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W Leibold

Publications and source records attributed to W Leibold.

At least 37 records · Page 2Linked to original sources
PubMed2001

Flow cytometric analysis of mitogen-induced activation of rainbow trout (Oncorhynchus mykiss) peripheral blood leucocytes.

Proliferation of rainbow trout peripheral blood leucocytes in vitro is usually assessed by measuring incorporated tritiated thymidine. In this report we monitored the in vitro proliferative response to the mitogen Concanavalin A (Con A) by means of flow cytometry (FCM) and 3H-thymidine incorporation. When analysed by FCM, blood leucocytes displayed two main cell populations with distinct forward and side scatter (FSC/SSC) characteristics: lymphocytes with low FSC/SSC values and non-lymphoid leucocytes (NLL) with increased FSC/SSC values. The nature of these cell types were confirmed by microscopy. Interestingly, the FSC/SSC pattern of lymphocytes remained unchanged after in vitro stimulation with Con A, whereas cells from the NLL population showed a marked shift towards increased FSC values. In stimulated cultures, the increase of FSC values of the NLL population significantly correlated with contemporarily measured 3H-thymidine incorporation (r = 0.7, P < 0.001). The mitogenic response of blood leucocytes originating from different individual fish varied over wide ranges. It was found to be related to the numbers of NLL present in the leucocyte sample. The present results show that qualitative and quantitative FCM analysis of morphological parameters (FSC/SSC) of blood leucocytes makes it possible to discriminate between leucocyte populations of the rainbow trout and to monitor cell proliferation experiments.

Animals↗
PubMed2001

Products of fourth-stage larvae of Oesophagostomum dentatum induce proliferation in naïve porcine mononuclear cells.

Infection of pigs with Oesophagostomum dentatum is a major cause of economic losses in pig productions. Whether infection with this nematode results in a protective immunity is still in debate and information about immune-modulating properties of O. dentatum are lacking. The present study investigated the question whether products of O. dentatum larvae modulate the proliferative response of porcine blood mononuclear cells (poMNC) in vitro. The poMNC of naïve and O. dentatum-infected pigs were cultured for 72 h in the presence of products (total homogenates and culture supernates) derived from third- (L3) and fourth-stage larvae (L4) of O. dentatum. Numbers of vital cells and blast-transformed cells were determined flow cytometrically. No larvae product induced an accelerated death of poMNC in vitro. In contrast, products of L4 (but not L3) significantly increased the numbers of vital poMNC in vitro (up to 187%). In addition, L4 products (homogenates and supernates, 0.1-10 microg/ml) but not those of L3 induced significant blastogenesis of poMNC. This was seen with poMNC from naïve and from O. dentatum-infected animals. In spite of these effects, the larvae products were not able to modulate the mitogen-induced (Concanavalin A) poMNC proliferation of naïve and infected animals. In summary, larvae of O. dentatum contain and secrete products with potential immunomodulatory capacity for porcine peripheral blood mononuclear cells. The differential effects of L3 and indicate that the parasite alters its set immunomodulatory substances during its development. This has to be considered in further studies and may help to identify the mediators involved.

Animals↗
PubMed2000

Expression and characterisation of equine interleukin 2 and interleukin 4.

In the present study, we describe the expression of equine IL2 and IL4. The cDNA of equine IL2 or IL4 was cloned in a mammalian expression vector, containing c-terminal myc- and six histidines His(6)-epitopes for recognition and purification of equine cytokines. The vector constructs were used for transfection of chinese hamster ovary (CHO) cells. Purified equine cytokines were characterised by western blotting. Equine IL2 was secreted with a molecular weight of approximately 17.1kDa, whereas IL4 was expressed in three different sizes of 17.1, 19.6 and 22.1kDa, probably due to different glycosylation modifications. The biological activities of both cytokines were tested by proliferation assays using leukoagglutinin (LAG) prestimulated equine PBMC. Both, equine IL2 and IL4 induced dose-dependent lymphocyte proliferation. In contrast to IL4, IL2 supported the proliferation of B cells.

