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A Roos

Publications and source records attributed to A Roos.

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

Both IgG and IgM anti-pig antibodies induce complement activation and cytotoxicity.

Hyperacute rejection of pig xenografts transplanted in humans is caused by endothelial cell binding of pre-formed xenoreactive antibodies (XAb) and activation of the classical pathway of complement. Human XAb mainly consist of anti-Galalpha1 3Gal antibodies, which occur in IgM, IgG and IgA classes. Whereas IgM anti-Galalpha1 3Gal antibodies have an established role in hyperacute rejection, the potential role of IgG XAb in this process is still controversial. The aim of the present study was to assess the specificity and functional properties of IgG and IgM XAb. Both classes were present in all human plasma samples tested, with a high inter-individual variability. Levels of IgG XAb did not correlate with levels of IgM XAb. Binding to Galalpha1 3Gal is strongly correlated with binding to the pig cell line PK15, both for IgG and for IgM, pointing to Galalpha1 3Gal as the major antigen recognized. Both purified IgM and IgG induced C3 deposition on PK15 cells and complement-dependent cytotoxicity in a dose-dependent way. The combination of IgG and IgM XAb resulted in an additive effect on cytotoxicity. Affinity-purified IgG anti-Galalpha1 3Gal antibodies were 22 times less potent than IgM in induction of cytotoxicity. These results indicate a quantitative, but not a qualitative, difference between IgM and IgG anti-pig antibodies concerning their complement-activating properties. Therefore, both classes of XAb are of importance in the pathogenesis of hyperacute rejection, and the relative importance of each class may differ considerably between individual patients, depending on the ratio of IgG and IgM XAb present in serum.

Animals↗
PubMed2001

Low molecular weight dextran sulfate prevents complement activation and delays hyperacute rejection in pig-to-human xenotransplantation models.

Dextran sulfate of 5000 molecular weight (DXS 5000) is known to block complement activation as well as the intrinsic coagulation cascade by potentiation of C inhibitor. The effect of DXS 5000 on hyperacute rejection (HAR) was tested in pig-to-human xenotransplantation models. For in vitro testing, a cytotoxicity assay was used with the pig kidney cell line PK15 as target cells and fresh, undiluted human serum as antibody and complement source. Ex vivo pig lung perfusion was chosen to assess DXS 5000 in a physiologic model. Pig lungs were perfused with fresh, citrate-anticoagulated whole human blood to which 1 or 2 mg/ml DXS 5000 were added; the lungs were ventilated and the blood de-oxygenated. Pulmonary vascular resistance (PVR) and blood oxygenation (deltapO2) were monitored throughout the experiment. Autologous pig blood and human blood without DXS 5000 served as controls. In the PK 15 assay DXS 5000 led to a complete, dose-dependent inhibition of human serum cytotoxicity with an average IC50 of 43 +/- 18 microg/ml (n=8). Pig lungs perfused with untreated human blood (n=2) underwent HAR within 105 +/- 64 min, characterized by increased PVR, decrease of deltapO2, and generalized edema. Microscopically, capillary bleeding as well as deposition of human antibodies, complement and fibrin could be observed. Addition of DXS 5000 (n=4) prolonged lung survival to 170 +/- 14 min for 1 mg/ml and 250 +/- 42 min for 2 mg/ml. and PVR values as well as edema formation were comparable to control lungs that were perfused with autologous pig blood (n=2). Activation of complement (activation products in serum, deposition on lung tissue) and the coagulation system (fibrin monomers) were significantly diminished as compared to human blood without DXS 5000. Binding of anti-Gal antibodies was not influenced, and in vitro experiments showed no evidence of complement depletion by DXS 5000. In conclusion, DXS 5000 is an efficient complement inhibitor in pig-to-human xenotransplantation models and therefore a candidate for complement-inhibitory/anti-inflammatory therapy either alone or in combination with other substances and warrants further investigation.

Animals↗
PubMed2001

The immune dysregulatory compound mercuric chloride induces integrin-mediated T-lymphocyte adhesion.

