bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of aminopeptidase N (APN) on peripheral blood mononuclear cells′ surface as a marker of these cells′ transendothelial migration properties in the course of multiple sclerosis
Authors: Ziąber J. , Baj Z. , Paśnik J. , Chmielewski H. , Tchórzewski H.
Published: July 2002

CD13 AG and CD11a, CD11b, CD18 molecule expression on peripheral blood mononuclear cells (PBMC) were studied as these cells’ adherent or transendothelial migration properties in three different multiple sclerosis (MS) patients groups (total 38): with clinically active MS (acute exacerbation of MS and primary chronic progressive MS (CP‐MS)) and with MS remission. The control group consisted of patients, suffering from other non‐inflammatory neurological diseases (OND). The results of our study suggest that CD11a/CD18 molecules expression on PB lymphocytes, although higher on these cells’ surface in the course of MS as compared to OND, does not differentiate clinical forms of MS. CD11a molecule expression on monocytes did not differ significantly in all tested MS patient groups in comparison to OND. Although the expression of CD11b/CD18 molecules on monocytes’ surface shows their activation in the course of MS, it does not differentiate them either. However, CD13 Ag of APN expression on PBMC surface may be an immunological marker of MS clinical form. CD13 Ag expression may also be a sensitive marker of these cells’ transendothelial migration properties.
Syntheses of two deacetyl‐thymosin β4 analogs and their anti‐inflammatory effect on carrageenin‐induced edema in the mouse paw
Authors: Abiko T. , Ogawa R.
Published: July 2002

Two {Met(0)6}deacetyl‐thymosin β4 analogs containing Phe(4F) or Tyr(Me) at position 12 were synthesized by the manual solid‐phase method, and their anti‐inflammatory effect on carrageenin‐induced edema in the mouse paw was studied. Fluorination of the para‐position of Phe12 resulted in a marked antiinflammatory effect on carrageenin‐induced edema in the mouse paw compared with that of our synthetic {Met(0)6}deacetyl‐thymosin β4, but the other analog,
{Met(0)6, Tyr(Me)12}deacetyl‐thymosin β4, showed
a marked reduction of the anti‐inflammatory effect.
Erythromycin and clarithromycin modulation of growth factor‐induced expression of heparanase mRNA on human lung cancer cells in vitro
Authors: Sasaki Masahiro , Ito Takefumi , Kashima Masayuki , Fukui Shin , Izumiyama Noriko , Watanabe Akiko , Sano Masaaki , Fujiwara Yosimasa , Miura Mamoru
Published: July 2002

Heparanase activity is correlated with the metastatic potential of several cancer cells and is a key enzyme in the breakdown of tissue barriers. It is also involved in the regulation of growth factor and cytokine activity. However, little is known about the factors that induce heparanase in cancer cells. We investigated the effect of three growth factors, plateletderived growth factor (PDGF), hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF), on heparanase mRNA induction in lung cancer cells in vitro. In addition, we examined the effect of erythromycin (EM) and clarithromycin (CAM), which are 14‐membered ring macrolide antibiotics that act as biological response modifiers, on the expression of heparanase mRNA induced by growth factors.

PDGF, HGF and bFGF stimulated cell migration activity and enhanced the expression of heparanase mRNA in the human lung adenocarcinoma cell line A549. Via different mechanisms, EM and CAM modulate the induction by these factors of heparanase mRNA expression on A549 cells. EM also significantly suppressed A549 cell migration induced by PDGF and HGF, and CAM significantly suppressed A549 cell migration induced by bFGF.

The results suggest that the growth factors PDGF, HGF and bFGF are important inducers of heparanase in potentially invasive and metastatic cancer cells. The suppressive effect of heparanase mRNA expression by EM and CAM may have interesting therapeutic applications in the prevention of metastasis.
Role of ICAM‐1 in the aggregation and adhesion of human alveolar macrophages in response to TNF‐α and INF‐γ
Authors: Sasaki Masahiro , Namioka Yuriko , Ito Takefumi , Izumiyama Noriko , Fukui Shin , Watanabe Akiko , Kashima Masayuki , Sano Masaaki , Shioya Takanobu , Miura Mamoru
Published: July 2002

Intracellular adhesion molecule‐1 (ICAM‐1)‐mediated cell‐cell adhesion is thought to play an important role at sites of inflammation. Recent evidence suggests that ICAM‐1 surface expression on alveolar macrophages is increased in pulmonary sarcoidosis and that inflammatory granuloma formation is characterized by the aggregation of macrophages. The present study shows that ICAM‐1 expression is significantly elevated on alveolar macrophages from patients with sarcoidosis in response to tumor necrosis factor‐α (TNF‐α) and interferon‐ γ (INF‐γ) compared with healthy controls. Aggregation and adhesion were significantly increased in alveolar macrophages treated with TNF‐α and INF‐γ, and significantly inhibited in those pretreated with a monoclonal antibody to ICAM‐1. Similarly, aggregation and adhesion were inhibited in macrophages treated with heparin, which then exhibited a wide range of biological activities relevant to inflammation. These results suggested that the surface expression of ICAM‐1 on alveolar macrophages in response to TNF‐α and INF‐γ is important in mediating aggregation and adhesion. Additionally, heparin may be useful for developing novel therapeutic agents for fibrotic lung disease.
Elevation of serum soluble E‐selectin and VCAM‐1 in severe asthma
Authors: Hamzaoui Agnes , Ammar Jamel , El Mekki Fethi , Borgi Olfa , Ghrairi Hédia , Ben Brahim Mounira , Hamzaoui Kamel
Published: July 2002

Objective: To investigate the significance of circulating adhesion molecules associated with leucocyteendothelial cell interactions in asthma, serum levels of soluble E (sE)‐selectin, soluble P (sP)‐selectin, soluble L (sL)‐selectin, and soluble vascular cell adhesion molecule‐1 (sVCAM‐1) were measured in mild, moderate and severe asthma.

