Enzymatic activity and thermoresistance of improved microbial transglutaminase variants.

Microbial transglutaminase (MTG, EC 2.3.2.13) of Streptomyces mobaraensis is widely used in industry for its ability to synthesize isopeptide bonds between the proteinogenic side chains of glutamine and lysine. The activated wild-type enzyme irreversibly denatures at 60 °C with a pseudo-first-order kinetics and a half-life time (t1/2) of 2 min. To increase the thermoresistance of MTG for higher temperature applications, we generated 31 variants based on previous results obtained by random mutagenesis, DNA shuffling and saturation mutagenesis. The best variant TG16 with a specific combination of five of seven substitutions (S2P, S23Y, S24 N, H289Y, K294L) shows a 19-fold increased half-life at 60 °C (t1/2 = 38 min). As measured by differential scanning fluorimetry, the transition point of thermal unfolding was increased by 7.9 °C. Also for the thermoresistant variants, it was shown that inactivation process follows a pseudo-first-order reaction which is accompanied by irreversible aggregation and intramolecular self-crosslinking of the enzyme. Although the mutations are mostly located on the surface of the enzyme, kinetic constants determined with the standard substrate CBZ-Gln-Gly-OH revealed a decrease in KM from 8.6 mM (± 0.1) to 3.5 mM (± 0.1) for the recombinant wild-type MTG and TG16, respectively. The improved performance of TG16 at higher temperatures is exemplary demonstrated with the crosslinking of the substrate protein ß-casein at 60 °C. Using molecular dynamics simulations, it was shown that the increased thermoresistance is caused by a higher backbone rigidity as well as increased hydrophobic interactions and newly formed hydrogen bridges.

Prolonged cardiac arrest and resuscitation by extracorporeal life support: favourable outcome without preceding anticoagulation in an experimental setting.

State-of-the-art cardiopulmonary resuscitation (CPR) restores circulation with inconsistent blood-flow and pressure. Extracorporeal life support (ECLS) following CPR opens the opportunity for "controlled reperfusion". In animal experiments investigating CPR with ECLS, systemic anticoagulation before induced cardiac arrest is normal, but a major point of dispute, since preliminary heparinization in patients undergoing unwitnessed cardiac arrest is impossible. In this study, we investigated options for ECLS after an experimental 15 minutes normothermic cardiac arrest, without preceding anticoagulation, in pigs. Neurological recovery was assessed by a scoring system, electroencephalography and brain magnetic resonance imaging. Additionally, brain histology was performed on day seven after cardiac arrest. We demonstrated that preliminary heparin administration was not necessary for survival or neurological recovery in this setting. Heparin flushing of the cannulae seemed sufficient to avoid thrombus formation. These findings may ease the way to using ECLS in patients with sudden cardiac arrest.

Molecular epidemiology of respiratory syncytial virus in hospitalised children in Heidelberg, Southern Germany, 2014-2017.

BACKGROUND: Respiratory syncytial virus (RSV) is the leading cause of hopitalisation in young children with respiratory tract infections (RTI). The aim of this research project was to analyse RSV genotypes and the diversification of RSV strains among hospitalised children in Heidelberg, Germany. METHODS: We prospectively analysed nasopharyngeal swabs (NPS) from children who were hospitalised with acute RTI at the University Hospital Heidelberg, Germany, during winter seasons 2014 to 2017. RSV RT-PCR and RSV sequence analysis of the G gene coding for the attachment glycoprotein were performed. Clinical data was obtained using a standardised questionnaire. RESULTS: RSV was detected in 405 out of 946 samples from hospitalised children. Most RSV positive children were below the age of two years (84.4%) and had a lower RTI (78.8%). The majority of RSV positive children was male, significantly younger than RSV negative children with a median age of 0.39 years and with more severe respiratory symptoms. Out of 405 positive samples, 317 RSV strains were successfully sub-grouped into RSV subtypes A (57.4%; 182/317) and B (42.6%; 135/317). Both RSV subtypes cocirculated in all analysed winter seasons. Phylogenetic analysis of 317 isolates revealed that the majority of RSV-A strains (180/182) belonged to the ON1 genotype, most RSV-B strains could be attributed to the BAIX genotype (132/135). ON1 and BAIX strains showed a sub-differentiation into different lineages and we were able to identify new (sub)genotypes. CONCLUSION: Analysis of the molecular epidemiology of RSV from different seasons revealed the cocirculation and diversification of RSV genotypes ON1 and BAIX.

