Effect of cyclosporine on different porcine lymphoid tissues.
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Animals↗
Biomedical subjects
A Saalmüller
Publications and source records attributed to A Saalmüller.
Immunologic monitoring after islet allotransplantation in immunosuppressed pigs.
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Animals↗
Molecular cloning of porcine T cell receptor alpha, beta, gamma and delta chains using polymerase chain reaction fragments of the constant regions.
Fragments of the constant regions of porcine alpha, beta, delta and two types of gamma T cell receptor (TcR) chains were obtained by reverse transcription and polymerase chain reaction (PCR). Screening of a porcine peripheral T cell cDNA library with these PCR fragments led to the isolation of porcine TcR alpha, beta, gamma and delta chain clones. Sequence analysis of these clones and the respective PCR fragments demonstrated the existence of one alpha, one beta, three gamma and one delta chain isotype. Comparisons of the deduced amino acid sequences of the constant region with other species revealed a significant homology. For two of the three identified porcine gamma chain insertions of 38 and 40 amino acids were found within the hinge region. In addition, our sequence data demonstrate a high variability in the cytoplasmic C gamma domain among the three porcine TcR gamma isotypes as well as between species, which might be of structural and/or functional significance. Comparison with biochemical data indicate the existence of four porcine TcR gamma isotypes.
Amino Acid Sequence↗
Rapid identification and quantitation of cells infected by recombinant herpesvirus (pseudorabies virus) using a fluorescence-based beta-galactosidase assay and flow cytometry.
We recently described construction and use of a beta-galactosidase expression cassette in isolating recombinant pseudorabies virus (PrV) mutants (Mettenleiter and Rauh, 1990). We report here the identification and exact quantitation of cells infected by these mutants using an assay based on the reaction of intracellular beta-galactosidase expressed during infection by the recombinant viruses with the fluorogenic substrate fluorescein di-beta-D-galactopyranoside (FDG) followed by detection of positive cells in flow cytometry (FACS-Gal assay; Nolan et al., 1988). The detection method is fast, sensitive, and reliable, and yields quantitative results on single cell basis.
Animals↗
Expression of gamma/delta T cell receptors in porcine thymus.
Swine possess an extraordinarily high number of T lymphocytes with the phenotype CD4-CD8- in peripheral blood as well as in lymphoid tissues. This subpopulation is subdivided into at least four subsets defined by the expression of CD2 and three biochemically distinct gamma/delta T cell receptors. The four subsets differ largely in their pattern of lymphoid homing in that CD2- subsets, historically referred to as null lymphocytes, predominate in the circulating pool, whereas CD2+ subsets are enriched in lymphoid tissues. Here we document the expression of all three types of gamma/delta T cell receptors by CD4-CD8- porcine thymocytes, which provides the first evidence for a thymic origination of all subsets of porcine gamma/delta T lymphocytes. The biochemical analysis shows that three distinct gamma-chains form disulfide-bonded cell surface heterodimers with a common delta-chain and that glycosylation of all chains is already completed within the thymus. Surprisingly, CD2- subsets, which are known to be enriched among thymic emigrants and which numerically predominate in peripheral blood, are underrepresented in the thymus, suggesting a high export rate.
Aging↗
Production and characterization of monoclonal antibodies differentiating subpopulations of porcine B lymphocytes in blood and lymphoid tissues.
Monoclonal antibodies (mAbs) against various subclasses of immunoglobulin molecules are important reagents for the characterization and differentiation of serum immunoglobulins (Ig) generated during immune responses. Furthermore, Ig-specific mAbs can be powerful tools for the detection of B lymphocytes in blood and lymphoid organs. Here we describe two mAbs generated in our laboratories, named 1G6 and 2E8, which react with distinct epitopes on porcine immunoglobulin molecules. MAb 1G6 recognizes an epitope of an immunoglobulin chain with apparent molecular mass of 25 Kd. This chain represents an immunoglobulin light chain and might be the porcine equivalent of the murine and human kappa chain. MAb 2E8 is directed against porcine IgM molecules, recognizing an epitope of the porcine mu chain. The use of these mAbs was shown to avoid some common disadvantages of anti-Ig polyclonal antisera, like the high background staining of cells and tissue culture sections in immunohistochemistry. Furthermore, the use of these mAbs in two-color flow cytometry (FCM) versus a polyclonal anti-porcine Ig antiserum enables distinct B-lymphocyte subpopulations in blood and lymphoid organs to be detected. Our mAbs seem therefore to represent important and powerful reagents to identify and characterize porcine Ig isotypes and surface-Ig positive porcine B lymphocytes; their discriminating power between distinct B lymphocyte subpopulations could prove useful in different fields of both applied and fundamental immunological research.
