Interleukin-15 plays an essential role in the pathogenesis of autoimmune diabetes in the NOD mouse.

AIMS/HYPOTHESIS: IL-15, induced by innate immune stimuli, promotes rheumatoid arthritis and inflammatory bowel disease. However, its role in autoimmune type 1 diabetes is unclear. Our aim is to define the role of IL-15 in the pathogenesis of diabetes in the NOD mouse model. METHODS: We generated NOD.Il15(-/-) mice expressing a polyclonal repertoire of T cell antigen receptor (TCR) or a transgenic TCR and monitored diabetes onset and insulitis. NOD Scid.Il15(-/-) (full name NOD.CB17-Prkdc (scid)/NCrCrl) and NOD Scid.gamma (full name NOD.Cg-Prkdc(scid) Il2rg ( tm1Wjl )/SzJ) mice were used to distinguish the requirement for IL-15 signalling in CD8(+) T cells and antigen-presenting cells (APCs) to induce disease. We examined the effect of blocking IL-15 signalling on diabetes onset in NOD mice. RESULTS: At 7 months of age, more than 75% of the NOD Il15(-/-) female mice remained diabetes free compared with only 30% in the control group. Diabetes incidence was also decreased in 8.3-NOD (full name NOD Cg-Tg[TcraTcrbNY8.3]-1Pesa/DvsJ).Il15(-/-) mice expressing a highly pathogenic transgenic TCR on CD8(+) T cells. Adoptive transfer of splenocytes from diabetic NOD and 8.3-NOD donors induced disease in NOD Scid recipients but not in NOD Scid.Il15(-/-) or NOD Scid.gamma mice. Transient blockade of IL-15 signalling at the onset of insulitis prevented diabetes in NOD mice. CONCLUSIONS/INTERPRETATION: Our results show that IL-15 is needed for the initial activation of diabetogenic CD8(+) T cells as well as for sustaining the diabetogenic potential of antigen-stimulated cells, acting on both CD8(+) T cells and on APCs. Our findings demonstrate a critical role for IL-15 in the pathogenesis of autoimmune diabetes and suggest that IL-15 is a promising therapeutic target.

Cysteinyl-leukotrienes induce vascular endothelial growth factor production in human monocytes and bronchial smooth muscle cells.

BACKGROUND: Cysteinyl leukotrienes (cysLTs) are suggested to be implicated in the process of airway remodelling in asthma. OBJECTIVE: We investigated the potential for cysLTs to modulate vascular endothelial growth factor (VEGF) expression, a growth factor involved in the angiogenesis of airway remodelling. METHODS: VEGF mRNA and protein were quantified by real-time PCR and ELISA, respectively. VEGF promoter activation was assessed using luciferase gene-tagged promoter constructs. RESULTS: We found that LTD(4) induction of VEGF in human monocytes and bronchial smooth muscle cells is cysLT1 dependent. Stimulation of HEK293 cells stably expressing cysLT1 or cysLT2 with cysLTs showed a concentration-dependent activation of the VEGF promoter and a time-dependent increase in VEGF mRNA and protein. For the cysLT1-mediated response, mutations of hypoxia-induced factor-1 (HIF-1) sites failed to reduce cysLT-induced VEGF promoter activation and 5' deletions showed that the proximal region containing one AP-1 and four specificity protein 1 (Sp1) sites was necessary. Pretreatment with inhibitors of c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), but not p38, and an overexpression of dominant negative forms of c-Jun, c-Fos or Ras suggested the implication of mitogen-activated protein kinases and AP-1. Mutation of the AP-1-binding element failed to prevent VEGF transactivation suggesting that AP-1 might not act directly on the promoter. Moreover, inhibition of Sp1-dependent transcription by mithramycin completely inhibited VEGF promoter transactivation and VEGF mRNA expression by LTD(4) . Finally, mutations of Sp1 binding elements prevented VEGF promoter transactivation. CONCLUSION AND CLINICAL RELEVANCE: Our data indicate for the first time that cysLTs can transcriptionally activate VEGF production via cysLT1 receptors, with the involvement of JNK, ERK, the AP-1 complex and Sp1. These findings suggest that cysLTs may be important in the angiogenic process of airway remodelling and potentially provide a previously unknown benefit of using cysLT1 receptor antagonists in the prevention or treatment of airway remodelling in asthma.

