Prognostic factors of disease-free and overall survival in patients with hepatocellular carcinoma undergoing partial hepatectomy in curative intent.

PURPOSE: Tumor recurrence after liver resection continues to pose a major problem in hepatocellular carcinoma (HCC). Here we aimed to evaluate prognostic markers for disease-free (DFS) and overall survival (OS) in HCC-patients who underwent liver resection in curative intent. Additionally, we investigated the effects of HCC-recurrence in a subgroup of patients. METHODS: Between 2010 and 2016, 111 patients underwent surgical resection for HCC at our institution. A subgroup of 50 patients showed tumor recurrence (n = 50) during follow-up. The associations of DFS and OS with histopathologic characteristics were assessed using univariable and multivariable Cox regression analyses. RESULTS: Median DFS was 31 months and median OS was 27 months. Milan criteria (p = 0.045), macrovascular invasion (p = 0.044) and UICC tumor stage (p = 0.003) were independently associated with DFS while macrovascular invasion (p = 0.001) and MELD score (p = 0.010) were independently associated with OS. Tumor recurrence did not show an association with OS (p = 0.228). However, patients with HCC-recurrence who underwent repeat-surgical or interventional treatment showed improved OS compared to patients treated with palliative or sorafenib treatment alone (OS 18 months vs. 2 months; p < 0.001). CONCLUSION: Tumor recurrence alone is not associated with poor oncological outcome and repeat liver resections as well as local-ablative procedures may help to improve OS in HCC.

Microparticles in patients undergoing transcatheter aortic valve implantation (TAVI).

Degenerative aortic stenosis (AS) is the most frequent form of acquired valvular heart disease. AS is known to entail endothelial dysfunction caused by increased mechanical shear stress leading to elevated circulatory levels of microparticles. Endothelial and platelet microparticles (EMP and PMP) are small vesicles that originate from activated cells and thrombocytes. We sought to evaluate whether transcatheter aortic valve implantation (TAVI) procedure would elicit effects on circulating EMP and PMP. 92 patients undergoing TAVI procedure for severe AS were included in this study. Samples were obtained at each visit before TAVI, 1 week post-procedure and at 1, 3 and after 6 months after TAVI and were evaluated using flow cytometry. A 12 month clinical follow-up was also performed. CD62E+ EMP concentration before TAVI was 21.11 % (±6.6 % SD) and declined to 20.99 % (±6.8 % SD) after 1 week, to 16.63 % (±5.4 % SD, p < 0.0001) after 1 month, to 17.08 % (±4.6 % SD, p < 0.0001) after 3 months and to 15.94 % (±5.4 % SD, p < 0.0001) after 6 months. CD31+/CD42b-, CD31+/Annexin+/- EMP remained unchanged. CD31+/CD41b+ PMP evidenced a slight, but statistically significant increase after TAVI and remained elevated during the entire follow-up. Apart from a procedure-related improvement in echocardiographic parameters, TAVI procedure led also to a decline in CD62E+ EMP. The reduction in pressure gradients with less hemodynamic shear stress seems also to have beneficially affected endothelial homeostasis.

Toward a spectroscopic hemogram: Raman spectroscopic differentiation of the two most abundant leukocytes from peripheral blood.

The first response to infection in the blood is mediated by leukocytes. As a result crucial information can be gained from a hemogram. Conventional methods such as blood smears and automated sorting procedures are not capable of recording detailed biochemical information of the different leukocytes. In this study, Raman spectroscopy has been applied to investigate the differences between the leukocyte subtypes which have been obtained from healthy donors. Raman imaging was able to visualize the same morphological features as standard staining methods without the need of any label. Unsupervised statistical methods such as principal component analysis and hierarchical cluster analysis were able to separate Raman spectra of the two most abundant leukocytes, the neutrophils and lymphocytes (with a special focus on CD4(+) T-lymphocytes). For the same cells a classification model was built to allow an automated Raman-based differentiation of the cell type in the future. The classification model could achieve an accuracy of 94% in the validation step and could predict the identity of unknown cells from a completely different donor with an accuracy of 81% when using single spectra and with an accuracy of 97% when using the majority vote from all individual spectra of the cell. This marks a promising step toward automated Raman spectroscopic blood analysis which holds the potential not only to assign the numbers of the cells but also to yield important biochemical information.

