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Biomedical subjects

R Fischer

Publications and source records attributed to R Fischer.

At least 163 records · Page 9Linked to original sources
PubMed1999

Focal lymphoid aggregates (nodules) in bone marrow biopsies: differentiation between benign hyperplasia and malignant lymphoma--a practical guideline.

AIMS: To provide practical guidelines for the differentiation between benign and malignant focal lymphoid aggregates (lymphoid nodules) in routinely referred bone marrow trephine biopsies, using a synoptic approach including clinical data and histological workup. METHODS: For easy identification of very small lymphoid infiltrates the chloroacetate esterase stain was applied as a screening procedure. This allowed the identification of 491 formalin fixed, paraffin wax embedded specimens with one or more lymphoid nodules. Examination of lymphoid infiltrates included such variables as histotopography, demarcation, cytology, reticulin fibres, and immunohistochemistry with a set of monoclonal antibodies (CD20, CD45R, CD45R0, CD3, CD43). Evaluation of clinical and morphological data was carried out independently. In case of malignant lymphomas, a correlation with corresponding lymph node findings was made. RESULTS: 352 patients had benign focal lymphoid aggregates usually associated with systemic autoimmune diseases, chronic myeloproliferative disorders, toxic myelopathy, and viral infections. Discrete nodular infiltrates of (small cell) malignant lymphomas (n = 93) simulating benign hyperplasia were found in chronic lymphocytic leukaemia, germinal centre cell lymphomas (CB-CC), and lymphoplasmacytic/cytoid lymphomas (LPI). In addition to immunoreactivity, certain histological variables proved distinctive. These were: (1) histotopography, that is, localisation of the lymphoid aggregates within the bone marrow space; (2) relation to the surrounding tissue: margination or interstitial spillage of lymphoid cells; and (3) increase in reticulin fibres. CONCLUSIONS: A combined diagnostic procedure identifying several distinctive features, in particular histotopography and immunohistochemistry, provides a most promising way of discriminating reactive from neoplastic lymphoid nodules in the bone marrow.

Bone Marrow↗
PubMed1999

Bone marrow histopathology in chronic myelogenous leukemia (CML)--evaluation of distinctive features with clinical impact.

Bone marrow features in stable-phase chronic myelogenous leukemia (CML) are characterized by a striking heterogeneity which is determinable by appropriate means including representative pre-treatment trephine biopsies, immunohistochemistry and morphometry. Cell lineages involved to a variable extent consist not only of neutrophil granulopoiesis, but include also megakaryocytes, erythroid precursors, resident macrophages and lymphocytes. Moreover, the stromal compartment, in particular reticulin and collagen fibers, plays a pivotal role in the disease process. Following morphometric analysis significant correlations may be calculated between histological parameters and clinical-laboratory findings. Relevant interactions are detectable between number of megakaryocytes and their precursors with fiber density. This findings is in line with the close functional relationships between megakaryopoiesis and fibroblasts regarding the complex pathomechanisms of myelofibrosis. Moreover, other correlations are observable between reduction of erythropoiesis or increase in fibers with clinical features like anemia, percentages of myelo- and erythroblasts in the peripheral blood, spleen size or LDH level. These variables are in keeping with more advanced stages of CML which indicate a transition to myeloid metaplasia and thus exert a significant impact on survival. Consequently, the different risk profiles of patients are determined by both clinical and morphological parameters of predictive value. Regarding the latter, extent of myelofibrosis, amount of erythroid precursors and numbers of myeloerythroblasts in the peripheral blood are significantly associated with prognosis. For this reason, it should be mandatory to enter morphological criteria into prospective clinical trials on CML, not only for diagnostic purpose, but also for a proper evaluation of different survival patterns.

Animals↗
PubMed1999

Clinicopathological diagnosis and differential criteria of thrombocythemias in various myeloproliferative disorders by histopathology, histochemistry and immunostaining from bone marrow biopsies.

