Polycomb group (PcG) proteins function as multiprotein complexes and are part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. Several studies have implicated the deregulation of different PcG proteins in neoplastic progression. Pancreatic ductal adenocarcinoma is an aggressive neoplasm that follows a multistep model of progression through precursor lesions called pancreatic intraepithelial neoplasia (PanIN). Aim of this study was to investigate the role of PcG protein CBX7 in pancreatic carcinogenesis and to evaluate its possible diagnostic and prognostic significance. We analysed by immunohistochemistry the expression of CBX7 in 210 ductal pancreatic adenocarcinomas from resection specimens, combined on a tissue microarray (TMA) including additional 40 PanIN cases and 40 normal controls. The results were evaluated by using receiver operating characteristic (ROC) curve analysis for the selection of cut-off scores and correlated to the clinicopathological parameters of the tumours and the outcome of the patients. Expression of E-cadherin, a protein positively regulated by CBX7, was also assessed. A significantly differential, and progressively decreasing CBX7 protein expression was found between normal pancreatic tissue, PanINs and invasive ductal adenocarcinoma. Loss of CBX7 expression was associated with increasing malignancy grade in pancreatic adenocarcinoma, whereas the maintenance of CBX7 expression showed a trend toward a longer survival. Moreover, loss of E-cadherin expression was associated with loss of CBX7 and with a trend towards worse patient survival. These results suggest that CBX7 plays a role in pancreatic carcinogenesis and that its loss of expression correlates to a more aggressive phenotype.

Introduction. Myocardial fibrosis contributes to hemodynamic and cardiac functional alterations commonly observed posttransplantation. Cardiac mast cells (MC) have been linked to fibrosis in posttransplantation hearts. Eotaxin, which has been shown to be involved in fibrogenesis, has been demonstrated to be increased in production in cardiac macrophages. The aim of our study was to correlate myocardial fibrosis during heart transplant rejection in the rat with eotaxin/chemokine [c-c motif] ligand 11 (CCL11) expression, and with various subtypes of infiltrating cardiac MC, namely connective-type MC (CTMC) and mucosa-type MC (MMC).Methods. We used tissues from 2 previous studies of ongoing acute rejection in allogeneic Brown-Norway to Lewis rat and an isogeneic Brown-Norway to Brown-Norway heterotopic heart transplantation models under cyclosporin/prednisolone immunosuppression. Collagen fibrils were stained with Masson's trichrome with myocardial fibrosis expressed as percent fibrotic area per total section area. Eotaxin/CCL11 previously measured in heart tissue using enzyme-linked immunosorbent assay (ELISA) was correlated with the extent of myocardial fibrosis. We compared values from native hearts (n = 4) as well as transplants on days 5, 16, and 28 (n = 4 in each group).Results. The area of myocardial fibrosis was significantly increased in the allogeneic compared with the isogeneic group at day 16 (38% vs 21%) and at day 28 (49% vs 22%) after transplantation. Myocardial fibrosis correlated significantly with eotaxin/CCL11 concentrations and the density of MMC, but not with CTMC in heart tissue.Conclusions. Eotaxin-triggered MC infiltration of the heart may contribute to myocardial fibrosis after transplantation. Targeting eotaxin/CCL11 with monoclonal antibodies, such as bertilimumab, could reduce MC infiltration, possibly resulting in decreased myocardial fibrosis and improved contractile function after heart transplantation.