Method: Serum levels of sE‐selectin, sP‐selectin, sLselectin, and sVCAM‐1 were measured in 32 women with asthma and 30 healthy donors using an enzymelinked immunosorbent assay method. Twenty patients were suffering from severe asthma, and 12 from mild/moderate asthma.

Results: Serum sE‐selectin and sVCAM‐1 levels from patients with asthma were significantly higher than those observed in healthy donors (p < 0.01). The levels of sP‐selectin were the same as those of controls. The level of sE‐selectin exhibited an important increase in the severe asthmatic patients compared with mild/moderate asthma (p < 0.01). The sVCAM‐1 level was increased in severe asthma when compared with healthy controls. There was no correlation between the levels of soluble selectins and the age of the patients. A significant correlation was found between sE‐selectin and sVCAM‐1 levels.

Conclusion: These data indicate that circulating soluble forms of the selectins may have different kinetics during the clinical course of asthma, suggesting that they may reflect different inflammatory pathways in severe asthma. Both sVCAM‐1 and sE‐selectin may be useful immunological markers for monitoring disease activity in asthma.
Sequential release of TNFα and phospholipase A2 in a rat model of LPS‐induced pleurisy
Authors: Cicala C. , Bucci M. , D′Acquisto F. , Parente L. , Cirino G.
Published: July 2002

The levels of extracellular phospholipase A2 (sPLA2) and TNFα, and cell accumulation were measured in the pleural washings obtained at different times following the induction of Escherichia coli lipopolysaccharide (LPS, 100 μg/cavity) pleurisy in rats. TNFα peaked at 2 hours (3036 ± 160.3 units/ml) and decreased thereafter. Conversely, levels of sPLA2 peaked at 48 hours (1.97 ± 0.64 ng/ml) and were increased further (14.02 ± 4.16 ng/ml) by pretreatment with anti‐TNFα antibody. Cell accumulation was not affected by antibody pretreatment. These data indicate that the sPLA2 enzyme is involved in LPS‐induced pleurisy. The enzyme seems not to be stimulated by TNFα which may be involved in the downregulation of
sPLA2 in this model of inflammation.
Prostanoids in bronchoalveolar lavage fluid do not predict outcome in congenital diaphragmatic hernia patients
Authors: Ijsselstijn H. , Zijlstra F. J. , De Jongste J. C. , Tibboel D.
Published: July 2002

Vasoactive prostanoids may be involved in persistent pulmonary hypertension (PPH) in infants with a congenital diaphragmatic hernia (CDH). We hypothesized that increased levels of prostanoids in bronchoalveolar lavage (BAL) fluid would predict clinical outcome. We measured the concentrations of 6‐keto‐prostaglandin F1α (6‐keto‐PGF1α), thromboxane B2 (TxB2), protein, albumin, total cell count, and elastase‐α1‐proteinase‐inhibitor complex in BAL fluid of 18 CDH patients and of 13 control subjects without PPH. We found different concentrations of prostanoids in BAL fluid of CDH patients with PPH: infants with a poor prognosis had either high levels of both 6‐keto‐PGF1α and TxB2 compared to controls, or high levels of
6‐keto‐PGF1α only. TxB2 levels showed a large variability in all CDH patients irrespective of outcome. We conclude that prostanoid levels in BAL fluid do not predict clinical outcome in CDH patients.
Human colonic intraepithelial lymphocytes regulate the cytokines produced by lamina propria mononuclear cells
Authors: Hoang P. , Dehennin J. P. , Li Li , Sibille C. , Geubel A. , Vaerman J. P.
Published: July 2002

Using an in vitro autologous human system, the immunomodulatory function of colonic intraepithelial lymphocytes (IEL) on cytokine production by lamina propria mononuclear cells (LPMNC) has been investigated. In contrast to LPMNC, colonic IEL produced only low amounts of IL‐10, interferon‐γ and interleukin‐2. However, co‐culture experiments (IEL + LPMNC) have shown that IEL can enhance the PHA‐induced synthesis of IL‐2 and interferon‐γ, but not IL‐10 by LPMNC. Using a transwell filter culture system apparatus, this effect was shown not to require a cell‐to‐cell interaction. Thus, IEL in vitro may modulate the cytokine synthesis of LPMNC, through the production of soluble factors. This may prove highly relevant in the in vivo immune activation of the gastrointestinal mucosa.
Perpetuation of inflammation associated with experimental arthritis: the role of macrophage activation by neutrophilic myeloperoxidase
Authors: Gelderman Monique P. , Stuart Rodney , Vigerust David , Fuhrmann Steven , Lefkowitz Doris L. , Allen Robert C. , Lefkowitz Stanley S. , Graham Susan
Published: July 2002

Rheumatoid arthritis (RA) is characterized by an abnormal cellular and cytokine infiltration of inflamed joints. This study addresses a previously unrecognized interaction between neutrophilic‐myeloperoxidase (MPO) and macrophages (MΦ) which could explain the perpetuation of inflammation associated with RA. A monoarticular arthritis was induced in female Lewis rats by injection of streptococcal cell wall extracts (PG‐APS). After swelling and erythema subsided, joints were re‐injected with one of the following: porcine MPO or partially inactivated MPO (iMPO). Injection with either MPO or iMPO induced a ′flare′ of experimental RA. Blocking the MΦ‐mannose receptor by mannans, ablated exacerbation of disease. These results indicate that MPO or iMPO can play a pivotal role in the perpetuation but not initiation of this RA model.
Transient degradation of NF‐κB proteins in macrophages after interaction with mast cell granules
Authors: Ito Noriko , Li Yuai , Suzuki Tsuneo , Stechschulte Daniel J. , Dileepan Kottarappat N.
Published: July 2002