Evidence that wild-type p53 in neuroblastoma cells is in a conformation refractory to integration into the transcriptional complex.

Neuroblastoma (NB) cells reportedly accumulate wild-type p53 exclusively in the cytoplasm. However, immunofluorescence assays with five different antibodies showed that p53 accumulates in the nucleus of up to 10% of NB cells. PAb1801 detected cytoplasmic 'punctate structures' which were also found in p53-null cells, rendering this antibody unsuitable for p53 detection. A comparison of DO-1 and PAb1801 staining in NB tissue sections confirmed the results obtained with NB cells. Nuclear accumulation of p53 was induced in NB cells using substances which disturb p53's tertiary structure at its zinc finger motif, or by treatment with mitomycin C. Constitutive nuclear accumulation was observed in an SK-N-SH variant, AW-1, which has a point mutation in p53 at Cys176>Ser, disturbing the same motif. Even though p53 showed DNA-binding capability after mitomycin C treatment of NB cells, the target gene products MDM2 and p21(WAF1,CIP1,SDI1) were not synthesized and no p53 transactivating activity measured in a reporter gene assay. Therefore we suggest that p53 in NB cells might be predominantly in a conformation refractory to integration into the transcriptional complex, resulting in at least partial transcriptional inactivity, hyperactive nuclear export and resistance to degradation by exogenously expressed MDM2.

Coexpression of endothelin-converting enzyme-1 and endothelin-1 in different stages of human atherosclerosis.

BACKGROUND: Endothelin-converting enzyme (ECE)-1 activates endothelin-1 (ET-1) and may thus contribute to the regulation of vascular tone and cell growth during atherosclerosis. METHODS AND RESULTS: To evaluate ECE-1 immunoreactivity concerning big ET-1/ET-1, we performed qualitative and quantitative immunohistochemistry in normal internal mammary arteries (n=10), in coronary arteries with adaptive intimal fibrosis (n=10), in aortic fatty streaks (n=10), and in distinct regions of advanced carotid plaques (n=15). Furthermore, we determined ECE-1 activity in the control specimens and in the inflammatory intimal regions of carotid plaques. Double immunolabeling showed that ECE-1 was present in endothelial cells, vascular smooth muscle cells, and macrophages. All ET-1(+) cells were simultaneously ECE-1(+). Most importantly, there were significantly more ET-1(+) cells in the intima and media when atherosclerosis was in an inflammatory stage than when it was in a noninflammatory stage. Moreover, ECE-1 activity was upregulated in the intima of carotid plaques, although immunohistochemically, there were no significant differences between the number of ECE(+) cells in the different compartments of the arterial wall. CONCLUSION: Together with ET-1, ECE-1 is abundantly present in human arteries and at different stages of atherosclerotic plaque evolution. The upregulation of the ECE-1/ET-1 system is closely linked to the presence of chronic inflammation and is present in very early stages of plaque evolution. Therefore, enhanced production of active ET-1 may substantially contribute to cell growth and the regulation of vascular tone in advanced atherosclerotic lesions and in the very early stages of plaque evolution, when a plaque is still imperceptible clinically.

Immunopathogenesis of atherosclerosis: endotoxin accelerates atherosclerosis in rabbits on hypercholesterolemic diet.

BACKGROUND: On the basis of our concept that atherosclerosis has an immunopathological background, we tested whether activation of the innate immune system influences its progression. METHODS AND RESULTS: Hypercholesterolemic (0.5% wt/wt diet) rabbits received either repeated intravenous injections of endotoxin (Escherichia coli lipopolysaccharide 1.25 to 2.5 microg, once per week) or a self-limiting cutaneous Staphylococcus aureus infection with or without a quinolone antibiotic. Measured laboratory parameters, including LDL and HDL cholesterols, were similar in the different groups of hypercholesterolemic animals. All endotoxin-treated animals developed transient episodes of fever after endotoxin administration. The extent of atherosclerosis was evaluated by computer-assisted morphometry in the aortas en face (Sudan IV) and by histology at 8 weeks after start of the experiments. Endotoxin-treated animals exhibited significantly accelerated atherosclerosis compared with control animals (141+/-38 versus 45+/-16 mm(3) total lesion volume, n=7 to 9 rabbits each, P<0.001). CONCLUSIONS: Nonspecific stimulation of the innate immune system accelerates cholesterol-induced atherosclerosis. These data support the concept that atherosclerosis has an immunopathological component and render it improbable that a single infectious agent should assume particular importance in its initiation or progression.