Animals↗
Pseudorabies virus protein homologous to herpes simplex virus type 1 ICP18.5 is necessary for capsid maturation.
In pseudorabies virus (PrV), an open reading frame that partially overlaps the gene for the essential glycoprotein gII has been shown to encode a protein homologous to the ICP18.5 polypeptide of herpes simplex virus type 1 (N. Pederson and L. Enquist, Nucleic Acids Res. 17:3597, 1989). To study the function of this protein during the viral replicative cycle, a PrV mutant which carries a beta-galactosidase expression cassette interrupting the ICP18.5(PrV) gene was constructed. This mutant could be propagated only on cell lines that were able to provide ICP18.5(PrV) in trans after transformation with a corresponding genomic PrV DNA fragment. Detailed analysis showed that inactivation of the ICP18.5(PrV) gene did not impair infection of noncomplementing cells, nor did it impair early or late gene expression, as shown by immunoprecipitation of glycoproteins gII, gIII, and gp50. Surface localization of glycoproteins as demonstrated by fluorescence-activated cell sorting analyses was also not affected. Southern blot hybridizations, however, showed that cleavage of replicative concatemeric viral DNA did not occur in noncomplementing cells infected by the ICP18.5 mutant PrV. In addition, electron microscopic analysis revealed an accumulation of empty capsids in the nucleus of mutant-infected noncomplementing cells. We conclude that the ICP18.5(PrV) protein is necessary for viral replication and plays an essential role in the process of mature capsid formation.
Animals↗
Pathogenesis of classical swine fever: B-lymphocyte deficiency caused by hog cholera virus.
Hog cholera, also known as classical or European swine fever, is caused by hog cholera virus, a member of the genus Pestivirus. It is shown here that the end stage of lethal infection in the natural host is associated with a dramatic depletion preferentially of B lymphocytes in the circulatory system as well as in lymphoid tissues. Already at the onset of disease, viral replication in lymphoid tissues demarcates the germinal centers, and the viral genome remains localized to that site as the disease progresses even after morphologic disintegration of the follicular structure. A block in B-lymphocyte maturation by infection and destruction of germinal centers is discussed as a key event in the pathogenesis of acute, lethal hog cholera.
Animals↗
Glycoprotein IV of bovine herpesvirus 1-expressing cell line complements and rescues a conditionally lethal viral mutant.
Glycoprotein IV (gIV) of bovine herpesvirus 1 (BHV-1), a homolog of herpes simplex virus glycoprotein D, represents a major component of the viral envelope and a dominant immunogen. To analyze the functional role of gIV during BHV-1 replication, cell line BUIV3-7, which constitutively expresses gIV, was constructed and used for the isolation of gIV- BHV-1 mutant 80-221, in which the gIV gene was replaced by a lacZ expression cassette. On complementing gIV-expressing cells, the gIV- BHV-1 replicated normally but was unable to form plaques and infectious progeny on noncomplementing cells. Further analysis showed that gIV is essential for BHV-1 entry into target cells, whereas viral gene expression, DNA replication, and envelopment appear unchanged in both noncomplementing and complementing cells infected with phenotypically complemented gIV- BHV-1. The block in entry could be overcome by polyethylene glycol-induced membrane fusion. After passaging of gIV- BHV-1 on complementing cells, a rescued variant, BHV-1res, was isolated and shown to underexpress gIV in comparison with its wild-type parent. Comparison of the penetration kinetics of BHV-1 wild type, phenotypically complemented gIV- BHV-1, and BHV-1res indicated that penetration efficiency correlated with the amount of gIV present in virus particles. In conclusion, we show that gIV of BHV-1 is an essential component of the virion involved in virus entry and that the amount of gIV in the viral envelope modulates the penetration efficiency of the virus.
Animals↗
Gamma/delta T-lymphocyte subsets in swine.
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Animals↗
Resting porcine T lymphocytes expressing class II major histocompatibility antigen.