Transforming growth factor-beta1 in asthmatic airway smooth muscle enlargement: is fibroblast growth factor-2 required?

Enlargement of airway smooth muscle (ASM) tissue around the bronchi/bronchioles is a histopathological signature of asthmatic airway remodelling and has been suggested to play a critical role in the increased lung resistance and airway hyperresponsiveness seen in asthmatic patients. The pleiotropic cytokine, TGF-beta1, is believed to contribute to several aspects of asthmatic airway remodelling and is known to influence the growth of many cell types. Increased TGF-beta1 expression/signalling and ASM growth have been shown to occur concurrently in animal models of asthma. Abundant studies further substantiate this association by showing that therapeutic strategies that reduce or prevent TGF-beta1 overexpression/signalling lead to a parallel decrease or prevention of ASM enlargement. Finally, recent findings have supported a direct link of causality between TGF-beta1 overexpression/signalling and the overgrowth of ASM tissue. To follow-up on these in vivo studies, many investigators have pursued detailed investigation of ASM in cell culture conditions, assessing the direct role of TGF-beta1 on cellular proliferation and/or hypertrophy. Inconsistencies among the in vitro studies suggest that the effect of TGF-beta1 on ASM cell proliferation/hypertrophy is contextual. A hypothesis focusing on fibroblast growth factor-2 is presented at the end of this review, which could potentially reconcile the apparent discrepancy between the conflicting in vitro findings with the consistent in vivo finding that TGF-beta1 is required for ASM enlargement in asthma.

Methylenetetrahydrofolate reductase (MTHFR): a novel target for cancer therapy.

Tumor cells have an enhanced requirement for glucose, amino acids and DNA precursors. Since folates are required for the synthesis of thymidine and purines, the metabolism of folate has been exploited as an anti-cancer target for over 6 decades, with emphasis on the inhibition of DNA synthesis. However, folate is also used to generate methionine, which is essential for proliferation by virtue of its role in protein synthesis, polyamine synthesis and transmethylation reactions. Tumor-derived cell lines and human tumor xenografts have been shown to be methionine dependent i.e., they are unable to survive without methionine and are unable to efficiently utilize homocysteine, the immediate metabolic precursor of methionine. Since non-transformed cells are methionine-independent, the targeting of methionine metabolism presents an opportunity to selectively disrupt the unique metabolic networks in cancer cells. This chapter provides an overview of the critical role of folate and methionine metabolism in tumor cells and summarizes the current anti-folate and anti-methionine strategies to inhibit growth of transformed lines and tumors. We also present our work on the development of a novel anti-cancer target, methylenetetrahydrofolate reductase (MTHFR), a key enzyme of both folate and methionine metabolism. Our data demonstrate that antisense-mediated inhibition of MTHFR is associated with increased cytotoxicity in vitro and with decreased growth of tumors in vivo. These findings warrant further investigation of this enzyme and the methionine biosynthetic pathway in exploring new strategies for cancer chemotherapy.

Leukotriene D4-induced, epithelial cell-derived transforming growth factor beta1 in human bronchial smooth muscle cell proliferation.