Animals↗
PubMed2000

Altered functional and immunophenotypical properties of neutrophilic granulocytes in postpartum cows associated with fatty liver.

The intention of the study was to analyze the relationship between liver triacyl glycerol content (liver TAG content) and immunophenotypical and functional properties of polymorphonuclear neutrophilic granulocytes (PMN) of dairy cows in the peripartum period. We investigated characteristics of bovine PMN from the blood and uterus of clinically healthy cows in the periparturient period. The numbers of circulating leukocytes and segmented granulocytes continuously increased until parturition and declined afterwards to starting values. This was independent of the liver TAG content and mainly affected neutrophils. The liver TAG content exceeded 40 mg/g liver, the reference value, in 12 of 19 cows in the first two weeks postpartum. Increased liver TAG content, > 40 mg/g, went in parallel with a reduced expression of function-associated surface molecules on blood neutrophils (e.g. CD11b/CD18 = CR3 and CD11c/CD18 = CR4). Moreover, in cows with high liver TAG levels the antibody-independent and -dependent cellular cytotoxicity (AICC, ADCC) of blood PMN was markedly reduced. PMN also were less capable of ROS generation after stimulation with Phorbol Myristate Acetate (PMA). In comparison with contemporarily harvested blood PMN, neutrophils recovered from the uterine lumen showed a decreased expression of 4/6 examined surface structures. Only the expression densities of CR3 molecules and those detected by mAb IL-A110 were enhanced on uterine PMN. The cytotoxic capacity and the ROS generation were significantly lower for uterine PMN than for blood PMN. The results suggest that increased liver TAG content in the first and second week after calving is associated with decreased functional capacities of PMN derived from blood and uterus. This may help to explain why cows who are too fat at calving (who therefore have an increased liver TAG content) have a higher incidence of infectious diseases such as endometritis

3-Hydroxybutyric Acid↗
PubMed2000

In vitro cultivation of Trypanoplasma borreli (protozoa: kinetoplastida), a parasite from the blood of common carp Cyprinus carpio.

An in vitro culture system was developed for Trypanoplasma borreli, a pathogenic flagellate from the blood of European cyprinids. Trypanoplasms multiplied rapidly in a mixture of Hanks' balanced salt solution (HBSS, 45%), L15 (22.5%), Earle's minimum essential medium (MEM, 22.5%) and 10% distilled water, which was supplemented with 5 to 10% heat-inactivated pooled carp serum. In medium supplemented with fetal bovine serum, multiplication of T. borreli seemed to be inhibited. Cultures initiated with less than 100 000 T. borreli per ml culture medium did not survive, and a substantial multiplication of trypanoplasms was found at inocula beginning with 630 000 flagellates ml(-1). Trypanoplasms multiplied at 15, 20 and 25 degrees C. In cultures incubated at 4 degrees C the trypanoplasms remained viable but the number of flagellates did not increase. Trypanoplasms from in vitro cultures retained their infectivity for carp for at least 90 d (5 passages). The trypanoplasms survived in culture over a period of up to 5 mo (10 passages). The established culture system allows the propagation of high numbers of fish-infective trypanoplasms, which are required to study parasite-host relationships in carp.

Animals↗
PubMed2000

Flow cytometric analysis of proliferative responses of carp Cyprinus carpio peripheral blood leukocytes to mitogens and to the hemoflagellate Trypanoplasma borreli.

The activation of carp peripheral blood leukocytes (PBL) was analysed radiometrically and by means of flow cytometry (FCM) in order to compare the results obtained with both methods. The qualitative and quantitative FCM analyses of cellular morphology and viability resulted in a further characterisation of proliferative responses of carp PBL to Trypanoplasma borreli in vivo and in vitro. The lymphocyte population of PBL from T. borreli-infected carp exhibited a marked shift in forward scattered light (FSC; cell size). When PBL from healthy carp were stimulated with mitogens in vitro, a lymphoid population with increased FSC profiles was also observed. The number of these cells coincided to ratios of 3H-thymidine incorporation, recorded from corresponding cultures. Thus, it was concluded that the increase in size of stimulated lymphocytes could be due to blastogenic transformation. The advantage of the FCM procedure is that activation and proliferation of carp lymphocytes can be monitored without labelling the cells. Cocultures of mitogen-stimulated carp PBL and T. borreli revealed the ability of the parasite to suppress lymphocyte proliferation in vitro.

