Biomedical subjects
Publications and source records attributed to C J De Groot.
Mice with a severe metastasized tumour burden can be cured with a single local injection of interleukin-2. Such a treatment can also be effective against ocular squamous cell carcinoma in cows and transmissible venereal tumours in dogs. We did not notice any toxic effects of this treatment.
OBJECTIVE: Recently, it has been proposed that hereditary coagulation abnormalities leading to an increased venous thrombosis risk may play a role in the development of preeclampsia. We tested this hypothesis in women who have had preeclampsia compared with matched control subjects. STUDY DESIGN: We conducted a case-control study of 163 women with preeclampsia during 1991-1996. Control subjects were matched for age and delivery date. Patients and control subjects were tested for the presence of factor V Leiden, prothrombin 20210A allele, protein C, protein S, and antithrombin deficiency. Logistic regression methods were used for data analysis. RESULTS: The prevalence of these genetic risk factors was similar in the patient group (12.9%) and the control group (12.9%; odds ratio, 1.0; 95% confidence interval, 0.5-3.9). Unexpectedly, we found a high prevalence of factor V Leiden in the control group (9.2%). CONCLUSION: We found no differences in the prevalence of genetic risk factors of thrombosis in women with preeclampsia compared with control subjects.
The pathology of multiple sclerosis (MS) is characterized by breakdown of the blood-brain barrier (BBB), accompanied by infiltration of macrophages and T lymphocytes into the central nervous system (CNS). The migration of these cells into the CNS parenchyma may be partly regulated by chemokines. The aim of this study was therefore to investigate the cellular localization of the potent monocyte- and T-cell-attracting chemokine monocyte chemoattractant protein (MCP)-1 by immunohistochemistry on postmortem brain tissue from MS and normal control cases. Brain tissue samples of six MS patients and four patients without a history of brain disease were neuropathologically classified according to characteristic (immuno)histochemical staining patterns. Frozen tissue sections of active demyelinating MS lesions, chronic active demyelinating MS lesions, and normal control brain were immunohistochemically stained with a monoclonal antibody directed against MCP-1. In active demyelinating MS lesions as well as in chronic active MS lesions, reactive hypertrophic astrocytes were strongly immunoreactive for MCP-1, whereas perivascular and parenchymal foamy macrophages did not express MCP-1 protein. These results suggest a significant role for the beta-chemokine MCP-1, synthesized in vivo by reactive hypertrophic astrocytes, in the recruitment and activation of myelin-degrading macrophages and thereby contributing to the evolution of MS lesions.
BACKGROUND: Because IgG antibodies to foods can be detected before IgE antibodies to inhalants, increased levels of IgG antibodies to foods might be used as a predictor of IgE-mediated allergy in initially nonatopic children. OBJECTIVE: To examine the cross-sectional relation between IgG to foods (i.e. mixture of wheat and rice, mixture of soybean and peanut, egg white, cow's milk, meat, orange and potato) and specific IgE to cat, dog, mite, milk and egg white in 1-year-old children. METHODS: All atopic children (n = 120; 58 with and 62 without eczema) and a random sample of the nonatopic children (n = 144) of the Bokaal study were tested on their IgG response to foods. The IgG results of the food assays were dichotomized high or low using the 66th centile as a cut-off value. RESULTS: Atopic children more often had high IgG levels to foods than nonatopic children. IgG to egg white (OR = 7.50) and mixture of wheat and rice (OR = 4.79) were most strongly associated with positive specific IgE. In a stepwise logistic regression analysis egg white, mixture of wheat and rice, and orange were selected (OR = 3.76, OR = 2.43, and OR = 2.11, respectively). In children without eczema higher levels of IgG to foods were still significantly associated with atopy, which was most prominent for egg white, orange and cow's milk. CONCLUSION: An increased IgG antibody level to foods, especially to egg white, orange, and mixture of wheat and rice, indicates an increased risk of having IgE to cat, dog, mite, egg and/or milk allergens, even in the noneczematous group. Therefore, in another prospective study we are currently investigating the usefulness of IgG in early identification, i.e. before IgE antibodies can be detected, of children with an increased risk of developing allergic diseases in the future.