BACKGROUND: Cysteinyl-leukotrienes (cys-LTs) orchestrate many pathognomonic features of asthma in animal models of allergic airway inflammation, including bronchial smooth muscle cell (BSMC) hyperplasia. However, because cys-LTs alone do not induce mitogenesis in monocultures of human BSMC, the effect observed in vivo seemingly involves indirect mechanisms, which are still undefined. OBJECTIVE: This study aims to investigate the regulatory role of leukotriene (LT)D(4) on TGF-beta1 expression in airway epithelial cells and the consequence of this interplay on BSMC proliferation. METHODS: HEK293 cells stably transfected with cys-LT receptor 1 (CysLT1) (293LT1) were stimulated with LTD(4) and TGF-beta1 mRNA and protein expression was measured using Northern blot and ELISA, respectively. Conditioned medium (CM) harvested from LTD(4)-treated cells was then assayed for its proliferative effect on primary human BSMC. TGF-beta1 mRNA expression was also determined in tumoural type II pneumocytes A549 and in normal human bronchial epithelial cells (NHBE) following LTD(4) stimulation. RESULTS: The results demonstrated that LTD(4)-induced TGF-beta1 mRNA production in a time- and concentration-dependent manner in 293LT1. TGF-beta1 secretion was also up-regulated and CM from LTD(4)-treated 293LT1 was shown to increase BSMC proliferation in a TGF-beta1-dependent manner. The increased expression of TGF-beta1 mRNA by LTD(4) also occured in A549 and NHBE cells via a CysLT1-dependent mechanism. CONCLUSION: In conclusion, elevated expression of cys-LTs in asthmatic airways might contribute to BSMC hyperplasia and concomitant clinical features of asthma such as airway hyperresponsiveness via a paracrine loop involving TGF-beta1 production by airway epithelial cells.

Leukotrienes and isocyanate-induced asthma: a pilot study.

BACKGROUND: The role of leukotrienes (LTs) in the pathophysiology of isocyanate-induced asthma is not well known. OBJECTIVE: We sought to characterize the type of airway inflammation induced by exposure to isocyanates and to investigate whether exposure to isocyanates induced an increase in LT receptor cysteinyl leukotriene ((CysLT)(1), CysLT(2) and leukotriene B(4) receptor (BLT(1))) expression, as well as a release of LT (LTC(4) and leukotriene B(4) (LTB(4))) and IL-8 in both asthmatics with isocyanate-induced asthma and healthy subjects. METHODS: We investigated eight subjects with isocyanate-induced asthma and eight healthy subjects. Both groups underwent specific inhalation challenges to isocyanates in the laboratory. Induced sputum was collected before and after exposure to isocyanates. CysLT(1), CysLT(2) and BLT(1) expression was assessed by flow cytometry, whereas LTC(4), LTB(4) and IL-8 were measured in the sputum supernatants by enzyme immunoassay. RESULTS: Exposure to isocyanates induced an increase in sputum neutrophils only in subjects with occupational asthma. There was a significant increase in CysLT(1) and BLT(1) receptor expression, as well as a release of LTB(4) and IL-8 after exposure to isocyanates compared with the baseline, only in subjects with isocyanate-induced asthma, whereas there was no increase in LTC(4). Exposure to isocyanates did not induce any change in LT receptor expression nor in the levels of LTC(4), LTB(4) and IL-8, in healthy subjects. CONCLUSION: The neutrophilia observed after exposure to isocyanates is likely to be related to the release of LTB(4), probably enhanced by the increased expression of BLT(1) on neutrophils as well as by the release of IL-8. The significance of the increase of CysLT1 receptor expression on neutrophils is unknown and needs further investigation.

[Effect of amifostine on reducing acute toxicity of megachemotherapy of tumors in children]. / Vliv amifostinu na redukci akutní toxicity pri protinádorové megachemoterapii detí.

BACKGROUND: Amifostine (WR-2721, Ethyol) is a chemoprotective agent. There is little experiences with amifostine application in megachemotherapy in children. We evaluated amifostine effect on the reduction of the acute toxicity. METHODS AND RESULTS: Retrospective comparison of patients who received amifostine with the control group (72 vs. 72). Amifostine 750 mg/m2 was given 15 minute before cytostatic dose and regularly each eight hours if we administered cytostatics continuously. Megachemotherapy schedule included melfalan, carboplatin, cyklophosphamid, vepesid, busulfan, thiotepa and karmustin. Type of graft: peripheral stem cells 56 vs. 29, bone marrow 8 vs. 30, combination 8 vs. 13. Nonhematological toxicity: mucositis p = 0.047, hepatotoxicity p < 0.001, nephrotoxicity p = 0.005. Hematological toxicity: engraftment D + 12 vs. D + 15 (p < 0.001), amount of erythrocyte transfusions 3 vs. 6 (p < 0.001), platelet transfusions 7 vs. 9 (p = 0.06), days when number of platelets reaches 20,000 without substitution D + 15 vs. D + 22 (p < 0.001). The only statistically difference was in the in total amount of platelets (p = 0.032), when we calculated patients, who received peripheral stem cells. Number of hospitalization days 14 vs. 18 (p = 0.016), days with antibiotics 14 vs. 18 (p = 0.016), number of febrile days 6 vs. 7 (p = 0.51). CONCLUSIONS: Amifostine reduces mucosal, liver and kidney damage. The graft type could affect hematological results.

