Association of αvβ3 integrin expression with the metastatic potential and migratory and chemotactic ability of human osteosarcoma cells

Introduction. Expression of adhesion molecules such as α _v β ₃ integrin has been associated with the metastatic potential of tumor cells. The purpose of this study was to determine whether α _v β ₃ expression correlated with the metastatic potential of human osteosarcoma cells. Materials and methods. We developed a series of sublines (LM2–LM7) from human osteosarcoma SAOS parental cells, with progressively increasing potential to form lung metastases in nude mice after intravenous injection. SAOS parental and LM2 cells were poorly metastatic, but LM7 cells resulted in visible metastatic lung nodules by 6–8 weeks. We quantified α _v β ₃ integrin expression using flow cytometry. Results. α _v β ₃ expression correlated with the metastatic potential of the cells, with LM7 cells showing the highest expression. LM7 cell adhesion to vitronectin decreased after treatment with echistatin, a RGD-containing peptide antagonist of α _v β ₃. LM7 cells demonstrated higher chemotactic activity than SAOS cells to a homogenate made from lung tissue. This chemotactic activity was also inhibited by echistatin. These data indicated that α _v β ₃ was critical for the migration of LM7 cells to the lung homogenate. Chemotaxis to a liver homogenate was the same for LM7 and SAOS cells. Migration of LM7 cells through lung endothelial cells was higher than that through liver endothelial cells, and echistatin again inhibited this migration. Conclusions. α _v β ₃ integrin expression may play a role in the metastatic potential of osteosarcoma cells by enhancing the ability of the cells to migrate specifically to the lung. α _v β ₃ integrin may therefore be a potential new target for osteosarcoma.This revised version was published online in August 2005 with a corrected cover date.     

The role of the phoPQ operon in the pathogenesis of the fully virulent CO92 strain of Yersinia pestis and the IP32953 strain of Yersinia pseudotuberculosis

At the genomic level, Yersinia pestis and Yersinia pseudotuberculosis are nearly identical but cause very different diseases. Y. pestis is the etiologic agent of plague; whereas Y. pseudotuberculosis causes a gastrointestinal infection primarily after the consumption of contaminated food. In many gram-negative pathogenic bacteria, PhoP is part of a two-component global regulatory system in which PhoQ serves as the sensor kinase, and PhoP is the response regulator. PhoP is known to activate a number of genes in many bacteria related to virulence. To determine the role of the PhoPQ proteins in Yersinia infections, primarily using aerosol challenge models, the phoP gene was deleted from the chromosome of the CO92 strain of Y. pestis and the IP32953 strain of Y. pseudotuberculosis, leading to a polar mutation of the phoPQ operon. We demonstrated that loss of phoPQ from both strains leads to a defect in intracellular growth and/or survival within macrophages. These in vitro data would suggest that the phoPQ mutants would be attenuated in vivo. However, the LD(50) for the Y. pestis mutant did not differ from the calculated LD(50) for the wild-type CO92 strain for either the bubonic or pneumonic murine models of infection. In contrast, mice challenged by aerosol with the Y. pseudotuberculosis mutant had a LD(50) value 40× higher than the wild-type strain. These results demonstrate that phoPQ are necessary for full virulence by aerosol infection with the IP32953 strain of Y. pseudotuberculosis. However, the PhoPQ proteins do not play a significant role in infection with a fully virulent strain of Y. pestis.     

Anti-beta 2-glycoprotein I antibodies of IgM class are linked to thrombotic disorders in young women without autoimmune disease

Antiphospholipid autoantibodies particularly antibodies against beta2-glycoprotein I (anti-beta2GPI) are casually associated with thromboses in patients with autoimmune diseases. However, their exact prevalence and role in the pathogenesis of thromboses in the absence of autoimmune disease is still inconclusive. They might be particularly important when other risk factors of thrombosis are absent. We investigated antiphospholipid antibodies in 68 young women (aged <45yr at onset of the event, without autoimmune disease and with an otherwise low risk of thrombosis) in the stable period following myocardial infarction (MI), lacunar cerebral infarction (LACI) or deep vein thrombosis (DVT) and in 37 healthy age-matched controls. Patients had increased IgM anti-beta2GPI compared to controls (36.0, 11.5-49.5 vs. 17.50, 3.50-30.0 arbitrary units (AU), p<0.001), whereas no difference was obtained in other measured antibodies (anticardiolipin and antiphosphatidylserine (aPS) antibodies of IgG and IgM). IgM anti-beta2GPI positively correlated with some markers of increased coagulation potential and negatively with BMI (r=-039, p<0.005) and other parameters of the metabolic syndrome. In conclusion, we found that levels of IgM anti-beta2GPI are increased in young women suffering arterial or venous thromboses in the absence of other known autoimmune diseases and also in the absence of pronounced classical risk factors. We found that IgM anti-beta2GPI positively correlated with some markers of increased coagulation potential and negatively with parameters of the metabolic syndrome. Thus, it appears that elevated levels of IgM anti-beta2GPI are linked to thrombotic disorders in young women (without autoimmune disease) particularly when classical risk factors or the metabolic syndrome are absent.     