The exposure of the macrophage cell line, J774 to mast cell granules (MCG) led to the form ation of altered nuclear transcription factor proteins (NFκBx), which had faster electrophoretic mobility than the p50 homodimer of NF‐κB, but retained comparable DNA binding capacity. Antibodies to N‐terminal peptides of p50, p52, p65 or c‐Rel supershifted only a fraction of NF‐κBx. Western blot analyses revealed that nuclear p65 and c‐Rel were progressively degraded after exposure to MCG, whereas nuclear p50 appeared to be unaffected. In contrast, cytoplasmic p50, p65, c‐Rel as well as IkBα remained intact after MCG treatment, although p52 was clearly degraded. In comparison to J774 cells, incubation of m ouse peritoneal macrophages with MCG resulted in more extensive alterations to NF‐κB proteins. The alterations in NF‐κB proteins did not affect the expression of inducible nitric oxide synthase (iNOS) or TNF‐α mRNA in J774 cells. These data indicate that exposure of J774 cells to MCG leads to generation of altered nuclear p52, p65 and c‐Rel, which retain intact N‐terminal peptides, specific oligonucleotide binding and transactivating activity. On the other hand, in peritoneal macrophages, MCG induce more extensive modifications to NF‐κB proteins with associated inhibition of iNOS or TNF‐α mRNA expression.
Epithelium‐dependent regulation of airways smooth muscle function. A histamine‐nitric oxide pathway
Authors: Gourgoulianis Konstantinos , Iliodromitis Zoe , Hatziefthimiou Apostolia , Molyvdas Paschalis-Adam
Published: July 2002

The airway epithelium is responsible for the production of a number of arachidonic acid and nonprostanoid inhibitory factors. Epithelium synthesises nitric oxide (NO) which may be important in regulating the function of airways smooth muscles. We studied in vitro the effect of histamine (100 nM100 μ M) which increases the NO release on rabbit airway smooth muscles induced by 80 mM KCl in the presence or not of 10-5 Methylene blue (MB) (inactivator of guanylate cyclase) or N(G)‐monomethyl L‐arginine (L‐NMMA), a NOS inhibitor. All experiments were done in tracheal muscle strips from 28 rabbits with epithelium and after epithelium removal. The additional use of histamine (1 μ M) on KCl contraction induced a relaxation of 10% of the initial contraction. The additional use of L‐NMMA decreased the relaxation to 5% of initial contraction. MB rather than L‐NMMA increased the contraction significantly (p < 0.01). Epithelium removal increased the contraction induced by KCl (80 mM) and histamine (1 μ M) by about 30% (p < 0.001). NO release especially from epithelium regulates the airways smooth muscle functions. Damage to the epithelium may contribute to an increase in airways sensitivity, observed in asthma.
Effect of mast cell granules on the gene expression of nitric oxide synthase and tumour necrosis factor‐α in macrophages
Authors: Li Y. , Nguyen T. D. , Stechschulte A. C. , Stechschulte D. J. , Dileepan K. N.
Published: July 2002

Previous studies have shown that mast cell granules (MCG) inhibit numerous macrophage functions including tumour cytotoxicity, superoxide and nitric oxide (NO) production, and FCγ2a receptor‐mediated phagocytosis. In this study, the effect of MCG on macrophage TNFα and nitric oxide synthase (iNOS) mRNA expression, and the production and fate of TNFα were examined. Upon activation with LPS+IFNγ, macrophages expressed both TNFα and iNOS mRNA and produced both TNFα and NO. Co‐incubation of LPS+IFNγ‐activated macrophages with MCG resulted in dose‐dependent inhibition of iNOS mRNA expression. TNFα production in the activated macrophages was decreased by MCG, which was associated with a reduction in TNFα mRNA expression. MCG were also capable of degrading both macrophage‐generated and recombinant TNFα. The direct effect of MCG on TNFα was partially reversed by a mixture of protease inhibitors. These results demonstrate that MCG decrease the production of NO and TNFα by inhibiting macrophage iNOS and TNFα gene expression. Furthermore, MCG post‐transcriptionally alter TNFα levels via proteolytic degradation.
Effects of acanthoic acid on TNF‐α gene expression and haptoglobin synthesis
Authors: Kang H-S. , Song H. K. , Lee J-J. , Pyun K-H. , Choi I.
Published: July 2002

Tumour necrosis factor‐α (TNF‐α) is a major proinflammatory cytokine inducing the synthesis and release of many inflammatory mediators. It is involved in immune regulation, autoim mune diseases, and inflammation. Our previous study demonstrated that acanthoic acid, (‐)‐pimara‐9(11), 15‐dien19‐oic acid, a pimaradiene diterpene isolated from Acanth opanax koreanum, inhibited TNF‐α production. To extend our understanding of inhibitory effects of acanthoic acid on TNF‐α production, its effects on TNF‐α gene expression was tested. Based on the results from RT‐PCR and promoter analysis of TNF‐α, it was found that acanthoic acid suppressed TNF‐α gene expression. But the same concentration of acanthoic acid had no effect on IL‐6 gene expression. Haptoglobin is an acute phase protein which is induced by TNF‐α. When liver cells were treated with acanthoic acid, haptoglobin synthesis was blocked by acanthoic acid. These data confirmed that acanthoic acid inhibited gene expression and biological function of TNF‐α.
Diminished nitroprusside‐induced relaxation of inflamed colonic smooth muscle in mice
Authors: van Bergeijk J. D. , van Westreenen H. , Adhien P. , Zijlstra F. J.
Published: July 2002