RationaleSSE is recognized as a risk factor for the development of childhood asthma. Previous studies have reported an association between SSE and certain immune responses during infancy. The purpose of this study was to determine whether there is an association between SSE and immune cell phenotypes during early childhood.MethodsTwo ml of peripheral blood was obtained from 110 healthy subjects aged 1-6 years, and assayed for serum cotinine levels by EIA and for immune cell phenotypes by flow cytometry.ResultsSixty-one subjects had no exposure to SSE (serum cotinine level < 0.05 ng/ml) and 49 had exposure to SSE. In subjects with and without SSE, the number/ul blood (mean ± SEM) of dendritic cells and activated CD4+ cells were 205.6 ± 18.6 and 135.5 ± 9.2, and 164.7 ± 12.4 and 108.3 ± 5.5, respectively. There was no difference in the number of activated CD8+ lymphocytes between the two groups.ConclusionsThese results demonstrate increased numbers of dendritic cells and activated CD4+ lymphocytes, but not activated CD8+ lymphocytes, in young children exposed to SSE. Future studies need to determine whether SSE-induced immune responses have an impact upon the development of childhood asthma and/or atopy. RationaleSSE is recognized as a risk factor for the development of childhood asthma. Previous studies have reported an association between SSE and certain immune responses during infancy. The purpose of this study was to determine whether there is an association between SSE and immune cell phenotypes during early childhood. SSE is recognized as a risk factor for the development of childhood asthma. Previous studies have reported an association between SSE and certain immune responses during infancy. The purpose of this study was to determine whether there is an association between SSE and immune cell phenotypes during early childhood. MethodsTwo ml of peripheral blood was obtained from 110 healthy subjects aged 1-6 years, and assayed for serum cotinine levels by EIA and for immune cell phenotypes by flow cytometry. Two ml of peripheral blood was obtained from 110 healthy subjects aged 1-6 years, and assayed for serum cotinine levels by EIA and for immune cell phenotypes by flow cytometry. ResultsSixty-one subjects had no exposure to SSE (serum cotinine level < 0.05 ng/ml) and 49 had exposure to SSE. In subjects with and without SSE, the number/ul blood (mean ± SEM) of dendritic cells and activated CD4+ cells were 205.6 ± 18.6 and 135.5 ± 9.2, and 164.7 ± 12.4 and 108.3 ± 5.5, respectively. There was no difference in the number of activated CD8+ lymphocytes between the two groups. Sixty-one subjects had no exposure to SSE (serum cotinine level < 0.05 ng/ml) and 49 had exposure to SSE. In subjects with and without SSE, the number/ul blood (mean ± SEM) of dendritic cells and activated CD4+ cells were 205.6 ± 18.6 and 135.5 ± 9.2, and 164.7 ± 12.4 and 108.3 ± 5.5, respectively. There was no difference in the number of activated CD8+ lymphocytes between the two groups. ConclusionsThese results demonstrate increased numbers of dendritic cells and activated CD4+ lymphocytes, but not activated CD8+ lymphocytes, in young children exposed to SSE. Future studies need to determine whether SSE-induced immune responses have an impact upon the development of childhood asthma and/or atopy. These results demonstrate increased numbers of dendritic cells and activated CD4+ lymphocytes, but not activated CD8+ lymphocytes, in young children exposed to SSE. Future studies need to determine whether SSE-induced immune responses have an impact upon the development of childhood asthma and/or atopy.

Cytokine gene polymorphisms moderate illness severity in a variety of inflammatory disorders. The purpose of this study was to determine whether certain TGF-β1 gene polymorphisms are associated with the development of OM during RSV infection. TGF-β1 genotyping was performed on 228 young children prospectively evaluated for the development of OM during RSV season. DNA was extracted from buccal brushings and assayed for TGF-β1 genotypes using PCR-SSP. The presence of an upper respiratory infection (URI) was assessed by signs and symptoms, OM was diagnosed by validated pneumatic otoscopy, and RSV infection was confirmed using rapid antigen detection and RT-PCR. 171 subjects developed a URI; 103 of those were diagnosed with OM and 37 had confirmed RSV infection. In subjects without RSV, the genotype for high as compared to low TGF-β1 cytokine production was associated with a decreased frequency of OM (35% versus 75%, respectively; p < 0.05). However, in subjects with RSV, the genotype for high TGF-β1 was associated with an OM frequency of 100%. These results suggest a role for TGF-β1 genotypes in the pathogenesis of OM during URIs. Moreover, they suggest a differential effect of this genotype in response to infection with RSV as compared to other respiratory viruses. If confirmed by future studies, cytokine genotyping may become a tool for identifying "at risk" children who may benefit from the selective use of preventative and/or early intervention treatments for these disorders.

