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A Saalmüller

Publications and source records attributed to A Saalmüller.

At least 55 records · Page 3Linked to original sources

Antiviral activity of an extract from leaves of the tropical plant Acanthospermum hispidum.

Incubation of the alphaherpesviruses pseudorabiesvirus (PRV) and bovine herpesvirus 1 during infection of cell cultures with an extract prepared from the leaves of Acanthospermum hispidum impaired productive replication of these viruses in a concentration-dependent manner whereas propagation of classical swine fever virus, foot-and-mouth disease virus and vaccinia virus was not affected. The 50% inhibitory concentration for cell growth (IC50) was 107 +/- 5 microliters/ml, and the concentration reducing PRV yield by 1 log10 (90% effective concentration, EC90) was 8 +/- 3 microliters/ml. The selectivity index calculated as the IC50/EC90 ration was 13 +/- 4. Delineation of the mechanism of the antiviral activity demonstrated inhibition of alphaherpesvirus attachment to and, to a lesser extent, penetration into the cells. In contrast, viral gene expression was not inhibited by the extract when added after entry of virions into the target cells. Reduced antiviral activity of A.h. against PRV deletion mutants lacking glycoprotein C (gC) or glycoproteins gC, gE, gG and gI altogether indicated that gC alone and/or viral attachment complexes of which gC is a component constitute the target structures for A. hispidum.

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Functional characterization of porcine CD4+CD8+ extrathymic T lymphocytes.

The porcine immune system is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting, extrathymic (CD1-) T lymphocytes. In addition to CD4-CD8+ and CD4+CD8- T lymphocytes, CD4-CD8- and CD4+CD8+ lymphocyte subpopulations are prominent in blood as well as in lymphoid tissues. In the present study, a functional comparison was made between CD4+CD8- and CD4+CD8+ T lymphocyte subpopulations. In a primary in vitro immune response against alloantigenic stimulator cells, both subpopulations proliferated without significant differences in their reactivity. Different results were obtained when analyzing the antigen-specific functions of the two CD4+ subpopulations in a secondary response against recall viral antigen; these experiments were performed with T lymphocytes from pseudorabies virus-immunized pigs. The proliferative response against viral antigens could be assigned to the CD4+CD8+ subpopulation, whereas the CD4+CD8- subpopulation remained nonreactive. Further analyses of the virus-specific in vitro immune response revealed a major histocompatibility complex (MHC) class II restricted helper T lymphocyte reaction involving CD4 but not CD8 molecules as restriction elements. Taken together, these results demonstrate that only the extrathymic CD4+CD8+ T lymphocyte subpopulation of swine contains MHC class II-restricted antigen-specific memory T helper cells.

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Isotype and antibody specificity of spontaneously formed immunoglobulins in pig fetuses and germ-free piglets: production by CD5- B cells.

Pig fetuses, colostrum-deprived newborns and germ-free (GF) piglets, animals in which B-cell development is not influenced by maternal regulatory factors, were employed to study the occurrence and specificity of natural antibodies (NAb). Serum immunoglobulins of all isotypes were found in 44-day-old fetuses (the gestation period in pigs lasts 114 days) and their level, with predominating IgM, was increased during fetal ontogeny. In sera of fetuses at the end of embryonic life as well as of newborns and older GF piglets, antibody activity against autoantigens (thyroglobulin, hormones, ssDNA), phylogenetically conserved proteins (myosin), haptens (trinitrophenyl; TNP) and bacterial components (Escherichia coli O86, tetanic anatoxin) was detected by enzyme-linked immunosorbent assay. The antigen-biding activity of IgM NAb increased after isolation of the serum immunoglobulins on a Staphylococcus Protein A (SPA)-Sepharose column. IgM reactivity similar to that detected in serum was found in supernatants from polyclonally stimulated cultures of spleen of 8- and 12-day-old GF piglets. Pig fetal liver IgM+ B cells, which were able to produce IgM after polyclonal stimulation, did not express the CD5 molecule. Our results indicate that pig preimmune repertoire is comparable to that described in humans and mice, although in contrast to these species pig B-1 cells do not express CD5.

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A porcine CD8+ T cell clone with heterotypic specificity for foot-and-mouth disease virus.

