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Biomedical subjects

E P Lillehoj

Publications and source records attributed to E P Lillehoj.

At least 19 recordsLinked to original sources
PubMed2001

Molecular, cellular, and functional characterization of chicken cytokines homologous to mammalian IL-15 and IL-2.

DNA sequence analysis of a chicken interleukin (IL)-15 cDNA identified a 187 amino acid open reading frame encoding a protein with a predicted molecular weight of 21,964Da, two potential N-linked glycosylation sites, four highly conserved Cys residues, two out-of-frame AUG initiation codons in the 5' untranslated region, and an unusually long (66 amino acid) signal peptide such that the expected size of the mature protein is 14,462Da. Chicken IL-15 and IL-2 were compared with regard to their molecular, cellular, and functional characteristics. The predicted amino acid sequences of both chicken cytokines showed greater homologies with mammalian IL-15s compared with mammalian IL-2s. Northern hybridization and RT-PCR demonstrated chicken IL-15 gene transcripts in a wide variety of tissues and cell types while the chicken IL-2 gene was expressed only in concanavalin A (con A)-activated spleen cells. Both recombinant cytokines stimulated the growth of spleen T-cells and enhanced the activity of natural killer (NK) cells in vitro. Subcutaneous injection with an expression plasmid encoding IL-15 increased the percentage of CD3+ spleen T-lymphocytes whereas injection of an IL-2 cDNA augmented CD3+, CD4+, CD8+, T-cell receptor (TCR)1+, and TCR2+ T-cells. Collectively, these results indicate that chicken IL-15 and IL-2 are T-cell growth factors potentially capable of enhancing cell-mediated immunity in vivo.

Amino Acid Sequence↗
PubMed2001

Muc1 mucins on the cell surface are adhesion sites for Pseudomonas aeruginosa.

Recently, we cloned and characterized a full-length cDNA of the hamster Muc1 gene, the expression of which appears to be associated with secretory cell differentiation (Park HR, Hyun SW, and Kim KC. Am J Respir Cell Mol Biol 15: 237-244, 1996). The role of Muc1 mucins in the airway, however, is unknown. In this study, we investigated whether cell surface mucins are adhesion sites for Pseudomonas aeruginosa. Chinese hamster ovary (CHO) cells not normally expressing Muc1 mucin were stably transfected with the hamster Muc1 cDNA, and binding to P. aeruginosa was examined. Our results showed that 1) stably transfected CHO cells expressed both Muc1 mRNA and Muc1 mucins based on Northern and Western blot analyses, 2) Muc1 mucins present on the cell surface were degraded by neutrophil elastase, and 3) expression of Muc1 mucins on the cell surface resulted in a significant increase in adhesion of P. aeruginosa that was completely abolished by either proteolytic cleavage with neutrophil elastase or deletion of the extracellular domain by mutation. We conclude that Muc1 mucins expressed on the surface of CHO cells serve as adhesion sites for P. aeruginosa, suggesting a possible role for these glycoproteins in the early stage of airway infection and providing a model system for studying epithelial cell responses to bacterial adhesion that leads to airway inflammation in general and cystic fibrosis in particular.

Animals↗
PubMed2000

Vaccines against the avian enteropathogens Eimeria, Cryptosporidium and Salmonella.

The worldwide poultry industry provides a substantial proportion of the nutritional requirement of the human population. To keep pace with the increasing demand for the high-quality, low-cost protein source that poultry provides, intensive rearing practices have been developed within the past few decades. For example, chickens are housed routinely in crowded environments under adverse conditions, and genetic strains have been selected for rapid growth, high protein-to-fat content and superior egg-laying characteristics. A major negative consequence of these practices has been an increase in the incidence of diseases. Enteric diseases in particular have emerged as a major problem threatening the future viability of the poultry industry. A variety of methods have been used to combat avian diseases in the commercial setting, including improved farm management practices, the use of antibiotic drugs, the selection of disease-resistant strains of chickens, and the manipulation of the chicken's immune system. In the latter category, the development of vaccines against the major avian diseases has become a priority in the poultry industry. This review will highlight recent progress in vaccine development against three major avian enteric pathogens: Eimeria, Cryptosporidium and Salmonella.