Recent studies have demonstrated a strong epidemiologic relationship between obesity and asthma. Other studies have shown elevated levels of pro-inflammatory cytokines in obese subjects. To date, no studies have examined the relationship between BMI and Th2 cytokines in allergic subjects. The purpose of this study was to determine if Th2 cytokine levels are elevated in overweight and obese subjects as compared to normal weight allergic subjects. Forty-six adult subjects with seasonal allergic rhinitis were enrolled and studied outside of their allergy season. BMI was calculated and subjects were classified as normal weight (BMI < 25), overweight (BMI =25 to <30) or obese (BMI ≥ 30). Whole blood was stimulated with mitogen and lymphocyte subsets and intracellular cytokine levels were assessed by flow cytometry. Sixteen subjects were normal weight [(mean (± SEM) BMI of 21.8 ± 1.8], 14 were overweight (27.0 ± 1.3) and 16 were obese (34.5 ± 4.4). The absolute number of CD8+ cells/ul producing IL-4 and IL-13 were 2.8 ± 0.4 and 3.1 ± 0.4 in normal weight, 7.1 ± 3.1 and 9.4 ± 4.1 in overweight and 9.6 ± 4.3 and 10.2 ± 4.1 in obese allergic subjects, respectively (p < 0.05). These results demonstrate elevated levels of Th2 cytokines in overweight and obese allergic subjects. Future studies need to elucidate the mechanism by which this occurs and to determine whether this response contributes to increased disease severity or refractoriness to treatment in allergic subjects.

Cerebral revascularization may be indicated either for blood flow preservation or flow augmentation, often in clinical situations where neither endovascular nor standard surgical intervention can be performed. Cerebral revascularization can be performed by using a temporary occlusive or a non-occlusive technique. Both of these possibilities have their specific range of feasibility. Therefore non-occlusive revascularization techniques have been developed. To further reduce the risks for patients, less time consuming, sutureless techniques such as laser tissue soldering are currently being investigated.

RATIONALE: Elevated levels of leukotrienes (LTs) have been detected in nasal secretions obtained from subjects with vURIs and intranasal administration of LTs has been shown to provoke signs/symptoms that accompany vURIs. These results suggest that locally produced LTs may play a role in the expression of symptoms of vURIs. The purpose of this study was to determine whether treatment with montelukast, a leukotriene antagonist, versus placebo has an effect on nasal secretion weights during naturally acquired vURIs in adults. METHODS: Eighty adult subjects with vURI signs/symptoms were enrolled and randomized (study day 0) to receive treatment with either montelukast (n = 40) 10 mg PO QD x 5 days or matching placebo (n = 40) in a double-blind manner. Following randomization, nasal secretion weights were monitored on a daily basis for the next 5 days (study days 1-6). RESULTS: The mean age of subjects was 32 ± 10 years; 71% were female and 46% were non-white. Nasal secretion weights were significantly decreased in subjects treated with montelukast versus placebo. On day 3, mean ± SEM nasal secretion weights (grams) were 2.6 ± 0.4 and 1.2 ± 0.4 in the placebo and montelukast groups, respectively (p ≤ 0.05). CONCLUSIONS: The results demonstrate that treatment with montelukast decreases nasal secretions during naturally acquired vURIs in adults. Moreover, they provide additional evidence that suggests that LTs play a role in the pathogenesis of vURIs. Future studies should expand this sample size and determine whether treatment with leukotriene modifiers attenuates complications of vURIs including otitis media, sinusitis and airway hyperreactivity.