The dextran sodium sulphate (DSS) induced colitis in mice was used as a experimental model to study the contractility of murine longitudinal colonic smooth muscle during inflammation. Smooth muscle segments
of proximal, middle and distal colon were mounted in organ baths . Smooth muscle contraction was induced by carbachol, showing an aboral increase in activity, where as in the inflamed middle colonics egment a marked decr ease in activity was observed. The dilatative effect of sodium‐nitroprus ‐side (SNP) as a nitric oxide donor was investigated after precontraction by carbachol. Both in normal and DSS segments administration of SNP to isolated
mouse colonic smooth muscle preparations caused regional differences in relaxation, the highest relaxation seen in normal proximal colonic tissue. However, this relaxation was markedly reduced in inflamed proximal preparations , associated with a diminished cGMP contents .
BN 52021 (a platelet activating factor‐receptor antagonist) decreases alveolar macrophage‐mediated lung injury in experimental extrinsic allergic alveolitis
Authors: Pérez-Arellano J-L. , Martón T. , López-Novoa J-M. , Sánchez M-L. , Montero A. , Jiménez A.
Published: July 2002

Several lines of research indirectly suggest that platelet activating factor (PAF) may intervene in the pathogenesis of extrinsic allergic alveolitis (EAA). The specific aim of our study was to evaluate the participation of PAF on macrophage activation during the acute phase of EAA in an experimental model of this disease developed in guinea pigs. Initially we measured the concentration of PAF in bronchoalvedar lavage fluid, blood and lung tissue. In a second phase we evaluate the participation of PAF on alveolar macrophage activation and parenchymal lung injury. The effect of PAF on parenchymal lung injury was evaluated by m easuring several lung parenchymatous lesion indices (lung index, bronchoalvedar lavage fluid (BALF) lactic hydrogenase activity and BALF alkaline phosphatase activity) and parameters of systemic response to the challenge (acute phase reagents). We observed that induction of the experimental EAA gave rise to an increase in the concentration of PAF in blood and in lung tissue. The use of the PAF‐receptor antagonist BN52021 decreases the release of lysosomal enzymes (β‐glucuronidase and tartrate‐sensitive acid phosphatase) to the extracellular environment both in vivo and in vitro. Furthermore, antagonism of the PAF receptors notably decreases pulmonary parenchymatous lesion. These data suggest that lung lesions from acute EAA are partly mediated by local production of PAF.
Abnormal directed migration of blood polymorphonuclear leukocytes in rheumatoid arthritis. Potential role in increased susceptibility to bacterial infections
Authors: Aglas F. , Hermann J. , Egger G.
Published: July 2002

Rheumatoid arthritis (RA) patients are at higher risks of bacterial infection than healthy subjects. Polymorphonuclear leukocytes (PMN) are the first line of nonspecific cellular defence against these infections. We tested the hypothesis that abnormal directed migration of PMN may be one reason for the increased infection rate of RA patients. PMN migration was investigated in 68 peripheral blood samples of 15 RA patients compared with 64 samples of healthy controls in a novel whole blood in vitro membrane filter assay. The migration of PMNs from RA patients and controls was stimulated using the bacterial chemoattractant N‐formyl‐methionyl‐leucylphenylalanine (fMLP). Unstimulated PMN migration of RA patients was increased compared with healthy controls as measured by the following parameters: (a) absolute number of migrant PMNs (1954 ± 87 vs. 1238 ± 58 PMN/mm2), (b) percentage of PMNs migrated into the filter (total migration index, TMI) (28.6 ± 0.9 vs. 24.0 ± 0.8 %), (c) the distance half the migrating PMNs had covered (distribution characteristic, DC) (22.6 ± 1.1 vs. 16.1 ± 0.6 mm) and (d) the product of TMI and DC (neutrophil migratory activity, NMA) (669.0 ± 45.0 vs. 389.0 ± 18.9). fMLP stimulated PMNs of RA patients showed defective migration compared to unstimulated samples as shown by (a) a reduced number of migrant PMNs (1799 ± 93 PMN/mm2), (b) lower TMI (26.1 ± 0.9 %), (c) unremarkable altered distribution characteristic (22.9 ± 0.8 mm) and (d) significant reduced migratory activity (600.0 ± 30.0). Our data suggest that the high incidence of infections in RA patients may partly be caused by defective migratory activity of PMNs to bacterial chemoattractants as demonstrated by fMLP.
Circulating TCR γδ Cells in the Patients with Systemic Lupus Erythematosus
Authors: Robak Ewa , Błoński Jerzy Z. , Bartkowiak Jacek , Niewiadomska Hanna , Sysa-Jędrzejowska Anna , Robak Tadeusz
Published: July 2002