High efficiency transient transfection of genes in human umbilical vein endothelial cells by electroporation.

Endothelial cells derived from the human umbilical vein (HUVEC) are used to study the mechanisms involved in EC response to various stimuli as well as to investigate the basis of pathological conditions of the vascular system such as altered endothelium permeability, tumor-induced angiogenesis, atherosclerosis and leukocyte extravasation in chronic inflammatory responses. However, investigations of gene involvement related to these conditions have progressed slowly because of the difficulty of transfecting HUVEC with high efficiency. Whereas several technical approaches have been described, they usually result in low levels of transfected cells or they require several steps or sophisticated instrumentation. We describe here a straightforward protocol of transfection of freshly isolated HUVEC that is based on the simple technique of electroporation. Efficiencies of gene transfection greater than 40% were routinely obtained by using a combination of optimized conditions of HUVEC isolation, composition of the electroporation medium and homogeneity of the plasmids. The protocol has been applied to the functional transient transfection of functional genes in HUVEC as illustrated in the case of the cDNA encoding GFP, protein kinase C (alpha and epsilon isotypes) and beta-galactosidase.

Inverse agonist activity of selected ligands of platelet-activating factor receptor.

The receptor for platelet-activating factor (PAFR) is a member of the G protein-coupled receptor (GPCR) family. According to the allosteric ternary complex model, GPCRs exist in an equilibrium between different conformations. Agonist binding promotes and stabilizes the receptor in an active conformation. On the other hand, ligands that stabilize the inactive conformation are known as inverse agonists. Due to the association of platelet-activating factor (PAF) with diverse physiological and pathological processes, considerable efforts have been invested in the development of antagonists to PAFR. A large number of these molecules has been shown to specifically interact with PAFR but, surprisingly, little is known about their impact on the conformation of the receptor and its activity. By using a constitutively active mutant (L231R) of the human PAFR and by transiently coexpressing the wild-type (WT) receptor with the G(alpha)q subunit of the trimeric G protein, we were able to address this issue with ligands of diverse structures such as phospholipids, benzodiazepines, furans, and others. We demonstrated that some of these molecules are potent inverse agonists. For example, when cells (WT PAFR + G(alpha)q) were exposed to WEB2086, SM10661, or alprazolam, the basal inositol phosphate production was reduced by 53 +/- 6, 44 +/- 3, and 54 +/- 4%, respectively. The decrease in basal inositol phosphate production by WEB2086 was significantly inhibited by a more neutral antagonist BN52021, confirming the specificity of the reaction. We demonstrate here that WEB2086 and other known ligands previously considered as antagonists can act as inverse agonists on the human PAF receptor.

IL-13 and IL-4 up-regulate cysteinyl leukotriene 1 receptor expression in human monocytes and macrophages.

The cysteinyl (Cys) leukotrienes (LT)C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of asthma. The human LTD(4) receptor (CysLT(1)R) was recently cloned and characterized. The present work was undertaken to study the potential modulation of CysLT(1)R expression by the Th2 cytokines IL-13 and IL-4. In this study, we report that IL-13 up-regulates CysLT(1)R mRNA levels, with consequently enhanced CysLT(1)R protein expression and function in human monocytes and monocyte-derived macrophages. CysLT(1)R mRNA expression was augmented 2- to 5-fold following treatment with IL-13 and was due to enhanced transcriptional activity. The effect was observed after 4 h, was maximal by 8 h, and maintained at 24 h. IL-4, but not IFN-gamma, induced a similar pattern of CysLT(1)R up-regulation. Monocytes pretreated with IL-13 or IL-4 for 24 h showed enhanced CysLT(1)R protein expression, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4), in terms of Ca(2+) mobilization, as well as augmented chemotactic activity. Our findings suggest a possible mechanism by which IL-13 and IL-4 can modulate CysLT(1)R expression on monocytes and macrophages, and consequently their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.