RATIONALE: Leukotrienes have been detected in middle ear effusions obtained from children with naturally-acquired OM. Animal studies have shown that instillation of leukotrienes into the middle ear results in the development of OM and that treatment with a leukotriene modifier results in resolution of OM. Since these results suggest that locally produced leukotrienes may play a role in the pathogenesis of OM, we hypothesized that treatment with montelukast would result in less recurrence of OM in susceptible children. METHODS: Seventy-seven subjects aged 18 months to 5 years were treated with standard anti-microbial therapy x 10 days and randomized to treatment with either montelukast 5 mg PO QD x 4 months or matching placebo in double-blinded manner. Subjects were evaluated for resolution and recurrence of OM at 4 post enrollment visits (2, 4, 8, and 16 weeks). RESULTS: Treatment with montelukast versus placebo had no effect on persistence or recurrence of OM. At visit 2, 52% and 51% (p > 0.05) of the subjects in the placebo and montelukast groups, respectively, had persistence of OM. At visit 5, 25% and 43% (p > 0.05)of the subjects in the placebo and montelukast groups, respectively, had recurrence of OM. CONCLUSIONS: These results demonstrate that treatment with montelukast versus placebo had no impact on the persistence or recurrence of OM. However, these results do not definitively exclude a role for leukotrienes in the development of OM. Future studies should be designed to determine whether pretreatment with montelukast can prevent the development of OM in susceptible children.

ABSTRACT Daptomycin monotherapy was superior to ceftriaxone monotherapy and was highly efficacious in experimental pneumococcal meningitis, sterilizing the cerebrospinal fluid (CSF) of three of three rabbits after 4 to 6 h. With daptomycin therapy only a negligible release of [ 3 H]choline as marker of cell wall lysis was detectable in the CSF, peaking around 250 cpm/min after 4 h, compared to a peak of around 2,400 cpm/min after 4 to 6 h for the ceftriaxone-treated rabbits.

RATIONALE: SSE is recognized as a risk factor for the development of childhood asthma and previous studies have reported an association between SSE and IL-13 production during infancy. The purpose of this study was to determine whether there is an association between SSE and IL-13 production from CD4+ cells during early childhood. METHODS: Two ml of peripheral blood was obtained from 67 healthy subjects aged 1-6 years, and assayed for serum cotinine levels by EIA and for IL-13 production from CD4+ cells by flow cytometry. RESULTS: Twenty-eight subjects had no exposure to SSE (serum cotinine level < 0.05 ng/ml) and 39 had exposure to SSE. There were no significant differences in absolute numbers (cells/ul blood) of activated and IL-13 producing CD4+ cells in subjects with SSE as compared to those without SSE. In subjects with and without SSE, the number (mean ± SEM) of CD4+CD25+ cells were 141 ± 10 and 117 ± 10, and the number of CD4+ IL-13 producing cells was 24 ± 3 and 20 ± 3, respectively. CONCLUSIONS: These results do not demonstrate an association between SSE and enhanced IL-13 production from activated CD4+ cells during early childhood. Future studies need to characterize the impact of other environmental and genetic factors on these responses.

We recently reported a complete change in the endothelial ABO histo-blood group phenotype of a cardiac allograft long term after B to O mismatched transplantation. In the context of the current controversy on graft recolonization with recipient endothelial cells and its importance in the development of immunological unresponsiveness, we monitored the expression of endothelial ABH histo-blood group antigens of 10 ABO-compatible, non-identical cardiac allografts over an observation period of at least 30 months. ABH antigens as well as markers for endothelial cells, erythrocytes and thrombocytes were investigated retrospectively by immunohistochemistry using monoclonal antibodies on sections of formalin-fixed, paraffin-embedded biopsies and were evaluated semi-quantitatively by microscopy. In contrast to our earlier finding of the change in the endothelial ABO histo-blood group phenotype long term after ABO- mismatched transplantation, we could not confirm this change in 10 compatible but non-identical cases.

Most of the 29 blood group systems known today are not restricted to erythroid tissues hence their more recent identification as histo-blood group systems. Beyond the uncontested importance of the HLA system in human allograft survival, some of the histo-blood group systems might increasingly become recognised to play a role in graft-host interaction and peritransplant transfusion therapy. At least the ABO histo-blood group system has drawn a lot of interest since both, elective ABO-mismatch with living kidney donor/recipient pairs and infant heart recipients have been described as radical, but effective treatments of end-stage organ dysfunction. More recently, at least in part successful efforts to overcome unintentional ABO-mismatched lung and heart grafts spark interest in more precisely avoiding hyperacute transplant rejection due to complement-activating anti-A/B antibodies of the recipients. Such options as to prepare the recipient with plasma exchange and following him up with polyspecific intravenous immunoglobulins, monoclonal antibodies and targeted immunosuppression using mycophenolate, rabbit antithymocyte globulin and anti-CD20 antibody rituximab are bound to efficiently remove anti-A/B antibodies and apparently inhibit their resynthesis. The present contribution overviews recently acquired knowledge on the ABO histo blood group system and the role it plays in solid organ transplantation leant against a patient observed at our institution.

