Enhanced ovalbumin-induced airway inflammation in CD26-/- mice.

In this study, we investigated the potential role of CD26 in ovalbumin (OVA)-induced airway inflammation using CD26 gene knockout mice. Compared with WT counterparts, CD26(-/-) mice showed an obviously enhanced tissue response and denser pulmonary infiltrates containing eosinophils around vessels and in the parenchyma after OVA sensitization and challenge. Serum IgG, including subclasses IgG1 and IgG2a, was greatly reduced in CD26(-/-) mice, but serum IgE remained unchanged. CD26(-/-) mice had increased mRNA expression of the Th2 cytokines IL-4, IL-5, and IL-13 in the lungs compared with WT mice, whereas the levels of the pro-Th1 cytokine IL-12p40 were similar in both strains. Consequently, enhanced protein secretion of IL-4, IL-5, and IL-13 was detected in bronchoalveolar lavage (BAL) fluid from CD26(-/-) mice. In agreement with overexpressed Th2 cytokines, both mRNA transcript and protein levels of chemokines eotaxin and RANTES, as well as their receptors CC chemokine receptor 3 (CCR3) and CCR5, were elevated in CD26(-/-) mice. These results suggest a protective role for CD26 in restricting OVA-induced airway inflammation.

Impact of microvessel density on lymph node metastasis and survival after curative resection of pancreatic cancer.

PURPOSE: The roles of angiogenesis and the most prominent angiogenic vascular endothelial growth factor (VEGF) in diseases of the pancreas remain controversial. We compared microvessel density (MVD) and VEGF status in normal pancreatic, chronic pancreatic, and pancreatic cancer (PC) tissues to establish their prognostic relevance. METHODS: Eighty samples of PC tissue, 32 samples of normal pancreatic tissue, and 20 samples of chronic pancreatitis (cP) tissue were immunostained with monoclonal anti-CD31 and polyclonal anti-VEGF antibody. The MVD was correlated with clinicopathological features and survival. RESULTS: Microvessel density was higher in PC than in cP (P < 0.001). Residual tumor status was highly predictive for survival (P < 0.001). After stratification for residual tumor status, we identified lymph node metastasis (LNM) in more than two lymph nodes (P < 0.04) and high MVD (P < 0.03) as risk factors for mortality. Multivariate analysis revealed only a high MVD (P = 0.03, odds ratio 0.441, 95% confidence interval 0.211-0.821) as an independent predictor of poor survival. Vascular endothelial growth factor was found over stromal cells in cP and over ductal adenocarcinoma cells in PC. Vascular endothelial growth factor expression status was not predictive of survival (P < 0.07). CONCLUSION: This study confirms the role of angiogenesis in PC and identifies MVD as an independent prognostic factor in patients with curatively resected PC.

Las cadherinas en el diagnostico histopatológico y pronostico del carcinoma de células renales / The cadherins in the histopathology diagnosis and prognosis of renal cell carcinoma

El carcinoma de células renales es el cáncer renal más común en el adulto. Se describen 5 tipos histológicos principales, que, en general, muestran al microscopio óptico características que permiten diagnosticarlos. Sin embargo, el solapamiento morfológico observado entre los diferentes tipos y la heterogeneidad histológica dentro del mismo tumor, continúan generando problemas en su clasificación. Algunos marcadores inmunohistoquímicos son empleados en el diagnóstico diferencial de estos carcinomas, pero tienen sus limitaciones. En el presente trabajo, se hizo una revisión de varios estudios, en los que se identificaron alteraciones en la expresión de algunas cadherinas en tejidos renales y líneas celulares con carcinoma de células renales. Estas alteraciones estuvieron asociadas con diferentes estados del desarrollo y progresión del tumor, y sirvieron para predecir el comportamiento neoplásico. Incrementar nuestro conocimiento en este campo, contribuiría al hallazgo de marcadores mucho más específicos para el diagnóstico y pronóstico de estos tumores.

