The p38 MAPK pathway mediates the growth inhibitory effects of interferon-alpha in BCR-ABL-expressing cells.

The mechanisms by which interferon-alpha (IFN-alpha) mediates its anti-leukemic effects in chronic myelogenous leukemia (CML) cells are not known. We determined whether p38 MAPK is activated by IFN-alpha in BCR-ABL-expressing cells and whether its function is required for the generation of growth inhibitory responses. IFN-alpha treatment induced phosphorylation/activation of p38 in the IFN-alpha-sensitive KT-1 cell line, but not in IFN-alpha-resistant K562 cells. Consistent with this, IFN-alpha treatment of KT-1 (but not K562) cells induced activation of the small GTPase Rac1, which functions as an upstream regulator of p38. In addition, IFN-alpha-dependent phosphorylation/activation of p38 was induced by treatment of primary granulocytes isolated from the peripheral blood of patients with CML. To define the functional role of the Rac1/p38 MAPK pathway in IFN-alpha signaling, the effects of pharmacological inhibition of p38 on the induction of IFN-alpha responses were determined. Treatment of KT-1 cells with the p38-specific inhibitors SB203580 and SB202190 reversed the growth inhibitory effects of IFN-alpha. On the other hand, the MEK kinase inhibitor PD098059 had no effects, further demonstrating the specificity of these findings. To directly determine the significance of IFN-alpha-dependent activation of p38 in the induction of the anti-leukemic effects of IFN-alpha, we evaluated the effects of p38 inhibition on leukemic colony formation in bone marrow samples of patients with CML. IFN-alpha inhibited leukemic granulocyte/macrophage colony formation in a dose-dependent manner, whereas concomitant treatment with p38 inhibitors reversed such an inhibition. Thus, the Rac1/p38 MAPK pathway is activated by IFN-alpha in BCR-ABL-expressing cells and appears to play a key role in the generation of the growth inhibitory effects of IFN-alpha in CML cells.

Engagement of the CrkL adaptor in interferon alpha signalling in BCR-ABL-expressing cells.

Interferon alpha (IFNalpha) has significant clinical activity in the treatment of patients with chronic myelogenous leukaemia (CML), but the mechanisms of its selective efficacy in the treatment of the disease are unknown. The CrkL adaptor protein interacts directly with the BCR-ABL fusion protein that causes the malignant transformation and is constitutively phosphorylated in BCR-ABL-expressing cells. In the present study, we provide evidence that CrkL was engaged in IFNalpha-signalling in the CML-derived KT-1 cell line, which expresses BCR-ABL and is sensitive to the growth inhibitory effects of IFNalpha. CrkL is constitutively associated with BCR-ABL in these cells and treatment with IFNalpha had no effect on the BCR-ABL/CrkL interaction. After IFNalpha stimulation, CrkL associated with Stat5, which also underwent phosphorylation in an IFNalpha-dependent manner. The interaction of CrkL with Stat5 was facilitated by the function of both the SH2 and the N-terminus SH3 domains of CrkL. The resulting CrkL-Stat5 complex translocated to the nucleus and could be detected in gel shift assays using elements derived from either the beta-casein promoter or the promoter of the PML gene, an IFNalpha-inducible gene that mediates growth inhibitory responses. In addition to its interaction with Stat5, CrkL interacts with C3G in KT-1 cells and such an interaction regulates the downstream activation of the small GTPase Rap1, which also mediates inhibition of cell proliferation. Thus, despite its engagement by BCR-ABL in CML-derived cells, CrkL mediates activation of downstream signalling pathways in response to the activated type I IFN receptor and such signals may contribute to the generation of the anti-proliferative effects of IFNalpha in CML.

Transient induction of cytokine production in human myocardial fibroblasts by coxsackievirus B3.