Immunohistochemical assessment of fibronectin and tenascin and their integrin receptors alpha5beta1 and alpha9beta1 in gastric and colorectal cancers with lymph node and liver metastases

The aim of the current study was to assess immunohistochemically and compare the level of expression of tenascin (TN) and fibronectin (FN) and their integrin receptors alpha9beta1 and alpha5beta1 in the primary colorectal and gastric tumors, and in corresponding lymph node and liver metastases from 53 patients. We detected similar high deposition of the studied ECM proteins and their receptors in the stroma of primary tumors and in liver metastases and a lower deposition in lymph node metastases. Cytoplasmic immune reaction for FN and TN was also seen in the tumor cells. A pronounced co-localization of immune deposits for FN and TN and their receptors was found in the stroma of the center and the invasion front (IF) (p<0.0001). A significant decrease of FN immune signal was observed in the IF in primary tumors and liver metastases (p<0.0001). The levels of immunolabeling of FN and TN correlated with the differentiation grade of primary tumors (p<0.0001). In conclusion, we may say that there is heterogeneous deposition of TN, FN and their integrin receptors in the different areas of primary colorectal and gastric tumors and of their metastases. These findings imply that the studied proteins may be involved in cell processes such as growth, adhesion, migration and apoptosis.     

The effect of vitamin A on CCl4-induced hepatic injuries in rats: a histochemical, immunohistochemical and ultrastructural study

In this study, we aimed to investigate the effect of vitamin A on the transformation of the Ito cells to fibrogenic form and suppression of the development of fibrosis. Carbon tetrachloride intoxication was performed on rats for 2, 8, 12 or 20 weeks and 5x10(4) IU vitamin A (as retinol palmitate) was injected subcutaneously once every 4 weeks. Ito cells were detected by gold chloride impregnation, as well as desmin and alpha-smooth muscle actin (alpha-SMA) immunohistochemistry. Additionally, all groups were examined ultrastructurally. The number of Ito cells that were labelled positively with gold impregnation decreased in the fibrotic groups; however, alpha-SMA and desmin immunopositive Ito cells increased. The samples from animals that were treated with vitamin A showed an increase in labelling with gold impregnation but a decrease in alpha-SMA immunopositivity. The data showed that vitamin A can prevent hepatic injury, by suppressing the transformation of Ito cells to fibrogenic form. We conclude that vitamin A has potential for the treatment of hepatic fibrotic diseases. Alpha-SMA immunohistochemistry was found to be more informative than desmin immunohistochemistry for monitoring liver fibrosis.     

Cyclin D1 overexpression in N-methyl-N′-nitro-N-nitrosoguanidine-induced rat gastric adenocarcinomas

Changes in cell cycle regulation are involved in many human cancers, including gastric cancer. In the present study, cyclin D1 expression and localization were immunohistochemically analyzed in 23 N-methyl-N'-nitro-N-nitrosoguanidine-induced rat gastric adenocarcinomas and compared with findings for beta-catenin. Cyclin D1 nuclear overexpression was more frequently observed in tumors displaying nuclear (4/4=100%) and cytoplasmic (3/4=75%) beta-catenin accumulation than those with membranous (3/15=20%) localization (nuclear vs. membranous, P<0.02). In the former cases it was considered that cyclin D1 was induced with beta-catenin activation; in the latter, a direct or indirect pathway for cyclin D1 accumulation bypassing Wnt pathway might be involved. Cyclin D1 was also found to be accumulated in gastric glands within normal-looking mucosa, these perhaps representing preneoplastic lesions for cancers with membranous beta-catenin accumulation.     