Inhibition of caspase-3 improves contractile recovery of stunned myocardium, independent of apoptosis-inhibitory effects.

OBJECTIVES: The aim of this study was to investigate whether the caspase-3 inhibitor Ac-DEVD-CHO functionally improves stunned myocardium. BACKGROUND: Degradation of troponin I contributes to the pathogenesis of myocardial stunning, whereas the role of apoptosis is unknown. Caspase-3 is an essential apoptotic protease that is specifically inhibited by Ac-DEVD-CHO. METHODS: Isolated working hearts of rats were exposed to 30 min of low-flow ischemia, followed by 30 min of reperfusion. Ac-DEVD-CHO (0.1 to 1 micromol/l) was added 15 min before ischemia/reperfusion or 5 min before reperfusion. Cardiac output, external heart power, left ventricular (LV) developing pressure and contractility (dp/dt(max)) were measured. Apoptosis was assessed by TUNEL staining and internucleosomal deoxyribonucleic acid fragmentation. Caspase-3 processing and troponin I cleavage were determined by immunoblotting. Caspase-3 activity was measured using a fluorogenic substrate. RESULTS: The addition of Ac-DEVD-CHO before ischemia/reperfusion or before reperfusion dose-dependently and significantly (p < 0.05) improved post-ischemic recovery of cardiac output, external heart power, LV developing pressure and dp/dt(max), compared with the vehicle (0.01% dimethyl sulfoxide). Ac-DEVD-CHO was similarly effective when given before reperfusion. Ac-DEVD-CHO blocked ischemia/reperfusion-induced caspase-3 activation, but cardiomyocyte apoptosis was unaffected. Troponin I cleavage was not inhibited by Ac-DEVD-CHO. CONCLUSIONS: Caspase-3 is activated in stunned myocardium. Inhibition of caspase-3 by Ac-DEVD-CHO significantly improves post-ischemic contractile recovery of stunned myocardium, even when given after the onset of ischemia. The mechanism(s) of protection by Ac-DEVD-CHO appear to be independent of apoptosis. Inhibition of caspase-3 is a novel therapeutic strategy to improve functional recovery of stunned myocardium.

Inhibition of endogenous nitric oxide synthase potentiates ischemia-reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway.

OBJECTIVE: Apoptosis of cardiomyocytes may contribute to ischemia-reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. METHODS AND RESULTS: Isolated perfused rat hearts (n = 6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia-reperfusion. CONCLUSION: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

Concordant upregulation of type II-TGF-beta-receptor, the cyclin-dependent kinases inhibitor P27Kip1 and cyclin E in human atherosclerotic tissue: implications for lesion cellularity.

