on the measurement of low affinity antibody.

The affinities of 9 IgG1 monoclonal anti-dinitrophenol (DNP) antibodies for 3H-epsilon-DNP-L-lysine, 125I-HOP-DNP-L-lysine and 125I-DNP-human serum albumin (HSA) were determined. 3H-DNP-lysine was used in equilibrium dialysis and ammonium sulphate globulin precipitation assays; 125I-HOP-DNP-lysine was used in equilibrium dialysis and polyethylene glycol precipitation; and 125I-DNP5-HSA in the polyethylene glycol precipitation assay for affinity. The ranking order of the monoclonal antibodies in terms of affinity by the assays was significantly correlated. Of particular importance was the observation that the simple and widely applicable globulin precipitation assay utilizing a protein antigen produced affinity values which showed concordance with the least equivocal but cumbersome assay, equilibrium dialysis. Mixing of antibodies of high and low affinity demonstrated that even a low proportion of high affinity antibody had marked effect on measurements of the amount and affinity of a predominantly low affinity antibody preparation.

Animals↗
PubMed1980

Immunologic aspects of combined immunodeficiency disease in Arabian foals.

Tests for T- and B-cell quantitation and immune function were developed, and their application in the diagnosis of primary severe combined immunodeficiency disease (CID) in Arabian foals was investigated. Foals with CID had severe lymphopenia and had small or zero numbers of B cells, as shown by immunofluorescence of surface immunoglobulin (Ig), erythrocyte-antibody-complement rosetting, and staphylococcal protein A rosetting. Serum IgM was undetectable in four CID foals 25 to 71 days old. Demonstrable antibody responses were not elicited in CID foals by phage phi X-174, a potent antigen in normal foals. Nonspecific esterase (NSE) staining in the pattern of a single vesicle was investigated as a possible marker for equine T cells. For normal foals, 64.0% of peripheral blood lymphocytes stained NSE positive. The CID foal 1 had only 4.0% NSE-positive lymphocytes, whereas CID foals 2, 3, and 4 had 75%, 68%, and 77.5%, respectively. In an in vitro T-cell function test, lymphocytes from 12 normal foals did not show a response. In normal foals, intradermal injection of 50 micrograms of phytohemagglutinin induced visible reactions, and skin grafting induced a pronounced mononuclear cell response at the base of the graft. In contrast, there was little or no response in the foals with CID.

Animals↗
PubMedDate not supplied

Enhanced survival of grafts genetically endowed with the ability to block CD2 and B7.

In a model of transplantation rejection, we have tested whether a graft manipulated to secrete immunomodulators could protect itself from immune destruction. An insulinoma cell line having the NOD genotype but also expressing the neoantigen, SV40 T antigen, was transfected with CTLA4Ig or LFA3Ig to block signals in the co-stimulatory/adhesion pathways. This neoantigen is potent at inducing graft rejection. Secretion of CTLA4Ig and LFA3Ig by transfectants promoted survival of the insulinoma graft in young NOD mice. In immunodeficient mice, cell growth was similar for all transfectants. However, in immunocompetent NOD mice the survival/growth of test grafts was significantly better than that of the controls. Graft survival was enhanced additively, when the two test transfectants were cotransplanted. Endowing the graft the ability to secrete immunomodulators that block individual co-stimulatory/adhesion signals can contribute to transplantation success. Blockade of two signals (CD2 and CD28) in these pathways enhances this success.

Abatacept↗
PubMedDate not supplied

Protection of xenografts by a combination of immunoisolation and a single dose of anti-CD4 antibody.

Immunoisolation is the separation of transplanted cells from cells of the immune system using a semipermeable membrane. Using one such immunoisolation capsule-the TheraCyte device-we have assessed the survival of encapsulated xenogeneic tissue in vivo as well as the contribution of CD4+ve T cells to encapsulated xenograft rejection. The foreign body reaction to the TheraCyte capsule in vivo was assessed by transplanting empty capsules into normal mice. These capsules elicit a foreign body response by the host animal. Encapsulated CHO, NIT-1, and PK-15 cells were placed in culture and in immunodeficient mice to investigate their growth characteristics in the TheraCyte device. These cell lines survive both in culture and in immunodeficient SCID mice. Xenogeneic PK cells were also transplanted into normal C57BL/6 mice. These cells do not survive in normal mice despite the absence of direct contact between infiltrating and encapsulated cells. In addition, the survival of encapsulated cells in mice treated with a single dose of anti-CD4 antibody was examined. This was assessed using two systems: 1) histological analysis of capsule sections; 2) a quantitative luciferase reporter system using PK cells transfected to express luciferase. In both cases, anti-CD4 antibody contributed to prolonged encapsulated xenogeneic cell survival. Encapsulated xenogeneic cells survive in immunodeficient mice but not normal mice. Treatment of normal mice with anti-CD4 antibody results in prolonged survival of xenogeneic cells that can be measured using a luciferase reporter system. These results highlight the contribution of CD4+ve T cells to encapsulated xenograft rejection.

Animals↗