Biomedical subjects
Publications and source records attributed to R Fischer.
The case report illustrates the successful application of a new method of sperm extraction from frozen-thawed testicular biopsy specimen within an established programme of intracytoplasmic sperm injection.
OBJECTIVE: The aim of this study was to investigate alterations of the surfactant system in multiple-trauma patients (MTP) with lung contusion and the influence of single- or multiple-organ dysfunction syndrome (OF/MOF) on the surfactant system. SETTING: University hospital, trauma-intensive care unit. DESIGN: Prospective, nonrandomized study. METHODS: MTP with an Injury Severity Score > 19 points have been recorded prospectively since 1992. Bronchoalveolar lavages were obtained daily either until day 14 or extubation. Three groups of MTP were compared: noL: MTP, no lung contusion (n = 14); LuCo-: MTP, lung contusion, no OF/MOF (n = 17); LuCo+: MTP, lung contusion, with OF/MOF (n = 10). Also, surfactant samples of 11 healthy volunteers (Con) were investigated and compared with MTP. All data were presented as mean +/- SEM. Statistical analysis were performed using programs of SPSS 6.0.1. (univariate ANOVA, Fisher's Exact Test, p < = 0.05). RESULTS: There were no differences in sex and age. Injury Severity Score was significantly impaired in group LuCo+ (44 +/- 4), compared with groups noL (31 +/- 3) and LuCo- (34 +/- 3). Group noL showed no statistical differences for lung function, total protein, and total phospholipid content of the bronchoalveolar lavage compared with group LuCo-. Furthermore, the relative content of phosphatidylcholine and phosphatidylglycerol in total phospholipids and surfactant-associated protein A were not significantly altered compared with group LuCo-. Lung function in group LuCo+ was significantly impaired and led to hypoxemia on the day of trauma. Total protein content and total phospholipids were significantly elevated in group LuCo+ compared with groups noL and LuCo- on the first day. Also, the relative content of phosphatidylcholine was significantly increased in group LuCo+ up to day 4, compared with groups noL and LuCo-. In comparison with groups noL and LuCo-, a significant decrease of the relative content of phosphatidylglycerol was obtained in group LuCo+ up to day 7. The surfactant-associated protein A was increased in group LuCo+ during the whole observation time, compared with the other groups. CONCLUSIONS: Multiple trauma leads to alterations in the surfactant system. The composition of surfactant was not further influenced by lung contusion alone. Only MTP with OF/MOF during the intensive care unit treatment showed significant alterations in surfactant composition and a decrease in lung function.
Large-cell anaplastic lymphomas (LCAL) are characterized by their distinctive morphology together with expression of the CD30 antigen. In addition, a chromosomal translocation, t(2;5) (p23; q35), can be detected in most cases. A significant proportion of LCALs carry rearrangements of the T-cell receptor-gamma (TCR-gamma) locus and display a T-cell phenotype. In about a third of the cases, another type of non-Hodgkin-lymphoma precedes LCAL. Early transformations of non-Hodgkin's lymphoma into LCAL might escape clinical detection in a significant number of cases. The existence of clonally related lymphoid cells within the lymph node infiltrates must be claimed in these cases. Recently, a small-cell-predominant variant of LCAL was described in which only few large tumor cells expressing the CD30 antigen are found together with numerous small lymphocytes, which are frequently CD30-. This observation in particular prompted us to investigate the clonal relationship of the tumor cell compartment and admixed small lymphocytes in one case of common LCAL with T-cell genotype. For this purpose, we chose to amplify rearranged TCR-gamma sequences from single cells isolated from immunostained frozen sections by using a micromanipulator. A total of 119 cells were investigated. Amplification products were obtained in 17 of 79 CD3+ cells, 12 of 30 CD30+ cells, and three of 10 CD20+ cells. The nucleotide sequences were determined in 28 cells by nonradioactive sequencing. In 11 CD30+ cells, the predominant rearrangement of TCR-gamma was identified. No clonal diversity was observed. The small CD3+ lymphocytes were unrelated to the anaplastic CD30+ tumor cells. This report describes a method to analyze rearrangements of the TCR-gamma in single cells isolated from immunostained frozen sections. Application of this technique revealed an absence of clonal diversity in a case of LCAL and documented the polyclonal nature of admixed small CD3+ lymphocytes.
