Biomedical subjects
S Fischer
Publications and source records attributed to S Fischer.
Oligoclonal in vitro response of CD4 T cells to vesicles of mistletoe extracts in mistletoe-treated cancer patients.
Mistletoe (Viscum album) extracts are widely used in adjuvant cancer therapy. We have investigated the in vitro responsiveness of T cells from mistletoe-treated cancer patients and untreated healthy donors to various preparations of mistletoe extracts. Proliferation of peripheral blood mononuclear cells from treated but not from untreated patients was observed in response to therapeutically used mistletoe extracts prepared from apple (mali) or pine (pini) host trees. The strongest proliferation was induced by a vesicle preparation of mali extract. Activation was strongly inhibited by interleukin-10. Using a newly developed flow-cytometry assay, we determined that cell growth was restricted to CD4 T cells. Analysis with a panel of monoclonal antibodies against the variable region of the T cell receptor beta chain (V beta) revealed an oligoclonal pattern of CD4 T cell activation. These results indicate that therapeutic administration of mistletoe extracts sensitizes a restricted set of CD4 T lymphocytes in mistletoe-treated patients.
Acute leukemias express a functional receptor for the human growth hormone.
The potential influence of the human growth hormone (hGH) on the behavior of acute leukemias is a matter of controversy. We investigated primary childhood and adult leukemias (n = 44) and leukemic cell lines (n = 13) for the expression of the hGH receptor (hGHR) by immunohistochemistry and flow cytometry. All leukemias expressed the hGHR in the cytoplasm; expression on the surface was undetectable in some of the leukemias. In leukemic cell lines, hGHR expression on the surface was demonstrated in a dose-dependent manner after incubation with rhGH. Physiologic concentrations of hGH were more efficient than higher doses in increasing hGHR surface expression. A proliferative response to hGH was accomplished in cell lines REH, Molt4, and K562. However, only one of 19 primary leukemias (ALL, n = 12; AML, n = 7) showed increased cell counts after the addition of 50-800 ng/ml recombinant hGH (rhGH). These cells were of an immature T-cell phenotype. We thus conclude that acute leukemias can be stimulated by hGH to up-regulate its receptor, but that most primary leukemias may require additional signals for the induction of proliferation.
Amperometric lactate oxidase catheter for real-time lactate monitoring based on thin film technology.
An amperometric lactate oxidase catheter has been developed for in vivo application to real-time lactate monitoring. The electrochemical behaviour of the 1 x 3 mm Pt-Ag/AgCl thin film electrode is not significantly influenced by lactate oxidase-polyurethane covering. Gamma-irradiation (25 kGy) is suitable for the sterilization procedure. The final lactate catheter is characterized by a linear concentration range between 0.5 and 20 mmol/l lactate with a sensitivity around 2 nA mmol-1 l-1 lactate. The accuracy is demonstrated by the measurement of control sera. Both physiological and pathological materials correlate well with the declared values. The dry stored lactate catheter needs about 10 min for hydration and is characterized by response times t98% of less than 2 min. Ex vivo whole blood measurements using the lactate catheter (y) give a correlation with the BIOSEN Med L (x) of y = (1.010x + 0.513) mmol/l (r = 0.9748). Lactate values obtained by continuous catheter operation ex vivo correlate well with those obtained by BIOSEN Med L. First subcutaneous implantation (dog) underlines the characteristics obtained ex vivo: after 30 min hydration the lactate catheter follows the lactate concentration measured ex vivo with samples from the leg vein by BIOSEN Med L.
Crosslineage TCR delta rearrangements occur shortly after the DJ joinings of the IgH genes in childhood precursor B ALL and display age-specific characteristics.