It is known that the pleiotropic cytokine transforming growth factor beta (TGF-beta) has a regulatory role in the process of tissue repair and remodelling following injury. As reports on these molecules in multiple sclerosis (MS) lesion with different lesional activity are rare, we studied the cellular localization of TGF-beta1, -beta2, and -beta3 isoforms, and TGF-beta receptor type I (TGF-betaR-I) and TGF-betaR-II expression by immunohistochemistry on postmortem brain tissue from MS and normal control cases. To validate the TGF-beta staining results we demonstrated that cultured human adult astrocytes that produce biological active TGF-beta2, and to a lesser extent TGF-beta1, were immunoreactive for all 3 TGF-beta isoforms. Moreover, at mRNA level TGF-beta1 was detected in MS and normal control brain tissue. In normal control brain tissue, TGF-beta isoforms were expressed in ramified microglia and TGF-beta2, and -beta3 on neuronal cells in the gray matter TGF-betaR-I and TGF-betaR-II expression was found on endothelial cells, astrocytes, microglia, and neurons. In active demyelinating MS lesions a strong to intense immunoreactivity was detected for all 3 TGF-beta isoforms in perivascular and parenchymal (foamy) macrophages and in hypertrophic astrocytes. Strong immunoreactivity for TGF-betaR-I and TGF-betaR-II was found on macrophages in both parenchymal and perivascular areas and on hypertrophic astrocytes and endothelial cells in active demyelinating MS lesions. In chronic active and inactive MS lesions, all 3 TGF-beta isoforms and their receptors were strongly expressed in hypertrophic astrocytes. Our findings strongly suggest that the expression of the various TGF-beta isoforms and their receptor types found in MS lesions with different cellular activity participate in reactive processes leading to the formation of chronic MS lesions.
In situ and in vitro studies suggest that activation of locally produced complement factors may act as a mediator between amyloid deposits and neurodegenerative changes seen in Alzheimer's disease (AD). C1-esterase inhibitor (C1-Inh), which regulates activation of C1 of the complement classical pathway, can be detected immunohistochemically in its inactivated form in activated astrocytes and dystrophic neurites in AD plaque areas. In this study, designed to investigate the cellular source of C1-Inh, C1-Inh was found to be secreted in a functionally active form by astrocytes cultured from postmortem human brain specimens as well as by neuroblastoma cell lines. Recombinant human interferon-gamma (IFN-gamma), which stimulates C1-Inh synthesis in various cell types, several-fold stimulated C1-Inh protein secretion by cultured human astrocytes derived from different regions of the central nervous system and by one (SK-N-SH) of two neuroblastoma cell lines (SK-N-SH and IMR-32) included in this study. In contrast to IFN-gamma, other cytokines [interleukin (IL)-1beta, IL-6 and tumor necrosis factor (TNF)-alpha] that can be found in brain areas affected by AD, did not stimulate C1-Inh secretion by astrocytes or neuroblastomas in vitro. This inability to secrete C1-Inh is probably due to unresponsiveness at the transcriptional level, since C1-Inh secretion paralleled the expression of the 2.1-kb C1-Inh mRNA. In situ hybridization with a C1-Inh RNA antisense probe labeled neurons rather than astrocytes, suggesting a role for neurons as producers of complement regulatory proteins in vivo. Since IFN-gamma is apparently lacking in the brain parenchyma, and amyloid plaque-associated cytokines (IL-1beta, IL-6, TNF-alpha) do not stimulate C1-Inh expression in vitro, the nature of the stimulus responsible for neuronal C1-Inh expression in AD brains remains to be investigated.
Primary cultures of human astrocytes, expressing glial fibrillary acidic protein (GFAP), were obtained from postmortem brain tissue samples. These cultured astrocytes produced an extracellular matrix (ECM), containing laminin (Ln) and fibronectin (Fn), as shown with specific antibodies. The perinuclear staining observed in these cells indicated that these proteins were de novo synthesized. Monoclonal antibody (mAb) 90.45, which recognizes the CS1 sequence found in an alternatively spliced form of Fn, also stained cultured astrocytes. Immunohistochemical analysis of normal human brain tissue showed positive staining for the CS1 domain, both on protoplasmic and fibrous astrocytes located in the gray and white matter. In contrast to cultured astrocytes, no immunoreactivity for Ln or Fn was found on astrocytes in normal human brain tissue. These in situ data indicate that the CS1 domain expressed by astrocytes is not part of a splicing variant of Fn. Western blot analysis confirmed that the CS1 domain expressed by cultured human astrocytes is part of an astrocyte protein which is different from human Fn. The CS1 domain is a known ligand for the adhesion receptor alpha4beta1 (VLA-4). We found that the human lymphoma cell lines Jurkat and Ramos, which express alpha4beta1, bound to cultured human astrocytes, and that this interaction could be partly blocked by mAb 90.45 or a synthetic CS1 peptide. Thus, the novel CS1-containing surface protein expressed by astrocytes in vitro and in vivo, contributes to binding of lymphoblasts, and therefore may be a relevant adhesion molecule for the recruitment of alpha4-integrin expressing leukocytes into the central nervous system (CNS).