Cytokine expression in enterovirus infections of the heart may trigger inflammation and have detrimental effects on myocytes. However, the induction of cytokines in human myocardial cells by cardiotropic enteroviruses, for example, Coxsackievirus B3 (CVB3), was not yet demonstrated. Fibroblasts are the predominant cell type of the myocardial interstitium before inflammatory infiltration develops. Hence, we investigated, by enzyme immunoassays, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and nucleic acid sequence-based amplification (NASBA), whether CVB3 induces cytokine expression in cultured human myocardial fibroblasts. As early as 3 hours after infection, RT-qPCR demonstrated a 2-fold increase of interleukin (IL)-6 and IL-8 mRNA compared with basal transcription, resulting in a significant increase of IL-6 and IL-8 to a median level of 1500 pg/mL (range, 1246 to 1858) and 529 pg/mL (range, 428 to 601) in culture supernatants, respectively. IL-6 and IL-8 expression returned to basal levels within 3 and 5 days, respectively, despite a persistent (carrier-state) CVB3 infection. For comparison, IL-6 and IL-8 were induced in dermal fibroblasts later than 3 days after CVB3 infection. Although the low-level IL-1alpha transcription of myocardial fibroblasts was not significantly increased, IL-1alpha was released from cells to culture supernatants 5 days after infection. Furthermore, a suppression of interferon-beta transcription was demonstrated up to 24 hours after CVB3 infection of myocardial fibroblasts by highly sensitive NASBA. In conclusion, our results demonstrate a heart-specific pattern of a rapid and transient induction of proinflammatory cytokines after CVB3 infection, whereas the expression of protective interferon-beta was suppressed by CVB3.

Activation of the Jak-Stat pathway in cells that exhibit selective sensitivity to the antiviral effects of IFN-beta compared with IFN-alpha.

We determined whether selective activation of components of the Jak-Stat pathway by different type I interferons (IFN) occurs in human myocardial fibroblasts that exhibit much higher sensitivity to the antiviral effects of IFN-beta than of IFN-alpha. Similar levels of activation of the Tyk2 kinase and the Stat3 transcription factor were induced in response to either IFN-beta or IFN-alpha treatment. However, activation of the Jak1 tyrosine kinase was detectable only in IFN-beta-treated but not IFN-alpha-treated cells. Consistent with this, tyrosine phosphorylation of Stat1 and Stat2 and formation of the IFN-stimulated gene factor 3 (ISGF3) complex occurred to a much higher degree in response to IFN-beta stimulation. These findings demonstrate that differential activation of distinct components of the Jak-Stat pathway by different type I IFN can occur. Furthermore, they strongly suggest that such selective activation accounts for the occurrence of differences in the antiviral properties of distinct type I IFN in certain cell types.

Adenoviruses and enteroviruses as pathogens in myocarditis and dilated cardiomyopathy.

BACKGROUND: Enteroviruses were detected in up to 50% in myocardium of patients with myocarditis and dilated cardiomyopathy, the latter being considered as a result of a prior subclinical myocarditis. A wide range of other infectious agents are being discussed as pathogens, often only based on reports of single cases. Adenovirus genome was recently identified in a significant number in the myocardium of paediatric patients with myocarditis. However, data on the role of adenoviruses for the aetiopathogenesis of myocarditis in adult patients is missing so far. Therefore, we studied the prevalence of adenoviral and enteroviral genome in myocardium of adults with myocarditis and dilated cardiomyopathy. METHODS: 15 patients were diagnosed at baseline with myocarditis, 16 patients with dilated cardiomyopathy according to clinical and histological criteria. Endomyocardial biopsies of these patients and 8 control patients with non-infectious heart diseases were evaluated by polymerase chain reactions for enterovirus and adenovirus genome. RESULTS: Enteroviral genome was detected in 27.3% patients with myocarditis or dilated cardiomyopathy, whereas adenoviral genome was not identified in any patient. Samples from control subjects systematically yielded negative results. CONCLUSIONS: From our data, it seems doubtful that adenoviruses are major pathogens of myocarditis or DCM, whereas enterovirus genome was identified in a significant number of patients with both diseases.

Low prevalence of hepatitis C virus antibodies and RNA in patients with myocarditis and dilated cardiomyopathy.

We investigated the prevalence of hepatitis C virus in patients with dilated cardiomyopathy (DCM) and myocarditis in comparison to a control group of patients suffering from noninflammatory cardiac diseases such as aortic stenosis. In contrast to the results of previous studies on small numbers of patients, no significant difference in the prevalence of hepatitis C infections was observed. Our data suggest that HCV is not an important causal agent for myocarditis and DCM.