In order to test the hypothesis that the most immature T-cell receptor (TCR) rearrangements occur after the DJ joining of the immunoglobulin heavy chain genes (IgH), we analysed the TCR Vdelta2-Ddelta3 rearrangements in precursor B-cell leukaemias (PBC ALL) from 25 children younger than 3 years at disease onset and found that most of the junctional regions had N nucleotides inserted. We then selected 14 of these PCB ALLs for DJH (DJ joining of the IgH) characterization. These joining regions showed homology-directed recombination and lack of N regions, indicating absence of terminal deoxynucleotidyl transferase (TdT) activity during their rearrangement. Most leukaemias with a DJH rearrangement without N region have no, or only one, nucleotide in the joining regions of their Vdelta2-Ddelta3 rearrangements. The N regions of the TCR delta rearrangements displayed 'age-specific' differences: in children younger than 3 years of age the N regions were shorter than in those older than 3 years, and the rearrangements frequently contained complete segments. We conclude that the Vdelta2-Ddelta3 rearrangement in childhood PCB ALLs is an early event following DJH rearrangement and that it occurs shortly before or after the first hit, leading to malignant transformation.
[Hypoxia protection of the blood-brain barrier: effects of barbiturates on hypoxic cultures of microvascular endothelial cells].
Explore the source record for details and available documents.
Identification and characterization of a novel hsc70-like gene in the human lung tumor cell line HS24.
We determined the cDNA sequence and analyzed the genomic structure of a novel human gene designated HS24/p52, which shows significant similarity to the ATP-binding domain of stress-70 proteins in the human lung tumor cell line HS24. The 2,203-nucleotide-long cDNA sequence is divided into an incomplete 10-nucleotide 5' nontranslated region, a 1,425-nucleotide open reading frame which codes for 474 amino acids and a 768-nucleotide 3' nontranslated region. The first 404 of the deduced 474 amino acids resemble the amino-terminal regions of Hsp70 proteins from different species. Furthermore, single amino acid and short amino acid stretches, which are thought to be essential for the ATPase mechanism and ATP-binding activity in Hsp70 proteins, are conserved in this sequence, too. The carboxy-terminal 70 amino acids exhibit no significant similarity to hsp70 nor to any other known protein sequences. The HS24/p52 gene contains at least five introns, which differ significantly from hsc70 genes with regard to their size and location within the coding sequences. The total size of this gene is more than 15 kbp. Polymerase chain reaction (PCR) experiments showed that this gene is expressed in different human cell lines and tissues and it also seems to be highly conserved between human and mouse.
The interaction of recombinant tissue type plasminogen activator and recombinant plasminogen activator (r-PA/BM 06.022) with human endothelial cells.
The Escherichia coli-expressed recombinant plasminogen activator (r-PA) comprising the kringle 2 and protease domains of human tissue-type plasminogen activator (t-PA) has a four-fold longer half-life time in the circulation than t-PA, possibly resulting in an increased opportunity for r-PA to interact with the endothelial lining. In the present study we investigated the interaction of r-PA and t-PA with human umbilical vein endothelial cells (HUVEC). Specific binding of 125I-t-PA and 125I-r-PA were similar at 4 degrees C (Kd 6 nmol/l; Bmax about 120 fmol/mg cell protein). About half of the specific binding sites were shared by t-PA and r-PA, because unlabeled t-PA and r-PA competed equally with 125I-labeled t-PA and r-PA for binding to HUVEC. The low affinity interaction of 125I-t-PA was several-fold higher than that of 125I-r-PA. When PA binding was studied at 37 degrees C, HUVEC bound more t-PA than r-PA to both specific and non-specific binding sites. Both t-PA and r-PA were internalized and degraded, but t-PA internalization proceeded more efficiently than that of r-PA. In the presence of 100 microM chloroquine, the degradation of t-PA and r-PA was inhibited by 75% and 40%, respectively, indicating lysosomal degradation. When the active sites of t-PA and r-PA were blocked by PPACK, part of the cell association and most of the degradation of both t-PA and r-PA were inhibited. This points to plasminogen activator inhibitor-1 (PAI-1) as one of the specific binding sites. A possible role of LDL-receptor related protein (LRP) or related members of this receptor family was investigated by using the 39 kD receptor associated protein (RAP) which prevents interaction of ligands with these receptors. RAP reduced the association of 125I-t-PA by 25% and the degradation of 125I-t-PA and 125I-r-PA by 65% and 50%, respectively. Our data show that both t-PA and r-PA bind to HUVEC and can subsequently be internalized and degraded. However, r-PA interacts less effectively with HUVEC than t-PA. This indicates that binding to the endothelium does not prevent the clearance of r-PA and is not the cause of its long half-life.