Asexual fruiting body development in Aspergillus nidulans requires a precise spatial and temporal coordinated expression of many genes. Insertional mutagenesis was used to isolate and characterize a new mutant of A. nidulans in which hyphal growth was slightly reduced and conidiophore development was specifically blocked at the metula stage. In contrast to the uninucleate metulae of the wild-type, in the mutant these structures were elongated, multinucleate and septate. Further differentiation and production of phialides by a budding-like process was not observed. The mutant metulae thus resembled hyphae rather than metulae and the gene was therefore named hypha-like metulae (hymA). The hymA gene was mapped to linkage group VI. The integrated vector was rescued with border sequences from the integration site. The border sequences were used to isolate a cosmid from a wild-type library which was subcloned to a 5 kb fragment able to complement the mutation in trans. This fragment encoded a 1.8 kb transcript expressed in hyphae and throughout development. It is proposed that hymA is involved in budding processes, and is required for the formation of metulae and for their further differentiation.
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To identify calmodulin-binding proteins in cellular extracts and tissue homogenates and to analyze purified calmodulin target proteins, overlay procedures using 125I-calmodulin or, more recently, nonradioactive biotinylated calmodulin have been widely used. Here we describe a rapid, alternative method for detecting calmodulin-binding proteins with a 32P-labeled calmodulin probe generated as a glutathione-S-transferase (GST)-fusion protein. We used a modified pGEX-2TK vector, which contains the flag epitope and the consensus sequence R-R-A-S, that can be phosphorylated by the cAMP-dependent protein kinase A. The fusion protein is easily purified from bacterial bysates by affinity chromatography using glutathione-Sepharose 4B beads. Phosphorylation of GST-calmodulin is performed directly on the beads and, after elution with reduced glutathione, the labeled calmodulin probe can be used for overlay experiments. We also describe a rapid renaturation protocol that enhances the signal for some but not all calmodulin-binding proteins and is used after the proteins have been transferred to nitrocellulose filters. Furthermore, we have compared the specificity and sensitivity of the 32P-labeled GST-calmodulin overlay with those of 125I-calmodulin and biotinylated calmodulin, clearly indicating that our newly developed protocol is a suitable alternative to conventionally used calmodulin overlay procedures.
The objective of this project was to determine the prevalence of hyperprolactinemia (HPRL) in systemic lupus erythematosus (SLE) and to evaluate the role of prolactin (PRL) as a disease-activity marker. We determined PRL in a cohort of 182 patients with SLE. Basal prolactin levels and clinical data (disease-activity index, sero-activity, medications) were analyzed in all patients. The findings show only mildly elevated PRL levels in four SLE-patients and normaprolactinemia in 98% (n = 178), without any association with disease activity. Our results differ from previous studies. HPRL appears not to be prevalent in SLE, nor to be associated with disease activity.
A clinicopathological study on 87 adult patients presenting with "de novo" acute myeloblastic leukemia (AML) was performed to assess the rate of apoptosis before and during chemotherapy and its predictive impact on clinical course. Evaluation included trephine biopsies of the bone marrow and the situ end-labeling technic (ISEL) for the identification of programmed cell death in large and intact hemopoietic tissue areas. In comparison with a control group of 21 patients without any hematological disorder, morphometric analysis revealed no significantly different numbers of apoptotic cells in AML at the onset of disease and following sequential examinations at intervals ranging between 10 to 19 months. Moreover, the incidence of programmed cell death was not associated with the subgroups of the FAB classification and statistics failed to show a relationship with survival or remission status. In conclusion, these findings are in keeping with the assumption that apoptosis occurs with the same frequency in recovering normal hemopoiesis in complete or partial remission, in manifest AML and relapse. In the latter conditions, enhancement of proliferation is not associated with an increase in the apoptotic index.