[Percutaneous balloon pericardiotomy for the treatment of a malignant pericardial tamponade]. / Perkutane Ballonperikardiotomie zur Behandlung einer malignen Perikardtamponade.

HISTORY AND CLINICAL FINDINGS: A 72-year old man was admitted because of increasing dyspnoea, failing fitness and weight loss. Physical examination was unremarkable except for decreased heart sounds. INVESTIGATIONS: He was anaemic (haemoglobin 11.7 g/dl), erythrocyte sedimentation rate was raised (80/90 mm) as was C-reactive protein (169 mg/dl). Chest radiogram showed tent-like widening of the cardiac silhouette and a 6 cm space-occupying lesion in the left hilus. Echocardiography revealed pericardial effusion of up to 2.3 cm (epi- to pericardium). TREATMENT AND COURSE: Because of an increase in dyspnoea and pericardial effusion a pericardiocentesis was performed. This fluid and needle biopsy of the space-occupying lesion revealed small-cell carcinoma. 6 days after the pericardiocentesis the dyspnoea further increased as did the pericardial effusion (to 3.9 cm). Percutaneous balloon pericardiotomy was uneventfully performed. During a follow-up period of 14 months there were no further pericardial effusions. CONCLUSION: Percutaneous balloon pericardiotomy can be undertaken as a minimally invasive treatment of symptomatic pericardial effusion. It should be considered as an alternative to surgical creation of a pericardial window, especially in very ill patients.

Highly sensitive detection of gene expression of an intronless gene: amplification of mRNA, but not genomic DNA by nucleic acid sequence based amplification (NASBA).

NASBA is an isothermal nucleic acid amplification reaction that amplifies mRNA in a dsDNA background. Although similar to the sensitive reverse transcription/polymerase chain reaction (RT-PCR) in mRNA detection, NASBA is not prone to give false positive results caused by genomic dsDNA. Therefore, NASBA is unique for sensitive detection of transcription of intronless genes, which preclude strategies such as intron spanning primer pairs to control false positive results in RT-PCR. Using NASBA, mRNA of the intronless human interferon-beta gene was demonstrated with a sensitivity of 10 copies, whereas 100 ng genomic DNA gave a negative result.

Coxsackievirus genome in myocardium of patients with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Enteroviruses are known as major infectious agents for inflammatory heart diseases such as myocarditis and dilated cardiomyopathy (DCM). Arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVC) is characterized by replacement of right ventricular myocardium by fatty and fibrous tissue. In about 65% of patients inflammatory infiltrates suggest an inflammatory or infectious etiopathogenesis. To test this hypothesis, we investigated endomyocardial biopsies of patients with ARVC, with myocarditis or DCM, and from patients with non-inflammatory cardiac disorders for the presence of enteroviral genome. Enteroviral RNA with homology to coxsackieviruses type B was detected in 3 of 8 patients with ARVC (37.5%), in 7 of 23 patients with myocarditis or DCM (30.4%), but in none of 5 patient with non-infectious myocardial diseases (p < 0.05 compared to ARVC patients). These results support earlier suggestions that coxsackievirus infection of the myocardium is possibly related to the pathogenesis of ARVC.

Interferon alpha activates the tyrosine kinase Lyn in haemopoietic cells.

We investigated whether the src-family tyrosine kinase Lyn is involved in the generation of interferon alpha (IFN alpha) signals in haemopoietic cells. In vitro kinase assays using IFN alpha-sensitive cells of B-cell origin demonstrated the presence of IFN alpha-dependent kinase activity in anti-Lyn immunoprecipitates. Further studies demonstrated that Lyn associates via its src homology 2 (SH2) domain with the Janus family tyrosine kinase Tyk-2. This interaction was IFN alpha-dependent and involved direct binding of the SH2 domain of Lyn to the IFN alpha-activated form of Tyk-2. Thus, during binding of IFN alpha to its receptor in malignant haemopoietic cells, Lyn is engaged in an IFN alpha-signalling pathway, probably downstream of Tyk-2.