Systemic lupus erythematosus (SLE) is a disorder with a wide range of immunological abnormalities. The results of the studies undertaken in the last decade indicated that SLE pathogenesis was mainly connected with the breakdown of the activation control of B and T cells, generating humoral or cell‐mediated responses against several self‐antigens of affected cells. The last studies demonstrate that the role of γδ T lymphocytes in autoimmune diseases can be especially important. Flow cytometry techniques were used to investigate the number and percentage of TCR γδ T cells and their most frequent subtypes in peripheral blood of 32 patients with SLE and 16 healthy volunteers. We also correlated TCR γδ cells number with the level of T CD3+, T CD4+, T CD8+, and NK (CD16) cells (cytometric measurements) and SLE activity (on the basis of clinical investigations). Our studies were preliminary attempts to evaluate the role of that minor T cell subpopulation in SLE. Absolute numbers of cells expressing γδ TCR in most SLE blood specimens were significantly lower than in the control group (P < 0.006). However, since the level of total T cell population was also decreased in the case of SLE, the mean values of the percentage γδ T cells of pan T lymphocytes were almost the same in both analysed populations (7.1% vs 6.3%, respectively). In contrast to Vδ2+ and Vγ9+ subtypes of pan γδ T cells, Vδ3+ T cells number was higher in SLE patients (20 × 10 cells/μl) than in healthy control group (2 × 2 cells/μl) (P = 0.001). However, we found no differences between the numbers of pan γδ T lymphocytes and studied their subtypes in the patients with active and inactive disease. These cell subpopulations were doubled in the treated patients with immunosuppressive agents in comparison with untreated ones; however, data were not statistically significant. Our study indicated that Vδ3+ subtype of γδ T cells seems to be involved in SLE pathogenesis; however, we accept the idea that the autoimmunity does not develop from a single abnormality, but rather from a number of different events.
Killer‐Cell Inhibitory Receptors, CD158a/b, are Upregulated by Interleukin‐2, but not Interferon‐γ or Interleukin‐4
Authors: Kogure Toshiaki , Fujinaga Hiroshi , Niizawa Atsushi , Hai Le Xuan , Shimada Yutaka , Ochiai Hiroshi , Terasawa Katsutoshi
Published: July 2002

Although it is now accepted that killer‐cell inhibitory receptors (KIRs), which were molecularly cloned in 1995, deliver negative signals to natural killer (NK) cells regarding the recognition of target cells, it is still unclear how the expression of these receptors on lymphocytes is regulated. Therefore, we investigated the regulation of expression of representative KIRs, CD158a and CD158b, by cytokines such as interleukin‐2 (IL‐2), IL‐4 and interferon‐γ (IFN‐γ). Neither IL‐4 nor IFN‐γ affected the expression of CD158a/b, but incubation for 48 h with IL‐2, which enhances the killer activity of NK cells, upregulated the expression of the KIRs. This upregulation by IL‐2 was also observed in CD16‐positive cells sorted from total lymphocytes. In contrast, IL‐4, which is a downregulator of IL‐2‐induced killer responses, did not change the level of CD158a/b expression when added after the IL‐2 treatment. These findings suggest that IL‐2 plays an important role in the regulation of CD158a/b expression, and might be involved in controlling NK activity via regulating expression of these molecules.
A Possible Involvement of Ion Transporter in Tumor Necrosis Factor α and Cycloheximide‐Induced Apoptosis of Endothelial Cells
Authors: Fujita Hiroshi , Morita Ikuo , Murota Sei-itsu
Published: July 2002

We examined the tumor necrosis factor α (TNFα)‐induced apoptosis of vascular endothelial cells from the standpoint of ion channels. Cultured vascular endothelial cells from bovine carotid artery were used. Apoptosis was determined by a propidium iodide assay. Treatment of the endothelial cells with TNFα and cycloheximide for 6h induced nuclear fragmentation in a TNFα dose‐dependent manner (1‐10 ng/ml). Concomitant treatment of endothelial cells with TNFα at a dose of 10ng/ml and cycloheximide at a dose of 10 micro g/ml elicited endothelial cell apoptosis as high as 23.4±4.1% at 6h after administration. However, 10 ng/ml TNFα alone elicited a little apoptosis at 6h after its administration (% apoptosis=4.1±0.8%). Cycloheximide (10 μg/ml) did not induce apoptosis at all. Concomitant treatment of endothelial cells with 1 m mol/l of 4,4‐diisothiocyanatostilbene‐2,2‐disulfonic acid, which is a chloride bicarbonate exchanger blocker, partially inhibited the TNFα and cycloheximide‐induced endothelial cell apoptosis. On the other hand, endothelial cell apoptosis due to TNFα and cycloheximide was completely inhibited by benzyloxycarbonyl‐Asp‐CH2OC(O)‐2,6‐dichlorobenzene (50 μmol/l), an inhibitor of caspase. Moreover, pyrrolidine dithiocarbanate, an inhibitor of nuclear factor kappa B (NF‐κB), also suppressed endothelial cell apoptosis induced by TNFα and cycloheximide completely. These findings suggest that the endothelial cell apoptosis induced by TNFα and cycloheximide is closely related to not only chloride ions, but also both NF‐κB and caspase activation. That is to say, there is a possibility that chloride ions or bicarbonate (pH) may play an important role in signal transduction such as NF‐κB and caspase activation in the apoptosis induced by TNFα and cycloheximide.
Interrelationship of the Kinin System, Nitric Oxide and Eicosanoids in the Antigen‐Induced Arthritis in Rabbits
Authors: Palácios Fernando Alfonso Salamea , Novaes Gilberto Santos , Guzzo Maria Luiza , Laurindo Ieda Maria Magalhães , de Mello Suzana Beatriz Verissimo
Published: July 2002