Animal Husbandry↗
PubMed2000

Serum antibodies reactive with non-human primate retroviruses identified in acute onset schizophrenia.

Schizophrenia is a pervasive neuropsychiatric disease of uncertain etiology. Previous studies have postulated that retroviruses may contribute to the etiology of some cases of schizophrenia. We examined the possible relationship between retroviral infection and schizophrenia by measuring antibodies to a number of different primate retroviruses in the sera of individuals undergoing their first hospitalization for this disease. Sera from patients with first onset schizophrenia and matched healthy controls were analyzed by immunoblot and enzyme linked immunosorbent assays using purified retrovirus antigens to identify and quantify antibodies reactive with retrovirus proteins. A significantly increased incidence of antibodies reactive to gag encoded proteins of Mason-Pfizer monkey virus (MPMV), baboon endogenous virus (BaEV) and simian retrovirus type 5 (SRV-5) was observed in the sera of schizophrenia patients compared to controls. The reactivity of the cases and controls displayed the greatest differences in terms of antibodies to the proteins of Mason-Pfizer monkey virus. Employing an algorithm of enzyme linked immunosorbent assay reactivity followed by immunoblot confirmation, we found that MPMV antibodies in 28.9% of the individuals with first episode schizophrenia patients as compared to 3.7% of the unaffected controls (P<0.009, Fisher's Exact Test). These studies are consistent with the occurrence of retrovirus replication in some individuals who are undergoing their first episode of schizophrenia.

Acute Disease↗
PubMed1993

Chicken macrophages and thrombocytes share a common cell surface antigen defined by a monoclonal antibody.

A monoclonal antibody (mAb), designated K1, reacted with a cell surface antigen shared on chicken macrophages and thrombocytes. By immunofluorescence staining, the mAb K1 was reactive with 31.8% of peripheral blood lymphocytes (PBL) separated on Histopaque. In contrast, only 3.2% of PBL separated by slow-speed centrifugation were K1 positive. This antibody did not react with B- or T-lymphocytes, as demonstrated by the very small percentage of positive cells in thymus, bursa and spleen. Furthermore, no staining was observed with avian T-cell (MDCC-RP1 and SK3) or B-cell (LSCC-RP9) lines. Adherent cells derived from PBL separated on Histopaque and cultured for 48 h in plastic cell culture dishes were 81.5% positive with K1. These cells were also 82.6% positive with an antibody detecting the major histocompatibility complex (MHC) Class II antigen in chickens, indicating that they were monocyte-derived macrophages. Sheep red blood cell phagocytosis by these cells could be demonstrated, further supporting their macrophage lineage. In addition, K1 stained virtually 100% of HD11 cells, a chicken macrophage cell line, as well as 86.7% of peritoneal exudate cells. Eighty five percent of plastic adherent cells from PBL collected after 2 h of adherence reacted with the mAb K1, but only 8% of these cells were MHC Class II positive. These cells were morphologically identified as thrombocytes. Immunoprecipitation analysis demonstrated that the K1-reactive antigen consisted of a heterodimer with constituent polypeptide chains of 135 kDa and 61-68 kDa.

Animals↗
PubMed1992

Virion-associated trans-regulatory protein of human T-cell leukemia virus type I.

Western blot analysis of HTLV-I virus particles from HUT-102 cells revealed a 40-kD protein strongly reactive with Tax-specific rabbit antisera. This protein subsequently was isolated from density gradient purified virions by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), purified from comigrating Gag and human cellular proteins by reversed-phase high-performance liquid chromatography (HPLC) and identified as the tax-encoded gene product by amino acid composition analysis. Among extracellular virions from five HTLV-I producing cell lines, only those from HUT-102 and C10MJ cells contained a detectable Tax protein, although all cells expressed Tax mRNA and protein intracellularly. To investigate the diagnostic implications of virion-associated Tax protein, sera from HTLV-I-infected individuals were compared on HUT-102 and MT-2 virus Western blots. The seroprevalence of antibodies to Tax, but not Gag or Env proteins, was substantially higher among adult T-cell leukemia and tropical spastic paraparesis patients using HUT-102 viral proteins. Thus, immunoassays utilizing HUT-102 virus are most sensitive for detection of Tax-reactive antibodies.