Renal cell carcinoma is the most common adult renal cancer. Five main histological types are described, that in general display characteristic light microscopic features that lead to a correct diagnosis. However, the morphologic overlap between tumors and the histologic heterogeneity within a single tumor continue to create problems in the classification. Some immunohistochemical markers are used in the differential diagnosis of these carcinomas, but they have limitations. In the present work, a review of several studies was made, in which alterations in expression of some cadherins in renal tissue and cell lines with renal cell carcinoma were identified. These alterations were associated with different stages of tumor development and progression, and used to predict the neoplastic behaviour. To increase our knowledge in this field, will contribute to find more specific markers for the diagnosis and prognosis of these tumors.

Tumor-associated angiogenesis and lymphangiogenesis correlate with progression of intrahepatic cholangiocarcinoma.

OBJECTIVES: Little is known about the function of tumor-associated neovascularization in the progression of intrahepatic cholangiocarcinoma (IHC). This study was conducted to evaluate the influence of tumor-associated angiogenesis and lymphangiogenesis on progression of IHC. METHODS: We analyzed tissue specimens of IHC (N=114) by immunohistochemistry using the endothelial-specific antibody CD31 and the lymphendothelial-specific antibody D2-40 and subsequently quantified microvessel density (MVD) and lymphatic microvessel density (LVD). To analyze the influence of tumor-associated angiogenesis and lymphangiogenesis on tumor progression, tumors were allocated according to mean MVD and LVD, respectively, into groups of "high" and "low" MVD and LVD, respectively, and various clinicopathological characteristics as well as recurrence and survival data were analyzed. RESULTS: IHC revealed an induction of tumor-associated angiogenesis and lymphangiogenesis. Tumors of "high" MVD displayed more frequently advanced primary tumor stages and multiple tumor nodes. Furthermore, patients with tumors of "high" MVD had an inferior curative resection rate and suffered more frequently from recurrence. A "high" LVD was correlated with increased nodal spread, and patients with "high" LVD tumors more frequently developed recurrence. In the univariate analysis, MVD and LVD revealed significant influence on survival, and MVD was identified as an independent prognostic factor for survival in the multivariate analysis. The 5-year survival of patients with "low" MVD tumors was 42.1%, compared with 2.2% in patients with "high" MVD tumors (P<0.001). CONCLUSIONS: This study suggests a critical function of tumor-associated angiogenesis and lymphangiogenesis for progression of IHC. Therefore, antiangiogenic and antilymphangiogenic approaches may have therapeutic potency in this tumor entity.

High thrombopoietin concentrations in the cerebrospinal fluid of neonates with sepsis and intraventricular hemorrhage may contribute to brain damage.

Thrombopoietin (TPO) and its receptor (TPOR) are expressed in the central nervous system (CNS). Although TPO shares significant homology with various neurotrophins, recent data indicate a proapoptotic function of TPO in the CNS. In this study, TPO concentrations were analyzed in the cerebrospinal fluid (CSF) of neonates. Human neuroblastoma-derived SH-SY5Y cells were established to elucidate the effects of inflammation and hypoxia on neuronal Tpo expression. TPO was detectable in the CSF of 6 of 15 neonates with bacterial infection/sepsis (median 140, range 2-613 pg/mL), 5 of 9 neonates with posthemorrhagic hydrocephalus (median 31, range 1.4-469 pg/mL), 3 of 4 neonates with posthemorrhagic hydrocephalus plus bacterial infection/sepsis or meningitis (median 97, range 6-397 pg/mL), but not in controls ( n = 3). Neither the presence of detectable TPO nor its level in the CSF significantly correlated with any clinical or laboratory parameter. In SH-SY5Y cells, TPO and TPOR expression was detected by RT-PCR and Western blot analysis. In vitro, interleukin-6 (IL-6) did not significantly change Tpo gene expression. In contrast, Tpo mRNA expression significantly decreased under hypoxia, whereas erythropoietin (EPO) mRNA expression increased. In conclusion, our data provide evidence that in neuronal cells, TPO production is regulated by different mechanisms than in hepatocytes.

Chemokine IL-8 and chemokine receptor CXCR3 and CXCR4 gene expression in childhood acute lymphoblastic leukemia at first relapse.