Exposure of Brown Norway rats to mercuric chloride induces systemic autoimmunity, involving T- and B-lymphocyte activation, (auto-)antibody production and multiorgan inflammation. Several divalent metal ions, such as Mg2+ and Mn2+, can activate binding of integrins to their ligands, thus causing lymphocyte adhesion. To test the hypothesis that Hg2+ acts in a similar way, we studied the effect of HgCl2 on integrin-mediated T-cell adhesion. HgCl2 induced cell-cell aggregation of human T lymphoblasts. Exposure of a human T-cell clone to HgCl2 for 1 hr enhanced, in a dose-dependent way, cell binding to fibronectin (FN) and to intercellular adhesion molecules (ICAM) -1, -2 and -3. Furthermore, HgCl2 induced strong binding of Jurkat T cells to FN. These effects of HgCl2 were of similar magnitude as the effects of phorbol 12-myristate 13-acetate (PMA) or MnCl2. Studies using blocking antibodies indicated the involvement of CD11a in binding to ICAMs, and of CD49d, CD49e, and CD29 in binding to FN. Adhesion to FN induced by HgCl2 or by PMA, but not by MnCl2, was dependent on temperature and on extracellular Ca2+ or Mg2+. Addition of cytochalasin B enhanced synergistically the FN adhesion induced by MnCl2, whereas the effects of PMA and HgCl2 were not modified. These results indicate that Hg2+ is a potent activator of T-cell adhesion, mediated by several integrins and ligands. In contrast to the effect of MnCl2, HgCl2-induced cell adhesion probably involves an intracellular pathway. Activation of integrins by HgCl2 may play an important role in activation and migration of leucocytes involved in HgCl2-induced immune dysregulation in vivo.

Actins↗
PubMed2000

Biochemical and structural analysis of the NS5B RNA-dependent RNA polymerase of the hepatitis C virus.

Hepatitis C virus (HCV), the major causative agent of chronic and sporadic non-A, non-B hepatitis worldwide, is a distinct member of the Flaviviridae virus family. These viruses have in common a plus-strand RNA genome that is replicated in the cytoplasm of the infected cell via minus-strand RNA intermediates. Owing to the lack of reliable cell culture systems and convenient animal models for HCV, the mechanisms governing RNA replication are not known. As a first step towards the development of appropriate in vitro systems, we expressed the NS5B RNA-dependent RNA polymerase (RdRp) in insect cells, purified the protein to near homogeneity and studied its biochemical properties. It is a primer- and RNA template-dependent RNA polymerase able to copy long heteropolymeric templates without additional viral or cellular cofactors. We determined the optimal reaction parameters, the kinetic constants and the substrate specificity of the enzyme, which turned out to be similar to those described for the 3D polymerase of poliovirus. By analysing a series of nucleosidic and non-nucleosidic compounds for their effect on RdRp activity, we found that ribavirin triphosphates have no inhibitory effect, providing direct experimental proof that the therapeutic effect observed in patients is not related to a direct inhibition of the viral polymerase. Finally, mutation analysis was performed to map the minimal NS5B sequence required for enzymatic activity and to identify the 'classical' polymerase motifs important for template and NTP binding and catalysis.

Baculoviridae↗
PubMed1999

Immunoglobulin M-enriched human intravenous immunoglobulin prevents complement activation in vitro and in vivo in a rat model of acute inflammation.