Amino Acids↗
PubMed1990

Development and evaluation of a human T-cell leukemia virus type I serologic confirmatory assay incorporating a recombinant envelope polypeptide.

A recombinant protein derived from the gp21 region of the human T-cell leukemia virus type I (HTLV-I) env gene was synthesized in Escherichia coli and purified by reversed-phase high-performance liquid chromatography. The purified protein was free of contaminating bacterial proteins and retained reactivity with human HTLV-I- and HTLV-II-positive sera and a gp21 monoclonal antibody. An immunoblot procedure using the recombinant polypeptide in conjunction with native viral proteins was more sensitive than the conventional immunoblot and radioimmunoprecipitation confirmatory assays for detection of antibodies to HTLV-I and HTLV-II env-encoded gene products. The recombinant protein was equally reactive with sera from polymerase chain reaction-confirmed HTLV-I or HTLV-II infections. Furthermore, on the basis of the differential reactivities of gp21-positive sera with the HTLV-I p19 and p24 gag-encoded proteins, an algorithm was proposed to distinguish exposure to HTLV-I from exposure to HTLV-II. These results establish the utility of a modified immunoblot assay incorporating a recombinant envelope polypeptide as an alternative to existing HTLV-I-confirmatory assays.

Antibodies, Viral↗
PubMed1989

Protein purification.

This monograph summarizes recent developments in the purification and analysis of natural and recombinant proteins. The basic strategies employed in protein purification are reviewed with regards to the characteristics of the protein of interest that may aid its isolation, choice of the starting material, and use of denaturants. Preparation of cell-free extracts followed by bulk precipitation and/or phase partition constitute the initial steps of many purification schemes. Chromatographic methods (size exclusion, ion exchange, hydroxylapatite, reversed phase, hydrophobic interaction and affinity based) utilizing either traditional, low pressure or high-performance liquid chromatography instrumentation are discussed. Electrophoretic techniques used to analyze the homogeneity of the protein product include SDS-PAGE, isotachophoresis, IEF and two dimensional gel electrophoresis.

Biotechnology↗
PubMed1988

Functional and biochemical characterizations of avian T lymphocyte antigens identified by monoclonal antibodies.

Seven monoclonal antibodies (mAb) were used to characterize antigens present on chicken T lymphocytes and on natural killer cells by flow cytometry, radioimmunoprecipitation and by effects on cell-mediated cytotoxicity and mitogen-induced proliferation. mAb CTLA8 and 5 stained 73% of thymus, 44% of spleen and 51% of peripheral blood lymphocytes (PBL), respectively, and immunoprecipitated 65- and 45-kDa proteins from detergent extracts of 125I surface-labeled thymocytes. Pretreatment of splenic lymphocytes with mAb CTLA5 and 8 in the presence of rabbit complement (C) eliminated the concanavalin A (Con A)-induced T cell proliferative responses. mAb CTLA3, 4 and 9 stained 43% of thymus, 36% of spleen and 18% of PBL, and immunoprecipitated 33-35-kDa proteins. Pretreatment of spleen cells with mAb 4 or 9 plus C reduced, but did not eliminate, the Con A-induced proliferative response and significantly reduced both major histocompatibility complex (MHC)-restricted and non-MHC-restricted cellular cytotoxicity. mAb CTLA1 and 6 stained 58% of thymus, 13% of spleen and 19% of PBL. mAb CTLA1 and 6 immunoprecipitated a 65-kDa protein. mAb CTLA1 and 6 had no effect on the Con A-induced blastogenesis and CTLA6 caused no decrease in virus-specific cytotoxic T lymphocyte and natural killer activity. These results indicate that (a) mAb CTLA5 and 8 identify antigens on mature T lymphocytes that are similar in tissue distribution, molecular mass and function to the mammalian CD5 antigen; (b) mAb CTLA3, 4 and 9 detect the avian homologue of CD8 antigen; and (c) mAb CTLA1 and 6 identify the avian homologue of CD4 antigen.