Differentiation of essential thrombocythemia (ET) from thrombocythemias occurring in various subtypes of chronic myeloproliferative disorders (MPDs) is controversial, because of the lack of uniform clinical and morphological criteria. A retrospective clinicopathologic study was performed on 375 patients presenting with a MPD and a platelet count exceeding 500 x 10(9/)l. For comparison 35 patients with reactive thrombocytosis (RT) and five patients with a myelodysplastic syndrome (MDS-5q(-) syndrome) were enrolled into this study. In addition to a complete clinicopathological work-up, procedures included histochemical and immunological staining techniques and morphometry of bone marrow biopsies for proper evaluation of megakaryocytes (CD61) and erythroid precursors (Ret40f). Because of the high patient's age on admission, relative survival rates with corresponding disease-specific loss of life expectancy were calculated. Analysis of clinical and morphological characteristics, in particular megakaryopoiesis revealed features which enabled a clear-cut distinction between thrombocythemias in MPDs and thrombocythemic states in MDS. This rationale proved to be most important for the diagnostic discrimination of the 33 patients with initial (prefibrotic) stages of idiopathic myelofibrosis (IMF) from ET (40 patients). A new set of relevant criteria for the diagnosis of IMF with special regard to early stages and its distinction from ET has been proposed. Hemorrhagic episodes were more frequently observed in ET than in thrombocythemias associated with polycythemia vera (PV). Computation of specific loss of life expectancy revealed two extremes: thrombocythemia in CML (81%) and ET (3%), whereas thrombocythemias in PV and IMF did not show a significantly different life loss (19-22%). The revised criteria for ET, PV and IMF are reliable by taking histopathological features from bone marrow biopsies into consideration, particularly for the diagnosis of ET and its differentiation from thrombocythemias as a presenting symptom accompanying the various subtypes of MPDs.

Aged↗
PubMed1999

The influence of hemochromatosis mutations on iron overload of thalassemia major.

BACKGROUND AND OBJECTIVE: Hemochromatosis is a genetic form of iron overload due to a defective HFE gene. Secondary iron overload is the main complication in transfusion-dependent thalassemia patients. In this work we have examined the prevalence of HFE mutations in thalassemia major and evaluated the degree of iron overload of patients with and without HFE mutations. DESIGN AND METHODS: HFE mutations were studied in 71 Italian thalassemic patients and in 189 normal controls, using PCR and restriction enzyme analysis. The degree of iron overload, assessed by serum ferritin and liver iron concentration (LIC), was compared in 17 patients with mutations in the HFE gene, and in 17 subjects with wild type HFE genotype. The two groups of patients had comparable globin gene mutations, were matched for age and were homogeneous for transfusion and chelation history. In all cases the iron balance calculated on the basis of transfusion regimen and iron excreted by chelation was available. RESULTS: The allele frequencies of C282Y and H63D were respectively 1.4% and 12.7% in patients and 1.1% and 11.4% in controls. No case of C282Y homozygosity was recorded among patients. No significant difference was found in terms of serum ferritin, LIC, or the age at chelation start between patients with and without HFE mutations. The single patient with H63D homozygosity was severely iron-loaded. INTERPRETATION AND CONCLUSIONS: Our data suggest that the presence of a single mutation in the HFE gene does not influence the severity of iron loading in thalassemia patients following a regular transfusion and chelation program.

Adolescent↗
PubMed1999

Towards molecular farming in the future: moving from diagnostic protein and antibody production in microbes to plants.

Molecular farming of pharmaceuticals in plants has the potential to provide almost unlimited amounts of recombinant proteins for use in disease diagnosis and therapy. Transgenic plants are attracting interest as bioreactors for the inexpensive production of large amounts of safe, functional, recombinant macromolecules, such as blood substitutes, vaccines and antibodies. In some cases, the function of expressed recombinant proteins can be rapidly analysed by expression in microbes or by transient expression in intact or virally infected plants. Protein production can be increased by upscaling production in fermenters, using yeast- or plant-suspension cells or by using transient-expression systems. Stable transgenic plants can be used to produce leaves or seeds rich in the recombinant protein for long-term storage or direct processing. This demonstrates the promise for using plants as bioreactors for the molecular farming of recombinant therapeutics, diagnostics, blood substitutes and antibodies. We anticipate that this technology has the potential to greatly benefit human health by making safe recombinant pharmaceuticals widely available.