A retrospective clinico-pathological study was performed on 208 consecutively recruited patients (94 males, 114 females, median age 67 years) with idiopathic (primary) osteo-/ myelofibrosis (IMF). According to bone marrow histology (cellularity) as well as extent (semiquantitative grading) and quality (reticulin/collagen) of myelofibrosis, stages of the disease process were determined. At closure of this study (observation time 65 months) 133 patients were dead and 75 alive and median survival was 56 months. The wide spectrum of clinical signs and symptoms and laboratory data on admission was reflected by a corresponding variety of histological features. Significant differences of hematological values could be calculated between patients with or without early reticulin fibrosis (fiber scores 0 and 1) and advanced fibro-osteosclerotic stages (fiber scores 2 and 3). Evolution of disease features was elicited by longitudinal follow-up studies and sequential bone marrow biopsies. Morphometric assessment of fiber density in patients without preceding chemotherapy revealed an unpredictable and varying progression of myelofibrosis associated with alterations of certain laboratory parameters (hemoglobin level, spleensize, thrombocytosis). Differentiation from essential (primary) thrombocythemia (ET) was required in 25 patients who fulfilled the postulated diagnostic criteria. In fact, this group was consistent with hypercellular, early stages of IMF without relevant reticulin fibrosis and an excessively raised platelet count (> or = 1000 x 10(9)/1). Discrimination was only feasible by regarding histology carefully, particularly abnormalities of megakaryopoiesis and follow-up data. Parameters of predictive value indicating a significant loss in life expectancy in comparison with a sex- and age-adjusted normal population included: age (> or = 60 years), hemoglobin levels (< or = 10 g/dl), thrombocyte count (< or = 600 x 10(9)/1) and the presence of myeloblasts and promyelocytes. Statistical analysis disclosed that in the so-called early stages of IMF without relevant myelofibrosis, findings indicative for extramedullary hemopoiesis or generalization of the disease process exerted an unfavourable influence on survival.
Midfacial T-cell lymphomas are more prevalent in Asia than in Europe or North America. Clinically, these lymphomas are noted as one major differential diagnosis in the malignant midline granuloma syndrome. During the past years, the group of nasal T/NK cell lymphomas has been recognized that is frequently associated with EBV-infection. The aim of the current publication was to describe the clinical presentation and course of 30 patients attending the West China University of Medical Sciences, Chengdu, P.R. China, between 1991 and 1994. Clinical records were assessed and the patients were followed for 6 to 29 (mean 12.4) months. Several microscopic features thought to be associated with this entity were carefully evaluated together with immunohistochemical data. The proliferation of the tumour cells was assessed by determining the mitotic index and the ratio of MIB-1 labelled cells. In addition, the incidence of apoptotic cells was investigated by means of the in-situ end labelling (ISEL) technique. Our data confirm the expression of T-cell markers by T/NK cell lymphomas as determined by the immunohistochemistry. The apoptotic index was found to correlate with the ratio of MIB-1 labelled cells. Expression of the bcl-2 oncoprotein was not associated with increased or diminished proliferation or cell death, respectively. Eight of the thirty patients succumbed to their disease during the follow-up period. Kaplan-Meier cumulative survivals and log-rank tests revealed a significant impact of MIB-1 labelling on mean survival times.
We report on new stripper guides that facilitate the introduction of the stripper from the proximal side. At the same time they allow for smaller skin incisions and fewer nerve lesions. The 60-cm-long cable instead of the conventional one (90 cm long) is adequate for partial stripping. Due to its shortness, it is less annoying in the operation field and it allows rotational control of the Tiemann guide. The "dolphin" and the "torpedo" help to remove segments of the saphenous vein that are left behind. All these instruments are available as a set in a small box, which is handy for the scrub nurse.
Amphibian oocyte nucleoli are a particular suited object for research on nucleolar chromatin organization. By selective rDNA amplification each pachytene oocyte nucleus accumulates 30 pg of extrachromosomal rDNA, this amount corresponds to 2 million rDNA copies. Following the selective amplification stage, the amplified gene copies are finally distributed within more than thousand extrachromosomal nucleoli per individual oocyte nucleus. The aim of the present study was first to obtain a precise documentation of the fate of amplified rDNA during early Xenopus oogenesis until the final functional integration of these copies into individual oocyte nucleoli, and, second, a close correlation of the structural data with determination of rDNA transcription rates by S1 transcript analysis for the subsequent stages of oocyte differentiation. In order to investigate the structural complexity of the intranuclear rDNA translocation process in detail, a confocal laser scan microscope (CLSM) was used, equipped with an external UV-laser. This instrumentation unambiguously allowed (i) the detection of small clusters of rDNA copies and (ii) the precise spatial documentation of the intranuclear position of rDNA clusters in relation to the protein-free pre-nucleolar protein bodies, a specific characteristic of late pachytene/early diplotene amphibian oocyte nuclei. Our results indicate that the major rDNA translocation processes, e.g. the association of rDNA clusters with pre-nucleolar protein bodies, the formation of ribbon-like pre-nucleolar units sensu Van Gansen and Schramm (J. Cell Sci. 10: 339-367, 1972), and, finally, the translocation of fused rDNA units into the interior of pre-nucleolar protein bodies, occur--for the most part--in absence of massive rDNA transcription. As shown by the S1 transcript analysis, the onset of massive rDNA transcription starts concomitantly with an unraveling of the densely packed rDNA clusters into finely dispersed rDNA units, which were shown by CLSM analysis to be distributed throughout the entire nucleolar volume.