Animals↗
PubMed1988

The gag gene products of human immunodeficiency virus type 1: alignment within the gag open reading frame, identification of posttranslational modifications, and evidence for alternative gag precursors.

Seven human immunodeficiency virus gag polypeptides were identified in the purified virus and in infected CD4+ lymphocytes by peptide mapping and limited amino acid sequencing of immune-purified proteins. Two gag polyproteins of 55,000 (p55) and 41,000 (p41) daltons were rapidly labeled and readily processed into the major internal gag proteins that were aligned within the gag open reading frame (ORF) as NH2-p16 (MA)-p24 (CA)-p9 (NC)-p7-COOH. The myristoylated p16 (matrix, MA) protein was processed from the myristoylated p55 gag precursor protein. The immunoreactivity of the p16 (MA) protein with region-specific gag antisera and the conservation of the N-terminal myristyl group of the p55 precursor protein in p16 (MA) confirmed its position as the N-terminal-most protein. The p9 (nucleocapsid, NC) protein was localized to residue 378 of the gag ORF, next to the C terminus of the p24/p25 (core antigen, CA) protein. The p9 protein had a repeating Cys residue containing motif which is found in the nucleic acid-binding Cys residue-containing proteins of retroviruses. The p24 (CA) protein, which was localized to residue 133 of the gag ORF, was apparently derived by C-terminal processing of an intermediate polypeptide, p25. Both the mature p24 (CA) and p16 (MA) proteins were phosphorylated at Ser residue(s). We also identified two forms of gag p41 species, one resulting from the C-terminal processing of p55 and the other originating either from N-terminal processing of p55 or from de novo synthesis.

Amino Acid Sequence↗
PubMed1988

Purification and structural characterization of the putative gag-pol protease of human immunodeficiency virus.

We have purified a 10,774-dalton protein from human immunodeficiency virus (HIV) type 1 that is encoded in the protease domain of the pol open reading frame (ORF). Radiochemical amino acid microsequencing identified 12 amino acids from the stretch of 39 N-terminal residues of this protein, beginning with a PQITLW sequence at position 69 of the pol ORF. Radiosequencing of selected tryptic peptides of the protein identified 11 additional residues (Leu-9 and Val-2) in six peptides encompassing the entire molecule of 99 residues. A protein of similar size and identical N-terminal sequence (determined through the first 39 residues) was present among the processed HIV pol gene products in Escherichia coli which expressed the entire HIV pol ORF. The C terminus of both the viral and E. coli-expressed proteins was inferred to be contiguous with the N terminus of the p64-p51 reverse transcriptase on the basis of tryptic mapping and specific immunoreactivity with an antiserum against a dodecapeptide located upstream of the reverse transcriptase. Thus, the initial processing of the pol precursor that generates the native protease is apparently preserved across phylogenetic barriers. Although the purified viral protease lacked measurable proteolytic activity, the bacterial extracts were capable of processing an HIV gag precursor protein synthesized in E. coli.

Amino Acid Sequence↗
PubMed1988

Quantitative differences in Ia antigen expression in the spleens of 15I5-B congenic and inbred chickens as defined by a new monoclonal antibody.