In this study, we examined the gene expression of interleukin (IL)-8, CXCR3, and CXCR4 in leukemic cells from 100 children with relapsed B-cell progenitors (BCP) acute lymphoblastic leukemia (ALL), using quantitative real-time polymerase chain reaction (RT-PCR). IL-8, CXCR3, and CXCR4 were expressed in almost all bone marrow (BM) samples. The CXCR4 expression significantly correlated with known prognostic factors at relapse: time point and site of relapse. Patients who had a combined BM relapse (n=21) had lower IL-8 and CXCR4 expression than those who had an isolated BM relapse (n=79). The CXCR3 expression was higher in female patients (n=39) than in male patients (n=61). However, this did not reach prognostic relevance in relapsed ALL.

Cardiac troponin C-L29Q, related to hypertrophic cardiomyopathy, hinders the transduction of the protein kinase A dependent phosphorylation signal from cardiac troponin I to C.

We investigated structural and functional aspects of the first mutation in TNNC1, coding for the calcium-binding subunit (cTnC) of cardiac troponin, which was detected in a patient with hypertrophic cardiomyopathy [ Hoffmann B, Schmidt-Traub H, Perrot A, Osterziel KJ & Gessner R (2001) Hum Mut17, 524]. This mutation leads to a leucine-glutamine exchange at position 29 in the nonfunctional calcium-binding site of cTnC. Interestingly, the mutation is located in a putative interaction site for the nonphosphorylated N-terminal arm of cardiac troponin I (cTnI) [ Finley NL, Abbott MB, Abusamhadneh E, Gaponenko V, Dong W, Seabrook G, Howarth JW, Rana M, Solaro RJ, Cheung HC et al. (1999) EJB Lett453, 107-112]. According to peptide array experiments, the nonphosphorylated cTnI arm interacts with cTnC around L29. This interaction is almost abolished by L29Q, as observed upon protein kinase A-dependent phosphorylation of cTnI at serine 22 and serine 23 in wild-type troponin. With CD spectroscopy, minor changes are observed in the backbone of Ca2+-free and Ca2+-saturated cTnC upon the L29Q replacement. A small, but significant, reduction in calcium sensitivity was detected upon measuring the Ca2+-dependent actomyosin subfragment 1 (actoS1)-ATPase activity and the sliding velocity of thin filaments. The maximum actoS1-ATPase activity, but not the maximum sliding velocity, was significantly enhanced. In addition, we performed our investigations at different levels of protein kinase A-dependent phosphorylation of cTnI. The in vitro assays mainly showed that the Ca2+ sensitivity of the actoS1-ATPase activity, and the mean sliding velocity of thin filaments, were no longer affected by protein kinase A-dependent phosphorylation of cTnI owing to the L29Q exchange in cTnC. The findings imply a hindered transduction of the phosphorylation signal from cTnI to cTnC.

Activation of AP-1 through reactive oxygen species by angiotensin II in rat cardiomyocytes.

Cardiovascular pathogenesis induced by angiotensin II (Ang-II) is a complex process often connected to oxidative stress. In the present study we show that, 4 h after addition, Ang-II induces a four- to fivefold increase in AP-1 activity in cultured neonatal rat cardiomyocytes and that the intracellular level of reactive oxygen species (ROS) correlates with the extent of AP-1 binding activity. Ang-II stimulated ROS generation in rat cardiomyocytes in a dose- and time-dependent manner. These effects of Ang-II were suppressed by the Ang-II receptor type I (AT1) inhibitor CV-11974 as well as by the antioxidants diphenylene iodonium (DPI) and N-acetyl-L-cysteine (NAC), but not by AT2 antagonist PD 122319. Furthermore, Ang-II induced a two- to threefold increase in protein synthesis and cell size during 12-24 h, which could be inhibited by CV-11974 as well as by DPI and NAC. Because the rat cardiomyocytes strongly expressed gp91(phox), this suggests that ROS generated in a gp91-containing NADPH oxidase are involved in signal transduction leading to AP-1 activation. Together, these findings indicate that Ang-II elicits the activation of the redox-sensitive AP-1 via ROS through AT1, resulting in effects on cardiomyocyte function such as hypertrophy.

Expression of interleukin-10 splicing variants is a positive prognostic feature in relapsed childhood acute lymphoblastic leukemia.