Aims Patency failure of small vascular synthetic grafts is still a major problem for coronary and peripheral revascularization. Thus, three new surface coatings of small synthetic grafts were tested in an acute pig model to evaluate their thrombogenicity (extracorporeal arterio-venous shunt) and in a chronic rat model to evaluate the tissue reaction they induced (subcutaneous implantation). Methods In five domestic pigs (25–30 kg) an extracorporeal femoro-femoral arterio-venous shunt model was used. The study protocol included first a non-heparinized perfusion sequence followed by graft perfusion after 10,000 UI iv heparin. Grafts were perfused for 3 and 9 minutes. The following coatings were tested on ePTFE grafts: poly-propylene sulphide (PPS) – poly-ethylene glycol (PEG) (wet and dry applications) as well as carbon. Two sets of control were used, one dry and one wet (vehicle only). After perfusion grafts were examined by scanning electron microscopy for semiquantitative assessment (score 0–3) of cellular and microthrombi deposition. To assess tissue compatibility, pieces of each material were implanted subcutaneously in 16 Wistar rats. At 2, 4, 8, 12 weeks four animals each were sacrificed for semi-quantitative (score 0–3) histologic evaluation of tissue reaction. Results In the pig model, cellular deposition and microthrombi formation increased over time. In non-heparinized animals, the coatings did not improve the surface characteristics, since they did not prevent microthrombi formation and cellular deposition. In heparinized animals, thrombogenicity was lowest in coated grafts, especially in PPS–PEG dry (P&0.05), and highest in controls. Cell deposition was lowest in PPS–PEG dry, but this difference was not statistically significant vs. controls. In the rat model, no significant differences of the tissue reaction could be shown between materials. Conclusion While all coatings failed to add any benefit for lowering tissue reaction, surface coating with PPS–PEG (dry application) reduced thrombogenicity significantly (in heparinized animals) and thus appears to be promising for improving graft patency of small synthetic vascular prostheses.

Mast cells (MC) can undergo significant changes in number and phenotype; these alterations result in the differential expression of growth factors and cytokines. Kit ligand (KL; stem cell factor) is produced by mesenchymal cells, and in the liver by biliary epithelial cells. Recent studies suggest that KL, and its receptor c‐kit, may be involved in liver regeneration after loss of liver mass. However, KL is also the major growth, differentiating, chemotactic, and activating factor for MC. The aim of our study was to elucidate the dynamics and phenotype of hepatic MC and KL/c‐kit expression during liver regeneration after partial (70%) hepatectomy in the rat. Regenerating livers were harvested after 1, 3, 7, and 14 days, respectively (n = 6 each day). MC were stained for naphthol‐AS/D‐chloroacetate esterase and counted as MC per bile ductule. MC phenotype was assessed by rat MC protease (RMCP)‐1 and ‐2 immunofluorescence staining, in order to distinguish RMCP‐1 positive connective tissue MC (CTMC) from RMCP‐2 positive mucosa MC (MMC). mRNA expression of RMCP, c‐kit, and the differentially spliced variants of KL was quantified by RT‐PCR. MC counts per bile ductule decreased in regenerating rat liver tissue at day 3, compared with native livers, and became normal thereafter. Hepatic MC were predominantly of a CTMC phenotype expressing RMCP‐1, as previously published; after hepatectomy, between 76 and 99% of all MC double‐expressed RMCP‐1 and ‐2, compatible with an MMC phenotype. The ratio of the two alternatively spliced mRNAs for KL (KL‐1 : KL‐2), and c‐kit mRNA expression did not differ significantly between regenerating livers and the livers of sham operated animals. These results suggest that hepatic mast cells are restored during liver regeneration after partial hepatectomy in the rat. Restored MC express an MMC phenotype, suggesting migration from outside into the regenerating liver. Alternative splicing of KL is affected by the surgical procedure in general, and, together with its receptor c‐kit, doesn't seem to be involved in liver regeneration after partial hepatectomy in the rat. Further functional studies, and studies in regenerating human livers might offer the possibility of elucidating the role of the hepatic mast cell, and its different protease phenotypes during liver regeneration after surgical loss of liver mass.