Stimulation of the specific immune system by mistletoe extracts.
We have investigated the in vitro responsiveness of T cells from mistletoe-treated cancer patients and untreated donors to various preparations of mistletoe extracts. Proliferation of peripheral blood mononuclear cells from treated but not from untreated patients was observed in response to therapeutically used mistletoe extracts prepared from apple (mali) or pine (pini) host trees. The strongest proliferation was induced by vesicle preparation of mali extract. Using a newly developed flow cytometry assay (standard cell dilution assay), we determined that cell growth was restricted to CD4+ T cells. Analysis with a panel of monoclonal antibodies against variable regions of the T cell receptor beta chain (V beta) revealed an oligoclonal pattern of CD4+ T cell activation. These results indicate that therapeutic administration of mistletoe extracts sensitized a restricted set of CD4+ T lymphocytes in mistletoe-treated patients. Lymphocytes from untreated donors are only stimulated with heat-treated mistletoe extracts. The responding T cells are gamma delta T cells and express variable T cell receptor elements V gamma 9 and V delta 2. The gamma delta-stimulating activity of heat-treated mistletoe extracts is sensitive to treatment with alkaline phosphatase but not with proteinase K, indicating that the ligands are non-proteinaceous phosphate-containing compounds.
Significance of the paralemniscal tegmental area for audio-motor control in the moustached bat, Pteronotus p. parnellii: the afferent off efferent connections of the paralemniscal area.
The paralemniscal tegmental area has been determined in the brain of the New World moustached bat, Pteronotus p. parnellii, by electrical microstimulation eliciting echolocation calls and pinna movements. It is located in the dorsal tegmentum rostral and medial to the dorsal nucleus of the lateral lemniscus and is characterized by medium sized and large neurons. Tracer injections (WGA-HRP) showed that the most intense input to the paralemniscal tegmental area originates in the intermediate and deep layers of the homolateral superior colliculus. The strong projections from the ipsi- and contralateral nucleus praepositus hypoglossus most probably contributes vestibular information. Further inputs in descending order of intensity are from the substantia nigra, the contralateral paralemniscal tegmental area, the putamen, the ventral reticular formation in its lateral portions, the medial cerebellar nucleus and the dorsal reticular formation. Efferent projections of the paralemniscal tegmental area reach the putamen bilaterally, the nucleus accumbens and other parts of the basal ganglia, the pretectal area, the substantia nigra, the intermediate and deep layers of the superior colliculus bilaterally and the tegmental area ventral to it. Connections to the dorsal part of the periaqueductal grey, the cuneiform nucleus and the parabrachial region are important in the context of vocal control, whereas projections to the medial portion of the contralateral facial nucleus may interfere with the control of pinna movement. The findings suggest that the paralemniscal tegmental area is involved in audio-motor control of vocalization and pinna movements in bats; connectional and functional similarities and disparities to tegmental regions described in other mammals are discussed.
Pronounced cytoplasmic pH gradients are not required for tip growth in plant and fungal cells.