Animals↗
PubMed1999

Towards molecular farming in the future: using plant-cell-suspension cultures as bioreactors.

Plant-suspension cells are an in vitro system that can be used for recombinant protein production under carefully controlled certified conditions. Plant-suspension cells can be grown in shake flasks or fermenters to produce secondary metabolites, like vincristine and vinblastine, and to produce recombinant proteins after transformation. This review article focuses on discussing the generation of transformed suspension-cell lines expressing recombinant proteins, like antibodies, and recombinant-protein downstream processing and purification.

Bioreactors↗
PubMed1999

Towards molecular farming in the future: transient protein expression in plants.

Molecular farming in plants can be achieved by stable or transient expression of a recombinant protein. Transient expression of recombinant proteins in plants can rapidly provide large amounts of the proteins for detailed characterization. It is fast, flexible and can be carried out at field scale using viral vectors, but it lacks the increases in production volume that can be achieved easily with stable transgenic crops. This review article focuses on discussing the applications of transient expression using viral vectors, biolistic methods or agroinfiltration.

Antibodies↗
PubMed1999

Towards molecular farming in the future: pichia pastoris-based production of single-chain antibody fragments.

This review article focuses on the use of the methylotrophic yeast Pichia pastoris as a recombinant protein-expression system. P. pastoris is a useful system for the expression of milligram-to-gram quantities of a protein, which can be scaled up to fermentation to meet greater demands. Compared with mammalian cells, Pichia do not require a complex growth medium or culture conditions, they are as easy to manipulate genetically as Escherichia coli and have a eukaryotic protein-synthesis pathway. They seem suited to laboratory-scale production of recombinant proteins for in-house use or, in some cases, molecular farming of recombinant products. This review article focuses on the use of P. pastoris, describes a fermentation production run of a single-chain antibody fragment and includes a discussion of fermentation as a production strategy.

Cloning, Molecular↗
PubMed1999

Production of carcinoembryonic antigen (CEA) N-A3 domain in Pichia pastoris by fermentation.

Carcinoembryonic antigen (CEA) is a 180-kDa glycoprotein found on the surface of normal colon and malignant human adenocarcinomas. Recently, a fusion protein containing two of the seven Ig-like domains present in CEA (N and A3) has been constructed and expressed in Pichia pastoris [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193-3201]. Here, we report the generation and selection of a multi-copy clone expressing this fusion protein, the optimization of the shake-flask expression protocol and the upscaled production of CEA N-A3 using fermentation technology. P. pastoris transformants secreting the CEA N-A3 domain were generated by electrotransformation of the GS115 host strain with the pPIC9K vector containing the CEA N-A3 cDNA [You, Hefta, Yazaki, Wu and Shively (1998) Anticancer Res. 18, 3193-3201] then screened for CEA N-A3 expression and G418 resistance. The recombinant CEA N-A3 domain was detected in the culture supernatant using the monoclonal anti-CEA antibody T84.66. Optimization of methanol-induction conditions resulted in a high-methanol shake-flask expression protocol yielding significantly increased CEA N-A3 levels. Fermentation and culture conditions were optimized for 5-l working-volume fermentations and CEA N-A3 was affinity purified using Ni-IDA (imino di-acetic acid) affinity chromatography from the clarified fermentation supernatant. Peptide N-glycosidase F treatment revealed that the recombinant protein was heavily glycosylated but expressed as a single polypeptide of 28 kDa with no evidence of proteolytic degradation. Our results demonstrate that functional CEA N-A3 domain can be produced in sufficient quantities in P. pastoris for structural analysis or diagnostic applications. To our knowledge, this article represents the first report on the production of a human tumour antigen through fermentation.

Base Sequence↗
PubMed1999

[Evaluation of the effect of professional dental cleaning and education in dental hygiene in type 1 diabetic patients].