G-protein-independent activation of Tyk2 by the platelet-activating factor receptor.

Platelet-activating factor (PAF) is a potent pro-inflammatory phospholipid with multiple physiological and pathological effects. PAF exerts its activity through a specific heptohelical G-protein coupled receptor, expressed on a variety of cell types, including leukocytes. In this study, we showed that PAF induced a rapid tyrosine phosphorylation of the Tyk2 kinase in the monocytic cell lines U937 and MonoMac-1. PAF-initiated Tyk2 phosphorylation was also observed in COS-7 cells transiently transfected with the human PAF receptor (PAFR) and Tyk2 cDNAs. In addition, we found that Tyk2 co-immunoprecipitated and co-localized with PAFR, independently of ligand binding. Deletion mutants of Tyk2 indicated that the N terminus of the kinase was important for the binding to PAFR. Activation of Tyk2 was followed by a time-dependent 2-4-fold increase in the level of tyrosine phosphorylation of signal transducers and activators of transcription 1 (STAT1), STAT2, and STAT3 and a sustained 2.5-fold increase in STAT5 tyrosine phosphorylation. In MonoMac-1 cells, STAT1 and STAT3 translocated to the nucleus following PAF stimulation, and their translocation in transiently transfected COS-7 cells was shown to be dependent on the presence of Tyk2. In addition, when COS-7 cells were transfected with PAFR and constructs containing PAFR promoter 1, coupled to the luciferase reporter gene, PAF induced a 3.6-fold increase in promoter activation in the presence of Tyk2. Finally, PAFR mutants that could not couple to G-proteins were found to effectively mediate Tyk2 activation and signaling. Taken together, these findings suggest an important role for the Janus kinase/STAT pathway in PAFR signaling, independent of G-proteins, and in the regulation of PAF receptor expression by its ligand.

[131I meta-iodobenzylguanidine in combination with hyperbaric oxygen therapy in the treatment of prognostically high-risk forms of neuroblastoma]. / 131I metajodobenzylguanidin v kombinaci s hyperbarickou oxygenoterapií v lécbe prognosticky vysoce nepríznivých forem neuroblastomu.

BACKGROUND: Despite of improving diagnostics, development of new drugs and treatment strategies, patients with biologically unfavourable, advanced or relapsed neuroblastoma remain practically incurable. Treatment related toxicity, requirement for personnel and financial costs have became limiting. Tumor specific therapy represented by 131I-meta-iodobenzylguanidine (MIBG) administration could become an alternative improving the overall survival. In comparison with standard external radiotherapy the targeted therapy enables to achieve radiation 5 to 10 times higher with lower organ toxicity. Data published by European and American colleagues brought evidence of high efficacy of this method. It motivated us to set and develop the method at our department. TYPE OF STUDY: Retrospective analysis of therapeutic results and side effects of the administration of 131I-meta-iodobenzylguanidine in high-risk neuroblastoma patients cured at the Department of Pediatric Oncology in Prague since 1997 till 2000. METHOD AND RESULTS: 131I-meta-iodobenzylguanidine was fourteen times therapeutically administered in seven high-risk relapsed neuroblastoma patients. Four children received a single dose of 131I-meta-iodobenzylguanidine, three patients were treated repeatedly. The first dose represented 5.5 GBq, repeated dose 3.7 GBq, irrespective to the body weight. Each MIBG administration was followed by four days hyperbaric oxygen therapy. The treatment was well tolerated, acute and late side effects were not serious and only rarely reached grade 3 or 4 according to the International North American Children's Cancer Group Classification. Three of the seven children have survived with no evidence of the disease. Four children died of the disease progress. CONCLUSIONS: 131I-meta-iodobenzylguanidine treatment combined with hyperbaric oxygen therapy becomes a well-tolerated therapy for high-risk neuroblastoma patients non-responding to the conventional treatment. Though the 131I-meta-iodobenzylguanidine administration probably cannot cure these patients, the repeated administration can bring long lasting remission.