Animals↗
PubMed2000

Prostaglandin E2 is variably induced by bacterial superantigens in bovine mononuclear cells and has a regulatory role for the T cell proliferative response.

Signal transduction in antigen presenting cells via MHC class II molecules induces production of prostaglandin E2 (PGE2) known to possess immunoregulatory potential. Since Staphylococcus aureus superantigens (SAgs) utilize MHC class II molecules as primary ligands, we wanted to know whether PGE2 is induced after in vitro SAg stimulation of bovine blood mononuclear cells (boMNC), and whether this arachidonic acid metabolite modulates the preferential SAg-induced proliferative response of bovine CD8+ T cells. SEB as well as SEA induced maximal amounts of PGE2 on day 2 of culture (1-2.5 x 10(-8) mol/l per 2 x 10(5) boMNC). PGE2 production could be inhibited completely by indomethacin (10(-5) mol/l) causing enhanced proliferation of boCD4+ T cells (174%) as well as of boCD8+ T cells (122%) between day 4 and 6 of the in vitro culture, however, only in a subset of the tested animals. Notably, the striking preference of proliferation of boCD8+ over boCD4+ T cells following SAg stimulation remained largely unchanged after inhibition of endogenous PGE2 synthesis or after addition of exogenous PGE2. Higher concentrations of exogenously added PGE2 (> or = 10(-8) mol/l) inhibited the proliferation reaction, mainly due to an increased death rate of both CD4+ and CD8+ blasts. In contrast, lower PGE2 concentrations between 10(-8)-10(-9) mol/l even slightly enhanced the proliferation of both T cell subsets, depending on the individual cell donor. Summing up: These data show that SAgs, indeed, can induce PGE2 production in boMNC which can enhance or reduce the proliferative response of bovine CD4+ and CD8+ T cells.

Animals↗
PubMed2000

Superantigen-dependent accelerated death of bovine neutrophilic granulocytes in vitro is mediated by blood mononuclear cells.

While classical interactions of bacterial superantigens (SAgs) with antigen presenting cells and T cells have been studied intensively, the potential interactions of SAgs with granulocytes (PMNs) have gained much less attention. We investigated if in the bovine system SAgs have any direct or indirect influence on the fate of granulocytes, which are among those cells primarily responsible for the elimination of superantigen-producing bacteria. The tested SAgs (SEA, SEB) had no apparent direct effect on PMN viability (neutrophils and eosinophils). However, in the presence of blood mononuclear cells (MNCs), SAgs led to an accelerated death of neutrophils but not of eosinophils. Compared to medium controls, in SAg-stimulated cultures only about 20-50% of the neutrophils survived after 24 hours in vitro. Accelerated death of neutrophils required the presence of at least 10% MNC and started between 2.5-24 h after initiation of the co-culture between MNC and PMN. Minimal effective SEA concentrations ranged between 10-100 pg/l (SEB 0.1-10 ng/l). The effect could be mimicked by culture supernatants of SAg-stimulated MNCs, suggesting that direct cell-cell interactions are not required for the killing. In the human system, where we tested the role of TNF-alpha, an antibody specific for this cytokine was not able to abolish the death of human neutrophils. Brefeldin A, an inhibitor of golgi transport and cytokine secretion, which blocked the SAg-induced activation of bovine MNC did not abolish the killing of neutrophils. Blocking of nitric oxide generation or PGE2 synthesis also could not alter the SAg-induced killing of bovine neutrophils. The observed indirect negative effects of SAgs on neutrophils may provide new insights in mechanisms by which superantigens modulate the hosts immune response.