Melatonin potentiates the GABAA receptor-mediated current in cultured chick spinal cord neurons

The effect of melatonin on the γ-aminobutyric acid_A (GABA_A) receptor-mediated response was studied in cultured chick spinal cord neurons using the whole-cell voltage-clamp recording technique. Melatonin rapidly and reversibly potentiated the GABA-induced current in a dose-dependent fashion, with an EC₅₀ of 766 μM and a maximal potentiation of 148%. Potentiation of the GABA response by melatonin was mediated by increasing the potency of GABA rather than the efficacy. Prolonged exposure to a saturating concentration of the disulfide-reducing agent dithiothreitol did not attentuate the effect of melatonin on the GABA response, indicating that melatonin does not act through the redox site. Furthermore, our results demonstrate that melatonin and 5α-pregnan-3α-ol-20-one (a positive steroid modulator of the GABA_A receptor) act through different sites.     

Immunomodulating effects of cefodizime on Klebsiella pneumoniae-stimulated neutrophils

To explore the immunoregulating effects of cefodizime on neutrophils and its mechanisms, we detected the expression of some cytokines secreted by neutrophils after heat-killed Klebsiella pneumoniae induced inflammation. We analyzed the changes of signal transduction in this process by detecting the mRNA expression of toll like receptor 4 (TLR4) and the inhibitor factor of kappaBalpha (I-kappaBalpha) expressed by neutrophils. The activity of nuclear factor kappaB (NF-kappaB) in neutrophils was also assayed by electrophoretic mobility shift assay (EMSA). We found cefodizime increased neutrophil production of TNF-alpha, IL-1beta in the early stage of inflammation, which was in agreement with the enhanced mRNA expression of TLR4 and DNA-binding activity of NF-kappaB. Taken together, the immunomodulating effects of cefodizime on cytokine production of K. pneumoniae stimulated neutrophils is possibly due to its regulation of TLR4 mRNA expression and DNA binding activities of NF-kappaB through the LPS-TLR4-NF-kappaB pathway.     

Immunohistochemical detection of estrogen receptor α in articular chondrocytes from cows, pigs and humans: in situ and in vitro results

Clinical observations suggest that estrogens are involved in the pathogenesis of postmenopausal osteoarthritis, but only little is known about the influence of these hormones on articular cartilage cells. The effect of estradiol is mediated by estrogen receptors α and β. The goal of the present study was to search for estrogen receptor α in articular tissue from cows, pigs and humans by immunohistochemistry to form a basis for in vitro studies. In addition, we also tried to detect estrogen receptor α in cultivated articular chondrocytes from cows and bulls under certain culture conditions. Estrogen receptor α is detected by the use of antibody 13H2 in articular chondrocytes from cows, bulls, pigs and humans. Chondrocytes are physiologically exposed to reduced oxygen tension. In isolated articular chondrocytes from cows and bulls incubated either with 21% O₂ or with 5% O₂ positive cells were also found. These positive results therefore encourage testing the influence of estradiol on cultivated articular cartilage cells in these species under different culture conditions.     

CD1d-restricted mouse Vα14 and human Vα24 T cells: lymphocytes of innate immunity

Mouse V α 14 T cells and their human homologs, V α 24 T cells, are prominent subsets of CD1d-restricted T cells. Here we discuss their striking similarities to B-1 B cells andγδ T cells and propose that these immune cells mediate various innate strategies in response to endogenous or exogenous danger signals.     

Three β-tubulin cDNAs from the parasitic nematode Haemonchus contortus

Experimental evidence indicates that tubulin is the site of action of the anthelmintic benzimidazoles. Furthermore, certain residues of β-tubulin seem to be critical for this mechanism. Although the benzimidazoles selectively affect nematode vs. mammalian β-tubulin, the molecular basis for this differential action is not known. To enhance our understanding of this phenomenon, and to provide the basis for investigating benzimidazole resistance in parasitic nematodes, we undertook the cloning of β-tubulin cDNAs from the ruminant parasite, Haemonchus contortus. We have cloned and sequenced three β-tubulin cDNAs from this organism, β12–16, β12–164, and β8–9. The first 2 differ at only 23 nucleotides, which give rise to 4 amino acid changes, β8–9 represents a different isotype class from the other two, since it differs extensively in the carboxyterminus. By comparing the sequences of these and other nematode β-tubulins with mammalian β-tubulins, several regions of consistent difference can be recognized; the functional significance of these regional differences has not been defined. Sequences very similar or identical to β8–9 and β12–16 are present in both benzimidazole-sensitive and benzimidazole-resistant populations of H. contortus. However, it appears that drug-resistant organisms may differ in the presence of a gene product which is closely related to β8–9.     