In the setting of a prospective, randomized trial the impact of professional oral hygiene and of intensive patient education in oral hygiene was investigated. It was studied, whether in young to middle-aged type 1 diabetic patients the risk or the progression of periodontal disease can be influenced during one year. Primary endpoint was bleeding at probing, secondary indices, the plaque index and probing pocket depth. 83 patients were enrolled, 70 completed the study according to the protocol. Oral cleansing was dramatically improved by education in the short term, however, the effect was much weaker after 6 and 12 months, respectively, although there remained some improvement compared to the basic level. Bleeding at probing increased in both groups, without reaching the level of statistical significance. Probing pocket depth increased in the intervention group weakly but significantly and remained unchanged in the control group. The tested intervention (professional oral hygiene, patient education in oral hygiene) could not improve the periodontal status in our type 1 patients.

Adolescent↗
PubMed1998

Transplantation of pediatric en bloc cadaver kidneys into adult recipients.

BACKGROUND: To maximize the renal donor pool, cadaveric pediatric en bloc kidneys have been transplanted as a dual unit by some transplant centers. We compared the short- and long-term outcomes of adult recipients of cadaveric pediatric en bloc renal transplants versus those of matched recipients of cadaveric adult kidneys. METHODS: Thirty-three adults who received pediatric en bloc kidney transplants between April 1990 and September 1997 were retrospectively identified and were compared with 33 matched adults who received adult cadaveric kidney transplants. The groups were identical for transplantation era, immunosuppression, recipient sex, race, cause of renal failure, mean weight, and follow-up duration (37.8 vs. 37.5 months). The mean recipient age study versus control was lower (36.3 vs. 48.9 years, P=0.0003). Results. There was no difference between the en bloc and adult donor groups in the 3-year patient survival rates (95% vs. 87%, P=0.16) or the 3-year graft survival rates (87.3% vs. 84.2%, P=0.35). Further, there was no difference in en bloc patient or en bloc graft survival time stratified by recipient age (14-44 vs. >45 years, P=0.11), en bloc donor age (<24 vs. >24 months, P=0.39), or recipient weight (<60, 61-75, >75 kg; P=0.60). Differences in serum creatinine (mg/dl) for the en bloc versus the control group at the time of discharge (3.0 vs. 7.8 mg/dl, P=0.06), at 1 year (1.4 vs. 2.0 mg/dl, P=0.06), and at 2 years (1.1 vs. 1.6 mg/dl, P=0.14) had dissipated by the time of the 5-year follow-up examination (1.1 vs. 1.6 mg/dl, P=0.14). Vascular complications were more prevalent in the en bloc group: renal vein thrombosis (one case), thrombosis of donor aorta (two cases), arterial thrombosis of one renal moiety (two cases), and renal artery stenosis (two cases). There were no differences between groups in delayed graft function, acute or chronic rejection, posttransplant hypertension, posttransplant protein-uria, or long-term graft function. CONCLUSIONS: Collectively, these data indicate that transplanting pediatric en bloc kidneys into adult recipients results in equivalent patient and graft survival compared with adult cadaveric kidneys. Further, the data also suggest that pediatric en bloc kidneys need not be strictly allocated based on recipient weight or age criteria.

Adolescent↗
PubMed1998

Peripheral-type benzodiazepine receptors in diagnostic subtypes of schizophrenic patients.

The peripheral-type benzodiazepine receptor (pBZD-R; also called the omega-3 receptor or the mitochondrial benzodiazepine receptor) seems to play a critical role in the production of neurosteroids, which are able to alter the electrical properties of neuronal membranes and thus the firing patterns of neurons. Putative endogenous ligands are the diazepam-binding inhibitor and its processing products, as well as porphyrins, some of them, in the case of porphyria, are well known to give rise to certain aspects of neuropsychiatric disorders, such as schizophrenic-like symptoms. Previous findings of altered benzodiazepine binding sites in post-mortem brain samples and platelets from small samples of schizophrenic patients have been inconclusive. Therefore we investigated characteristic binding parameters (Bmax, Kd) of the granulocytic pBZD-R by using the selective ligand PK11.195 in 53 subjects, fulfilling ICD-10 and DSM-IV criteria of schizophrenia. The binding parameters in our total group of 53 schizophrenic patients did not differ from those in healthy subjects. However, Bmax values were significantly reduced in schizophrenic patients with predominantly negative symptoms (residual type) compared to schizophrenic patients with predominantly positive symptoms, i.e. paranoid (-50%) and catatonic subtype (-38%). Moreover, only residual type schizophrenics exhibited a significantly reduced binding capacity compared to healthy subjects (-38%). More studies are warranted to clarify the functional significance of this binding site in the pathogenesis of negative symptoms.