An important antiinflammatory mechanism of intravenous immunoglobulin preparations (IVIG) is their ability to block complement activation. The purpose of this study was to compare the complement-inhibitory activity of four IVIG preparations differing in isotype composition. The preparations were: (1) IVIgG (48 g/L IgG, 2 g/L IgA; Intraglobin F); (2) Pentaglobin (38 g/L IgG, 6 g/L IgM, 6 g/L IgA); (3) IVIgM (35 g/L IgM, 12 g/L IgA, 3 g/L IgG); and (4) IVIgA (41 g/L IgA, 9 g/L IgG), all from Biotest Pharma GmbH, Dreieich, Germany. Their complement inhibitory activity was assessed in vitro by measurement of the blocking of C1q-, C4-, and C3 deposition on solid-phase aggregated rabbit IgG by enzyme-linked immunosorbent assay (ELISA). Complement inhibition in this ELISA was best for IVIgM, followed by Pentaglobin and IVIgG; IVIgA did not exhibit an inhibitory effect. Control experiments with excess concentrations of C1q as well as with C1q-depleted serum showed that the inhibitory effects of IVIG were not caused by complement activation and thus, consumption, but that C4 and C3 were scavenged by IgM and to a lesser extent by IgG. These results were confirmed in vivo in the rat anti-Thy 1 nephritis model, in which a single dose of 500 mg/kg of IVIgM prevented C3-, C6-, and C5b-9 deposition in the rat glomeruli, whereas the effect of IVIgG was much less pronounced. Reduction of complement deposition was paralleled by a diminished albuminuria, which was completely absent in the IVIgM-treated rats. IVIgM and to a lesser extent IVIgG also prevented rat C3 deposition on cultured rat glomerular mesangial cells in vitro, but did not influence anti-Thy 1 binding. Neither IVIgM nor Pentaglobin nor IVIgG negatively affected in vitro phagocytosis of Escherichia coli (E coli) by human granulocytes. In conclusion, we have shown that IgM enrichment of IVIG preparations enhances their effect to prevent the inflammatory effects of complement activation.

Animals↗
PubMed1999

Surgically created fourth-third cranial nerve communication: temporary success in a child with bilateral third nerve hamartomas. Case report.

Shortly after birth, an otherwise healthy infant developed eye deviation and ptosis due to a hamartomatous lesion of the interpeduncular segment of the right oculomotor nerve. The left nerve became similarly involved when the child was 1.5 years of age. Direct nerve repair was not possible. Instead, the trochlear nerve was divided and its proximal end was attached to the distal end of the third nerve. Elevation of the upper eyelid and partial adduction of the eye developed gradually over the ensuing 3 to 5 months. Both functions were lost after an additional 2 months, presumably as a result of tumor recurrence or neuroma formation. This case report shows that surgically created fourth-third cranial nerve communication is feasible and may merit consideration under similar circumstances.

Anastomosis, Surgical↗
PubMed1998

Biochemical and kinetic analyses of NS5B RNA-dependent RNA polymerase of the hepatitis C virus.

The biochemical properties of the RNA-dependent RNA polymerase (RdRp) of the hepatitis C virus were analyzed. A hexahistidine affinity-tagged NS5B fusion protein was expressed with recombinant baculoviruses in insect cells and purified to near homogeneity. Enzymatic activity of the purified protein was inhibited by KCl or high concentrations of NaCl and was absolutely dependent on Mg2+, which could be replaced by Mn2+. NS5B was found to be processive and able to copy long heteropolymeric templates with an elongation rate of 150-200 nucleotides/min at 22 degreesC. Kinetic constants were determined for all four nucleoside triphosphates and different templates. In case of a heteropolymeric RNA template corresponding to the last 319 nucleotides of the hepatitis C virus genome, Km values for UTP, GTP, ATP, and CTP were approximately 1.0, approximately 0.5, approximately 10, and approximately 0.3 microM, respectively. The profile of several inhibitors of RdRp activity and substrate analogs indicated that the enzyme has a strong preference for ribonucleoside 5'-triphosphates and that it closely resembles 3Dpol of picornaviruses.

Hepacivirus↗
PubMed1998

Strong expression of CD134 (OX40), a member of the TNF receptor family, in a T helper 2-type cytokine environment.

CD134 (OX40) is involved in T cell costimulation and T cell-dependent antibody production. We show strongly increased T cell expression of CD134 in a model of T helper 2-mediated systemic autoimmunity, induced by HgCl2. Regulation of CD134 expression on CD4+ T cells was further studied in vitro, identifying CD134 as an early marker of T cell activation. CD134 expression could be induced by interleukin-4, but not by interferon-gamma or tumor necrosis factor-alpha. Effects of interleukin-4 and of phorbol 12-myristate 13-acetate on CD134 expression could be blocked by the protein kinase inhibitor staurosporin. Combination of these stimuli with ionomycin resulted in a strongly synergistic increase of CD134 expression, which was blocked by the calcineurin-inhibitor cyclosporin A. The results demonstrate the involvement of two synergistically acting pathways in induction of CD134 expression. Furthermore, they suggest a role for interleukin-4 in induction of CD134 expression in vivo.