Immunomodulatory effects of cardiotrophin-1 on in vitro cytokine production of monocytes & CD4 + T-lymphocytes.

BACKGROUND & OBJECTIVES: In congestive heart failure (CHF), increased concentrations of several cytokines including cardiotrophin-1 (CT-1) and immunactivation are found. This study was performed to evaluate whether CT-1 can induce in vitro cytokines in monocytes and CD4 + T-lymphocytes of healthy volunteers. METHODS: The study was performed in vitro to see whether CT-1 can modulate monocyte or CD4 + T-lymphocyte interleukin (IL)-1ß, -2, -4, -5, -10, interferon γ (IFNγ), and tumour necrosis factor α (TNFα) expression by flow cytometry following stimulation with CT-1 alone or together with lipopolysaccharide (LPS) or phorbol myristate acetate (PMA)/ionomycine (iono). RESULTS: CT-1 increased the number of TNFα and IL-1ß positive monocytes. LPS induced IL-10, TNFα, and IL-1ß in monocytes but only IL-2 in CD4+ T-lymphocytes, whereas PMA/iono induced all cytokines besides IL-5 in monocytes and IL-1ß in CD4+ T-lymphocytes. In LPS activated monocytes, CT-1 induced a concentration-dependent reduction in the number of TNFα positive monocytes. After LPS activation, CT-1 decreased the number of CD4+ lymphocytes positive for IL-2, IL-4, and IL-5. In addition, following PMA/iono stimulation, CT-1 initiated a concentration-dependent decrease of CD4 + T-lymphocytes positive for TNFα, IL-4, IL-5, and IL-10. INTERPRETATION & CONCLUSIONS: The present data show that in vitro CT-1 can activate monocytes and modulate cytokine production of activated CD4 + T-lymphocytes. We speculate that CT-1 may at least be partly responsible for immunactivation in CHF.

BCG strain S4-Jena: An early BCG strain is capable to reduce the proliferation of bladder cancer cells by induction of apoptosis.

BACKGROUND: Intravesical immunotherapy with Mycobacterium bovis bacillus Calmette-Guérin has been established as the most effective adjuvant treatment for high risk non-muscle-invasive bladder cancer (NMIBC). We investigated the differences between the S4-Jena BCG strain and commercially available BCG strains. We tested the genotypic varieties between S4-Jena and other BCG strains and analysed the effect of the BCG strains TICE and S4-Jena on two bladder cancer cell lines. RESULTS: In contrast to commercially available BCG strains the S4-Jena strain shows genotypic differences. Spoligotyping verifies the S4-Jena strain as a BCG strain. Infection with viable S4-Jena or TICE decreased proliferation in the T24 cell line. Additionally, hallmarks of apoptosis were detectable. In contrast, Cal29 cells showed only a slightly decreased proliferation with TICE. Cal29 cells infected with S4-Jena, though, showed a significantly decreased proliferation in contrast to TICE. Concordantly with these results, infection with TICE had no effect on the morphology and hallmarks of apoptosis of Cal29 cells. However, S4-Jena strain led to clearly visible morphological changes and caspases 3/7 activation and PS flip. CONCLUSIONS: S4-Jena strain has a direct influence on bladder cancer cell lines as shown by inhibition of cell proliferation and induction of apoptosis. The data implicate that the T24 cells are responder for S4-Jena and TICE BCG. However, the Cal29 cells are only responder for S4-Jena and they are non-responder for TICE BCG. S4-Jena strain may represent an effective therapeutic agent for NMIBC.

Cardiotrophin-1 induces tumor necrosis factor alpha synthesis in human peripheral blood mononuclear cells.

Chronic heart failure (CHF) is associated with elevated concentrations of tumor necrosis factor (TNF) alpha and cardiotrophin-1 (CT-1) and altered peripheral blood mononuclear cell (PBMC) function. Therefore, we tested whether CT-1 induces TNFalpha in PBMC of healthy volunteers. CT-1 induced in PBMC TNFalpha protein in the supernatant and TNFalpha mRNA in a concentration- and time-dependent manner determined by ELISA and real-time PCR, respectively. Maximal TNFalpha protein was achieved with 100 ng/mL CT-1 after 3-6 hours and maximal TNFalpha mRNA induction after 1 hour. ELISA data were confirmed using immunofluorescent flow cytometry. Inhibitor studies with actinomycin D and brefeldin A showed that both protein synthesis and intracellular transport are essential for CT-1 induced TNFalpha expression. CT-1 caused a dose dependent nuclear factor (NF) kappaB translocation. Parthenolide inhibited both NFkappaB translocation and TNFalpha protein expression indicating that NFkappaB seems to be necessary. We revealed a new mechanism for elevated serum TNFalpha concentrations and PBMC activation in CHF besides the hypothesis of PBMC activation by bacterial translocation from the gut.