The aim of the present study was to investigate the interrelationship of the kinin system, nitric oxide and eicosanoids in the acute phase of antigen‐induced arthritis (AIA) in rabbits. The arthritis was induced in immunized rabbits and the following parameters were evaluated 24 hours later: leukocyte influx (total and differential white cell count), vascular permeability (Evans′s blue method), and synovial PMN cell infiltrate. PGE2 and LTB4 (radioimmunoassay) levels were quantified in the synovial fluid. The animals were pre‐treated with 20 mg/kg/day during 14 days with L‐NAME or D‐NAME and/or Enalapril (0.12 mg/ kg/day‐14 days), and/or the B2 antagonist of Bradykinin HOE 140 (0.9 mg/kg). Our results showed that L‐NAME was effective in the prevention of AIA with reduction of all inflammatory parameters analyzed. Enalapril partially reverted the L‐NAME anti‐inflammatory effects. The simultaneous treatment with HOE 140 abolished this reversion and returned the inflammatory parameters to the levels observed in L‐NAME treated animals. Our results suggest that pressoric alterations induced by L‐NAME could not account for all its anti‐inflammatory action in this model of experimental arthritis. Additionally the contribution of the kinin system in AIA was characterized as well as its interaction with eicosanoids and nitric oxide.
Prognostic Markers in Chondrosarcoma: Evaluation of Cell Proliferation and of Regulators of the Cell Cycle
Authors: Scully Sean P. , Layfield Lester J. , Harrelson John M.
Published: July 2002

Purpose. The prognosis, treatment principles and prediction of clinical outcome of patients with chondrosarcoma
currently rest on histologic grading which is somewhat ambiguous due to difficulty in pathologic interpretation of this
neoplasm. Immunohistochemistry, flow cytometry and oncogene/tumor suppressor gene expression have been examined
as alternative indices to predict the biologic behavior of these tumors. Because of partial successes obtained with flow
cytometry and because of the improvement in predicting recurrence offered by examining the S‐phase fraction, we
undertook the current study to determine if expression of specific regulators of the cell cycle would act as prognostic
indicators for these patients.

Subjects/methods. We examined archival pathologic specimens from 39 patients with at least 2 years′ clinical follow‐up for
the presence of p53, Rb, src and MIB‐1 by immunohistochemistry and correlated this with clinical histories and incidence
of recurrence.

Results. While Rb, p53 and src gene products were identified to a variable extent in these specimens, there was no
prognostic significance to their expression. In contrast, MIB‐1, an epitope expressed only during semiconservative
replication and an accepted marker of cell proliferation, served as a significant prognostic indicator. MIB‐1 staining was
present in 14.5% of tumor cells in all specimens (range 0–59%). When MIB‐1 staining was examined with respect to
disease recurrence, there was a statistically significant association between staining and histologic grade (p < 0.05) as well
as event‐free survival (p < 0.02). Comparing survival curves stratified by MIB‐1 expression, there was a significant decrease
in event‐free survival associated with increasing MIB‐1 indices (p < 0.003). Covariates that were associated with event‐free
survival include histologic grade (p = 0.025) and stage (Musculoskeletal Tumor Society) (p = 0.014). There was no
statistical association with patient age (p = 0.15), tumor size (p = 0.47), tumor histology (p = 0.62) or anatomic location
(p = 0.316).

Discussion. These results indicate that determination of the proliferation index by MIB‐1 immunostaining may serve as
a useful adjunct to current histopathologic classification. Patients with a high proliferation index may benefit from
established adjuvant therapies or experimental approaches including immunotherapy or biologic modulation.
Phase II Study of Paclitaxel in Patients With Soft Tissue Sarcomas
Authors: Patel Shreyaskumar R. , Linke Kaye A. , Burgess M. Andrew , Papadopoulos Nicholas E. , Plager Carl , Jenkins Jan , Benjamin Robert
Published: July 2002

Purpose. Patients with soft tissue sarcoma (STS) who have previously received standard chemotherapy including
adriamycin (doxorubicin), ifosfamide, cyclophosphamide and DTIC (dacarbazine) have very limited therapeutic options.
It is important to identify new drugs with some activity in this disease and we therefore undertook this trial to determine
the antitumor activity of paclitaxel (Taxol).
Methods. We conducted a phase II study of paclitaxel in patients with STS who had received prior standard
chemotherapy. Paclitaxel was administered at a starting dose of 200 mg m−2 as a 24‐h infusion with STS premedication,
every 21 days or upon hematologic recovery (absolute granulocyte count (AGC) ≥ 1500/μl,
platelets ≥ 100 000/μl).
Neupogen was not used routinely. The study was conducted based on a two‐stage design proposed by Simon. Responses
were assessed radiographically using standard criteria.
Results. Nineteen eligible patients were treated in the first stage of the study. The median age was 50 years (range 20–68
years), and there were nine females and 10 males with Zubrod performance status of 1 or 2. One patient achieved a minor
response. Median AGC nadir was 0.1/μl on day 12 with absolute neutropenia lasting 5 days. Median platelet nadir was
171 000/μl on day 9. There were no grade 3/4 non‐hematologic toxicities and no deaths related to treatment.
Discussion. Paclitaxel, at this dose and schedule, is well tolerated but inactive in this
patient population.
Chest Wall Sarcoma: Outcome in 22 Patients After Resection Requiring Thoracic Cage Reconstruction
Authors: Jönsson Per , Gyllstedt Erik , Hambraeus Göran , Lillogil Ramon , Rydholm Anders
Published: July 2002

Purpose. To evaluate the outcome after resection of malignant chest wall sarcoma, requiring reconstruction of the chest wall.

Subjects. Twenty‐two patients, 15 with primary tumours, were operated on in our institution between 1983 and 1996. Four patients underwent surgery after a previous intralesional or marginal excision and three patients because of a local recurrence.

Methods. The tumour was resected ‘en bloc’, including skin, muscle and thoracic skeleton. When necessary, adjacent organs invaded by the tumour, such as lung, pericardium and diaphragm, were also removed to obtain a wide margin. Reconstruction of the chest wall was performed with Marlex mesh (n=9), methylmethacrylate cement (n=2) or a Marlex methylmethacrylate ‘sandwich’ (n=11).