We have successfully established highly enriched astrocyte cultures upon passaging of primary cultures derived from various regions of postmortem human adult brain and spinal cord. Tissues were collected at autopsies with relatively short postmortem times (3-9 hr) from multiple sclerosis (MS) and (normal) control cases. Immunocytochemical analysis showed that primary cultures were composed of colonies of oligoclonal cells that expressed the intermediate filament proteins glial fibrillary acidic protein (GFAP), vimentin, as well as glutamine synthetase (GS). Passaging the astrocytes did not affect their proliferating capacity as monitored by bromodeoxyuridine (BrdU) incorporation. Astrocyte-specific markers were stably expressed for at least 12 passages per individual tissue sample. Large numbers of GFAP-positive astrocytes were obtained from each sample and could be stored frozen and recultured. Very few macrophages/microglial cells (1-3%) were present in the human adult astrocyte cultures, using a panel of macrophage-specific markers. However, the monoclonal antibodies (mAbs KP1, EBM1, 25F9) and lysozyme antiserum directed against lysosomal antigens strongly immunostained cultured astrocytes derived from MS and control cases, implicating that expression of these lysosomal antigens is not restricted to macrophages/ microglial cells in human glial cell cultures. Interestingly, astrocytes derived from active demyelinated MS lesions showed an increased proliferating capacity compared to astrocytes derived from non-lesioned and normal brain and spinal cord regions, as shown with a microculture tetrazolium assay (MTT assay).
The pathology of multiple sclerosis (MS) is characterised by breakdown of the blood-brain barrier accompanied by infiltration of macrophages and T cells into the central nervous system (CNS). Myelin is degraded and engulfed by the macrophages, producing lesions of demyelination. Some or all of these mechanisms might involve proteinases, and here we have studied the cellular localisation and distribution of two matrix metalloproteinases (MMPs), MMP-7 (matrilysin) and MMP-9 (92-kDa gelatinase), in the normal human CNS and active demyelinating MS lesions. Cryostat sections of CNS samples were immunostained with antisera to MMP-7 and MMP-9. In addition, non-radioactive in situ hybridisation (ISH) was performed using a digoxygenin-labelled riboprobe to detect the expression of MMP-7. MMP-7 immunoreactivity was weakly detected in microglial-like cells in normal brain tissue sections, and was very strong in parenchymal macrophages in active demyelinating MS lesions. This pattern of expression was confirmed using ISH. MMP-7 immunoreactivity was not detected in macrophages in spleen or tonsil indicating that it is specifically induced in infiltrating macrophages in active demyelinating MS lesions. MMP-9 immunoreactivity was detected in a few small blood vessels in normal brain tissue sections, whereas many blood vessels stained positive in CNS tissue sections of active demyelinating MS lesions. The up-regulation of MMPs in MS may contribute to the pathology of the disease.
The cellular localization and distribution of inducible and constitutive nitric oxide synthase (iNOS/cNOS) was determined in tissue sections from multiple sclerosis (MS) and control brain and spinal cord. Immunocytochemical techniques were applied using specific iNOS- and cNOS-directed antibodies. In addition, NADPH-diaphorase histochemistry was performed. To establish the identity of iNOS-, cNOS- and NADPH-diaphorase-positive cells single and double staining was performed on tissue sections with the macrophage marker KP1 (CD68) and with the astrocyte marker glial fibrillary acidic protein (GFAP). Areas of myelin breakdown and demyelination were determined using a staining for neutral lipids, Oil Red O (ORO). Furthermore, macrophages isolated from active demyelinating MS lesions were stained for iNOS, cNOS, KP1 and ORO. In active MS lesions strong iNOS immunoreactivity was found exclusively in perivascular and parenchymal macrophages distributed within regions of active demyelination. In these active MS lesions immunoreactivity for cNOS was also found in macrophages. Macrophages isolated from active MS lesions also showed immunoreactivity for iNOS and cNOS. Moreover, these isolated macrophages produced nitric oxide (NO; >30 microM) in vitro. NADPH-diaphorase activity was detected in KP1-positive perivascular and parenchymal macrophages and in GFAP-positive reactive astrocytes in active MS lesions and in reactive astrocytes located in the hypercellular rims of chronic active MS lesions. cNOS-positive reactive astrocytes were detected in both active and chronic active MS lesions. Inside chronic active lesions some residual macrophages were weakly iNOS-positive. In control brain and spinal cord no iNOS immunoreactivity could be detected. These results suggests an important role for human macrophages capable of producing the free radical nitric oxide (NO), which may contribute to the cytotoxicity of oligodendrocytes and destruction of myelin in MS brain and spinal cord.