Apart from ablation properties at the stapes footplate, the degree of thermal stress of the inner ear is important when considering the suitability of pulsed lasers for stapedotomy. The aim of the present study was to compare the heating of cochlear structures with presently available pulsed laser systems during stapedotomy under reproducible conditions. Temperature increases and heat-exchange processes were examined in a physiologic model of the cochlea using various laser parameters effective for footplate perforations. With all systems, increases in energy density, number of pulses and resultant total energy led to higher temperatures. In the effective energy density range, the highest temperature increases achieved with the requisite number of pulses at a distance of 2 mm behind the footplate perforation were 30 degrees and 26 degrees C with the pulsed CO2 and Ho:YAG lasers, respectively. The lowest temperature recorded was 5.5 degrees C with the Er:YAG and <5 degrees C with the Er:YSGG laser. The excimer laser investigated at only one energy density showed maximum temperatures of 10 degrees C. With regard to possible inner ear damage from thermal stress during laser stapedotomy, the Er:YSGG laser can be used safely over a relatively broad energy density range. The Er:YAG laser investigated also appears suitable for stapedotomy when considering thermal effects. In contrast, application of the pulsed CO2 laser at parameters effective for stapedotomy leads to high temperatures and wide scattering to compromise its use. The Ho:YAG laser also appears to be unsuitable because of the higher energy density and pulse rate required for sufficient perforation and the resultant higher temperature increases in the perilymph. Although footplate perforations can be achieved with only slight temperature increases in the fluid of the cochlea model, the excimer laser does not seem to be appropriate for stapedotomy because of the long period of heat exposure required and the lower ablation rates at the stapes.
To assess possible alterations of megakaryocytes associated with interferon (IFN) and busulfan (BU) therapy of Ph(1+)-CML, an immunohistochemical and morphometric study was performed on trephine biopsies of the bone marrow taken before and at varying intervals during treatment. For the identification of megakaryopoiesis and its endoreduplicative activity the monoclonal antibodies CD61 (anti-platelet glycoprotein IIIa) and PC10 raised against proliferating cell nuclear antigen (PCNA) were used. We compared 60 specimens from 20 patients following IFN alpha-2b administration (in combination with IFN gamma in seven patients) with 57 specimens from 22 patients after monotherapy with BU. A close correlation with clinical follow-up studies revealed that in the IFN-treated group the prevalence of atypical micro-megakaryocytes, usually characterizing CML, was conspicuously reduced in repeatedly taken bone marrow samples. Initially, even an increase in size which was levelled to normal values during maintenance therapy was observed. These features were most prominently expressed in the 13 patients with a complete hematologic and/or partial cytogenetic response. Associated with this phenomenon was a significant enhancement of the PCNA-labelling index which indicated a stimulation of endoreduplicative (endomitotic) activity necessary for achieving normal size and ploidy. In the second group of patients treated by BU these changes were absent. For this reason, our findings are in keeping with the assumption that during IFN treatment, there is at least partial recovery and expansion of a putative normal (Ph1-) megakaryopoiesis. In conclusion, megakaryocyte morphology, i.e. normalization in size, is thought to be a useful indicator to evaluate the response to IFN in CML patients.
In order to elucidate the involvement of adhesion mechanisms in the process of megakaryocyte-dependent fibroblast growth, we applied BSA-coupled polymers of glucose, galactose, fucose, mannose, and several lectins (AAA, LCA, LTA, UEA-I) to cocultures of CD61 -positive (CD61+)/MACS-enriched megakaryocytes and human bone marrow fibroblasts. Fibroblast monocultures served as controls. After 6 days, glucose, as well as galactose-treated cultures showed a significant reduction of fibroblast growth in cocultures and fibroblast monocultures. In contrast, application of mannose caused no reducing effect on fibroblast numbers. Administration of fucose, AAA, LTA or UEA-I revealed a strong impairment of fibroblast growth in the megakaryocyte-fibroblast cocultures. Adhesion experiments using MACS-enriched, fluorescein-labelled megakaryocytes cultured in the presence of carbohydrates and lectins on a near-confluent layer of fibroblasts were additionally performed. Following fucose-BSA, alpha Fuc-1,2Gal beta-HSA or UEA-I treatment a significant reduction of megakaryocyte adhesion to the fibroblast layer could be observed. In the case of AAA a weak impairment of megakaryocyte adhesion could be noticed. Selective pretreatment of either fibroblasts or megakaryocytes with fucose-BSA or alpha Fuc-1,2Gal beta-HSA was consistent with the finding of a prominent involvement of fucosylated residues located on megakaryocytes in this interaction. In conclusion, our studies are in keeping with the assumption that fucosylated and fucose-binding structures are playing a key role in adhesion mechanisms between megakaryocytes and fibroblasts and thus influence significantly the megakaryocyte-dependent growth of bone marrow fibroblasts.