A patient with blood group type O accidentally received a blood group B cardiac allograft. He died almost 5 years after transplantation because of transplant vasculopathy. We monitored the expression of ABO-type antigens on the vascular endothelial cells of the cardiac allograft for 44 months, using monoclonal antibodies for immunohistology. The antigenic profile of the graft endothelial cells changed progressively from B to O. This change was first detectable 14 months after transplantation and it was most prominent at the end of the observation period. Thus, we have shown that the allograft blood type of the endothelium of this patient's heart began to change from type B to his own, 1 year after transplantation. By 44 months post-transplantation, it had changed to O type.

The purpose of the current study was to compare undesired thermal and mechanical damage induced by Er:YAG laser irradiation (lambda = 2.94 mum) and orthopedic surgical instruments. Cartilage incisions were made either by a fiber-guided free-running Er:YAG laser (E = 100 and 150 mJ) at a fixed pulse length (tau = 400 mus) and repetition rate of 8 Hz, or by surgical instruments (Draenert drill, punch, trephine, rongeur, Spratt curette, scalpel blade). After treatment, the tissue samples were assessed by a cell viability test to investigate the undesired cartilage damage. The viability test revealed with the Er:YAG laser (150 mJ, double shot) a thermal damage depth of 66 +/- 11 mum aqjacent to a smoothly ablated surface (ablation depth 645 +/- 58 mum). In contrast, tissue damage induced by the surgical instruments was shown to be caused mainly by mechanical impact. The damage zone varied significantly in depth with the type of instrument, from 80 +/- 23 mum with the punch to as much as 579 +/- 63 mum with the Draenert drill. The Er:YAG laser seems to be an excellent tool for smoothing and remodeling rough cartilage surfaces and for cartilage ablation with comparable or even smaller acute tissue damage than that caused by surgical instruments.

Apoptosis plays a role in everything from early development to ageing and in a host of disease states. Studying this important process in the in vivo state is critical, to understand its varied role and to open further avenues of therapeutic intervention. The present paper presents an ex vivo bovine articular cartilage model to study apoptotic and necrotic processes following acute injury. Ex vivo bovine articular cartilage was assessed 1, 3 and 6 days following holmium : YAG laser treatment (780 mJ). Markers to visualize cell viability, caspase‐3 activity, changes in mitochondrial membrane potential and the degree of DNA fragmentation (TUNEL assay) were used alone or in various combinations. Standard histology and transmission electron microscopy (TEM) were also performed for a more comprehensive assessment. A significant progression ( p < 0.05) of ethidium/caspase‐3‐positive signal depth at day 3 preceded a significant increase ( p < 0.05) in TUNEL signal depth by day 6. The mitochondrial matrix marker CMXRos was shown to provide an alternative to calcein‐AM for assessing cell viability. The identification of chondrocyte apoptosis morphology by TEM was not conclusive. Nevertheless, TEM revealed that cells which were clearly necrotic also stained positively for TUNEL, thus indicating the risk of using TUNEL alone for the assessment of apoptosis. The model described here allows the rapid, spatial and temporal determination of cell viability and of apoptotic and necrotic processes in whole‐tissue specimens after acute injury, and permits study of the balance between these events. The assessment of healthy and diseased cartilage and of the effects of surgical, pharmaceutical or in vitro intervention are immediate applications of these protocols. Moreover, this model may be useful for the study of key mechanisms involved in apoptosis or for the establishment of other markers of apoptosis. Copyright © 2002 John Wiley & Sons, Ltd.