Monocyte activation in congestive heart failure due to coronary artery disease and idiopathic dilated cardiomyopathy.

OBJECTIVE: To investigate plasma tumor necrosis factor (TNF)alpha, tumor necrosis factor alpha soluble receptor I, interleukin-1beta and neopterin concentrations as markers of monocyte activation in patients with heart failure. STUDY DESIGN: The group consisted of patients with heart failure due to dilated cardiomyopathy (n=19) and coronary artery disease (n=11). Patients without cardiac failure served as controls (n=10). RESULTS: TNFalpha concentrations were elevated only in heart failure patients with coronary artery disease (2.9+/-0.3 pg/ml versus 1.7+/-0.3 pg/ml; P<0.05). When the patients were grouped according to acute and chronic failure, TNFalpha concentrations were significantly elevated in acute failure (3.1+/-0.4 pg/ml, n=6 versus 1.7+/-0.2 pg/ml, n=8; P<0.05). TNFalpha concentrations were elevated in patients with coronary artery disease and chronic heart failure compared to coronary artery disease patients without failure (2.0+/-0.4 pg/ml, n=6 versus 1.8+/-0.3 pg/ml, n=7; P<0.05). A higher proportion of patients with myocardial insufficiency showed increased lipopolysaccharide-inducible TNFalpha concentrations (10/30 versus 0/9, P<0.05). CONCLUSIONS: TNFalpha is elevated in patients with acute cardiac decompensation. Among patients with chronic heart failure only those with coronary artery disease exhibit increased levels. Cytokine concentrations are similar in heart failure due to dilated cardiomyopathy and coronary artery disease. Monocytes of patients suffering from cardiac insufficiency show an increased sensitivity towards stimuli such as lipopolysaccharides.

Antiviral activity of WIN 54954 in coxsackievirus B2 carrier state infected human myocardial fibroblasts.

Persistent infections with a cardiotropic enterovirus, e.g. coxsackievirus B2 (CVB2), cause chronic myocarditis and eventually congestive heart failure. Therefore, the antiviral activity of WIN 54954, a capsid binding antiviral agent that inhibits enterovirus uncoating, was studied in persistently CVB2-infected cultures of human myocardial fibroblasts. Cultures displayed a typical carrier state infection with virus titers of 3.9 +/- 1.6 x 10(5) plaque forming units (PFU)/ml and 0.99% infected cells. WIN 54954 (0.025-1 microg/ml) application was started 7 days after infection of the cultures. Compared to the WIN 54954 concentration resulting in a 90% plaque number reduction (EC90 = 0.197 microg/ml) in acutely infected Vero cells, WIN 54954 reduced virus yields of myocardial fibroblast cultures more efficiently, e.g. more than 100 fold (99%) with 0.025 microg/ml after 4 days of application. Antiviral effects of WIN 54954 increased with application time and at 0.025 microg/ml Win 54954 completely inhibited infectious virus progeny after 16 days. Increasing the WIN 54954 concentration up to 1 microg/ml did not cause a greater inhibition of virus replication. In situ hybridization demonstrated that at 0.1 microg/ml WIN 54954 reduced the number of infected cells from 0.99 to 0.18%, although a complete eradication of CVB2-infected cells was not achieved at concentrations as high as 1 microg/ml. In conclusion, the results indicate that low concentrations of WIN 54954 are effective in treating persistent enterovirus infections of myocardial fibroblasts, although a complete eradication of the infection is not achieved with WIN 54954 as a single antiviral agent.

Giant cell myocarditis due to coxsackie B2 virus infection.

Giant cell myocarditis is a rare disorder characterized by the histologic hallmark of diffuse inflammatory infiltrates with the appearance of multinucleated giant cells. We report on a 52-year-old man who died of rapidly progressive cardiogenic shock due to giant cell myocarditis. Serological and immunoblotting techniques revealed a myocardial infection with coxsackie B2 virus, suggesting a viral etiology of this disease. Here we present evidence for the involvement of autoimmune responses to the myocardium as numerous cardiomyocytes exhibited deposits of cell-adherent immunoglobulins. Although other causative factors may initiate giant cell myocarditis as well, our case suggests coxsackie B2 virus as one etiologic agent capable of triggering autoimmune reactions to altered heart tissue.