Cis-9,trans-11-CLA exerts anti-inflammatory effects in human bronchial epithelial cells and eosinophils: comparison to trans-10,cis-12-CLA and to linoleic acid.

Interaction of eosinophils and bronchial epithelial cells plays a pivotal role in maintaining inflammatory airway disease. Since conjugated linoleic acids (CLA) are suggested to exert anti-inflammatory effects, one purpose of this study was to compare cis-9,trans-11-CLA and trans-10,cis-12-CLA with regard to their influence on the stimulus-induced activation of eosinophils. ECP (eosinophil cationic protein) released in co-culture of stimulated and CLA-treated eosinophils with stimulated bronchial epithelial cells (BEAS-2B) was measured and cis-9,trans-11-CLA was found to be most potent in inhibiting ECP formation. Further, expression of the activation markers CD69 and CD13 induced by various stimuli (TNF-alpha, IL-5, IL-3) was significantly reduced in the presence of cis-9,trans-11-CLA. Subsequently, various concentrations of cis-9,trans-11-CLA vs. linoleic acid (LA, cis-9,cis-12-octadecadienoic acid) were tested for the effect on proliferative response and release of the pro-inflammatory cytokine IL-8 in stimulated BEAS-2B. Addition of cis-9,trans-11-CLA attenuated cell growth and significantly reduced IL-8 production at mRNA and protein levels. In contrast, LA had a slight stimulating effect on proliferation and was less effective in reducing the cytokine release. It was demonstrated that the inhibitory effect of cis-9,trans-11-CLA on IL-8 production is mediated through activation of the nuclear receptor PPARgamma, since blocking the receptor with a selective antagonist (GW9662) restored the stimulus-induced enhancement in IL-8 mRNA expression and protein secretion. PPARgamma has previously been shown to be closely involved in the downregulation of inflammation during hyperresponsiveness related to pulmonary immune responses. Thus, targeting PPARgamma, cis-9,trans-11-CLA might be of therapeutic value in the focus of airway disease while ameliorating inflammatory processes by affecting epithelial and eosinophil functions.

The impact of atomization on the surface composition of spray-dried milk droplets.

The dominant presence of fat at the surface of spray-dried milk powders has been widely reported in the literature and described as resulting in unfavourable powder properties. The mechanism(s) causing this phenomenon are yet to be clearly identified. A systematic investigation of the component distribution in atomized droplets and spray-dried particles consisting of model milk systems with different fat contents demonstrated that atomization strongly influences the final surface composition. Cryogenic flash-freezing of uniform droplets from a microfluidic jet nozzle directly after atomization helped to distinguish the influence of the atomization stage from the drying stage. It was confirmed that the overrepresentation of fat on the surface is independent of the atomization technique, including a pressure-swirl single-fluid spray nozzle and a pilot-scale rotary disk spray dryer commonly used in industry. It is proposed that during the atomization stage a disintegration mechanism along the oil-water interface of the fat globules causes the surface predominance of fat. X-ray photoelectron spectroscopic measurements detected the outermost fat layer and some adjacent protein present on both atomized droplets and spray-dried particles. Confocal laser scanning microscopy gave a qualitative insight into the protein and fat distribution throughout the cross-sections, and confirmed the presence of a fat film along the particle surface. The film remained on the surface in the subsequent drying stage, while protein accumulated underneath, driven by diffusion. The results demonstrated that atomization induces component segregation and fat-rich surfaces in spray-dried milk powders, and thus these cannot be prevented by adjusting the spray drying conditions.

Expression and synthesis of fibroblast growth factor-9 in human gammadelta T-lymphocytes. Response to isopentenyl pyrophosphate and TGF-beta1/IL-15.