Animals↗
PubMed2000

Assessment of antibody-independent cellular cytotoxicity (AICC) of porcine neutrophilic granulocytes by quantitative flow cytometry. Lack of modulation by larval products of Oesophagostomum dentatum.

After infection of pigs by the larvae of Oesophagostomum dentatum, granulomas are formed around the third-stage larvae in the submucosa of the gut which contain a considerable number of neutrophils. This has no obvious impact on the larvae, which develop to fourth-stage larvae within these granulomas. We therefore asked, whether the products of O. dentatum larvae modulate the functional capacity of porcine neutrophils. The antibody-independent cellular cytotoxicity (AICC) was chosen as a model system. This assay was developed for the pig and quantified using flow cytometry. Bovine lymphoblastoid cells (cell line Anna TA1) served as targets. The measurement of cytotoxicity was based on the determination of absolute numbers of vital target cells. This procedure proved to be reliable and required no additional labelling of target and/or effector cells. Porcine neutrophils, when stimulated with phorbol 12-myristate 13-acetate (PMA; > or = 10 nmol/l), killed target cells at effector: target ratios between 1:1 and 9:1. AICC was not demonstrable after 4 h but could be observed between 16 h and 20 h after in vitro co-culture. Killing of targets required close physical contact between effector and targets, since supernatants of PMA-stimulated polymorphonuclear cells were not able to lyse the target cells. Homogenates of third- and fourth-stage larvae of O. dentatum did not affect the vitality of porcine granulocytes or target cells in vitro, nor did they modulate the AICC capacity of porcine granulocytes.

Animals↗
PubMed2000

[Occurrence of various immunoglobulin isotopes in horses with equine recurrent uveitis (ERU)].

We investigated 30 healthy eyes and 41 eyes with ERU from 57 horses. The total immunoglobulin titers and titers of IgGa, IgGb, IgM were measured in aqueous humour, vitreous and serum using different ELISA techniques. Every sample investigated contained detectable amounts of immunoglobulins. Compared to control eyes significantly increased titers were found in the aqueous humour and vitreous of the ERU eyes for all immunoglobulin isotypes studied (p < or = 0.01). While IgM was detected in only 2 out of thirty aqueous humour and in none of the thirty vitreous samples of healthy eyes, 79.6% of samples of ERU eyes revealed considerable IgM titers. Changes of the IgGa/IgGb ratio in the eye as compared to that in the autologous serum was more frequent in affected than in healthy eyes. In contrast to the intraocular immunoglobulins there were no significant differences in immunoglobulin serum titers in healthy horses and those affected with ERU (p > 0.05). In conclusion, the results argue for a physiological appearance of immunoglobulins in the healthy eye. The increased titers of immunoglobulins in eyes stricken with ERU might be signs either of a local ocular production of antibodies and/or an increased permeability of intraocular barriers.

Animals↗
PubMed1998

Organisation of the equine immunoglobulin constant heavy chain genes. II. Equine cgamma genes.

The number of immunoglobulin G constant heavy chain genes (cgamma genes) varies broadly among mammalian species, reflecting structural and functional differences between expressed immunoglobulin G (IgG) isotypes and allotypes. Up to now equine IgG isotypes have been defined only at the biochemical and serological level. It is still not clear how many IgG isotypes exist in horses and whether there are any allotypes. Here, we describe the isolation and characterisation of equine cgamma genes. An equine genomic lambda phage library was screened with a human cgamma4 probe. Cross-hybridising equine cgamma sequences were cloned twice and characterised by restriction mapping with the human cgamma4 and a murine sgamma1 probe. Genomic equine DNA probes for both, cgamma genes and corresponding switch regions (sgamma), were isolated and used for a more detailed BamHI restriction analysis, comparing genomic DNA of various horses. This analysis reveals the existence of at least five, or probably six cgamma genes in the equine haploid genome. Beside the porcine system, this is the highest number of cgamma genes described for any mammalian species. Moreover, for two of these cgamma genes, BamHI restriction fragment length polymorphism became evident.