Oxidized low-density lipoprotein and peroxisome-proliferator-activated receptor alpha down-regulate platelet-activating-factor receptor expression in human macrophages.

Regulation of the expression of platelet-activating factor (PAF) receptor by atherogenic lipoproteins might contribute to atherogenesis. We show that progressive oxidation of low-density lipoprotein (LDL) gradually inhibits PAF receptor expression on the macrophage cell surface. We tested the effect of oxidized LDL (oxLDL) on PAF receptor expression in human monocytes that do not contain peroxisome-proliferator-activated receptor gamma (PPARgamma), a nuclear receptor activated by oxLDL. OxLDL decreased by 50% (P < or = 0.001) and by 29% (P < or = 0.05) the binding of PAF and the expression of PAF receptor mRNA respectively. Next we demonstrated that progressive oxidation of LDLs significantly activated PPARalpha-dependent transcription in transfected mouse aortic endothelial cells. Finally we demonstrated, in mature macrophages, that fenofibrate (20 microM), a specific PPARalpha agonist, but not the specific PPARgamma agonist BRL49653 (20 nM), significantly decreased both PAF binding and PAF receptor mRNA expression, by 65% and 40% (P < or = 0.001) respectively. Additionally, another PPARalpha agonist, Wy14,643, decreased PAF receptor promoter activity by 70% (P < or = 0.05) in transfected THP-1 cells, suggesting the involvement of the proximal promoter region (-980 to -500) containing a series of four nuclear factor (NF)-kappaB motifs. Thus PPARalpha might be involved in the down-regulation of PAF receptor gene expression by oxLDLs in human monocytes/macrophages. The oxidation of one or more lipid components of LDLs might result in the formation of natural activators of PPARalpha. It is hypothesized that such activators might modulate inflammation and apoptosis upon atherogenesis by decreasing the expression of PAF receptor.

Expression of platelet-activating factor receptor in human carotid atherosclerotic plaques: relevance to progression of atherosclerosis.

BACKGROUND: Human monocyte-derived macrophages synthesize numerous proinflammatory and prothrombotic substances, including lipid mediators, such as platelet-activating factor (PAF), which may play a major role in the onset and perpetuation of atherosclerotic lesions. In addition, both monocytes and macrophages express PAF receptors (PAF-R). The expression of PAF-R is transcriptionally downregulated by oxidized LDL in in vitro primary cultures of monocyte/macrophages. In this study, we evaluated the expression of PAF-R in human carotid plaque tissue, in foam cells isolated from human carotid plaques, and in primary cultures of umbilical smooth muscle cells (SMCs). METHODS AND RESULTS: We show that PAF-R was expressed at low levels in foam cells compared with monocyte/macrophages in plaques, as assessed by immunohistochemical staining and in situ hybridization. In addition, low levels of mRNA were also detected by RT-PCR in isolated human carotid foam cells. A prominent finding of our study was the demonstration that contractile SMCs were positive for PAF-R, and its mRNA was extracted from primary cultures of umbilical SMCs. CONCLUSIONS: As macrophages loose their inflammatory phenotype on transformation into foam cells, they may equally loose their capacity of defense against aggression. We postulate that the diminished expression of PAF-R may be deleterious in the context of plaque formation and progression. The observation that arterial SMCs of contractile phenotype express PAF-R opens new avenues concerning the migration of these cells from media to intima and atherosclerotic plaque formation.

IL-5 up-regulates cysteinyl leukotriene 1 receptor expression in HL-60 cells differentiated into eosinophils.