Animals↗
PubMed1998

Differential regulation of expression of the MHC class II molecules RT1.B and RT1.D on rat B lymphocytes: effects of interleukin-4, interleukin-13 and interferon-gamma.

Susceptibility to induction of both T helper 1- (Th1) and Th2-mediated autoimmunity is multifactorial and involves genetic linkage to the major histocompatibility complex (MHC) class II haplotype. Brown Norway (BN) rats exposed to mercuric chloride develop a Th2-dependent systemic autoimmunity, whereas Lewis rats, which are highly susceptible to Th1-mediated autoimmunity, develop immune suppression after mercuric chloride exposure. Exposure to mercuric chloride is known to enhance B-lymphocyte expression of the MHC class II molecule RT1.B, predominantly in BN rats. We demonstrate that, in contrast, expression of RT1.D was unmodified on these B cells, whereas both RT1.B and RT1.D were up-regulated on epithelial cells. Regulation of B-cell MHC class II isotype expression was further studied in vitro, using BN rat lymph node (LN) cells. Interleukin-4 (IL-4) strongly enhanced B-cell expression of RT1.B (2.8-fold), whereas RT1.D expression was only slightly, although significantly, modified (1.2-fold). B cells from Lewis rats showed a similar IL-4-induced enhancement of RT1.B expression (2.5-fold), whereas, in contrast, RT1.D expression was unmodified. Exposure of LN cells from BN rats to interferon-gamma induced a moderate increase of B-cell MHC class II expression, predominantly of RT1.B. Strong and rapid enhancement of B-cell RT1.D expression was observed after stimulation by phorbol 12-myristate 13-acetate and ionomycin. Rat IL-13 did not modify B-cell MHC class II expression; however, it induced typical morphological changes in peritoneal macrophages. These experiments demonstrate isotype-specific and strain-dependent regulation of MHC class II expression on rat B lymphocytes, which may be of pathophysiological relevance for the strain-dependent susceptibility for Th1- or Th2-mediated autoimmunity.

Animals↗
PubMed1998

Differential expression of T-cell adhesion molecules and LFA-1-dependent intercellular adhesion in HgCl2-induced autoimmunity and immune suppression.

Exposure of Brown Norway (BN) rats to HgCl2 induces Th2-mediated systemic autoimmunity. In contrast, in Lewis rats, HgCl2 induces immune suppression, mediated by CD8+ T cells. HgCl2 was previously found to enhance expression of LFA-1, ICAM-1 and CD134 (OX40) on T cells in BN rats. In the present study, T cells from Lewis rats were studied at day 4 after injection of HgCl2. CD8+ T lymphoblasts were significantly increased, which were predominantly CD45RC(hi), and which showed enhanced LFA-1 expression. Furthermore, CD4+CD45RC(hi) T cells showed increased numbers of ICAM-1+ cells, whereas expression of CD134 and CD26 was relatively decreased in CD4+ T lymphoblasts. Ex vivo experiments demonstrated that HgCl2-exposure of BN rats, but not of Lewis rats, significantly enhances PMA [phorbol 12-myristate 13-acetate]-induced lymphocyte aggregation, mediated by LFA-1 and ICAM-1. In conclusion, HgCl2-injected Lewis rats show early signs of T-lymphocyte activation, predominantly on CD8+ cells. Strain-dependent effects of HgCl2 on cell adhesion molecules and expression of CD134 may play an important role in development of either autoimmunity or immune suppression.

Animals↗
PubMed1998

Age variance of left ventricular diameters in dogs with cardiac disease.