The effects of increasing postmortem delay (PMD) times on morphological, immunological and functional characteristics of various brain cells both in situ and in vitro were studied in postmortem brain tissue derived from rats with acute experimental allergic encephalomyelitis (EAE). A decline of the brain tissue structure was first noted after a PMD of 6 h. Radial glia in the cerebellum were frequently interrupted and retractions artifacts appeared around brain cells. However, even after the longest PMD interval of 18 h the quality of the cell and tissue structure was still good enough for immunohistochemical characterization. Immunohistochemical staining of frozen and fixed rat brain tissue sections resulted in an enhancement of the immunoreactivity after a PMD of 4 h, using a panel of mono and polyclonal antibodies directed against glial fibrillary acidic protein (GFAP), basement membranes (laminin), brain macrophage antigens (ED1 and ED2), and various immunologically important surface molecules, such as major histocompatibility complex (MHC) class II (Ia) antigen (OX6), CR3 complement receptor (ED8), and leukocyte common antigen (OX1). No increase in staining intensities with the ED1, ED8 and OX6 mAbs specific for macrophage antigens could be detected on brain macrophages that were isolated from brain tissue of rats with EAE obtained after various PMD intervals. Irrespective of the PMD interval, viable astrocyte cell cultures were obtained with comparable staining intensities for GFAP. These cultured astrocytes were capable of ingesting Latex beads and were highly proliferative as measured by BrdU uptake, at all investigated PMDs. Thus, even after long PMD intervals, brain material can be used successfully. Other data suggest that the situation is similar to human brain material, even though the PMD times may be somewhat different.
We examined the effect of bromocriptine (BCR) treatment on the duration and severity of neurological symptoms of acute experimental allergic encephalomyelitis (EAE), an animal model for demyelinating diseases, particularly multiple sclerosis. To mimic the clinical situation, BCR treatment was started after the onset of clinical signs. Furthermore, the effect of BCR treatment on the course of a chronic relapsing form of EAE was studied. BCR was injected at daily intervals in a dose that resulted in sustained suppression of plasma concentrations of prolactin, a pituitary hormone that plays a role in immunoregulation. In acute EAE, BCR therapy reduced both severity and duration of the clinical signs. In chronic relapsing EAE, BCR treatment did not affect the severity and duration of the first attack, but reduced the duration of the subsequent, second attack. Thus, BCR treatment improves the clinical course in animals with ongoing disease. These findings may have implications for the search for new therapeutic approaches in multiple sclerosis.
BACKGROUND: Cyclosporin A is reported to impair endothelium-mediated vasorelaxation and induce endothelin release in some noncoronary vascular beds. We wished to determine whether acute cyclosporine administration induces endothelial dysfunction in coronary conductance or resistance arteries. METHODS AND RESULTS: We examined the effect of intracoronary acetylcholine, N omega-nitro-L-arginine methyl ester (L-NAME), L-arginine, nitroglycerin, and adenosine before and after acute cyclosporine administration (3 mg/kg IV over 30 minutes) in anesthetized dogs. Flow velocity was measured with a 0.014-in Doppler wire to assess resistance vessel responses, and epicardial coronary lumen area was simultaneously measured with a 4.3F, 30-MHz imaging catheter inserted over the Doppler wire. In 6 dogs, acetylcholine-induced increase in flow velocity was attenuated by cyclosporine in vehicle (137% to 55% at 10(-5) mol/L, P < .001), as was acetylcholine-induced epicardial vasodilation (14.1% to 6.7% at 10(-5) mol/L, P < .001). Vasodilation in response to intracoronary nitroglycerin (200 micrograms) and adenosine (6 mg) were unchanged by cyclosporine. Epicardial vasoconstriction with L-NAME (10(-4) mol/L) was reduced by cyclosporine (Pre, 7.4 +/- 0.9%; Post, 2.6 +/- 1.2%; P = .04), but L-arginine (10(-4) mol/L) had no effect after cyclosporine. In another 5 dogs, pure cyclosporine impaired acetylcholine-induced vasodilatation to the same degree as cyclosporine in vehicle (Cremophor); vehicle infusion did not impair endothelial function. In 5 more dogs, cyclosporine did not increase either arterial or coronary sinus concentrations of endothelin-1. CONCLUSIONS: The present study shows that cyclosporine acutely impairs release of endothelium-derived relaxing factor in canine conductance and resistance coronary arteries and provides evidence for decreased epicardial nitric oxide release after cyclosporine. The potential contribution of acute cyclosporine-induced coronary endothelial dysfunction to posttransplant vasculopathy needs further study.