PURPOSE: Biologic features of hematologic malignancies have prognostic implications and are essential elements in the design of current therapeutic trials. This study aimed to determine the expression of a splicing-derived variant of interleukin (IL) -10 in leukemic cells and its clinical relevance in children with acute lymphoblastic leukemia (ALL) at first relapse. PATIENTS AND METHODS: Between January 1997 and December 2001, bone marrow (BM) samples were collected from 98 children with first relapse of ALL at diagnosis. These patients were enrolled in the relapse trial ALL-REZ BFM (ALL-Relapse Berlin-Frankfurt-Munster) 95 and 96. The detection of IL-10 isoforms in leukemic cells of BM samples were performed by conventional reverse transcriptase polymerase chain reaction and by immunoblotting. RESULTS: IL-10 was detected in 93.9% BM samples. In addition to expressing full-length IL-10, a new splicing-derived IL-10 variant (termed IL-10delta3) that lacked the entire exon 3 was identified in leukemic cells. The IL-10delta3 variant was found in 80.4% of BM samples. Most importantly, expression of IL-10delta3 was associated with a significantly better response to chemotherapy (P = .001) and probability of event-free survival (P = .01) at 5 years. CONCLUSION: These results indicate that splicing-derived IL-10 isoforms may modulate IL-10-mediated biologic effects and therapeutic efficacy in lymphatic disease, and expression of IL-10delta3 is a positive prognostic feature in relapsed childhood ALL.

Cytokine/cytokine receptor gene expression in childhood acute lymphoblastic leukemia: correlation of expression and clinical outcome at first disease recurrence.

BACKGROUND: Recent studies have shown that cytokines/cytokine receptors (C/CR) affect leukemic cell growth and survival. The goal of the current study was to investigate possible correlations between gene expression patterns of C/CR in leukemic cells, clinical features, and outcome in children with acute lymphoblastic leukemia (ALL) at first disease recurrence. METHODS: Between January 1997 and December 2000, bone marrow (BM) samples were collected from 68 children with first ALL recurrence at diagnosis. These patients were enrolled in the ALL-REZ 95-96 disease recurrence trials of the Berlin-Frankurt-Munster study group. C/CR gene expression (interleukin [IL]-7, IL-10, IL-12p40, IL-15, IL-18, IL-7Ralpha, IL-10R1, IL-15Ralpha, interferon-gamma [IFN-gamma], vascular epithelial growth factor [VEGF], Flt1, and transforming growth factor-beta) was quantified by real-time reverse transcriptase-polymerase chain reaction and correlated with protein expression by immunofluorescence. RESULTS: In comparison with T-lineage ALL specimens, expression of IL-10, IFN-gamma, IL-15Ralpha, and Flt1 was significantly higher in B-cell precursor (BCP) ALL specimens (P <0.01). Among BCP ALL samples, gene expression of IL-7Ralpha and Flt1 was higher in pre-B than in common or pro-B leukemic cells. Moreover, expression levels of VEGF, IL-7Ralpha, IL-10R1, and IL-15Ralpha were lower in lymphoblasts of patients with a combined BM recurrence than in those with an isolated recurrence (P <0.05). Children with IL-15Ralpha expression above the median level had a significantly better probability of event-free survival (0.65 vs. 0.34, P=0.04) and survival (0.71 vs. 0.37, P=0.02) at 5 years. CONCLUSIONS: Expression of distinct C/CR in ALL cells was associated with lineage commitment and differentiation of leukemic cells, as well as with prognosis. It remains to be evaluated whether these prognostic and biologic findings of distinct C/CR expression in leukemic cells also have therapeutical implications for future antileukemic strategies.

Interaction of bone marrow stromal cells with lymphoblasts and effects of predinsolone on cytokine expression.

Cytokines play a key role in the differentiation, growth and survival of hematopoietic cells in the bone marrow (BM) stroma microenvironment. The mechanisms by which stromal derangements may contribute to the evolution of hematopoietic neoplasias are largely unknown. Here, we characterized BM stromal cells isolated from children with acute lymphoblastic leukemia and determined the effect of the interaction between stromal cells and lymphoblasts on cytokine expression as well as the effect of prednisolone using mono- and co-culture models. The analyses demonstrate that (1) stromal cells and lymphoblasts display different patterns of cytokine gene expression individually. (2) Stromal cells influence gene expression of cytokines in lymphoblasts and vice versa. (3) Glucocorticoid substitution inhibit cytokine gene expression in stromal cells. These findings indicate that stromal cells are important components involved in malignant hematopoiesis and also in response to therapy.

