Biomedical subjects
Publications and source records attributed to E Holler.
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Bispecific antibodies directed against tumor-associated target antigens and to surface receptors mediating T-cell activation, such as the TCR/CD3 complex and the costimulatory receptor CD28, are capable of mediating T-cell activation resulting in tumor cell killing. In this study, we used the B-cell-associated antigens CD19 and CD20 as target structures on human leukemic cells. We found that a combination of bispecific antibody fragments (bsFab2) with target x CD3 and target x CD28 specificity induces vigorous autologous T-cell activation and killing of malignant cells in peripheral blood and bone marrow cultures from patients with chronic lymphocytic leukemia and follicular lymphoma. The bsFab2 targeting CD20 were considerably more effective than those binding to CD19. The colony-forming capacity of treated bone marrow was impaired due to large amounts of tumor necrosis factor alpha produced during bsFab2-induced T-cell activation. Neutralizing tumor necrosis factor alpha antibodies were found to reverse this negative effect without affecting T-cell activation and tumor cell killing. CD20 x CD28 bsFab2, when used alone rather than in combination, markedly improved the recognition of leukemic cells by allogeneic T cells. Therefore, these reagents may be capable of enhancing the immunogenicity of leukemic cells in general and, in particular, of increasing the antileukemic activity of allogeneic donor buffy coat cells in relapsed bone marrow transplanted patients.
beta-Poly(L-malate) is supposed to function in the storage and transport of histones, DNA polymerases and other nuclear proteins in the giant syncytical cells (plasmodia) of myxomycetes. Here we report on the biosynthesis of [14C]beta-poly(L-malate) from injected L-[14C]malate in the plasmodium of Physarum polycephalum. The effects of KCN, arsenate, adenosine 5'-(alpha, beta-methylene)triphosphate, adenosine 5'-(beta, gamma-methylene)triphosphate, guanosine 5'-(beta, gamma-methylene)triphosphate, desulfo coenzyme A and phenylarsinoxid on beta-poly(L-malate) synthesis were studied after their coinjection with L-[14C]malate. The synthesis was not affected by KCN or desulfo coenzyme A, but was blocked by arsenate and adenosine 5'-(alpha,beta-methylene)triphosphate. The plasmodium lysate catalysed an L-malate-dependent ATP-[32P]pyrophosphate exchange, but was devoid of beta-poly(L-malate) synthetic activity under all experimental conditions tested. The results suggested an extramitochondrial synthesis of beta-poly(L-malate), involving the polymerization of beta-L-malyl-AMP. It is assumed that the lack of synthesis in the lysate is caused by the inactivation of beta-poly(L-malate) polymerase involving a cell injury kinase pathway. Because injected guanosine 5'-(beta, gamma-methylene)triphosphate blocks the synthesis, the injury signal is likely to be GTP dependent.
BACKGROUND AND PURPOSE: There is only limited information about late neurological complications after bone marrow transplantation (BMT). The purpose of this study is to describe a cerebral angiitis-like syndrome after allogeneic BMT. METHODS: Clinical and diagnostic findings of 5 BMT patients with chronic graft versus host disease and neuropathological data of 1 patient were reported. RESULTS: In the described patients, focal neurological signs and neuropsychological abnormalities occurred years after BMT. MRI revealed periventricular white matter lesions, lacunar or territorial infarctions, leukoencephalopathy, and hemorrhages. Angiitis of the central nervous system was confirmed in 1 patient at autopsy, and an angiitis-like syndrome was suspected in the other patients because of the clinical course and response to treatment. Three patients received cyclophosphamide and steroids (2 improved, 1 died), 1 patient improved after steroids alone, and 1 patient without immunosuppressive therapy deteriorated further. CONCLUSIONS: We propose that an angiitis-like syndrome of the central nervous system can be a neurological manifestation of graft versus host disease, which should be considered a possible cause of cerebral ischemic episodes and pathological MRI scans in BMT patients with graft versus host disease.