Gammadelta T-lymphocytes are believed to play a role in maintaining the normal configuration of epithelial tissue. As little is known about the factors mediating this function, we addressed the question of whether gammadelta T-lymphocytes produce fibroblast growth factor (FGF)-9 as well as two other growth factors associated with epithelial tissue reconstitution. Blood gammadelta T cells isolated from healthy donors were grown in the presence of isopentenyl pyrophosphate (IPP) or transforming growth factor-beta1 (TGF-beta1)/interleukin-15 (IL-15) for 24 h and were assessed for the expression and synthesis of FGF-9, keratinocyte growth factor (KGF), and epidermal growth factor (EGF). Resting human gammadelta T cells constitutively expressed KGF and FGF-9 mRNA but no EGF mRNA. In the presence of IPP, FGF-9 mRNA expression significantly increased in a dose-dependent manner, expression of KGF remained unaltered, and EGF mRNA could not be detected. In contrast to IPP, stimulation of the cells with TGF-beta1/IL-15 did not alter FGF-9 expression. Moreover, stimulation with anti-CD3 does not induce FGF-9 expression but triggers a high signal of interferon-gamma mRNA. Western blot analysis of gammadelta T cell lysates, prepared 4 days following stimulation with IPP, showed an increase of FGF-9 protein as compared with control cells. In conclusion, the results demonstrate for the first time that human blood and bronchoalveolar lavage gammadelta T-lymphocytes are capable of expressing FGF-9. The data also provide novel evidence that immunoregulatory cells can synthesize FGF-9.

The association between endothelial microparticles and inflammation in patients with systemic sclerosis and Raynaud's phenomenon as detected by functional imaging.

UNLABELLED: Systemic sclerosis (SSc) is a systemic, autoimmune connective tissue disease characterized by vasculopathy and microvascular changes. Fluorescence Optical Imaging (FOI) is a technique used to assess inflammation in patients with arthritis; in this study FOI is used to quantify inflammation in the hand. Endothelial Microparticle (EMP) can reflect damage or activation of the endothelium but also actively modulate processes of inflammation, coagulation and vascular function. The aim of the present study was to quantify EMP and FOI, to determine an association between these microparticles and inflammation and to endothelial function. METHODS: EMP were quantified in plasma samples of 25 patients (24 female, 1 male, age: 41 ± 9 years) with SSc using flow cytometry. EMP was defined as CD31+/CD42- MP, and CD62+ MP. Perivascular inflammation was assessed using fluorescence optical imaging (FOI) of the hand. Macrovascular endothelial function was non-invasively estimated using the Endopat system. RESULTS: Plasma levels of CD31+/CD42- EMP and CD62+ EMP were lower in patients with SSc compared to controls (both p <  0.05). An impaired endothelial function with an increased hyperemia index was observed. A strong association could be demonstrated between CD62+ EMP and perivascular soft tissue inflammation as assessed by the FOI global score (Spearman, p = 0.002, r = 0.61). CONCLUSIONS: EMP indicate molecular vascular damage in SSc; in this study a strong association between EMP and perivascular inflammation as quantified by FOI is demonstrated. Consequently EMP, using FOI, may be a potential marker benefitting the diagnosis and therapy monitoring of patients with SSc with associated Raynaud's phenomenon.

Phenotypic and molecular characterization of CD103+ CD4+ T cells in bronchoalveolar lavage from patients with interstitial lung diseases.

BACKGROUND: The integrin CD103 is preferentially expressed on intraepithelial T lymphocytes, and cells expressing this integrin may play a regulatory role in the microenvironment of the epithelial cell layer. METHODS: The relative number of CD103(+)/CD4(+) T cells in the bronchoalveolar lavage was significantly elevated in all patients diagnosed with interstitial lung diseases compared with patients with other non-fibrotic disorders of the lung. RESULTS: Analysis by flow cytometry showed that the CD103(+) and the CD103(-) subpopulations were memory T cells based on the high expression of CD45RO(+). However, the CD103(+)/CD4(+) T cells were CD25(low), CD27(-), CD28(low), and CD62L(-), whereas the CD103(-)/CD4(+) T cells expressed CD25 and CD62L and were CD27(high) and CD28(high). In addition, the CD103(+)/CD4(+) T cells expressed significantly higher quantities of VLA-1 and CD101 than did CD103(-)/CD4(+) T cells. Reverse transcriptase polymerase chain reaction analysis of purified CD103(+) and CD103(-) CD4(+) T cells showed production of tumor necrosis factor (TNF) alpha-R-1 (p55), TNF-alpha-R-2 (p75), interferon gamma, interleukin-10, and TNF-alpha mRNA in both subpopulations. No interleukin-4 mRNA was detected in either subpopulation. CONCLUSIONS: CD103(+)/CD4(+) T cells represent a T-helper 1-like subpopulation in human lungs with a distinct effector phenotype. Despite the lack of CD27 and the low CD25 and CD28 expression, these cells show a high degree of activation. These results suggest that CD103 expressing CD4 T cells in the lung are continuously activated, long-living cells.