Atherosclerosis is a 'response-to-injury' process associated with chronic inflammation, tissue repair and a considerable cell turnover. These growth-related processes are controlled by the 'cell cycle clock' which is composed of cyclin-dependent kinases (Cdks), their activating subunits, the cyclins, and by inhibitors of Cdks (Ckis). P27 is a Cki which associates with cyclin A-Cdk2, cyclin D-Cdk4 and with cyclin E (CE)-Cdk2 complexes thereby abrogating their catalytic activity leading to potent inhibition of late G1 to S-phase transition. Furthermore, TGF-beta1 mRNA and immunoreactivity are locally increased in atherosclerotic lesions. Since TGF-beta1 growth suppressive function in the late G1 phase may be mediated by p27, blocking the catalytic activity of CE-Cdk2 complexes, via the stimulation of TGF-beta-RI and TGF-beta-RII, we investigated the topographical association between TGF-beta-RI, TGF-beta-RII, P27Kip1 and CE by immunohistochemistry in coronary artery segments without atherosclerosis and carotid atheromatous plaques of 11 patients undergoing carotid endarterectomy. P27-immunoreactivity was present in 11/11 atherosclerotic (92.7 +/- 3.3% of the cells) and 5/5 control (80.9 +/- 3.7% of the cells; P < 0.002 versus control) specimens and localized to nuclei of macrophages (CD68-positive), vascular smooth muscle cells (alpha-actin positive), T-lymphocytes (CD3-positive) as well as to the nuclei of endothelial cells. In the atherosclerotic tissue, TGF-beta-RI and TGF-beta-RII-immunoreactivity was present in 11/11 specimens and localized to inflammatory cells and to cells with VSMC-like-morphology. TGF-beta-RI-immunoreactivity was present in 87.4 +/- 5.3% (controls 75.3 +/- 7.48%; n.s.) and TGF-beta-RII-immunoreactivity was present in 83.7 +/- 6.8% (controls 39.5 +/- 7.3%; P < 0.002) of the cells. Double immunolabeling, and investigation of serial sections revealed co-expression of TGF-beta-RI and TGF-beta-RII in virtually all cells positive for P27. In the atherosclerotic specimens, CE-immunoreactivity was present in all specimens in macrophages (CD68-positive), vascular smooth muscle cells (alpha-actin positive) and in endothelial cells in 12.58 +/- 13.58% of the nuclei whereas in the controls CE staining was restricted to 0.19 +/- 0.43% of the cells (P < 0.001). Importantly, as shown by immunofluorescent double-labeling, we found cells expressing P27 that were simultaneously positive for CE. In summary, the present study provides evidence that TGF-beta1 present in human atherosclerotic tissue may mediate its growth suppressive activity also by p27, blocking the activity of CE-Cdk2 complexes. Quantitative analysis revealed that TGF-beta-RII, p27 and CE are concordantly upregulated in the atherosclerotic tissue with chronic inflammation, supporting the view that TGF-beta1, p27 and CE may play an important role in the processes associated with chronic inflammation and cell turnover in advanced human atherosclerotic plaques. Taken together, these results provide a possible link between the chronic inflammation associated with advanced atherosclerosis, the effects of extracellular growth factors and cell cycle control.

Quantitative proteome analysis in benign thyroid nodular disease using the fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain.

Thyroid tumorigenesis involves qualitative and quantitative changes in protein expression, which can be comprehensively studied by proteome analysis. However, one of the technical bottlenecks of proteomics remains a reliable, sensitive and inexpensive method for quantification of differentially expressed proteins. This is due to the limited linear range of most available protein stains, i.e. silver and Coomassie blue, and high costs of commercially available fluorescent stains. In this paper we describe our experience with a lab-made ruthenium based fluorescent stain (ruthenium II tris(bathophenanthroline disulfonate) (RuBPs)) to perform proteome analysis of nodular thyroid disease. We first compared the properties of RuBPs with two highly sensitive protein stains: (1) silver staining and (2) the commercially available fluorescent dye Sypro Ruby. We show that in addition to its highly sensitive staining capabilities similar to Sypro Ruby and silver (2 ng), RuBPs offers several advantages such as a broad dynamic range (similar to Sypro Ruby and 500 times broader than the dynamic range of silver stain), low costs ( 0.03 per gel) and excellent compatibility with mass spectrometry. We then applied the inexpensive RuBPs stain to 2D gels (pH 4-7) of four benign thyroid nodules and normal thyroid tissue. We were able to detect approximately 1800 protein spots/gel in our thyroid samples. Quantitative changes in protein expression levels of at least 20-42 proteins were noted in the benign nodules compared with the normal thyroid tissue of the same patient. Differentially expressed spots were further characterised by nano-LC-FTICR and MALDI-TOF mass spectrometry. In summary we demonstrate, that the novel fluorescent ruthenium II tris(bathophenanthroline disulfonate) stain is a highly sensitive, reliable and inexpensive tool for quantitative proteome analysis in thyroid nodular disease.

Endothelin-1 and endothelin converting enzyme-1 in human atherosclerosis--novel targets for pharmacotherapy in atherosclerosis.