Animals↗
PubMed1998

Reactivity of workshop monoclonal antibodies on paraformaldehyde-fixed porcine blood mononuclear cells.

One hundred sixty-four monoclonal antibodies (mAbs) of the second international swine CD workshop were tested for their reactivity with porcine blood mononuclear cells before and after fixing the cells with varying concentrations of paraformaldehyde (PFA) (1, 5 and 10 g l-1). A total of 38 (out of 134) positive reacting mAbs were significantly affected in their binding behavior on fixed cells. Modulation was seen as reduction in binding (staining intensity and/or % positive cells, n = 18) or in elevated values (n = 20). Modified mAb binding occurred after fixing cells with 5 to 10 g l-1 PFA.

Animals↗
PubMed1998

There is no regulatory role for induced nitric oxide in the regulation of the in vitro proliferative response of bovine mononuclear cells to mitogens, alloantigens or superantigens.

Nitric oxide (NO) is a potent cellular mediator which has been shown to modulate several immune mechanisms. Between species, however, there are considerable differences regarding the signals required for induction of NO as well as the kind of cells capable of producing NO. The object of this study was to determine the kinetics of NO production of bovine blood mononuclear cells (boMNC) stimulated in vitro and to investigate whether it modulates their proliferative response following allogeneic (mixed leukocyte cultures, aMLC), mitogenic (PWM, Con A) or superantigenic (SEA, SEB) stimulation. NO production was indirectly determined with the Griess reagent measuring nitrite (NO2-). Significant but low amounts of NO could be detected as early as day 3 after in vitro stimulation and did noly slightly increase during the 6-8 day culture period. Superantigens (SEA, SEB) and aMLCs (4.3-5.2 microM NO2-) induced a significantly higher nitrite accumulation compared to Con A (2.6 microM NO2-). Generation of nitrite, most likely produced by monocytes/macrophages, could be inhibited by 1 mM N-monomethyl-L-arginine (NMLA). Flow cytometric characterization of various cellular responses revealed no differences between cultures with or without NMLA. This included the determination of blastogenesis, absolute numbers of viable cells, expression density of activation markers (MHC class II, IL-2R alpha) and cellular subpopulations (CD4+, CD8+, sIg+) among blasts. In addition, exogenously provided NO via SNOG in non-toxic concentrations (10(-5)-10(-4) M) did not alter the proliferative reaction of boMNC in vitro. The results suggest that NO is induced after in vitro stimulation of boMNC, however, at a low level, and without having any positive or suppressive effects on the so far tested cellular parameters of activation and proliferation.

Animals↗
PubMed1998

Organization of the equine immunoglobulin heavy chain constant region genes; III. Alignment of c mu, c gamma, c epsilon and c alpha genes.

Previous restriction analysis of cloned equine DNA and genomic DNA of equine peripheral blood mononuclear cells had indicated the existence of one c epsilon, one c alpha and up to six c gamma genes in the haploid equine genome. The c epsilon and c alpha genes have been aligned on a 30 kb DNA fragment in the order 5' c epsilon-c alpha 3'. Here we describe the alignment of the equine c mu and c gamma genes by deletion analysis of one IgM, four IgG and two equine light chain expressing heterohybridomas. This analysis establishes the existence of six c gamma genes per haploid genome. The genomic alignment of the cH-genes is 5' c mu/(/) c gamma 1/(/) c gamma 2/(/) c gamma 3/(/) c gamma 4/(/) c gamma 5/(/) c gamma 6/(/) c epsilon-c alpha 3', naming the c gamma genes according to their position relative to c mu. For three of the c gamma genes the corresponding IgG isotypes could be identified as IgGa for c gamma 1, IgG(T) for c gamma 3 and IgGb for c gamma 4.

Animals↗
PubMed1997

Organization of the equine immunoglobulin constant heavy chain genes. I. c epsilon and c alpha genes.