Cardiotrophin-1 induces intercellular adhesion molecule-1 expression by nuclear factor kappaB activation in human umbilical vein endothelial cells.

BACKGROUND: In addition to elevated concentrations of cytokines, patients with congestive heart failure (CHF) show endothelial dysfunction and increased plasma concentrations of adhesion molecules like intercellular adhesion molecule-1 (ICAM-1). Furthermore, the concentration of cardiotrophin-1 (CT-1)--a cytokine of the interleukin-6 superfamily--is increased in CHF. We tested the hypothesis whether CT-1 is able to induce ICAM-1 in human umbilical vein endothelial cells (HUVEC). Furthermore we examined the signalling mechanisms of CT-1 mediated ICAM-1 expression. METHODS: Confluent layers of HUVEC were incubated with increasing concentrations of CT-1 (5 to 100 ng/ml) for different periods. ICAM-1 mRNA was determined by real-time polymerase chain reaction (PCR) and ICAM-1 surface expression by fluorescence-activated cell sorter (FACS) analysis and soluble ICAM-1 (sICAM-1) in the culture supernatant by enzyme linked immunosorbent assay (ELISA). To clarify the signalling pathway of CT-1 induced ICAM-1 expression we used various inhibitors of possible signal transducing molecules, electromobility shift assay (EMSA) and Western blot analysis. RESULTS: CT-1 induced ICAM-1 mRNA (1.8 +/- 0.8 fold increase compared to unstimulated cells after 6 hours) and protein (1.4 +/- 0.2 fold increase compared to unstimulated cells after 48 hours) in HUVEC in a time- and concentration-dependent manner. EMSA experiments show that CT-1 causes nuclear factor (NF) kappaB activation. Because parthenolide could inhibit CT-1 induced ICAM-1 expression NFkappaB activation is required in this pathway. CT-1 did not activate extracellular signal regulated kinases (ERK), c-Jun N-terminal kinase (JNK) and p38. CONCLUSION: CT-1 is able to induce ICAM-1 in endothelial cells by NFkappaB activation. These results may explain in part elevated ICAM-1 concentrations in patients with CHF and endothelial dysfunction.

Phosphodiesterase-4 inhibitors as a novel approach for the treatment of respiratory disease: cilomilast.

Phosphodiesterase-4 (PDE4) is an important cAMP-metabolising enzyme in immune and inflammatory cells, airway smooth muscle and pulmonary nerves. The phosphodiesterase 4 (PDE4) enzyme plays a significant role in modulating the activity of cAMP, an important second messenger that mediates the relaxation of airway smooth muscle and suppresses inflammatory cell function, thereby attenuating the inflammatory response. Selective inhibitors of this enzyme show a broad spectrum of activity in animal models of COPD and asthma. These drugs block the hydrolysis of cAMP via inhibition of PDE4 and are attractive candidates for novel anti-inflammatory drugs. At present, two second-generation PDE4 inhibitors for the treatment of COPD and asthma patients are being tested in clinical Phase III trials. The most advanced compound is the orally active, selective PDE4 inhibitor cilomilast (Ariflo, SB-207499, cis-4-cyano-4-[3-cyclopentyloxy-4-methoxyphenyl]-cyclohexanecarboxylic acid; GlaxoSmithKline). Cilomilast shows high selectivity for cAMP-specific PDE4, an isoenzyme that predominates in pro-inflammatory and immune cells and that is 10-fold more selective for PDE4D than for PDE4A, -B or -C. In vitro, cilomilast suppresses the activity of several pro-inflammatory and immune cells that have been implicated in the pathogenesis of asthma and COPD. Moreover, it is highly active in animal models of these diseases. Cilomilast has been shown to exert potent anti-inflammatory effects both in vitro and in vivo. It is orally active and may be effective in the treatment of asthma and COPD; however, complete assessment of the therapeutic value of this novel compound class must await the outcome of longer-term clinical trials. This review presents a summary of the preclinical and clinical profile of cilomilast in patients with COPD.