The existence of pronounced cytoplasmic pH gradients within the apices of tip-growing cells, and the role of cytoplasmic pH in regulating tip growth, were investigated in three different cell types: vegetative hyphae of Neurospora crassa; pollen tubes of Agapanthus umbellatus; and rhizoids of Dryopteris affinis gametophytes. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual emission confocal ratio imaging of the pH-sensitive probe carboxy SNARF-1. Considerable attention was paid to the fine tuning of dye loading and imaging parameters to minimise cellular perturbation and assess the extent of dye partitioning into organelles. With optimal conditions, cytoplasmic pH was measured routinely with a precision of between +/-0.03 and +/-0.06 of a pH unit and a spatial resolution of 2.3 microm2. Based on in vitro calibration, estimated values of mean cytoplasmic pH for cells loaded with dye-ester were between 7.15 and 7.25 for the three cell types. After pressure injecting Neurospora hyphae with dextran-conjugated dye, however, the mean cytoplasmic pH was estimated to be 7.57. Dextran dyes are believed to give a better estimate of cytoplasmic pH because of their superior localisation and retention within the cytosol. No significant cytoplasmic pH gradient (delta pH of >0.1 unit) was observed within the apical 50 microm in growing cells of any of the three cell types. Acidification or alkalinisation of the cytoplasm in Neurospora hyphae, using a cell permeant weak acid (propionic acid at pH 7.0) or weak base (trimethylamine at pH 8.0), slowed down but did not abolish growth. However, similar manipulation of the cytoplasmic pH of Agapanthus pollen tubes and Dryopteris rhizoids completely inhibited growth. Modification of external pH affected the growth pattern of all cell types. In hyphae and pollen tubes, changes in external pH were found to have a small transient effect on cytoplasmic pH but the cells rapidly readjusted towards their original pH. Our results suggest that pronounced longitudinal gradients in cytoplasmic pH are not essential for the regulation of tip growth.
Glibenclamide, but not acarbose, increases leptin concentrations parallel to changes in insulin in subjects with NIDDM.
OBJECTIVE: To hypothesize if glibenclamide, which increases insulin levels, also increases leptin concentrations. RESEARCH DESIGN AND METHODS: Leptin is a hormone that regulates weight in mice. In obese humans, leptin concentrations are increased, suggesting resistance to the effects of this hormone. Although short-term infusion of insulin during the hyperinsulinemiceuglycemic clamp does not increase leptin concentration, the effect of oral antidiabetic agents on leptin concentration is unknown. Differing effects can be expected, since glibenclamide acts via stimulation of insulin secretion, whereas acarbose inhibits alpha-glucosidases of the small intestine and has no direct effect on insulin levels. We examined the effect of acarbose (n = 4), glibenclamide (n = 6), and placebo (n = 6) on insulin and leptin levels during 24-h periods before and after 16 weeks of therapy. RESULTS: We observed a significant diurnal variation in leptin concentrations. This was inversely related to insulin levels during the 24-h follow-up with usual diet. Neither the placebo nor acarbose altered leptin concentrations. However, glibenclamide increased leptin concentrations parallel to insulin levels. There were only minor changes in body weight during the l6-week follow-up: decrease in the placebo group (change -0.5 kg/m2, P = 0.07) and acarbose (change -0.7 kg/m2, P = 0.046) and increase in the glibenclamide group (change 0.8 kg/m2, P = 0.27). However, individual subjects who gained weight had increases in their leptin concentrations. The diurnal variation in leptin concentrations was preserved after glibenclamide. CONCLUSIONS: Glibenclamide increases circadian leptin and insulin concentrations, whereas acarbose does not. This observation may help to explain weight gain in subjects treated with glibenclamide and stable weight in those treated with acarbose in the long run.
Role of adenosine in the hypoxic induction of vascular endothelial growth factor in porcine brain derived microvascular endothelial cells.