We report on a 56-year-old woman with bilateral chronic venous insufficiency, a cavernous hemangioma at the right thigh and an aneurysm of the left femoral vein, without bone or soft tissue hypertrophy. Histopathology revealed a mucoid degeneration of the media musculature of the venous wall. Such a finding has not yet been described in the world literature. The differential diagnosis must consider a secondary hitherto unknown alteration of varicose veins, a variety of a vascular malformation not yet described, and a primary degeneration of the smooth muscles of the vascular wall followed by the development of varicose veins.
B-cell non-Hodgkin's lymphomas with a marked preponderance of reactive T cells, so-called T-cell rich B-cell lymphomas (TCRBCLs), can be morphologically confused with Hodgkin's disease (HD). To establish helpful distinguishing features in paraffin sections, 10 cases of L26-positive, CD15-negative HD and 10 cases of TCRBCL were compared; 4 cases of HD had morphologic features of the nodular lymphocyte predominant (LP) type. Nine of 10 cases of HD contained fewer than 20 mitoses/20 high power fields (hpf) and only 1 had pericapsular involvement. In contrast, 9 of 10 TCRBCL had greater than 20 mitoses/20 hpf and 7 had perinodal infiltration. HDLP was easily distinguished from TCRBCL by the expanded dendritic meshworks outlining the L & H nodules and the high content of CD57-positive lymphocytes. The remaining 6 cases of non-LP L26-positive HD had a relatively distinctive immunostaining pattern, with absence of CD45 and discordant reactivity for L26 and Ki-B5 in Reed-Sternberg cells and variants. Only 3 cases of TCRBCL had a similar CD45 and L26/Ki-B5 immunostaining pattern, and these could be distinguished by demonstrable cytoplasmic light-chain restriction. These results show that evaluation of the mitotic count, pericapsular involvement, and immunohistochemical staining patterns for Ki-M4p, CD57, L26/Ki-B5, and CD45 can help to discriminate TCRBCL from L26-positive HD when only fixed material is available.
There seems to be general agreement that AIDS-related bone marrow changes are consistent with myelodysplastic features. To re-evaluate this assumption more critically, a comparative study was performed on marrow smears and trephine biopsies in 20 patients presenting manifest stages of AIDS and in 30 patients with primary myelodysplastic syndromes (MDS). For an assessment of cytological atypias enzyme-(naphthol AS-D-chloroacetatesterase) and immuno-histochemical techniques including monoclonal antibodies directed against erythro-(Ret40f) and megakaryopoiesis (CD61), macrophages (PG-M1) and proliferating cell nuclear antigen-PCNA (PC10) were applied and staining results determined by morphometric analysis. The in situ end-labeling (ISEL) technique was used for the demonstration of cells undergoing apoptosis (programmed cell death). In the HIV-infected bone marrow the frequency of erythroid precursors and macrophages exceeded significantly the corresponding counts in MDS. In comparison with a control group, megakaryocytes were increased in both entities under study. However, MDS was characterized by a prevalence of atypical microforms. The small-sized megakaryocytes in MDS revealed striking abnormalities of nuclear-cytoplasmic organization. Severe defects of nuclear lobulation (pseudo-Pelger anomalies) of mature neutrophils or nuclear bridging of erythro-normoblasts were not conspicuous in AIDS. Whereas the incidence of apoptosis was significantly higher in MDS, no such differences could be found comparing the PCNA-labeling index of AIDS and MDS. On the other hand, both parameters were significantly increased in comparison with the controls. In the HIV-infected bone marrow alterations of the myeloid stroma were most prominently expressed. If well defined criteria for the diagnosis of (myelo-) dysplasia were applied, our findings in AIDS were not in keeping with the assumption of significant maturation defects comparable with MDS. Hence, bone marrow lessions accompanying AIDS should be separated from MDS and should be diagnosed as HIV-myelopathy.