A monoclonal antibody (MAb), designated P2M11, that detects a monomorphic determinant of chicken class II antigens was produced from the fusion of P3X63 myeloma cells with spleen cells from BALB/c mice immunized with chicken splenic lymphocytes. Flow cytometric analyses of lymphocytes from the SC and FP strains of chickens showed 30 to 50% staining of bursa cells, 15 to 20% staining cells, and less than 5% staining of thymus cells. Addition of MAb P2M11 to splenic of T cell cultures stimulated with allogeneic cells or concanavalin A resulted in a significant inhibition of the T cell proliferation responses. Immunoprecipitation of 35S-methionine-labeled spleen cell extracts using MAb P2M11 identified molecules with apparent molecular weights of approximately 28,000, 30,000, and 32,000 by sodium dodecyl-polyacryl-amide gel electrophoresis. Taken together, these data indicate that the antigens detected by MAb P2M11 are similar in cell distribution and structure to chicken Ia antigens encoded by B-L genes. Using this MAb, a strain difference was demonstrated in the tissue distribution of Ia antigen positive lymphocytes in the spleens but not the thymuses of 15I5-B congenic and inbred strains of chickens. This difference may be due to the genes associated with B-complex genes.

Animals↗
PubMed1987

Analysis of the D-region products of H-2q using monoclonal antibodies reveals the expression of a new class I-like molecule.

To analyze how many D-region-encoded molecules could be detected in H-2q, we produced a panel of nine monoclonal antibodies from AKR (KkDk) anti-AKR.M (KkDq) immunizations. All of the Dq region antibodies cross-reacted on Dd and/or Ld, and all except one cross-reacted on Db, confirming the previously observed serologic and amino acid sequence homology between the D-region products of H-2d, H-2b, and H-2q. All of these monoclonal antibodies precipitated 46,000 dalton molecules from both cell-surface-labeled and biosynthetically labeled B10.AKM spleen cells, indicating that all were reactive with class I-like molecules. Sequential immunoprecipitation analysis with one of these antibodies, 66-3-5, reveals the presence of a previously unidentified class I-like molecule. Tryptic peptide map analysis reveals that this molecule may be the product of a newly described H-2Dq-region gene.

Animals↗
PubMed1986

Three distinct H-2Ks molecules differing at the carboxy terminus are expressed on a tumor from SJL/J mice.

A dimethylbenzanthracene-induced leukemia of H-2s origin expressed at least two class I molecules on the cell surface that were precipitated by anti-H-2.19, an alloantiserum prepared against the private H-2Ks specificity. Mapping studies in recombinant inbred strains along with comparisons of tryptic peptide maps and N-terminal sequences indicated that the proteins were virtually identical and probably encoded by the same class I gene. When cells were labeled in the presence of tunicamycin, the proteins precipitated by anti-H-2.19 were further resolved into three distinct peptides. Experiments were performed to determine which of these various proteins were phosphorylated and which were recognized by an anti-synthetic peptide serum directed against the ultimate C-terminus of H-2K class I molecules. The results indicate that a single class I gene from the H-2Ks region may encode three class I molecules that differ only at the C-terminus due to alternative splicing of pre-mRNA.

Amino Acid Sequence↗
PubMed1986

Structural analyses define an additional H-2D region class I antigen that is expressed by a variant mouse strain.

The genetic complexity of the H-2D region includes haplotype disparities in apparent gene and product number. To probe the genetic basis of this complexity, the products of two independently derived mouse strains (STU and B10.SAA48) that express Dw3 antigens were compared. Serologic, fluorometric and peptide map comparisons were made using monoclonal antibodies. Although both STU and B10.SAA48 mice were found to express indistinguishable Dw3 molecules, only B10.SAA48 mice were found to express an additional antigen designated Lw3. Several lines of evidence are presented that suggest the gene encoding Lw3 maps to the D region. Furthermore peptide map comparisons of Dw3 with Lw3 molecules implied that they are products of separate genes; but Dw3 and Lw3 molecules were found to be more homologous to each other than Dd and Ld molecules are to each other. Inter-haplotype comparisons of Dw3 and Lw3 molecules with other D region molecules showed no striking homologies to Dd, Ld or eight other molecules compared. However, both Dw3 and Lw3 molecules were found to be unexpectedly homologous to the Ddx and Dw25 molecules, thus defining another family of structurally related D region antigens. This so called Dw3-family was found to be quite distinct from the previously defined Ld-family of molecules, since no joint members were found. The results of these studies of Dw3 encoded antigens are discussed as evidence for intra-D region recombination or mutation.

Animals↗