We provide a restriction map of the equine c epsilon and c alpha genes as a molecular basis for isotype classification. Human and murine DNA probes were used for identification of homologous equine DNA sequences and for isolation of the equine c epsilon and c alpha genes from a genomic DNA library. A detailed map of the equine 5'-s epsilon/c epsilon-s alpha/c alpha-3' gene region was obtained. Equine c epsilon and c alpha DNA probes were prepared and used for restriction analysis of immunoglobulin heavy chain gene loci from different horses. This analysis indicated the presence of only one equine c epsilon and one c alpha gene in the haploid equine genome. In addition, for the equine c alpha gene, four haplotypes were identified according to BamHI restriction fragment length polymorphism (RFLP) of genomic DNA. The relative location of the c epsilon and c alpha genes 3' of the equine c mu and c gamma genes was determined by restriction analysis of equi-murine heterohybridomas.

Animals↗
PubMed1997

[Demonstration of immunoglobulin isotypes in the vitreous body as a contribution to the etiology of recurrent equine uveitis].

The functional properties of different immunoglobulin isotypes in equine recurrent uveitis (ERU) has not been investigated yet. Here, we describe the quantitative determination of total immunoglobulin levels and isotype differentiation in the vitreous of four horses with ERU as compared to that of seven healthy horses. In contrast to almost equal amounts of total immunoglobulin in the vitreous of both groups, remarkable differences were found: All four of the horses with ERU had significantly higher IgA contents in their vitreous as compared to the control group. However, the other isotypes monitored (IgM, IgGa, IgGb, IgGc and IgG(T)) indicated no differences between both groups. Comparing the individual ratios of immunoglobulin isotypes in the vitreous and the autologous serum of two horses with ERU and two control animals provided informative results: IgM was only detected in serum but not at all in vitreous of all horses investigated. All four IgG isotypes monitored in the diseased animals as well as these IgG isotypes and the IgA in healthy animals were present in the same ratios in serum and in vitreous of the individual horses. In general, the content of such isotypes in the vitreous was about 1000 fold lower then the respective isotypes in the autologous serum. These results are compatible with a transfer of the IgG and IgA isotypes from the serum into the vitreous in healthy individuals. All four horses with ERU, however, had a selectively increased relative IgA content in the vitreous as compared to their serum. This argues for a preferential local IgA production within the framework of a local immunological reaction to antigens located within the eyes of horses with ERU.

Animals↗
PubMed1996

Biotinylation of cell surface MHC molecules: a complementary tool for the study of MHC class II polymorphism in cattle.

Biotinylation of cell surface proteins is often used as an alternative to radioactive labelling procedures, but very little is known about the labelling of the different allelic products of polymorphic antigenic systems. In this report, we demonstrate that NHS-LC-biotin labels bovine MHC class II molecules with different efficiencies for several allelic forms of this polymorphic system compared to conventional metabolic labelling with [35S]methionine. This was shown after immunoprecipitation and one-dimensional isoelectric focusing (1D-IEF). The avidity of the monoclonal antibody Bo139 (alpha-bovine MHC class II) was not affected after in situ biotinylated of bovine PBMC, as revealed by flow cytometric analyses, immunoblotting after SDS-PAGE and immunoprecipitation. The biotinylation did not affect the apparent isoelectric points of polymorphic bovine MHC class II beta chains. This was demonstrated by double labelling of cells with [35S]methionine and subsequent biotinylation and comparison of the banding pattern after immunoprecipitation and 1D-IEF. 1D-IEF of 11 unrelated animals resulted in the demonstration of 29 polymorphic bands of which eight were detected by both labelling procedures, six only after biotinylation and 15 only after metabolic labelling with [35S]methionine. Hence, biotinylation alone cannot serve as an alternative for radioactive labelling of bovine MHC class II molecules but can reveal expressed allelic forms not detectable by metabolic labelling with [35S]methionine.

Alleles↗