The immune system of swine is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting, extrathymic (CD1-) T lymphocytes in the circulation as well as in lymphoid tissue. Here it is documented that the specialty of the porcine T lymphocyte compartment extends to the expression of class II MHC (SLA) antigens. While the TCR gamma/delta CD4-CD8- as well as the TCR alpha/beta CD4+CD8- subpopulation both lack MHC II, the TCR alpha/beta CD4-CD8+ and the CD4+CD8+ subpopulation, the latter of which is private to swine, both do express MHC II. As opposed to human T lymphocytes, expression of porcine MHC II is not transient and restricted to lymphoblasts but is imminent in small, resting T lymphocytes of the two CD8-expressing subsets, even though also in swine activation can induce MHC II. Activation-induced extrathymic acquisition of MHC II without reversal can be discussed as one possible way of how MHC II+ T lymphocytes are generated. Alternatively, MHC II antigens should already be expressed by thymic progenitors. Remarkably, all CD1+CD4-CD8+ and most CD1+CD4+CD8+ thymocytes lack MHC II, yet, a minor subset with the phenotype CD4hiCD8hi expresses MHC II. One may speculate that these cells do not undergo thymic selection and represent the progenitors of the unusual, swine-typic MHC II+CD4+CD8+ extrathymic T lymphocytes.
Animals↗
The monoclonal antibody 11G7 recognizes a novel differentiation antigen expressed on hemopoietic precursor cells.
A monoclonal antibody (11G7) detecting a novel antigen on human hemopoietic progenitor cells (named 11G7R = 11G7 receptor) was raised by immunization of a Balb/c mouse with the leukemic blasts of a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). The antigen is expressed on most of MHC class II bearing peripheral blood leucocytes (PBL) and on a subpopulation of bone marrow mononuclear cells (BMMNC). By FACS-sorting and colony assays, it could be demonstrated that 11G7R is expressed on myelo-monocytic and myelo-granulocytic bone marrow precursor cells (GM-CFC, G-CFC, M-CFC) but is absent from erythroid precursor cells (BFU-E) and on cells exhibiting the capacity to form mixed colonies (GEMM-CFC). Double-fluorescence analysis on BMMNC revealed that 11G7R is expressed on a subset of B-cells, myeloid cells and cells carrying the HPCA-1 antigen (CD34). It has a similar distribution pattern to the myeloid antigens CD13 and CD33. However, in contrast to these antigens, 11G7R is also expressed on the blasts of several lymphoid leukemias (4/9 B-ALL, 1/2 T-ALL) and therefore it is not restricted to the myeloid lineage.
Animals↗
Endoglin is expressed on a subpopulation of immature erythroid cells of normal human bone marrow.
A monoclonal antibody (1G2) was raised by immunization of a Balb/c mouse with the leukemic blasts from a patient suffering from chronic myelogenous leukemia blast crisis (CML-BC). Sequential immunoprecipitation of the protein from human umbilical vein endothelial cells with 1G2 and the endoglin-specific monoclonal antibody 44G4 indicated that both antibodies react with the same molecule, a homodimer of molecular mass 180,000. This protein was first identified on acute lymphoblastic leukemia and was shown to be primarily associated with endothelial cells. In addition, 1G2 and 44G4 identified the same subpopulation of human bone marrow mononuclear cells (BMMNC), as established by two colour immunofluorescence analysis. By cell sorting and colony assays it could be demonstrated that endoglin is not expressed on hemopoietic precursor cells (CFU-G, CFU-GM, CFU-GEMM, BFU-E). May-Grünwald-Giemsa staining of sorted BMMNC revealed that 1G2 recognized immature proerythroblasts and double-fluorescence analysis showed that endoglin is present on a subset of glycophorin A-positive BMMNC. 1G2 was not reactive on bone marrow B-cells (CD19, CD20), T-cells (CD3, CD7), natural killer cells (CD56), myeloid cells (CD13, CD14, CD15, CD33), and on CD34-positive cells. Endoglin contains an arginine-glycine-aspartic acid sequence, a feature generally associated with extracellular matrix proteins which interact with integrins. It is suggested that proerythroblasts may utilize endoglin to interact with integrins in cell-cell adhesion events in the stromal or hemopoietic compartment of the bone marrow.
Animals↗
Distinct gamma/delta T cell receptors define two subsets of circulating porcine CD2-CD4-CD8- T lymphocytes.
The species swine provides the only example for CD2+ and CD2- subsets of Ig-CD4-CD8- lymphocytes with the propensity for homing to lymphoid tissue (Saalmüller et al., Eur. J. Immunol. 1989. 19: 2011). That the CD2-CD4-CD8- lymphocytes are bare of marker molecules that typify T lymphocytes raised the question of whether or not this cell type is descended from the T lymphocyte differentiation lineage. It is documented that expression of a phylogenetically conserved external epitope of T cell receptor gamma/delta subdivides porcine CD2- lymphocytes into an epitope 86D+ minor and an 86D- major subset. Expression of distinct forms of the T cell receptor gamma/delta, disulfide-bonded N-glycosylated surface heterodimers of under reducing conditions 38/40 and 37/40 kDa, respectively, hallmarks the CD2-86D+ and CD2-86D- subsets both as T lymphocytes.