Foot-and-mouth disease virus (FMDV)-specific T cell lines and clones have been obtained from a swine lymphocyte antigen (SLA) inbred miniature pig vaccinated with chemically inactivated virus. One of the clones obtained, CE3, showed a specific and heterotypic proliferation against infectious but not inactivated FMDV in the presence of syngeneic peripheral blood mononuclear cells (PBMC). Adherent cells from PBMC were sufficient to support specific activation of the clone and the proliferation was abolished when the contact between CE3 and adherent cells was prevented. Phenotypic characterization of CE3 cells revealed expression of CD2, CD25 (interleukin-2 receptor), SLA class I and SLA class II. Furthermore, the cells were highly CD8 positive but showed low expression of CD4. The expression of T cell receptor (TCR) alpha and beta genes confirmed their T cell nature. Consistent with the CD8 phenotype, the proliferative response of CE3 was inhibited with MAbs to SLA class I and CD8. Altogether, these results indicate that CE3 is a porcine SLA class I-restricted CD8+ T cell clone, that recognizes a heterotypic FMDV antigen.

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Differentiation between MHC-restricted and non-MHC-restricted porcine cytolytic T lymphocytes.

The immune system of swine is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of resting extrathymic T lymphocytes. Beyond phenotypic differences to other species, porcine T lymphocytes, particularly when derived from infected animals, are known to show high non-specific cytolytic in vitro activity. Here we describe the putative porcine CD6 antigen (workshop CD6; wCD6) which enables a phenotypic separation of T lymphocytes responsible for major histocompatibility complex (MHC)-restricted and non-MHC-restricted cytotoxicity. The putative porcine CD6 analogue, wCD6, a protein with a molecular mass of 110,000, shows high specificity for T lymphocytes and is neither expressed on B lymphocytes nor on cells of the myeloid lineage. In the extrathymic T-lymphocyte compartment wCD6 characterizes two T-lymphocyte fractions: wCD6+ T lymphocytes including both CD4+ T-helper cell subpopulations (CD4+CD8- and CD4+CD8+) and within the CD4-CD8+ fraction cells with high CD8 antigen density. In contrast the CD4-CD8- gamma/delta T-cell receptor (TCR) subset and CD4-CD8+ cells with low CD8 antigen density are included in the wCD6- T-lymphocyte fraction. Functional studies with separated wCD6 fractions revealed that the wCD6- cells can be characterized by spontaneous and non-MHC restricted cytolytic activity, whereas the wCD6+ T lymphocytes are responsible for MHC-restricted T-cell functions. Thus, the porcine wCD6 is an important antigen to discriminate between MHC-restricted and non-MHC-restricted cytotoxicity.

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Classical swine fever virus-specific cytotoxic T lymphocytes and identification of a T cell epitope.

Classical swine fever virus (CSFV)-specific cytotoxic T lymphocytes (CTL) were derived from peripheral blood mononuclear leukocytes of immunized NIH-minipigs (MHC d/d haplotype) after in vitro restimulation with infectious CSFV. Their cytotoxic activity was determined against CSFV-infected target cells obtained from simian virus 40 (SV40) large T antigen-transfected immortalized kidney cells of a syngeneic miniature swine. Experiments with separated effector cell populations revealed that the CSFV-specific cytotoxic activity was mediated by CD4(-)CD6+CD8+ MHC class I-restricted T lymphocytes. Infection of target cells with various vaccinia virus/CSFV recombinants led to the identification of a major antigenic site for CSFV-specific CTL near the cleavage site between the non-structural proteins p80 (NS3) and p10 (NS4a). Using synthetic overlapping nonapeptides which covered this protein region the sequence ENALLVALF is the first sequence to be identified as an MHC class I-restricted T cell epitope recognized by CSFV-specific CTL.

Amino Acid Sequence↗

Cell surface proteoglycans are not essential for infection by pseudorabies virus.

Cell surface proteoglycans, in particular those carrying heparan sulfate glycosaminoglycans, play a major role in primary attachment of herpesviruses to target cells. In pseudorabies virus (PrV), glycoprotein gC has been shown to represent the major heparan sulfate-binding virion envelope protein (T. C. Mettenleiter, L. Zsak, F. Zuckermann, N. Sugg, H. Kern, and T. Ben-Porat, J. Virol. 64:278-286, 1990). Since PrV gC is nonessential for viral infectivity in vitro and in vivo, either the interaction between virion envelope and cellular heparan sulfate is not necessary to mediate infection or other virion envelope proteins can substitute as heparan sulfate-binding components in the absence of gC. To answer these questions, we analyzed the infectivity of isogenic gC+ and gC- PrV on mouse L-cell derivatives with defects in glycosaminoglycan biosynthesis, using a rapid and sensitive fluorescence-based beta-galactosidase assay and single-cell counting in a fluorescence-activated cell sorter. Our data show that (i) in the virion, glycoprotein gC represents the only proteoglycan-binding envelope protein, and (ii) cellular proteoglycans are not essential for infectivity of PrV. Attachment studies using radiolabeled virions lacking either gC or the essential gD confirmed these results and demonstrated that PrV gD mainly contributes to binding of Pr virions to cell surface components other than proteoglycans. These data demonstrate the presence of a proteoglycan-independent mode of attachment for Pr virions leading to infectious entry into target cells.