The role of chronic inflammation in the pathogenesis of the acute coronary syndromes has received increasing attention since active plaques rich in macrophages (Mphi's) are more prone to rupture whereas plaques rich in myofibroblasts are considered to be stable. Functionally, active plaques show a locally enhanced vasoreactivity. Endothelin-1 (ET-1) a potent vasoconstrictor acts in a paracrine fashion to regulate vascular tone. ET-1 is also produced by inflammatory cells suggesting a role for ET-1 in inflammation. Additionally, ET-1 is a mitogen. Endothelin converting enzyme-1 (ECE-1) activates ET-1 and may thus contribute to the regulation of vascular tone and cell growth during atherosclerosis. We evaluated the presence of ECE-1 and big ET-1/ET-1 and the activity of ECE-1 in different plaque types. Together with ET-1, ECE-1 is present in endothelial cells (ECs), vascular smooth muscle cells (VSMCs) and Mphi's. ECE-1 activity and ET-1-immunoreactivity (IR) both are upregulated during the progression of atherosclerosis from a non-inflammatory to an inflammatory stage. Thus, enhanced production of active ET-1 may contribute to cell growth and regulation of vascular tone in advanced plaques and also in very early stages of atherosclerosis. Furthermore, we examined the presence of ET-1 in coronary plaque tissue obtained by directional coronary atherectomy. ET-1 IR localized to plaque components indicative of chronic inflammation. Semiquantitative analysis of ET-1 IR revealed significantly higher staining grades in active coronary lesions compared with nonactive lesions. The increased ET-1 content in active coronary lesions may be beneficial to the stabilization of the vessel wall after plaque rupture and disadvantageous because it may lead to vasospasm and to the progression of atherosclerosis.

Hepatitis C virus infection in intravenous drug users.

Intravenous drug use (IVDU) remains a major means of hepatitis C virus (HCV) transmission. In this study, 101 drug users were studied prospectively after cessation of IVDU. Of these, 75.8% were anti-HCV positive, and 71.4% had elevated levels of alanine aminotransferase. These levels decreased significantly within 1 month of IVDU cessation (p 0.02). Liver biopsies showed minimal or mild fibrosis in 32 (71%) of 45 subjects, and severe fibrosis in two (4.4%) subjects. Anti-HCV-positive intravenous drug users in this study presented with mild liver disease and variable stages of disease progression. Biochemical disease activity might be affected by IVDU.

Nucleotide-evoked relaxation of human coronary artery.

Endothelium-dependent dilation of coronary blood vessels in response to ATP and related nucleotides has been demonstrated in various animal species. The aim of the present study was to investigate a possible relaxant effect of ATP, the adenine nucleotides 2-methylthio ATP (MeSATP) and adenosine 5'-O-(2-thiodiphosphate) (ADPbetaS), and the pyrimidine nucleotide UTP in isolated human coronary artery. In endothelium-intact rings of human coronary artery precontracted with K+ (20-40 mM), the nucleotides caused relaxation. Average maximal percentage relaxations and average EC50 values (concentrations causing half-maximal relaxation) were 89% and 47.1 microM for ATP, 28% and 0.3 microM for MeSATP, 35% and 0.6 microM for ADPbetaS, and 49% and 1.6 microM for UTP. For each of the four agonists, the potency to elicit relaxation varied greatly between individual rings, so that equi-relaxing concentrations spanned several orders of magnitude. Moreover, the sensitivities to ATP and UTP, when tested in the same ring, were not correlated. Mechanical removal of the endothelium as well as NG-nitro-L-arginine methyl ester (L-NAME; 30 microM), an inhibitor of nitric oxide synthase, abolished the relaxation caused by MeSATP, ADPbetaS and UTP and greatly attenuated the response to lower concentrations of ATP (3.2-320 microM), but high concentrations of ATP (320 and 1000 microM) caused relaxation also in endothelium-denuded preparations and in the presence of L-NAME. High concentrations of ADPbetaS (32 and 100 microM) and UTP (320 and 1000 microM) caused contraction of endothelium-denuded preparations. Thus, extracellular nucleotides cause endothelium-dependent, primarily nitric oxide-mediated relaxation of human coronary artery. ATP in addition causes endothelium-independent relaxation. The receptors activated by the nucleotides appear to be unevenly distributed on the coronary endothelium.

Is MIB-1 proliferation index a predictor for response to neoadjuvant therapy in patients with esophageal cancer?