Hepatic veno-occlusive disease (VOD) is the third most important fatal complication in allogeneic bone marrow transplantation (BMT), the second most significant one in the autologous setting and the most severe of all the regimen related toxicities. A growing number of VOD cases has to be expected due to the increasing number of high dose chemotherapies given with consecutive stem cell transplantation in patients with solid tumors. Confirmation of the diagnosis of VOD by biopsy is associated with a high risk of severe bleeding complications and, unfortunately, until now reliable laboratory markers have not as yet been established. Recently, plasminogen activator inhibitor 1 (PAI-1), the main inhibitor of the fibrinolytic system, has been found to be significantly elevated in VOD patients probably reflecting hypofibrinolysis in these patients. Furthermore, PAI-1 was able to distinguish between patients with VOD and those with hyperbilirubinemia after BMT caused by graft-versus-host-disease (GVHD) or toxic effects, in which cases the PAI-1 levels were mostly within the normal range. In this overview we summarize the data strongly indicating that PAI-1 is a useful marker for the diagnosis of VOD and helps in the differential diagnosis of hyperbilirubinemia after BMT.
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Thrombotic complications are observed in patients undergoing bone marrow transplantation despite thrombocytopenia and impaired coagulation due to liver function disturbances. Endothelial cell damage which is involved in the pathogenesis of major transplant related complications like graft-versus-host disease, veno-occlusive disease, sepsis or microangiopathy may be a contributing factor. Little is known about platelet function in bone marrow transplant recipients. In order to study functional alterations in circulating platelets we investigated unstimulated and ADP-stimulated platelets of 10 bone marrow transplant recipients ex vivo by flow cytometry in a pilot study using a panel of monoclonal antibodies to characterize changes in membrane glycoproteins. Samples were collected before and during conditioning and at three timepoints after engraftment. 10 healthy volunteers served as controls. Platelets of bone marrow transplant recipients showed partly a significant, higher expression of surface bound fibrinogen, activated fibrinogen receptor, and glycoprotein Ib as compared to controls. P-selectin, a marker of platelet degranulation was significantly elevated after ADP-induced stimulation at all timepoints compared to controls. Only marginal differences were found for GP IIb/IIIa surface expression. The data point to an increased platelet activation state in bone marrow transplant recipients which might contribute to the thrombotic phenomena observed in these patients.
Plasmodia of Physarum polycephalum contain large amounts of the cell-type-specific polyanion beta-poly(L-malate) and of a corresponding specific hydrolase (polymalatase), both expressed in the plasmodial form of the organism. We have partially purified polymalatase, the preparation consisting of several polypeptides, which could not be separated without destroying the hydrolase activity. Polypeptides of 68 kDa and 97 kDa were identified as polymalatases. Both were glycosylated, the 68-kDa form giving rise to a 54-kDa form when deglycosylated, and the 97-kDa form giving rise to an 88-kDa polypeptide that was indistinguishable from an 88-kDa inactive species also contained in the enzyme preparation. Antisera against each of these proteins were used to detect the intracellular distribution of the proteins. We found that the antisera crossreacted with the three proteins and, furthermore, with a multiplicity of polypeptides ubiquitously distributed over the plasmodium. Results of a two-dimensional non-denaturing in the first dimension and SDS-denaturing polyacrylamide gel electrophoresis in the second dimension suggested that the proteins were derived from a 200-kDa 'precursor' protein by proteolytic fragmentation. Polymalatase activity could be generated from a high molecular-mass precursor. According to several pieces of evidence, the proteolytic nicking occurred within plasmodia. The fragments were sticky and gave rise to preferred sizes of nicked macromolecules. The observed multiplicity varied as a function of the age of the cultures. The cellular distribution and the intracellular pH value were not compatible with an in situ polymalatase activity and suggested other, presently unknown, function(s) such as in the transportation of beta-poly(L-malate) from the nucleus to the culture medium.