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Porcine T-cell receptors: molecular and biochemical characterization.

Two subclasses of CD3 associated T-cell receptors (TcR) have been described so far, consisting of either an alpha and beta chain (TcR alpha beta) or a gamma and delta chain (TcR gamma delta). Of the two subclasses, the TcR alpha beta is the one predominantly expressed on peripheral T lymphocytes of humans and rodents. TcR gamma delta T lymphocytes represent only a minor subset in these species. Among all mammalian species studied so far, swine showed the most diversified composition of the T-lymphocyte population characterized by the expression of CD4 and CD8 differentiation antigens. Besides CD4+CD8- and CD4-CD8+ T lymphocytes, CD4+CD8+ and CD4-CD8- T lymphocytes are prominent in the extrathymic T-lymphocyte compartment. Because of the lack of specific monoclonal antibodies (mAb), to date the porcine TcR can only be characterized with biochemical and molecular biological methods. TcR on porcine peripheral blood T lymphocytes with the phenotype CD4+ and/or CD8+ are characterized as 46-48 kDaR heterodimers which were supposed to represent the porcine TcR alpha beta. Biochemical analyses of the CD4-CD8- T lymphocytes revealed three distinct TcR gamma delta; all are characterized by a 40 kDa delta chain but differed in their gamma chains. One gamma chain with a molecular mass of 38 kDaR is preferentially expressed on CD4-CD8- T lymphocytes derived from peripheral blood; another chain with molecular mass of 37 kDaR is evenly distributed between CD4-CD8- T lymphocytes from blood and lymphoid tissues.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

Characteristics of porcine T lymphocytes and T-cell lines.

Monoclonal antibodies (mAb) against leucocyte differentiation antigens have altered the way in which immunologists examine the immune system. These mAb allow us to identify distinct surface molecules on leucocyte populations, by which these cells can be classified, isolated and studied for their functional properties. This review summarizes the knowledge about differentiation antigens useful in the characterization of porcine T lymphocytes. Furthermore, it focuses on several properties of porcine T lymphocytes, T-lymphocyte subpopulations and the generation of porcine antigen-specific T-cell lines.

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Summary of workshop findings for porcine T-lymphocyte antigens.

Fifty-four mAb preselected in the first round of the first porcine CD workshop for their possible reactivity with T-lymphocyte specific antigens and/or activation antigens were further analysed in a second round. PBMC, thymocytes and nylon-wool purified T lymphocytes derived from peripheral blood, mesenteric lymph nodes and spleen served as target cells for flow cytometric analyses. For the classification of activation antigens several experiments were performed with activated, mitogen-stimulated T lymphocytes and long-term T-lymphocyte cultures. Out of the 54 mAb, 35 mAb could be distributed to six different CD clusters and two swine workshop clusters (SWC). Five mAb could be distributed to the porcine CD2, four mAb to the CD4. Six mAb seemed to recognize the porcine CD5 and two mAb the porcine CD6 analogue. Six mAb were directed against the porcine CD8, whereas two different epitopes could be defined. One mAb was directed against the porcine CD25 analogue. Nine mAb could be clustered to the SWC1, defining an antigen on T lymphocytes and cells of the myeloic linage. Two mAb with high T-cell specificity were clustered to the SWC2.

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Analyses of monoclonal antibodies reactive with porcine CD5.

Among all monoclonal antibodies (mAbs) analyzed in the first porcine CD workshop, six mAbs showed reactivity to the porcine CD5 antigen (workshop mAbs 067, 068, 069, 070, 071 and 119). Because of lack of immunoprecipitation studies for five (067, 068, 069, 070 and 071) out of the six mAbs, only one mAb (119) could be definitely characterized as mAb against the monomeric 63 kDa porcine CD5 antigen. The five other mAbs included in this cluster are characterized by an identical labelling pattern in FCM and competition of the CD5 epitope recognized by mAb 119. These mAbs were allocated to the wCD5 subcluster.

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Analyses of monoclonal antibodies reactive with porcine CD6.

Amongst the monoclonal antibodies (mAbs) submitted to the first porcine CD workshop, two mAbs (workshop numbers 055 and 120) could be identified to recognize the porcine CD6 analogue. Both mAbs seemed to be highly T-cell specific and showed neither reactivity with cells of the myeloic lineage nor with B lymphocytes. The observed molecular mass of the antigen precipitated by mAb 120 of 110 kDa confirmed this classification. Without molecular analyses of the antigen recognized by mAb 055, but similar staining pattern in FCM compared with 120, mAb 055 was allocated to the wCD6 subcluster.