BACKGROUND: The overall survival rate for patients with an esophageal cancer remains poor. As a consequence, preoperative chemoradiation was introduced for patients with tumor stage T >1 M0 regardless of tumor histology or localization. However, factors predicting response to this therapy pretherapeutically are largely unknown. METHODS: Clinical results of preoperative chemoradiation were investigated. The rates of proliferation and apoptosis were determined in pretherapeutic tumor samples and correlated with tumor response and long-term survival after surgery. RESULTS: A complete tumor response due to chemoradiation (n = 42; cervically localized tumors excluded) was achieved in 11 patients (26%) after resection. Five-year survival rate was significantly improved in these patients compared with those who did not respond to chemoradiation (48% versus 5.5%; P = 0.003). Chemoradiation was performed without benefit in 43%. Perioperative hospital mortality rate was 14.3% in all patients. No correlation of apoptosis with response to chemoradiation or postoperative long-term survival was observed. However, there was a clear correlation between the proliferation rate as determined by MIB-1 immunohistology. Five-year survival rate of patients with a proliferation index (PI) >/=39% was 38% compared with 0% in tumors with a PI <39%. Tumors with a PI >/=39% responded to chemoradiation in 71.4%, but 100% of tumors with a PI <39% did not. Mean survival time of these patients was 33 months and 11 months, respectively (P = 0.015). CONCLUSIONS: The results indicate that the PI may be used for stratification of patients treatment prior surgery. However, these results need further validation in larger patient numbers in the search for factors indicating response pretherapeutically to preoperative chemoradiation in esophageal cancer.

Predictive factors for response to neoadjuvant therapy in patients with oesophageal cancer.

BACKGROUND: Preoperative radio-chemotherapy (RCX) was introduced to improve the outcome of patients with oesophageal cancer (EC), but conflicting results have been released. Some 20-30% of patients show a complete pathological response, however, the perioperative morbidity and mortality is increased. To search for factors indicating response prior to the onset of RCX we investigated the proliferative activity (MIB-1), the expression of vascular endothelial growth factor (VEGF), and the capillary density (CD34) in samples of EC obtained by endoscopy prior to the start of the treatment. METHODS: Forty-six (MIB-1) and 21 (VEGF, CD34) tissue specimens of ECs were available from 56 patients undergoing pretherapeutic endoscopy, RCX and surgery. Perioperative morbidity was divided into surgery and non-surgery related morbidity. MIB-1, VEGF and CD34 expression were investigated immunohistochemically. Multivariate analysis was carried out to prove independence of investigated variables. RESULTS: Postoperative morbidity was noticed in 54 of 56 operated patients. Eight of 56 patients who received RCX died in hospital. Survival was significantly different between the group of complete responders (n=14) and non-responders (n=23; P=0.0026). None of the investigated tumour samples from patients with a complete response (CR) had a proliferation index of less than 45. Tumour samples from patients with a CR showed a VEGF expression of 10.7 compared with 36.58 of tumours with no response (P=0.035). CD34 expression showed a correlation with VEGF expression. The relation of mean indices of VEGF expression and proliferative activity in tumours from patients with complete, partial or no response was 10.7:58.8, 18.3:53.8 and 36.6:43.5, respectively. CONCLUSIONS: According to these results, it may be expected that tumours with a VEGF/MIB-1 ratio of 1:6 or less prior to RCX will respond to this therapy.

Increased tissue endothelin-1-like immunoreactivity in the internal mammary artery of patients with diabetes or hypercholesterolemia modulates the graft flow in the peri-operative period.

OBJECTIVE: Peri-operative ischemic episodes following coronary artery bypass grafting with the internal mammary artery (IMA) are thought to be due to a vasospasm of this conduit. Endothelin-1 (ET-1) is a potent vasoconstrictor by itself and increases the response to other vasoconstrictor stimuli. This study focused on the possible role of an enhanced tissue ET-1-like immunoreactivity in the perioperative reaction of the IMA in patients with diabetes or hypercholesterolemia. METHODS: Specimens of the distal part of the IMA from 46 patients (mean age 58.5 years, four women, 42 men) were studied prospectively. Nine of those patients were diabetic and 26 had evidence of hypercholesterolemia. Another cohort of 20 IMA specimens was stained retrospectively; 10 of those biopsies were from patients that had experienced transient ischemic events peri-operatively in the myocardial area supplied by the IMA. The biopsies were examined histologically and immunohistochemically (rabbit polyclonal ET-1 antiserum, three-step avidin-biotin complex) with regard to their immunoreactivity to tissue ET-1. RESULTS: An immunoreactivity to ET-1 (graded 0-3) was present in 89% of the biopsies. The reactivity was significantly higher in patients with hypercholesterolemia ( 1.92+/-0.74) when compared to controls (1.0+/-0.63) (P = 0.04). The reactivity was also increased in patients with non-insulin-dependent diabetes mellitus (2.1+/-0.79), when compared to controls (P = 0.02). Mostly transient ischemic events in the area supplied by the IMA seemed to occur more frequently when the biopsies revealed a higher immunoreactivity to ET-1. They showed an increased reactivity to ET-1 (2.27+/-0.76) compared to 10 patients with an uneventful peri-operative course (1.66+/-0.71 ) (P = 0.04). CONCLUSIONS: This study provides evidence that the internal mammary artery is not a passive conduit. Vasospasm or vasoconstriction, in particular at its distal end, may occur more frequently in patients with hypercholesterolemia or diabetes, and may lead to post-operative ischemic events.