Cardiotrophin-1 induces interleukin-6 synthesis in human monocytes.

BACKGROUND: Patients with congestive heart failure (CHF) show increased serum concentrations of cytokines like interleukin-6 (IL-6) and cardiotrophin-1 (CT-1). Additionally, monocyte function is modulated in CHF. The aim of this study was to examine if CT-1 is able to induce IL-6 in human monocytes and to investigate the underlying pathway. METHODS: Separated peripheral blood monocytes of healthy volunteers were cultured with increasing concentrations of CT-1 for different periods. IL-6 mRNA was determined by RT-PCR or real-time PCR and IL-6 protein concentration in the supernatant by ELISA. Phosphorylation of signal transducer and activation of transcription (STAT) 3 was analyzed by western blot or by FACS analysis. To clarify the signalling pathway of CT-1 induced IL-6 expression various inhibitors of possible signal transducing molecules were used. RESULTS: CT-1 induced IL-6 mRNA in monocytes in a time- and concentration-dependent manner. Maximal mRNA induction was detectable after 6h with 100 ng/ml CT-1. IL-6 protein also increased in a time- and concentration-dependent manner with a maximum after 48 h with 100 ng/ml CT-1. AG490 as well as SB 203580 and parthenolide blocked CT-1 induced IL-6 expression completely. AG 490 was able to inhibit STAT3 phosphorylation in western blot analysis completely. This indicates that JAK2/STAT3, p38 and nuclear factor kappaB (NFkappaB) are involved in this pathway. To exclude a possible influence of plastic adherence of monocytes on CT-1 induced IL-6 expression, we determined intracellular STAT3 phosphorylation in whole blood samples by FACS analysis and observed independently of culture conditions a CT-1 concentration-dependent STAT3 phosphorylation. CONCLUSION: CT-1 induces IL-6 mRNA and protein expression in a time- and concentration-dependent manner. The underlying pathway is Janus kinase (JAK)2/STAT3, p38 and NFkappaB dependent. These data may explain increased IL-6 serum concentrations and altered monocyte function found in patients with CHF. Modulation of the CT-1 pathway might be a interesting strategy in the treatment of CHF.

A heuristic two-dimensional presentation of microsatellite-based data applied to dogs and wolves.

Methods based on genetic distance matrices usually lose information during the process of tree-building by converting a multi-dimensional matrix into a phylogenetic tree. We applied a heuristic method of two-dimensional presentation to achieve a better resolution of the relationship between breeds and individuals investigated. Four hundred and nine individuals from nine German dog breed populations and one free-living wolf population were analysed with a marker set of 23 microsatellites. The result of the two-dimensional presentation was partly comparable with and complemented a model-based analysis that uses genotype patterns. The assignment test and the neighbour-joining tree based on allele sharing estimate allocated 99% and 97% of the individuals according to their breed, respectively. The application of the two-dimensional presentation to distances on the basis of the proportion of shared alleles resulted in comparable and further complementary insight into inferred population structure by multilocus genotype data. We expect that the inference of population structure in domesticated species with complex breeding histories can be strongly supported by the two-dimensional presentation based on the described heuristic method.

Expression of metalloproteinase-7 (matrilysin) in human blood and bronchoalveolar gamma/delta T-lymphocytes. Selective upregulation by the soluble non-peptidic mycobacterial phosphoantigen (isopentenyl pyrophosphate).