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Analyses of mAb reactive with porcine CD8.

Among all mAb submitted to the first porcine CD workshop, based on FCM analyses six mAb could be identified to recognize the porcine CD8 analogue (workshop Nos. 004, 051, 052, 053, 108 and 109). In immunoprecipitation studies three mAb (Nos. 004, 108 and 109) recognized an antigen with an apparent molecular mass of about 35 kDa under reducing conditions and about 70 kDa under non-reducing conditions. The molecular masses of the antigens recognized by the three other mAb (Nos. 051, 052 and 053) are still unknown. Epitope analyses performed by blocking experiments led to the determination of two CD8 epitopes: CD8a and CD8b. CD8a is recognized by mAb Nos. 004, 051 and 052, and CD8b by Nos. 053, 108 and 109.

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Analysis of mAb reactive with the porcine SWC1.

Among 54 mAb determined to be reactive with porcine T lymphocytes and/or activation antigens, eight mAb (workshop Nos. 005, 031, 080, 091, 092, 093, 094 and 110) derived from different laboratories grouped together in the T11 cluster and were ordered into the SWC1. One mAb (No. 111) which belong also to this group was lost during the workshop. The SWC1 antigen is a molecule expressed on the majority of leukocytes, resting T lymphocytes, monocytes and granulocytes, but not on B lymphocytes. On T lymphocytes it is down-regulated after activation. The molecular mass of the antigen is unknown. Epitope analyses revealed that seven out of the nine mAb recognized similar epitopes on the SWC1 molecule.

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Major histocompatibility antigen class II expressing resting porcine T lymphocytes are potent antigen-presenting cells in mixed leukocyte culture.

The immune system of swine is unique in that the expression of CD4 and CD8 antigens defines four subpopulations of extrathymic resting T lymphocytes in blood as well as in lymphoid tissues. Recently, we documented an additional peculiarity of the porcine T lymphocyte population: a substantial proportion of resting T lymphocytes show constitutive surface expression of MHC II antigens (Saalmüller et al. 1991, Immunobiol. 183, 102). These cells belong predominantly either to the CD4-CD8+ or to the CD4+CD8+ T lymphocyte subpopulations. This report describes the antigen-presenting cell function of MHC II+ T lymphocytes in mixed leukocyte cultures. Experiments employing highly purified T lymphocytes as alloantigen-presenting stimulator cells resulted in a high proliferative and cytolytic alloantigen-specific T lymphocyte response. After separation of T lymphocyte stimulators into MHC II- and MHC II+ fractions, alloantigen-presenting cell function could be assigned to the MHC II+ T lymphocyte subset. Although the mechanisms involved in the response to alloantigen are not completely understood and may be different from conventional antigen presentation, this is the first report where naive resting MHC II+CD8+ T lymphocytes can be defined as antigen-presenting cells, presenting alloantigen to cells of their own lineage.

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Discrimination between two subsets of porcine CD8+ cytolytic T lymphocytes by the expression of CD5 antigen.

Previous work has revealed striking differences in the peripheral T-lymphocyte compartments of swine compared to other species. The key difference is the existence of four T-lymphocyte subpopulations defined by their CD4/CD8 expression. Besides this difference in the CD4/CD8 antigen expression, we report here another unusual antigen distribution on porcine extrathymic T lymphocytes: the expression of the 63,000 MW CD5 antigen. In porcine thymus the CD5 antigen shows a biphasic antigen density, whereas the majority of thymocytes are characterized by an intermediate CD5 expression. In the extrathymic T-lymphocyte compartment, CD5 also shows a heterogeneous antigen distribution as defined by three subsets: CD5high, CD5dim and CD5- T lymphocytes. Analyses of the CD5 expression on the four CD4/CD8-defined peripheral T-cell subpopulations revealed that all CD4+ T lymphocytes, CD4+ CD8+ as well as CD4+ CD8- T lymphocytes, belonged to the subset with high CD5 antigen density. The CD4- CD8- subpopulation, containing in the majority T-cell receptor (TcR) gamma delta T lymphocytes, was characterized by dim CD5 expression. The most notable difference was the division of the CD4- CD8+ cytolytic T-lymphocyte subpopulation into two CD5-defined subsets: CD4- CD5- CD8+ lymphocytes with spontaneous cytolytic activity against tumour cells, and CD4- CD5+ CD8+ T lymphocytes with major histocompatibility complex (MHC)-restricted cytolytic function. Thus, the porcine CD5 antigen is an important marker to discriminate between CD5- CD8+ natural killer (NK) cells and CD5+ CD8+ progenitors of MHC-restricted cytolytic T lymphocytes.

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