Results. The median tumour size was 9.5 (2–20) cm. The most common type of tumour was chondrosarcoma (12 cases). No patient died in hospital. Five patients required reoperation because of complications, two patients because of loosening of the acrylate prosthesis, two because of necrosis of soft tissue coverage and one was reoperated because of bleeding. Four patients died of generalized tumour disease between 5 and 77 months after surgery and one patient died of a local recurrence 32 months after the primary operation. Seventeen patients are alive, with a median follow‐up of 36 (4–162) months. Microscopic radicality (negative margin) was achieved in 17 patients but 5 of these had local recurrences. Two of five patients with positive margins had a local recurrence of the tumour. Of the seven patients with local recurrences, two also developed metastases.

Discussion. Large chest wall sarcomas can be successfully resected and the chest wall reconstructed with low morbidity and mortality.
A Comparison of Outcome of Osteoarticular Allograft Reconstruction and Shoulder Arthrodesis Following Resection of Primary Tumours of the Proximal Humerus
Authors: Probyn L. J. , Wunder J. S. , Bell R. S. , Griffin A. M. , Davis A. M.
Published: July 2002

Purpose. The purpose of this study was to compare the oncologic, reconstructive and functional outcomes of patients who underwent osteoarticular allograft reconstruction with those who underwent arthrodesis for a primary malignant bone tumour of the proximal humerus.

Patients. Eleven patients were treated with osteoarticular allograft reconstruction of the proximal humerus. Five of these reconstructions failed, necessitating revision to a secondary arthrodesis. Five patients underwent arthrodesis as a primary reconstruction, for a total of ten patients in the arthrodesis group.

Methods. End points included local and systemic disease recurrence, complications and functional outcome (measured using the 1987 and 1993 Musculoskeletal Tumour Society Rating Scales (MSTS) and the Toronto Extremity Salvage Score (TESS)).

Results. One patient died of systemic disease 2 years post‐operatively and one patient had an axillary node recurrence resected 10 months post‐operatively and remains free of disease 53 months later. The other 14 patients were alive with no evidence of disease at the time of the last follow‐up. Complications after the osteochondral allografts (n=11) included two infections, four fractures and three subluxations in eight patients. Six of these patients required removal of the allograft; one had a repeat osteochondral allograft and five were converted to an arthrodesis. Complications after arthrodesis in the ten patients (five primary and five secondary arthrodeses) included two non‐unions, one infection and one fracture in three patients. Patients who underwent shoulder arthrodesis scored better in all outcome measures and this was statistically significant in the MSTS 1993 (p=0.001, Mann–Whitney U Test).

Discussion. In this study, there was a trend towards improved function following arthrodesis compared to osteochondral allograft reconstruction following proximal humerus bone tumour resection.
Long‐Term Neurotoxicity of Chemotherapy in Adolescents and Young Adults Treated for Bone and Soft Tissue Sarcomas
Authors: Earl Helena M. , Connolly Sean , Latoufis Christos , Eagle Karen , Ash Catherine M. , Fowler Clare , Souhami Robert L.
Published: July 2002

Purpose. To study the long‐term neurotoxicity of chemotherapy in adolescents and young adults treated for bone and soft tissue sarcomas.

Patients and Methods. Thirty‐six adolescents and young adults (median age 17 years) were examined following chemotherapy for bone and soft tissue sarcomas. Twenty‐nine (29/36) had received cisplatin (median 400 mg/m2), 15/36 ifosfamide (median 20 g/m2), and 12/36 vincristine (median 16 mg). Neurotoxicity was assessed at a median of 8 months (range, 1–54 months) after completion of chemotherapy by clinical examination, nerve conduction studies, audiograms and autonomic function tests. The same nerve conduction studies were carried out in 20 normal volunteers to define normal ranges in this age group.

Results. Sixteen patients (44%) had a significant reduction in deep tendon reflexes, and this clinical parameter correlated well with abnormalities detected in nerve conduction studies. Vibration perception threshold (VPT) was raised in 20/36 patients (55%) and this was the most sensitive single test in the assessment of neuropathy. There was a significant
correlation between VPT and cumulative cisplatin dose received in mg/m2 (r=0.607, p<0.01). Ten of 29 patients (35%) had abnormal nerve conduction studies with a pattern characteristic of sensory axonal neuropathy. No patient complained of auditory symptoms, but minor high tone hearing loss was detected by audiograms in 5/28 patients who had received cisplatin. No patients had symptoms of autonomic neuropathy, but autonomic function tests showed minor abnormalities
in 4/22 patients tested, and all had received cisplatin.

Conclusions. This study demonstrates significant, although asymptomatic, long‐term neurotoxicity of cisplatin in adolescents and young adults receiving chemotherapy for bone and soft tissue sarcomas. Follow‐up studies are planned to assess whether these neurological deficits improve with time.
A study of idiom comprehension in children with semantic‐pragmatic difficulties. Part I: Task effects on the assessment of idiom comprehension in children
Authors: Kerbel Debra , Grunwell Pam
Published: July 2002