Human gammadelta T-lymphocytes are believed to regulate local immune defense and enhance resistance against invading microbes, although their precise function remains unknown. Herein, we addressed the question whether gammadelta T-lymphocytes mediate these processes via synthesis of MMP-7, a protease closely associated with both epithelial repair and mucosal defense. Blood and bronchoalveolar gammadelta T-lymphocytes were cultured in the absence and presence of isopentenyl pyrophosphate (IPP) or TGF-beta1/IL-15 for 24 h, and assessed for the expression and synthesis of MMP-1, MMP-7, and MMP-9. Resting human gammadelta T-lymphocytes constitutively expressed MMP-9 mRNA, a marginal or no MMP-7 and MMP-1 mRNA. In the presence of IPP (3 microg/ml), expression of MMP-7 mRNA significantly increased, whereas TGF-beta1/IL-15 had no effect. Further, quiescent gammadelta T-lymphocytes obtained from bronchoalveolar lavage (BAL) fluid showed a weak or no MMP-7 mRNA signal which was raised significantly following stimulation with IPP. In Western blot analysis, a 28-kDa pro-matrilysin could be detected both in cell lysates (2 days) and supernatants (5 days) with a four- to sevenfold increased signal following IPP-stimulation of the gammadelta T-lymphocytes. In conclusion, the data demonstrate for the first time that both human blood and BAL gammadelta T-lymphocytes express MMP-7 mRNA and synthesize MMP-7-protein. This unfolds a new perspective for the understanding of gammadelta T-lymphocyte function.

Interferon-alphacon-1 treatment of three patients with severe glucocorticoid-dependent asthma. Effect on disease control and systemic glucocorticosteroid dose.

We report herein the therapeutic effect of interferon (IFN)-alphacon in three patients with severe persistent asthma and long-term oral glucocorticoid treatment. IFN-alphacon (9 microg) administered subcutaneously thrice a week over a period of more than 24 months led to a substantial clinical improvement with regard to the number of daily asthma attacks, nighttime disturbance, emergency visits and hospitalizations. In addition, lung function and exercise capacity improved. At the same time, treatment with IFN allowed discontinuation of the daily glucocorticoid dose in all patients for the first time in more than 8 years. Our findings suggest that IFN-alphacon leads to a significant clinical improvement while at the same time allowing reduction and discontinuation of the glucocorticoid treatment in severe persistent glucocorticoid-dependent asthma.

Cardiotrophin-1 induces interleukin-6 synthesis in human umbilical vein endothelial cells.

In patients with chronic heart failure (CHF) increased plasma concentrations of proinflammatory cytokines are found. For example, the plasma interleukin-6 (IL-6) concentration correlates with disease severity. Beside IL-6 cardiotrophin-1 (CT-1), a member of the IL-6 superfamily, is also increased in CHF. We examined whether CT-1 is able to induce IL-6 in human umbilical vein endothelial cells (HUVEC) and characterised the underlying pathway. IL-6 mRNA was determined by real-time PCR and by RT-PCR in HUVEC which were stimulated with different CT-1 concentrations and for different time periods. IL-6 concentration in the supernatant was determined by ELISA. For the pathway determination following inhibitors were used: piceatannol (signal transducer and activation of transcription (STAT)3 phosphorylation), wortmannin (phosphatiylinositol 3-kinase (PI3K)), SB203580 (p38 mitogen-activated protein kinase (MAPK)), AG490 (Janus kinase (JAK)2), PD98059 (mitogen-activated protein kinase kinase (MEK) 1/2), parthenolide (nuclear factor kappaB) and cycloheximide (protein biosynthesis). CT-1 caused a concentration- and time-dependent increase in IL-6 mRNA in HUVEC with a maximal induction seen after 6 h (2-fold compared to control) with 100 ng/ml CT-1. In the supernatant of HUVEC a concentration- and time-dependent increase of IL-6 protein was found. A maximum effect with 100 ng/ml CT-1 was found after 24 h (11-fold compared to control). AG490, SB203580, piceatannol, parthenolide and cycloheximide inhibit CT-1 induced IL-6 mRNA and protein expression whereas wortmannin and PD98059 did not inhibit IL-6 expression. CT-1 induced both IL-6 mRNA and protein in a concentration- and time-dependent manner in HUVEC. The underlying pathway includes activation of JAK2, STAT3, p38 and NFkappaB. CT-1 induced IL-6 expression and requires protein synthesis and IL-6 is not stored intracellularly. We speculate that in CHF CT-1 might be in part responsible for increased IL-6 plasma concentrations. Modulation of the CT-1 pathway may be a further strategy in CHF treatment.

Immunopathogenesis of dilated cardiomyopathy. Evidence for the role of TH2-type CD4+T lymphocytes and association with myocardial HLA-DR expression.