The cysteinyl leukotrienes, leukotriene (LT) C(4), LTD(4), and LTE(4), are lipid mediators that have been implicated in the pathogenesis of several inflammatory processes, including asthma. The human LTD(4) receptor, CysLT(1)R, was recently cloned and characterized. We had previously shown that HL-60 cells differentiated toward the eosinophilic lineage (HL-60/eos) developed specific functional LTD(4) receptors. The present work was undertaken to study the potential modulation of CysLT(1)R expression in HL-60/eos by IL-5, an important regulator of eosinophil function. Here, we report that IL-5 rapidly up-regulates CysLT(1)R mRNA expression, with consequently enhanced CysLT(1)R protein expression and function in HL-60/eos. CysLT(1)R mRNA expression was augmented 2- to 15-fold following treatment with IL-5 (1-20 ng/ml). The effect was seen after 2 h, was maximal by 4 h, and maintained at 8 h. Although CysLT(1)R mRNA was constitutively expressed in undifferentiated HL-60 cells, its expression was not modulated by IL-5 in the absence of differentiation. Differentiated HL-60/eos cells pretreated with IL-5 (10 ng/ml) for 24 h showed enhanced CysLT(1)R expression on the cell surface, as assessed by flow cytometry using a polyclonal anti-CysLT(1)R Ab. They also showed enhanced responsiveness to LTD(4), but not to LTB(4) or platelet-activating factor, in terms of Ca(2+) mobilization, and augmented the chemotactic response to LTD(4). Our findings suggest a possible mechanism by which IL-5 can modulate eosinophil functions and particularly their responsiveness to LTD(4), and thus contribute to the pathogenesis of asthma and allergic diseases.

Increased micronuclei frequencies in couples with reproductive failure.

A higher incidence of chromosomal instability in the infertile population is widely recognized. An increased level of micronuclei has been shown to be a marker of chromosome damage. Therefore, micronuclei frequencies were assessed in cytokinesis-blocked lymphocytes of 130 patients (65 couples) with idiopathic infertility or with two or more spontaneous abortions, and 30 healthy fertile donors (15 couples). The frequency of micronucleated cells in the cohort with reproductive failure and healthy controls averaged 14.95+/-6.04 per 1000 and 10.60 +/-2.57 per 1000 (P<0.0001), respectively. When micronuclei frequency sums in particular couples (male + female) were analyzed in the same order, identical statistical significance was reached (P<0.0001). We found no effect of age or sex on micronuclei frequency. In summary, the cytokinesis-blocked micronuclei assay revealed increased micronucleus frequency in couples with infertility or two or more spontaneous abortions, suggesting a possible role of chromosomal instability in reproductive failure.

[Detection of minimal residual disease in Ewing's sarcoma using RT-PCR]. / Detekce minimální reziduální nemoci u Ewingova sarkomu pomocí RT-PCR.

BACKGROUND: More than 90% of Ewing's sarcomas (ES) contain a fusion of the EWS and FLI-1 genes, due to the t(11;22)(q23;q12) translocation. At the molecular level, the EWS-FLI-1 rearrangement shows great diversity. Specifically, many different combinations of exons from EWS-FLI-1 encode in-frame fusion transcripts and result in differences in length and composition of the chimeric protein, which function as an oncogenic aberrant transcription factor. The finding of this translocation gives evidence for the presence of ES cells. The aim of this prospective study was to verify applicability of the RT-PCR method for the detection of minimal residual disease in patients with ES. METHODS AND RESULTS: Conditions for the detection of Ewing's sarcoma cells by means of the reverse-transcriptase polymerase chain reaction (RT-PCR) at fusion transcripts in peripheral blood, bone marrow (BM) and autologous hematopoietic stem cell grafts in patients with ES were appointed. 31 samples of BM, 5 samples of blood and 7 peripheral blood grafts obtained from 23 patients were investigated. Presence of tumor cells was identified in 7 BM samples from 7 different patients (31 samples from 16 patients), all the peripheral blood and graft samples were negative. CONCLUSIONS: The high sensitivity of RT-PCR method in detection of cells bearing t(11;22)(q23;q12) was demonstrated in the experimental model and clinical samples. Likewise the literary statements, the RT-PCR method was found to be more sensitive than cytology.

[Familial hyperlipoproteinemias--correlations between phenotypes and genotypes]. / Familiární hyperlipoproteinémie--korelace nálezu ve fenotypu s genotypem.