In the apparent absence of suitable measures of idiom comprehension in normally developing and clinical populations, this study examined the relationship between a newly developed play task and a more conventional definition task. On the play task, children listened to a 1 5‐minute, tape‐recorded story into which were embedded 12 common idioms drawn from recordings of classroom teaching and children's television. As the story was then played again, sentence by sentence, the children were required to act it out using a play set and props. For each idiom, it was possible to act out either the idiomatic or literal meaning, but only the idiomatic meaning made sense in the context. The children's actions were video‐taped and then played back to the child during the definition task. For this task, the video was stopped after each idiom occurred and the children were asked what they thought each idiom meant. Four groups of children were included. Twenty‐six children (aged between 6‐11 years), considered to have semantic‐pragmatic difficulties, were compared with two groups of mainstream children (aged 6;6‐7;6 and 10;6‐11;6, respectively) and with a group of children (aged between 8‐11 years) diagnosed with (other) language disorders not primarily of a semantic or pragmatic nature. The results indicate that the definition task underestimated common‐idiom comprehension in normally developing children and, in particular, in children diagnosed with semantic‐pragmatic difficulties or (other)language disorders. Furthermore, a significant difference in idiom comprehension between the two clinical groups evidenced on the play task was entirely masked on the definition task. It appeared that the expressive and metalinguistic demands of the definition task had a greater negative effect on the group of children with language disorders than on the children with semantic‐pragmatic difficulties. Possible mechanisms through which the play task might have overestimated or underestimated idiom comprehension in these groups are examined and discussed. Although the nature of the play task probably facilitated idiom comprehension in these children, analysis supports the results of the play task being a reflection of true ability rather than an overestimation of idiom comprehension. A considerable incidence of false positive results on the definition task, coupled with its masking of significant differences in idiom comprehension across clinical groups militates againstthe use ofdefinition tasks to assess idiom comprehension in research and clinical settings. In comparison with definition and multiple choice methodologies, the play task emerged as a more valid measure of common‐ and concrete‐idiom comprehension in normally developing and clinical child populations.
Induction of Growth Cone Formation by Transient and Localized Increases of Intracellular Proteolytic Activity
Authors: Ziv Noam E. , Spira Micha E.
Published: July 2002

The formation of a growth cone at the tip of a transected axon is a crucial step in the subsequent regeneration of the amputated axon. During this process, the transected axon is transformed from a static segment into a motile growth cone. Despite the importance of this process for regeneration of the severed axon, little is known about the mechanisms underlying this transformation.

Recent studies have suggested that Ca2+-activated proteinases underlay the morphological remodeling of neurons after injury. However, this hypothesis was never tested directly. Here we tested the ability of transient and localized increases in intracellular proteolytic activity to induce growth cone formation and neuritogenesis. Minute amounts of the proteinase trypsin were microinjected into intact axonal segments or somata of cultured Aplysia neurons, transiently elevating the intracellular protease concentration to 13–130 nM in the vicinity of the injection site. Such microinjections were followed by the formation of ectopic growth cones and irreversible neuritogenesis. Growth cones were not formed after external application of trypsin, microinjection of the carrier solution, or inactivated trypsin. Growth cone formation was not preceded by increases in free intracellular Ca2+ or changes in passive membrane properties, and was blocked by inhibitors of actin and tubulin polymerization. Trypsin-induced neuritogenesis was associated with ultrastructural alterations similar to those observed by us after axotomy.

We conclude that local and transient elevations of cytoplasmic proteolytic activity can induce growth cone formation and neuritogenesis, and suggest that localized proteolytic activity plays a role in growth cone formation after axotomy.
Major Binding Sites for the Nuclear Import Receptor Are the Internal Nucleoporin Nup153 and the Adjacent Nuclear Filament Protein Tpr
Authors: Shah Sundeep , Tugendreich Stuart , Forbes Douglass
Published: July 2002

A major question in nuclear import concerns the identity of the nucleoporin(s) that interact with the nuclear localization sequences (NLS) receptor and its cargo as they traverse the nuclear pore. Ligand blotting and solution binding studies of isolated proteins have attempted to gain clues to the identities of these nucleoporins, but the studies have from necessity probed binding events far from an in vivo context. Here we have asked what binding events occur in the more physiological context of a Xenopus egg extract, which contains nuclear pore subcomplexes in an assembly competent state. We have then assessed our conclusions in the context of assembled nuclear pores themselves. We have used immunoprecipitation to identify physiologically relevant complexes of nucleoporins and importin subunits. In parallel, we have demonstrated that it is possible to obtain immunofluorescence localization of nucleoporins to subregions of the nuclear pore and its associated structures. By immunoprecipitation, we find the nucleoporin Nup153 and the pore-associated filament protein Tpr, previously shown to reside at distinct sites on the intranuclear side of assembled pores, are each in stable subcomplexes with importin α and β in Xenopus egg extracts. Importin subunits are not in stable complexes with nucleoporins Nup62, Nup93, Nup98, or Nup214/CAN, either in egg extracts or in extracts of assembled nuclear pores. In characterizing the Nup153 complex, we find that Nup153 can bind to a complete import complex containing importin α, β, and an NLS substrate, consistent with an involvement of this nucleoporin in a terminal step of nuclear import. Importin β binds directly to Nup153 and in vitro can do so at multiple sites in the Nup153 FXFG repeat region. Tpr, which has no FXFG repeats, binds to importin β and to importin α/β heterodimers, but only to those that do not carry an NLS substrate. That the complex of Tpr with importin β is fundamentally different from that of Nup153 is additionally demonstrated by the finding that recombinant β or β45–462 fragment freely exchanges with the endogenous importin β/Nup153 complex, but cannot displace endogenous importin β from a Tpr complex. However, the GTP analogue GMP-PNP is able to disassemble both Nup153– and Tpr–importin β complexes. Importantly, analysis of extracts of isolated nuclei indicates that Nup153– and Tpr–importin β complexes exist in assembled nuclear pores. Thus, Nup153 and Tpr are major physiological binding sites for importin β. Models for the roles of these interactions are discussed.