BACKGROUND: An immunological pathogenesis underlying dilated cardiomyopathy and myocarditis has been suggested on the basis of the subtype of lymphocyte infiltrates and the degree of HLA expression in cardiac tissue. In the present study, we investigated the relation between the peripheral CD4+T-cell subset and the degree of HLA expression in the heart. METHODS: Fifty-four patients with heart insufficiency included in the study were biopsied after coronary heart disease had been excluded. Immunohistological staining of the left ventricular tissue were performed employing anti-CD3, -CD4, -CD8, -CD14, and HLA-DR monoclonal antibodies. Intracellular expression of IL-2, IL-4, IL-5, IFN-gamma, and TNF-alpha in peripheral CD4+T lymphocytes was determined using flow cytometry. The severity of heart insufficiency was determined by measurement of brain natriuretic peptide (BNP) and the NYHA class. On the basis of HLA expression in the heart, the patients were divided into three groups: Group I (mild-to-none), Group II (moderate), and Group III (strong-to-very strong). RESULTS: Of the 54 patients included in this study, 33 (61%) patients were diagnosed as having idiopathic dilated cardiomyopathy and 10 (18.5%) borderline or healing myocarditis according to the Dallas criteria. Both patient groups were found in all three HLA-DR groups. There was no difference in BNP level or NYHA class between the three groups. However, a significant difference in the proportion of CD4+T lymphocytes producing IL-2 (39.2 versus 21.8%), IFN-gamma (19.5 versus 7.8%), and TNF-alpha (35.8 versus 16.1%) between Groups I and III could be detected, whereas the distribution of IL-4 and IL-5 producing CD4+T lymphocytes was similar. The myocardium of Group III patients exhibited a significant higher number of CD3+T cells (11.4 versus 4.3 per mm2) and CD4+T cells (4.7 versus 0.8 per mm2) compared to Group I patients, while no difference existed with respect to CD8+T cells. CONCLUSION: High myocardial expression of the HLA-DR antigen is associated with an increase of peripheral-blood CD4+T lymphocytes expressing cytokines of the TH2 subset. The degree of HLA-DR expression is not associated with the degree of heart insufficiency or underlying diagnosis, but correlates with an increase of activated T cells in the myocardium. The data suggest that CD4+T lymphocytes infiltrating cardiac tissue may play a pathogenic role in dilated cardiomyopathy.

Human gamma delta-T lymphocytes express and synthesize connective tissue growth factor: effect of IL-15 and TGF-beta 1 and comparison with alpha beta-T lymphocytes.

T lymphocytes bearing the gammadelta-TCR accumulate during wound healing and inflammation. However, the role of gammadelta-T lymphocytes in fibrogenic tissue reactions is not well understood. Therefore, we addressed the question of whether human gammadelta-T cells express and synthesize connective tissue growth factor (CTGF), a factor known to regulate fibrogenesis and wound healing. In addition, the lymphoblastic leukemia T cell line (Loucy) that possesses characteristics typical of gammadelta-T cells was used as a model to evaluate the regulation of CTGF gene expression. Blood gammadelta-T cells isolated from healthy donors were grown in the presence of IL-15/TGF-beta1 for 48 h and assessed for the expression and synthesis of CTGF. Nonstimulated human blood gammadelta-T cells and Loucy gammadelta-T cells expressed low levels of CTGF mRNA. Costimulation of the cells with IL-15 and TGF-beta1 resulted in a substantially increased level of CTGF mRNA expression within 4-8 h, and it remained elevated for at least 48 h. In contrast, no CTGF mRNA was detected when nonstimulated and stimulated human CD4+ alphabeta-T cells were analyzed. In addition, Western blot analysis of human gammadelta-T cell lysates prepared 4 days following stimulation with IL-15 and TGF-beta1 revealed a 38-kDa CTGF protein in cell lysates of human gammadelta-T cells. Detection was confirmed using Colo 849 fibroblasts, which can constitutively express high levels of CTGF. In conclusion, we herein present novel evidence that in contrast to CD4+ alphabeta-T cells human gammadelta-T cells are capable of expressing CTGF mRNA and synthesizing its corresponding protein, which supports the concept that gammadelta-T cells may contribute to wound healing or tissue fibrotic processes.