The intron 6 G/T polymorphism of c-myb oncogene and the risk for coronary in-stent restenosis.

BACKGROUND: The principle mechanisms leading to the development of atherosclerosis are long-term accumulation of lipids and cell proliferation. We have recently shown that a single nucleotide polymorphism in the c-myb gene is associated with the development of coronary artery disease in humans and intracellular lipid accumulation. C-myb expression has been further shown to be up-regulated during cell proliferation. The development of in-stent restenosis is predominantly driven by smooth muscle cell proliferation. METHODS: To study a possible association of c-myb with neointima formation in humans we genotyped 485 consecutive patients undergoing coronary stenting for a G/T-single nucleotide polymorphism in intron 6 of the cmyb gene. Restenosis was assessed by quantitative coronary angiography and angiographic follow-up after 6 months. To study the effect of c-myb on smooth muscle cell proliferation primary human smooth muscle cells were infected with recombinant adenovirus expressing c-myb, a dominant negative myb-engrailed fusion protein or control virus. RESULTS AND CONCLUSION: Restenosis > 50% occurred in 27.6% of patients with at least one G-allele and in 20.8% of those without (p = 0.10). Even after adjustment for the independent risk factors diabetes mellitus, reference lumen diameter, smoking, dyslipidemia and number of diseased vessels, the observed difference in the distribution of the c-myb alleles did not reach statistical significance (p = 0.08). Adenoviral gene transfer of c-myb did not increase proliferation of cultured smooth muscle compared to control virus or untransfected cells, while the expression of the dominant negative mutant reduced proliferation of VSMC as previously shown. Our results indicate that expression of c-myb, while being important for cell cycle is not sufficient to induce smooth muscle cell proliferation. The G/T-nucleotide transversion polymorphism in intron 6 of the c-myb oncogene that has been associated with atherosclerosis and lipid accumulation is not a risk factor for human in-stent restenosis.

Levels of the soluble, 55-kilodalton isoform of tumor necrosis factor receptor in bone marrow are correlated with the clinical outcome of children with acute lymphoblastic leukemia in first recurrence.

BACKGROUND: It has been shown that the soluble, 55-kilodalton isoform of tumor necrosis factor receptor (sTNFRp55) enhances tumor survival by exhibiting competitive ligand binding. The objective of the current study was to determine the levels of sTNFRp55 and their impact on outcome in 106 children with acute lymphoblastic leukemia (ALL) in first recurrence. METHODS: Between January 1997 and December 2001, bone marrow (BM) samples were collected from 106 children with a first recurrence of ALL at diagnosis. These patients were enrolled in the Berlin-Frankfurt-Münster (BFM) ALL recurrence trial, ALL-REZ BFM 90-96. Levels of sTNFRp55 in BM samples were determined with a commercially available enzyme-linked immunosorbent assay kit. Event-free survival (EFS) and overall survival were assessed from the date of study entry or the date of randomization, as appropriate. RESULTS: The mean sTNFRp55 level (+/- standard deviation) was 3.40 +/- 2.57 ng/mL. High levels of sTNFRp55 were associated with shorter duration of first complete remission and observation time as well as poor response to chemotherapy. Most importantly, the probability of EFS (pEFS) at 3 years was significantly worse for children with recurrent ALL who had sTNFRp55 levels greater than the median value (> 2.77 ng/mL) compared with patients who had levels that were less than the median value (pEFS: 0.44 +/- 0.10 ng/mL vs. 0.12 +/- 0.10 ng/mL; P = 0.006). It is noteworthy that the sTNFRp55 levels in 22 children with recurrent, TEL-AML1-positive ALL ([t(12;21)(p13;q22)]; 2.69 +/- 1.05 ng/mL) were significantly lower compared with the levels in children who had TEL-AML1-negative ALL (3.34 +/- 1.49 ng/mL; P < 0.05). CONCLUSIONS: The results indicated that a high sTNFRp55 level represents a negative prognostic factor for children with recurrent ALL in terms of EFS and overall survival.