Comparison of MR sequences in quantifying in vitro cartilage degeneration in osteoarthritis of the knee.

Magnetic resonance imaging of amputated human knees was performed to determine optimal sequences for depicting articular cartilage. 24 knees were examined with eight different sequences in a 1.0 T imager. Each cartilage lesion was graded from 1 to 4 (Outerbridge staging system). The results of each sequence were compared with the macroscopic findings and statistically tested against each other. The FLASH sequence (TR = 50 ms) with combination of flip angle of 40 degrees and echo time of 10 ms and the FISP sequence (TR = 40 ms) with combination of flip angle of 40 degrees and echo time of 11 ms were best for depicting cartilage structure and internal detail. There was no significant difference between fat-saturated three-dimensional FLASH (FS-3D-FLASH) and FS-3D-FISP (p = 0.05). These FS-3D sequences were significantly better than sequences without fat saturation (p = 0.05). There was no significant difference between magnetization transfer (MT) 3D-FLASH, MT-3D-FISP and 3D-FISP. All 3D sequences showed significantly (p = 0.05) better results than spin echo or fast spin echo sequences. The T1 weighted SE pulse sequence was significantly (p = 0.005) better than the T2 weighted TSE sequence. Fast T2 weighted spin echo was not suitable for early and accurate detection of cartilage lesions.

Amyloid tumors of the lung--an immunocytoma?

Amyloid tumors are nodular amyloid depositions usually limited to one organ, which often develop without a known cause. In most cases they may be observed as being situated in the lung, the larynx, the skin, the urinary bladder and in the region of the orbita, and are restricted to these organs. In the present study, we report on two cases of pulmonal amyloid tumors which after immunohistochemical investigation revealed a clonal evolution of light chain restricted plasma cells and lymphocytes corresponding to a localized primary extranodal lymphoplasmacytic immunocytoma, with only few vital tumor cells among abundant tumor-shaped amyloid. Additionally, using polymerase chain reaction (PCR) to investigate IgH gene rearrangement, clonality of the tumor cells could be demonstrated in both cases.

Immunoelectron microscopy of different forms of glomerulonephritis in routine biopsy material.

Renal biopsies were investigated of patients with IgA or membraneous glomerulonephritis or with systemic lupus erythematosus by light microscopy, electron microscopy, light microscopic immunohistology and by immunoelectron microscopy using the post-embedding technique applied to LR-White embedded tissue. Aim of the study was to explore whether immunoelectron microscopy is reproduced on routine biopsy material and in accordance with light microscopic immunohistological findings. The study shows that immunoelectron microscopy can be applied to routine biopsy material and gives reproducible results. The applied method proved to be reliable, and, hence, routine biopsy material may be used for further studies concerning subcellular mechanisms in immunocomplex deposition and removal.

An immunohistochemical study of endothelin-1 in the human eye.

PURPOSE: Endothelin-1 (ET-1) is a potent vasoconstrictive and neural peptide that has been demonstrated to be present and functionally active and important in the eye. This study was undertaken to examine for the first time the cellular distribution of ET-1 in the whole human eye. METHODS: Twelve human eyes were examined by immunohistochemical staining of paraffin sections, using an anti-ET-1 primary antibody and an ABC-detection system. RESULTS: Endothelin-1-immunoreactivity (ET-1-IR) was detected primarily in the fibrovascular stroma of the iris, ciliary body and choroid, in the retinal blood vessels, the ciliary and optic nerves, and in the corneal and the non-pigmented ciliary epithelium. CONCLUSION: In the eye, ET-1-IR is present in fibrovascular, neural and epithelial structures. Changes in the distribution and concentration of ET-1 may be relevant to a variety of ocular diseases including diabetes mellitus, hypertension, sickle cell disease, optic neuritis, AION, papilledema, corneal ulcer, corneal epithelial dystrophy or after keratoplasty.