Animals↗
Porcine gamma/delta T lymphocyte subsets differing in their propensity to home to lymphoid tissue.
Three types of T cell receptors (TcR) gamma/delta were identified in swine, all of which are disulfide-bonded cell surface heterodimers. The first, a 38/40-kDa molecule, is related to the TcR gamma/delta of sheep and cattle, and defines in swine a subset of CD4-CD8- T lymphocytes that is enriched in the circulating T lymphocyte pool. The other two receptors are private to swine. The second, a 37/40-kDa molecule, is expressed by the major subset of CD2-CD4-CD8- T lymphocytes that is neither enriched in nor excluded from lymphoid tissue. By contrast, the third type of TcR gamma/delta, a 40/46-kDa molecule, is found on CD2+CD4-CD8- T lymphocytes accumulating in lymphoid tissue. Thus, TcR gamma/delta T lymphocyte subsets differing in homing propensity are typified by the expression of distinct molecular forms of the TcR gamma/delta.
Animals↗
Phenotypic discrimination between thymic and extrathymic CD4-CD8- and CD4+CD8+ porcine T lymphocytes.
The composition of the T lymphocyte population in swine is special in that in addition to the CD4-CD8+ subpopulations, CD4-CD8- and CD4+CD8- and CD4+CD8+ subpopulations are prominent in the peripheral circulating as well as in the resident T lymphocyte pools. Since the same phenotypes are characteristic of thymic populations, it was asked whether the unusual distribution in swine may result from an emigration of thymic precursor phenotypes to the periphery. This explanation was refuted, as all thymic subpopulations were found to express CD1, albeit with differences in antigen density, whereas all extrathymic subpopulations lack CD1. The cellular distribution of CD2 in swine is without precedent among all species studied. Whereas in sheep and cattle the extrathymic CD4-CD8- subpopulation is known to entirely lack CD2 and to have a low propensity for homing to lymphoid tissues, the CD4-CD8- subpopulation in swine splits into CD2+ and CD2- subsets, both of which do reside in lymphoid tissues. While CD2+CD4-CD8- T lymphocytes are rare in the circulating pool, this subset accumulates in spleen and lymph nodes. This may indicate a role for CD2 in homing. Thus the species swine is immunologically unique, not only because of having CD1-CD2+CD4+CD8+ T lymphocytes in the periphery, but also with regard to subdivision and homing behavior of its CD4-CD8- T lymphocyte subpopulation.
Animals↗
Specific antibody response towards predicted epitopes of the epidermal growth factor receptor induced by a thermostable synthetic peptide adjuvant conjugate.
Applying computer-assisted epitope prediction to the amino-acid sequence of the epidermal growth factor receptor (EGFR), the extracytoplasmic domain EGFR(516-529) was selected as a putative antigenic region. EGFR(516-529) was synthesized on a solid-phase matrix and N-terminally linked to the low mol. wt adjuvant tripalmitoyl-S-glyceryl-cysteinyl-serine (Pam3 Cys-Ser). The conjugation to this B cell and macrophage-activating lipopeptide considerably enhanced the immunogenicity of the EGFR peptide. Using the conjugate Pam3 Cys-Ser-EGFR(516-529), a peptide-specific monoclonal antibody was produced. By flow cytometry and immunoprecipitation the antibody was demonstrated to recognize EGFR on A431 cells, expressing large numbers of EGFR. With this novel approach synthetic immunogens can be prepared which could serve as thermostable synthetic vaccines with great potential in countries where a functional cold chain cannot be maintained.
Adjuvants, Immunologic↗
Immune system of swine: dissection of mononuclear leucocyte subpopulations by means of two-colour cytofluorometric analysis.
Expression of the differentiation antigen 8/1, a porcine leucocyte marker for which no counterpart is known as yet in other species, was tested in relation to the expression of surface Ig and the monocyte marker 74-22-15. B lymphocytes and T lymphocytes can be typified by the exclusive expression of surface Ig and 8/1, respectively, while all monocytes defined by the expression of the 74-22-15 antigen co-express 8/1 with the highest staining density observed for this marker. 8/1+ T lymphocyte subsets defined by the expression of CD4 and CD8 cell surface antigens differed in the expression of 8/1 with the staining density being high in CD4+ T lymphocytes, intermediate in CD8+ as well as in CD4+CD8+ T lymphocytes, and low in the CD4-CD8- subset. Practically all mononuclear leucocytes present in peripheral blood are included in the subpopulations defined by this kit of reagents, and thus it can be predicted that if mononuclear leucocytes which do not belong to any of these subpopulations exist at all, these must be rare and will not be accessible to an analysis without enrichment.
Animals↗