Adult↗
PubMed1998

Dissociation between high anti-PR3 titers (c-ANCA) and the clinical course of disease in a case of Wegener granulomatosis.

In May 1984 a 58-year-old woman presented with a broad spectrum of clinical symptoms including malaise, arthralgia, hemoptysis and dyspnea, proteinuria and hematuria and a vasculitic necrotizing rash. Bronchial biopsies revealed subglottic granulomatous lesions and renal biopsies showed necrosis, extracapillary proliferation and crest formation, confirming the diagnosis of Wegener granulomatosis. Positive c-ANCA and anti-proteinase 3 subfraction (anti-PR3) titers were first analysed in 1991. Clinical remission was achieved by standard immunosuppressive therapy and renal function was stabilised. Several minor relapses were treated with pulsed intravenous cyclophosphamide but the symptoms could not be completely controlled. Eight years after the onset of disease, a dramatic increase in anti-PR3 titers was observed (34438 U/ml, normal range < 10, ELISA), followed 3 months later by a clinically apparent relapse. Immunosuppressive therapy was reinstituted without clinical improvement. At this point plasmapheresis resulted in an amelioration of clinical symptoms as well as a reduction in anti-PR3 titers. Concomitant immunosuppressive therapy was administered with oral corticosteroids. Forty days later anti-PR3 titers increased, reaching 75000 U/ml twelve months later, however this time without associated clinical symptoms. During the following months the patient had a further transient deterioration of pulmonary and renal function due to secondary bacterial infection which was successfully treated with antibiotics. A nephritic sediment was not present during these episodes. Curiously, the anti-PR3 titers have remained excessively elevated for the last three years.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1998

High affinity calmodulin target sequence in the signalling molecule PI 3-kinase.

In this study we report that phosphatidylinositol 3-kinase (PI 3-kinase), a lipid kinase which participates in downstream signalling events of heterotrimeric G protein-coupled receptors and receptor tyrosine kinases, contains a high affinity binding site for calmodulin (CaM). The putative CaM-binding peptide derived from the p110gamma isoform interacts with CaM in a calcium-dependent way. Using gel shift analysis and fluorescence spectrophotometry we discovered that the peptide forms a high affinity complex with CaM. Titration experiments using dansylated CaM gave an affinity constant of 5 nM. Furthermore, a sequence comparison among different PI 3-kinase isoforms revealed that the sequence which can bind CaM is highly conserved within different PI 3-kinase isoforms. These results indicate a novel mechanism for regulating PI 3-kinase and provide a new direct link between Ca2+ and phospholipid signalling pathways.

Amino Acid Sequence↗
PubMed1998

Integrity of a Zn finger-like domain in SamB is crucial for morphogenesis in ascomycetous fungi.

Genetic features determine the site of polarized growth in filamentous fungi and lead to hyphal tip extension or subapical branching. We have isolated the samB gene (suppressor of anucleate metulae) of Aspergillus nidulans which encodes a 66 kDa protein carrying an atypical Cys4 and an additional Cys2/His/Cys Zn finger motif at the carboxy-terminus. Such novel Zn finger-like domains have recently been found in several other developmental regulators in organisms ranging from yeast to man. Deletion of this domain at the carboxy-terminus of SamB led to premature hyphal ramification, mislocalization of septa and suppression of the asporogenous phenotype of the developmental mutant aps (anucleate primary sterigmata). A DeltasamB deletion strain displayed an identical phenotype. A homologous gene in Saccharomyces cerevisiae was also characterized whose deletion resulted in a multi-budding phenotype; thus it was named MUB1. An underlying common mechanism for both genes in determination of the onset of polarized growth and its links to other cellular developmental processes is discussed.

Amino Acid Sequence↗