Immunohistochemical localization of five members of the Kv1 channel subunits: contrasting subcellular locations and neuron-specific co-localizations in rat brain.

A large variety of potassium channels is involved in regulating integration and transmission of electrical signals in the nervous system. Different types of neurons, therefore, require specific patterns of potassium channel subunits expression and specific regulation of subunit coassembly into heteromultimeric channels, as well as subunit-specific sorting and segregation. This was investigated by studying in detail the expression of six different alpha-subunits of voltage-gated potassium channels in the rat hippocampus, cerebellum, olfactory bulb and spinal cord, combining in situ hybridization and immunocytochemistry. Specific polyclonal antibodies were prepared for five alpha-subunits (Kv1.1, Kv1.2, Kv1.3 Kv1.4, Kv1.6) of the Shaker-related subfamily of rat Kv channels, which encode delayed-rectifier type and rapidly inactivating A-type potassium channels. Their distribution was compared to that of an A-type potassium channel (Kv3.4), belonging to the Shaw-related subfamily of rat Kv channels. Our results show that these Kv channel alpha-subunits are differentially expressed in rat brain neurons. We did not observe in various neurons a stereotypical distribution of Kv channel alpha-subunits to dendritic and axonal compartments, but a complex differential subcellular subunit distribution. The different Kv channel subunits are targeted either to presynaptic or to postsynaptic domains, depending on neuronal cell type. Thus, distinct combinations of Kv1 alpha-subunits are co-localized in different neurons. The implications of these findings are that both differential expression and assembly as well as subcellular targeting of Kv channel alpha-subunits may contribute to Kv channel diversity and thereby to presynaptic and postsynaptic membrane excitability.

The SA/rABC technique: a new ABC procedure for detection of antigens at increased sensitivity.

Since its introduction, the avidin-biotin-peroxidase (ABC) complex has become an invaluable detection system for a wide variety of bioanalytical applications. In these techniques, avidin and biotin-peroxidase are mixed at a pre-determined ratio so that the soluble ABC complex retains biotin binding sites. Consequently, the complex contains an excess of avidin over biotinylated peroxidase residues. On theoretical considerations, however, an ABC complex designed for maximal signal intensity must consist of an excess of peroxidase over avidin molecules. Therefore, ABC complexes with reversed molar ratios of biotinylated peroxidase to avidin (rABC complexes) were prepared and an intermediate streptavidin step was introduced to bind the rABC complexes to biotinylated IgG molecules. The signal generating power of this new streptavidin-rABC sequence proved superior to that of the conventional ABC technique in ELISA assays and in immunostaining of tissue sections.

Sulpho-N-hydroxysuccinimide activated long chain biotin. A new microtitre plate assay for the determination of its stability at different pH values and its reaction rate with protein bound amino groups.

Biotinamidohexanoic acid N-hydroxysulphosuccinimide ester (N-hydroxysulphosuccinimide activated long chain biotin; sulpho-NHS-LC-biotin) has become an invaluable tool for the biotinylation of protein despite the absence of data concerning its stability and reaction velocity. A convenient, rapid and sensitive assay for this compound has been developed based on the sulpho-NHS-LC-biotin mediated biotinylation of bovine serum albumin following adsorption to the wells of a microtitre plate. Bound biotin was visualized by the sequential use of streptavidin and biotinylated horseradish peroxidase. This assay was used for the determination of the stability of sulpho-NHS-LC-biotin in aqueous solution of different pH values. Hydrolysis half lives were below 15 min at pH values above 8.0, but a pH values below 6.5 they exceeded 2 h. It is suggested, therefore, that biotinylations should be performed with sulpho-NHS-LC-biotin taken from a stock solution, prepared at pH values between 3.0 and 5.8. Reaction velocities with primary amino groups were also investigated by means of this ELISA procedure. As expected, biotinylation proceeds faster at pH 8.0 as compared to 7.2, but the increased reaction rate does not compensate for the decreased hydrolysis half life at the higher pH value. Thus, biotinylation with sulpho-NHS-LC-biotin at near neutral pH values appears to be optimal.