The aim of this study was to assess neurological, neuropsychological, and neuroradiological findings in long-term survivors of allogeneic bone marrow transplantation (BMT) who were recruited from a hematological outpatient clinic. In addition, risk factors for the development of late neurological complications were identified. In contrast to previous studies on autopsied patients, our study design provoked a bias away from increased neurological sequelae, because patients with early complications after BMT were excluded. Fifty-nine allogeneic patients and 7 autologous BMT patients underwent clinical examination, short neuropsychological testing, and cranial magnetic resonance imaging (MRI) 34 +/- 26 months after BMT. The pathological results of the neurological examination (abnormal 64%) and the MRI examination (white matter lesions, 54%; atrophy, 11%) were associated with the occurrence of chronic graft-versus-host disease (GvHD) evolving from acute GvHD, with corticosteroid therapy and with cyclosporine medication. Neuropsychological impairment (cognitive deficits, 37%) was associated with long-term cyclosporine medication and age. No influence of pre-BMT disease, BMT donor status, or the conditioning regimen was found. These results suggest that the frequent neurological abnormalities in long-term survivors of allogeneic BMT are associated with chronic GvHD and with the resulting immunosuppression as major risk factors.
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Recent reports have shown that pH could also be used as a melting factor to monitor helix-coil transitions in DNA; the results being comparable to those obtained by Tm studies. The rapidity with which the method can be performed to obtain similar transition curves, and elimination of the evaporation factor (at high temperatures as seen in Tm studies) is one of the advantages offered by this technique. With regard to its suitability in studying DNA-drug interactions, the addition of platinum (II) complexes changed the Pm (pH of melting) in a predictable manner thereby confirming the destabilization of bases in DNA. In the present study, melting profiles of calf thymus DNA modified by certain chloro substituted platinum complexes have been generated using pH as a denaturing factor. These novel platinum complexes have been recently shown to have potential tumour inhibiting properties too. Diammine diaqua platinum (active form of the anti tumour drug cisplatin) was coupled to beta poly-L-malate (a bioresorbable polymer synthesized by a myxomycete), L-malate and L-succinate. At a constant Pt:P ratio (0.2), the extent of damage to DNA by these complexes in comparison to cisplatin was cisPt>SuccPt>MalPt>PMA-Pt>carboplatin. Given the similarity of the side groups of these platinum compounds with that of carboplatin (a successful second generation analog of cisplatin), interesting variations have been obtained in the DNA melting profiles, the implications of which have been discussed in the present study.
Serum levels of Interleukin-6 (IL-6), a proinflammatory cytokine, are increased in early stages of inflammatory diseases such as infection and sepsis. Assay systems which permit its measurement within a few hours and as a single measurement have not been reported so far. We therefore evaluated a now commercially available automated method for IL-6 measurement on the Cobas Core immunological analyzer (Roche Diagnostic Systems) which enables single IL-6 measurement within about 1 hour. The automated assay correlates well with an established, manual microtiter plate assay (Biosource GmbH) which uses the same antibodies and reagents (r=0.98). Accuracy of the automated method was established by adding known amounts of IL-6 international reference preparation. Recovery of the international standard was in the range of 92104%. The automated assay had a precision of singletons below 6% and was linear up to 2800 pg/ml. This automated assay provides a suitable, convenient and time saving method for measurement of IL-6 serum levels in the routine clinical laboratory.
Beta-poly(L-malate), a major constituent of nuclei in plasmodia of Physarum polycephalum, is enzymatically degraded to L-malate after secretion into the culture medium. This depolymerization is specifically catalysed by an endogenous polymalatase. The mode of action and the specificity criteria have been investigated by employing various chemical derivatives of beta-poly(L-malate), including substitution at the hydroxy-terminus and carboxy-terminus of the polymer, esterification of the pending alpha-carboxylate, and beta-poly(DL-malate). The results of the investigation were summarized in a specificity model that involved recognition of the hydroxy-terminus and of the alpha-carboxylate as substituents of the asymmetric carbon in the malic acid unit. Depolymerization proceeded from the hydroxy-terminus towards the carboxy-terminus, thereby degrading the polymer to L-malate. When the terminal beta-carboxylate had been amidated with the fluorescent N-(1-naphthyl)ethylenediamine, degradation was normal but was arrested at the level of the terminal beta-carboxy-substituted dimer. It should be possible to employ polymalatase as a tool for the detection of branching and other modifications of beta-poly(L-malate).