Hypoxia induced the mRNA expression of vascular endothelial growth factor (VEGF) in porcine brain derived microvascular endothelial cells (BMEC) in a time-dependent manner. Corresponding to the mRNA induction the protein level of VEGF was elevated during hypoxia. The adenosine A1 receptor antagonist 8-phenyltheophylline (8-PT) reduced the hypoxia-induced VEGF mRNA and protein expression significantly. The treatment of BMEC with cobalt chloride-known to activate an oxygen sensing mechanism similar to the one used by the erythropoietin gene-also induced the VEGF mRNA expression, but 8-PT did not reduce this VEGF induction. Although, earlier studies revealed that agents like phorbolester induced the VEGF mRNA expression, the specific inhibitor of the proteinkinase C (PKC) bisindolylmaleimide (BIM) did not reduce but enhanced the hypoxia-induced VEGF mRNA expression. These results indicate that the VEGF induction in BMEC can proceed through PKC-dependent and -independent pathways (like those acting via the putative oxygen sensor). Hypoxia in BMEC probably activates the PKC-dependent pathway mainly via adenosine which might be formed during hypoxia and thereby inhibits activation of PKC-independent, oxygen sensing, pathways. This suggestion was supported by the fact that hypoxia as well as adenosine increased the VEGF mRNA expression post-transcriptionally by enhancing the stability of the VEGF mRNA [corrected].
Inhomogeneities in wall stress measured by microergometry in the heart muscle in situ.
Microergometry is a method which we have developed as a tool to measure local mesh-tension within the myocardial weave at any measuring site of both ventricles and the septum on the beating heart in situ. In a mapping procedure on pig and dog hearts, both in control conditions and in the hypertrophied state after aortic banding, local mesh-tension was measured in several areas and in up to eight depths proceeding from the epicardium to the endocardium: Probe-to-fibre coupling is definitely more stable in the canine myocardium than in the porcine heart muscle, probably due to a more effective connective tissue fettering of the canine myocardial weave. The observed longitudinal gradient, with the highest tension in the base, of control dog hearts was levelled out in the hypertrophied hearts. Furthermore, in control dog hearts mesh-tension in the subepi- and subendocardial layers was higher than in the midlayers. This pronounced midlayerhypotension was smoothed in the hypertrophied hearts. Further studies will be dedicated to the question of whether the impact of ventricular size and shape on intersegmental stress transmission is determined by the Frank-Starling mechanism alone or whether protracted remodelling processes on the level of the local fibre weave cause slow coupling alterations.
[Mayo duplication in treatment of incisional hernia of the abdominal wall after conventional laparotomy. Results of a retrospective analysis and comparison with the literature].
About 10% of patients undergoing conventional laparotomy will develop incisional hernias. Traditionally these hernias are in this country most often repaired by a Mayo-duplication. In this retrospective analysis we investigated a consecutive series of 114 patients (mean age: 53 (11-87) years, gender ratio m:f 1.2:1.97% electively operated) with 135 hernias, operated between 1/1985 and 12/1992 by a standard Mayo-procedure. Recurrence-rates and quality of life were evaluated by clinical examination. The mean follow-up time was 5.7 (2.5-10.2) years with a follow-up rate of 84.4%. The overall recurrence rate was 53.5%. Further uni- and multi-variate analysis was unable to find any clinically relevant risk factors for hernia development. Health related quality of life was evaluated with a validated index at the time of their follow-up visit. There were no differences in patients without a hernia recurrence (n = 52) when compared to those with an actually present recurrence (n = 36). However, physical function of all patients was significantly impaired when compared to healthy individuals. According to our results and those reported by others the Mayo-duplication when applied to all patients leads to non-acceptable results and implantation of auto- or alloplastic material should be considered more frequently.
Recruitment of activated p56lck on endosomes of CD2-triggered T cells, colocalization with ZAP-70.
We have previously established that upon CD2 activation of T cells, p56(lck) showed a transient increase in its kinase activity and was partially internalized. Here we studied the possibility that p56(lck) could retain its kinase activity in the endosomes of CD2-triggered cells. T cells were fractionated on a sucrose gradient, and the endosomal fraction was isolated. In CD2-triggered cells, part of Lck was internalized and presented a maximal kinase activity in the endosome-enriched fraction after 5 min, decreasing thereafter. In the endosomal fraction of activated cells, four tyrosine-phosphorylated proteins of apparent molecular masses of 30, 40, 56, and 70 kDa were detected. We demonstrated that the protein tyrosine kinase ZAP-70 was recruited to the endosomal fraction upon CD2 stimulation with kinetics similar to that of p56(lck), suggesting that recruitment of protein tyrosine kinases to endosomal vesicles could promote specific transduction signals at the intracellular level.