Ventricular size increases during growth, but often due to cardiac disease. This study aims to describe left ventricular dimension interrelations using a representation that is applicable to patients with cardiac disease, and subsequently to statistically study possible age and gender influences in a large population. In retrospect we analyzed echocardiographically obtained diameters of the left ventricle in 442 dogs of various breeds with congenital or acquired heart disease. Also, we compared our findings with published data on humans and other animals. Multivariate regression analysis was applied to assess possible influences of age and gender. A high correlation was found for end-systolic diameter (ESD) versus end-diastolic diameter (EDD): ESD (cm) = -1.01 cm + 0.93 x EDD (cm) with r = 0.94, p < .00001. Next, these patients were categorized into three age groups (divisions at 3 and 7 years). We detected a slight age dependent effect: the regression coefficients for the younger group differed from the two older groups. No significant gender-related influence was detected. The observation of a high correlation for the ESD versus EDD relationship could be confirmed by reanalysis of published data on normal individuals and human patients. The newly described relationship between ESD and EDD applies in particular to cardiac patients. This is a relevant finding, because clinically important indices of ventricular performance generally depend on ESD, EDD or both. Thus, ESD versus EDD offers a convenient framework for studies on cardiac volume regulation and performance in the cardiopathological spectrum, while permitting incorporation of modulating effects related to age.

Age Factors↗
PubMed1997

Thiol levels in CD134-defined subsets of rat T lymphocytes: possible implications for HgCl2-induced immune dysregulation.

CD134 (OX40), a member of the tumour necrosis factor receptor family, is expressed on activated T cells and mediates T and B cell costimulation. Its expression is increased after exposure to the thiol-binding compound HgCl2 in BN rats, but not in Lewis rats, in association with induction of a T cell-dependent systemic autoimmune syndrome only in BN rats. Intracellular thiols are involved in regulation of activation and death in T lymphocytes. Therefore, we examined intracellular thiol levels in CD134-defined T cell subsets from BN and Lewis rats. Levels of total thiols and glutathione (GSH) were significantly higher in CD134+CD4+ cells than in CD134+CD4+ cells in both strains. In Lewis rats, total thiol levels in CD4+CD134+ cells, but not in CD4+CD134+ cells, were higher than in BN rats. In contrast, BN rats showed higher GSH levels in CD4+CD134+ cells, but not in CD4+CD134+ cells. In vitro exposure to HgCl2 decreased intracellular thiol levels, predominantly in CD4+CD134+ cells. Furthermore, HgCl2-induced enrichment of CD134+ viable cells was inversely correlated to HgCl2-induced cell death. Strain-dependent differences in thiol levels in CD134-defined subsets of CD4+ lymphocytes and subset-specific modification of thiol levels may contribute to differential lymphocyte activation by oxidizing chemicals.

Animals↗
PubMed1996

Enhanced T lymphocyte expression of LFA-1, ICAM-1, and the TNF receptor family member OX40 in HgCl2-induced systemic autoimmunity.

Injection of mercuric chloride into Brown Norway (BN) rats induces a T lymphocyte-dependent autoimmune syndrome. In order to investigate whether modification of adhesion and costimulatory molecules on T lymphocytes may be involved in early T lymphocyte activation by HgCl2, the authors analysed expression of these molecules in peripheral lymph node cells from BN rats at day 4 after injection of HgCl2. Tri-colour flow cytometry was performed for expression analysis within CD45RC-defined subsets of CD4+ and CD8+ cells. Compared to control rats, HgCl2-exposed rats showed increased numbers of lymphocytes, especially of T lymphocyte blast cells. The levels of LFA-1 expression as well as the fractions of ICAM-1 + cells were significantly increased in all CD45RC-defined subsets of CD4+ and CD8+ cells. Within the CD4 + CD45RC10 T lymphocyte population, HgCl2-injected rats showed a highly significant increase in the number of cells expressing OX40, which is a member of the TNF receptor family. Moreover, only CD4 + CD45RC10 blast cells of HgCl2-exposed rats showed decreased expression of CD43, increased expression of CD49d and decreased numbers of CD26 + cells. The results indicate that induction of autoimmunity by HgCl2 in BN rats is associated with altered expression of T lymphocyte costimulatory molecules, predominantly on CD4+ CD45RC10 cells, which may be caused by a direct effect of HgCl2 on these cells, and may precipitate further activation of T and B lymphocytes by HgCl2.