Within the grant project patients with familial hyperlipoproteinaemias have been examined. The examination was performed in the oldest lipid clinic and research laboratory in the world. The classification of lipid metabolism disorders was based upon a detailed biochemical analysis of plasma lipids including electrophoresis and assessment of apolipoprotein levels. Then optimal treatment regimen could be established. The project was aimed to evaluate the efficacy of different treatment regimens in different types of hyperlipoproteinaemias. Biochemical parameters and mainly the impact of treatment of hyperlipoproteinaemia on morphology and function of the vessel wall was monitored. The non-invasive ultrasound measurement of the intima thickness of carotid arteries was used. For more precise diagnosis of genetically determined disorders of lipid metabolism a large scale of methods of molecular biology was introduced. These methods enable confirmation of familial hypercholesterolaemia, familial defective apolipoprotein B-100 or studying polymorphism of apolipoprotein E. The effort of the authors of the project was to maximally utilise the results of basic and applied research in formulating recommendations for everyday practice of physicians.

[High-dose chemotherapy in the treatment of children with malignant lymphoma]. / Vysokodávkovaná chemoterapie v lécbe detí s maligním lymfomem.

BACKGROUND: Children with primary refractory or recurrent malignant lymphoma have usually poor prognosis. Less than 10% of those, who were treated with conventional-dose regimens had survived for 2 years. In an attempt to improve the outcome for these patients, we explored the role of consolidation high-dose chemotherapy with autografting. METHODS AND RESULTS: Forty-five patients with poor-prognosis lymphoma, of whom 27 were males, underwent megatherapy between January 1992 and December 1999. High-dose chemotherapy was indicated in patients with poor initial response to first-line chemotherapy (14 cases) or in the relapse (31 cases). The group consisted of 27 patients with Hodgkin's disease and 18 patients with non-Hodgkin's lymphoma. The median age was 14.7 years. The conditioning for Hodgkin's disease patients contained cyclophosphamide, etoposide and busulfan or carmustine. Patients with non-Hodgkin's lymphomas received cyclophosphamide, etoposide and busulfan or total body irradiation. Bone marrow was used as the source of haemopoietic stem cells in ten patients, peripheral blood in twenty-eight, and both sources were used in seven patients. After the median follow-up of 47 months, the final survival was 61%. Eleven patients died of the disease progression, four of the infectious complications, one at a car accident. Median time to relapse after the transplantation was 7.5 months. CONCLUSIONS: Further improvement of these results will require earlier transplantation, improved preparative regiments or early posttransplant immunotherapy.

Heat-killed Streptococcus suis capsular type 2 strains stimulate tumor necrosis factor alpha and interleukin-6 production by murine macrophages.

Streptococcus suis capsular type 2 is an important etiological agent of swine meningitis, and it is also a zoonotic agent. Since mononuclear phagocytes have been suggested to play a central role in the pathogenesis of meningitis, the objective of the present study was to evaluate the capacity of whole killed S. suis type 2 organisms to induce the release of the proinflammatory cytokines tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6) by murine macrophages. Induction of cytokines was evaluated in the presence or absence of phorbol ester (phorbol 12-myristate 13-acetate [PMA]) costimulation. Results showed that S. suis type 2 stimulated the production of both cytokines in a concentration- and time-dependent fashion. Although large doses of bacteria were required for maximal cytokine release, titers were similar to those obtained with the lipopolysaccharide (LPS) positive control. An increase in cytokine release was observed with both S. suis and LPS with PMA costimulation. Experiments with cytochalasin-treated macrophages showed that the stimulation of cytokine production was phagocytosis independent. When macrophages were stimulated with an unencapsulated mutant, an increase in TNF production was observed, but the absence of the capsule had no effect on IL-6 production. In fact, whereas purified capsular polysaccharide of S. suis failed to induce cytokine release, purified S. suis cell wall induced both TNF and, to a lesser extent, IL-6. IL-6 secretion probably requires some distinct stimuli which differ from those of TNF. Finally, the S. suis putative virulence factors suilysin and extracellular protein EF showed no cytokine-stimulating activity. The ability of S. suis to trigger macrophages to produce proinflammatory cytokines may have an important role in the initiation and development of meningitis caused by this microorganism.