Hepatic levels of bile acids in end-stage chronic cholestatic liver disease.
In chronic cholestatic liver disease hydrophobic and potentially cytotoxic bile acids are assumed to accumulate in the liver. To test this hypothesis we investigated bile acid levels and pattern in livers and serum of patients with, (A) end-stage chronic cholestatic liver disease, and with (B) end-stage cirrhosis of alcoholic/chronic hepatitic origin who underwent liver transplantation. Bile acids were also analyzed in (C) normal liver tissue. Levels of bile acids were 215 +/- 39.1 nmol/g liver (wet weight) in chronic cholestasis and 120 +/- 32.7 and 56.1 +/- 24.2 nmol/g liver in group B and group C (P < 0.01 and P < 0.005), respectively. Cholic acid was the prevailing bile acid in chronic cholestasis (51%) and was elevated eight-fold as compared to group C (P < 0.005). Chenodeoxycholic acid contributed 41% to total bile acids and was elevated four-fold (P < 0.005). Deoxycholic acid contributed only 1.5% to bile acids in chronic cholestasis as compared to 27% in group C (P < 0.01) and was absent in group B. Levels of lithocholic acid tended to be increased in chronic cholestasis as compared to group C and its sulfation was impaired (P < 0.05). The pattern of serum bile acids in chronic cholestasis agreed well with the bile acid pattern in the explanted livers. We conclude that hepatic accumulation of hydrophobic chenodeoxycholic acid and impaired sulfation of lithocholic acid might contribute to tissue degeneration in chronic cholestatic liver disease due to the detergent effects of these bile acids.
The 2.3 A crystal structure of the catalytic domain of recombinant two-chain human tissue-type plasminogen activator.
Tissue-type plasminogen activator (t-PA), a multidomainal serine proteinase of the trypsin-family, catalyses the rate-limiting step in fibrinolysis, the activation of plasminogen to the fibrin-degrading proteinase plasmin. Trigonal crystals have been obtained of the recombinant catalytic domain of human-two-chain t-PA, consisting of a 17 residue A chain and the 252 residue B chain. Its X-ray crystal structure has been solved applying Patterson and isomorphous replacement methods, and has been crystallographically refined to an R-value of 0.184 at 2.3 A resolution. The chain fold, active-site geometry and Ile276-Asp477 salt bridge are similar to that observed for trypsin. A few surface-located insertion loops differ significantly, however. The disulfide bridge Cys315-Cys384, practically unique to the plasminogen activators, is incorporated without drastic conformational changes as the insertion loop preceding Cys384 makes a bulge on the molecular surface. The unique basic insertion loop Lys296-Arg304 flanking the primed subsites, which has been shown to be of importance for PAI-1 binding and for fibrin specificity, is partially disordered; it can therefore freely adapt to proteins docking to the active site. The S1 pocket of t-PA is almost identical to that of trypsin, whereas the S2 site is considerably reduced in size by the imposing Tyr368 side-chain, in agreement with the measured preference for P1 Arg and P2 Gly residues. The neighbouring S3-S4 hydrophobic groove is mainly hydrophobic in nature. The structure of the proteinase domain of two-chain t-PA suggests that the formation of a salt bridge between Lys429 and Asp477 may contribute to the unusually high catalytic activity of single-chain t-PA, thus stabilizing the catalytically active conformation without unmasking the Ile276 amino terminus. Modeling studies show that the covalently bound kringle 2 domain in full-length t-PA could interact with an extended hydrophobic groove in the catalytic domain; in such a docking geometry its "lysine binding site" and the "fibrin binding patch" of the catalytic domain are in close proximity.