Explore the source record for details and available documents.
Galactocerebrosidase-deficient oligodendrocytes of 'twitcher' (twi/twi) mice degenerate prematurely. Transplantation of normal bone marrow cells has been shown to alleviate symptoms and to prolong survival time. However, characteristic ataxia ('twitching') is not cured. In an attempt to improve further the condition of twitcher mice, allogeneic foetal liver cells were transplanted as a source of normal haemopoietic stem cells and supplemented with intracerebral transplantation of foetal brain cells. A reliable method was developed to detect donor-type cells in brain tissue. Bacteriophage lambda transgenic foetal mice were used as donors of both foetal liver and brain cells. Integrated copies of lambda DNA in donor cells were detected by in situ hybridization with biotinylated probes, which were then stained using streptavidin alkaline phosphatase. This technique was combined with immunohistochemistry to distinguish donor-type oligodendrocytes from macrophages. Immunoperoxidase staining with an antiserum to carbonic anhydrase-II produced dark perikarya of oligodendrocytes. The results demonstrated that local foetal brain cell grafts resulted in a wide dissemination of donor-type oligodendrocytes throughout the twitcher brain. The addition of a foetal brain cell graft to haemopoietic cell transplantation resulted in significantly prolonged survival of twitcher mice.
Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS). In particular in the CsA-induced chronic relapsing form (CREAE), pronounced demyelination occurs, in temporal association with relapses. It is still a matter of discussion which cell type ultimately is responsible for the actual process of demyelination. Macrophages, cytotoxic T lymphocytes and also astrocytes are possible candidates. In this short overview, the role of macrophages in the pathogenesis of EAE is discussed. It is shown that in particular, newly recruited macrophages play a crucial role in the generation of clinical signs. Possible mechanisms by which macrophages are involved in the pathogenesis of demyelinating diseases are presented.
The inducibility of major histocompatibility complex class II (Ia) antigens on glial cells of the brain suggests that neuroglia have immunoregulatory functions within the central nervous system (CNS), i.e., recognition and presentation of antigens. The aim of the present study was to investigate rat recombinant-interferon-gamma (r-IFN-gamma) induced Ia antigen expression in rat cerebral cultures containing type-1 astrocytes and macrophages, and in rat spinal cord cultures enriched in type-2 astrocytes or oligodendrocytes. We compared induction of Ia antigen expression in glial cell cultures derived from Lewis rats, which are very susceptible to experimental allergic encephalomyelitis (EAE), with those from Wistar rats, which are but modestly EAE susceptible. After 5 days in culture we found in Wistar rat type-1 astrocyte-enriched cultures that Ia antigens were expressed by 19% of the astrocytes, whereas we found that in Lewis rat type-1 astrocyte cultures a considerably higher number of astrocytes expressed Ia antigens (53%). However, no significant difference were found in Ia antigen expression between type-2 astrocytes derived from Wistar rat spinal cord (49%) and Lewis rat type-2 astrocytes (56%). In contrast, in oligodendrocyte-enriched cell cultures derived from either Lewis or Wistar rats no Ia antigen expression was found. Interestingly, we found in type-1 astrocyte-enriched cerebral cultures a large number (approx. 46% of the cells) of brain macrophages (amoeboid microglia), all expressing Ia antigens after treatment with r-IFN-gamma.
Experimental allergic encephalomyelitis (EAE) is an autoimmune inflammatory disease of the central nervous system (CNS) which causes paralysis. Several studies have reported the involvement of Ia antigen-expressing cells in the pathogenesis of EAE. Interferon-gamma (IFN-gamma) can induce Ia antigen expression on a wide range of cells. We examined the effect of IFN-gamma on EAE in Lewis rats. Systemically administered IFN-gamma did not change the disease course of EAE, whereas IFN-gamma applied locally into the ventricular system of the CNS resulted in complete suppression of clinical signs. Furthermore, we found that systemic administration of anti-IFN-gamma just prior to the onset of clinical symptoms resulted in a more severe disease course. We conclude that IFN-gamma is capable of exerting a suppressive action in EAE, possibly through induction of Ia antigen expression or through the induction of suppressive mechanisms locally in the CNS.