Animals↗
PubMed1996

Endothelial 5-HT receptors mediate relaxation of porcine pulmonary arteries in response to ergotamine and dihydroergotamine.

1. The aim of the present study was to investigate whether antimigraine ergot compounds may act at endothelial 5-hydroxytryptamine (5-HT) receptors which trigger the release of endothelium-derived relaxing factor (EDRF). Changes in tone of porcine isolated pulmonary arteries were measured isometrically. The integrity of the endothelium was assessed by the bradykinin-induced relaxation of prostaglandin F2 alpha (PGF2 alpha, 3 microM)-precontracted vessels. 2. The ergot derivatives ergotamine, dihydroergotamine (DHE) and dihydroergocristine, as well as 5-HT and (+/-)-alpha-methyl-5-HT, elicited a reversible endothelium-dependent relaxation of PGF2 alpha-precontracted arterial ring segments. The relaxation to both ergotamine and 5-HT was associated with an increase in cyclic GMP. After pretreatment of the vessels with NG-nitro-L-arginine methyl ester (200 microM), or removal of endothelium by mechanical rubbing, the relaxant responses were abolished. 3. The mean pEC50 values for relaxant responses followed the order: (+/-)-alpha-methyl-5-HT (8.80) > 5-HT (8.75) > ergotamine (8.17) > DHE (7.70) > 5-carboxamidotryptamine (7.62) > dihydroergocristine (7.17). 4. The relaxant effects of both ergotamine and dihydroergotamine were resistant to block by indomethacin (3 microM), prazosin (1 microM) and ketanserin (1 microM). However, the ergotamine-induced relaxation was highly susceptible to block by pizotifen (pA2 = 8.23), norclozapine (pA2 = 8.20), methiothepin (-log IC50 = 7.26), rauwolscine (pA2 = 7.24) and mesulergine (pA2 = 6.64). Each antagonist inhibited the relaxant responses to (+/-)-alpha-methyl-5-HT in the same manner with similar potency as that determined against ergotamine. 5. Recently, mRNA transcripts for 5-HT1D beta and 5-HT2B receptors have been demonstrated in porcine pulmonary arteries. The rank order of potencies of agonists and antagonists in the present study suggests that the relaxant responses to 5-HT and ergot derivatives are mediated through activation of endothelial 5-HT receptors which are similar to the 5-HT2B receptor subtypes.

Adrenergic alpha-Antagonists↗
PubMed1995

Correlation of interstitial pneumonia with human cytomegalovirus-induced lung infection and graft-versus-host disease after bone marrow transplantation.

In a retrospective analysis lung biopsy specimens obtained postmortem from 30 consecutive allogeneic bone marrow transplant recipients who had died of either either interstitial pneumonitis (IP; 18/30 patients) or various other causes (12/30 patients) were studied for the local presence of human cytomegalovirus (HCMV) by culture, in situ hybridization, polymerase chain reaction (PCR) and immunohistochemistry for HCMV proteins. All patients suffering from IP were found to be HCMV positive in the lung biopsy. PCR revealed the highest sensitivity for HCMV detection in lung biopsies, but in 15/18 PCR-positive samples local HCMV infection could be confirmed by at least one additional technique. All the lung biopsies obtained from the 12 patients without IP were negative for HCMV by all techniques applied, except one with a weak HCMV-DNA signal in the PCR assay. The severity of the clinical, as well as histological and immunohistological alterations in the lung did not correlate with the amount of HCMV-DNA or the number of HCMV-positive cells detected in the biopsy. An increase of HLA-class II antigen and of ICAM-1 expression on the alveolar epithelium, as well as presence of activated CD8+ or CD4+ lymphocytes infiltrating only HCMV-positive lung biopsies revealed T cell-mediated immune reactions to be involved in the pathogenesis of IP. Since all analyzed patients presented with severe acute or extensive chronic graft-versus-host disease (GvHD), but only those with pulmonary HCMV infection developed IP, dissemination of HCMV appears to be the primary requirement for the initiation of IP. GvHD, however, may interfere with normal control of subsequent antiviral immune response and, thus, provoke the immunopathology of IP.

Adolescent↗