To extend previous studies on the anti-inflammatory role of interleukin (IL)-10 in vivo, mice pretreated with IL-10 were subjected to ionizing radiation (IR), lipopolysaccharide (LPS), or both and assessed for the expression of the intercellular adhesion molecule 1 (ICAM-1) in immunohistochemical analyses. IL-10 was able to almost fully protect LPS+IR-treated animals against ICAM-1 up-regulation. Because LPS and IR also increased adhesion of peripheral blood mononuclear cells, transendothelial migration assays were performed to investigate the functional significance of these findings. IR was found to induce transendothelial migration, and this effect could be enhanced by cotreatment with LPS, in the same fashion as peripheral blood mononuclear cell adhesion. Also in this system, IL-10 proved to act as a potent LPS antagonist. Finally, in vivo immunohistochemical analyses revealed an infiltration of CD3+ T lymphocytes into organs that were the target of transplant-related complications after LPS+IR treatment. This infiltration could also be completely reversed by IL-10 pretreatment.
Human umbilical vein endothelial cells (HUVECs) undergo programmed cell death (apoptosis) after coculture with peripheral blood mononuclear cells (PBMCs) preactivated by ionizing radiation (IR) or by bacterial endotoxin (lipopolysaccharide [LPS]). Cell-to-cell contact-mediated apoptosis could be blocked in both cases by anti-tumor necrosis factor-alpha (anti-TNF-alpha) monoclonal antibody MAK195 and also by the antagonistic cytokine interleukin-10 (IL-10). Cell-free PBMC supernatants from both preactivation treatments were sufficient to trigger endothelial apoptosis. In contrast, MAK195 and IL-10 were found to be ineffective in this system, suggesting a TNF-alpha-independent mechanism. However, N-Acetylcystein, an antioxidant, fully abrogated programmed cell death mediated by the supernatant of IR-treated PBMCs, but not of LPS-treated PBMCs. Additionally, we found that coculture and cell-free supernatants of preactivated as well as untreated PBMCs caused cell cycle arrest in proliferating EC in G(0/1), which could be relieved by IL-10, but not by anti-TNF-alpha. Further analysis showed that transforming growth factor-beta, which was constitutively expressed in the supernatant of PBMCs, namely lymphocytes, was responsible for this. These data suggest a pathophysiologic model in which preactivated PBMCs cause EC damage and may prevent blood vessel repair by arresting the proliferation of ECs. This could contribute to the understanding of various clinical endothelial complications that occur after irradiation as well as in cases of endotoxemia or related inflammatory states.
Hepatic veno-occlusive disease (VOD) is a frequent and severe complication after bone marrow transplantation (BMT). We previously have described plasminogen activator inhibitor-1 (PAI-1) as a possible marker of VOD. To confirm the significance of this finding, we now determined PAI-1 levels in 31 of 186 consecutive patients undergoing BMT who developed hyperbilirubinemia greater than 3 mg/dL for various reasons. Diagnoses were made by clinical criteria and confirmed by biopsy in 23 of 31 patients. They included VOD (n = 7), acute graft-versus-host disease (GVHD) of the liver (n = 7), and other hepatic injury (n = 17). PAI-1 (mean +/- SD) was significantly (P < .001) elevated in patients with VOD (321.6 +/- 161.2 ng/mL) as compared with patients with GVHD (22.8 +/- 8.4 ng/mL) or other hepatic damage (32.8 +/- 30.8 ng/mL) at the timepoint of bilirubin increase. At the peak bilirubin concentration, the corresponding PAI-1 levels were 426.1 +/- 230.0 ng/mL in patients with VOD, 41.0 +/- 20.6 ng/ mL in patients with GVHD, and 44.6 +/- 32.9 ng/mL in patients with other hepatic injury (P < .001 VOD v GVHD/other hepatic injury). Our results underline the relevance of PAI-1 in the differential diagnosis of hyperbilirubinemia after BMT and its significance as a sensitive and specific marker of severe VOD.