Anti-inflammatory effect of β2-agonists: Inhibition of TNF-α release from human mast cells

β₂-Agonists inhibit the release of preformed mediators such as histamine and newly synthesized mediators such as prostaglandin D₂ from mast cells. However, although mast cells have been identified as an important source of several cytokines including tumor necrosis factor-α (TNF-α), there is no information about their regulation by β₂-agonists. Thus given the importance of TNF-α in inflammation and the widespread use of β₂-agonists, we investigated the effect of long-acting (salmeterol) and short-acting (salbutamol) β₂-agonists on the secretion of TNF-α from human skin mast cells. Treatment of mast cells with salmeterol or salbutamol (100 nmol/L) inhibited the IgE-dependent release of TNF-α (82% and 74%, respectively). Moreover, 2-hour treatment with salmeterol, isoproterenol, or salbutamol inhibited mast cell cytotoxicity against a TNF-α–sensitive cell line, WEHI-164, with an IC₅₀ of 71, 50, and 29 nmol/L, respectively. Specificity for β-adrenergic receptors was shown with propranolol. The inhibitory effect of β₂-agonists was observed after only 20 minutes of treatment but was lost by 24 hours after removal of salbutamol and isoproterenol (7% and 11% inhibition remaining, respectively). In contrast, the inhibition of TNF-α release was increased 1 hour after removal of salmeterol and remained significant 24 hours later. Furthermore, β₂-agonists did not show tachyphylaxis for the inhibition of TNF-α release. Thus selective β₂-agonists demonstrate anti-inflammatory activity by inhibiting the release of TNF-α from mast cells stimulated through their IgE receptor or by a tumor target cell. This inhibitory effect of β-agonists may be important in their mode of action in the treatment of allergic diseases. (J Allergy Clin Immunol 1997;100:825-31.)     

Intermediate-term toxicity of repeated orally administered doses of the anti-malarial β-artemether in dogs

The artemisinin derivative beta-artemether, an anti-malarial, was evaluated for its toxicity and tolerability in a 2-week, multiple-dose study in dogs. Eight beagle dogs (4 females, 4 males) were given beta-artemether by oral gavage 3 times daily at 45 mg/kg/dosing (a total daily dose-level of 135 mg/kg) for 2 weeks. This beta-artemether dose regime was well tolerated. Body weight changes were normal although feed consumption during the treatment period reduced compared to that of the pre-trial period. Clinical signs were transient spells of soft to liquid feces. On completion of the treatment period, the animals were sacrificed and submitted to a full macroscopic post-mortem examination. Designated organs were weighed and a complete light microscopic examination was performed on 43 selected tissues from 1 animal per sex, and on the liver, kidneys, thymus, mandibular lymph nodes and lungs of the three other animals per sex. Major findings were high liver weight and histopathologic findings of slight diffuse hepatocellular hypertrophy and distal tubular dilatation, together with flattened epithelium, in the kidneys. With the dose regime used in this trial beta-artemether produced no clinical or apparent histopathological signs of neurotoxicity in dogs.     

Innate immune system favors emergency monopoiesis at the expense of DC‐differentiation to control systemic bacterial infection in mice

DCs are professional APCs playing a crucial role in the initiation of T‐cell responses to combat infection. However, systemic bacterial infection with various pathogens leads to DC‐depletion in humans and mice. The mechanisms of pathogen‐induced DC‐depletion remain poorly understood. Previously, we showed that mice infected with Yersinia enterocolitica (Ye) had impaired de novo DC‐development, one reason for DC‐depletion. Here, we extend these studies to gain insight into the molecular mechanisms of DC‐depletion and the impact of different bacteria on DC‐development. We show that the number of bone marrow (BM) hematopoietic progenitors committed to the DC lineage is reduced following systemic infection with different Gram‐positive and Gram‐negative bacteria. This is associated with a TLR4‐ and IFN‐γ?signaling dependent increase of committed monocyte progenitors in the BM and mature monocytes in the spleen upon Ye‐infection. Adoptive transfer experiments revealed that infection‐induced monopoiesis occurs at the expense of DC‐development. Our data provide evidence for a general response of hematopoietic progenitors upon systemic bacterial infections to enhance monocyte production, thereby increasing the availability of innate immune cells for pathogen control, whereas impaired DC‐development leads to DC‐depletion, possibly driving transient immunosuppression in bacterial sepsis.     

Molecular and biochemical mechanisms ofPasteurella haemolyticaleukotoxin-induced cell death

Pasteurella haemolyticaleukotoxin (LKT) is a member of the RTX family of pore-forming toxins that kill bovine immune cells. Several studies have suggested that RTX toxins kill target cells by the induction of apoptosis. In the present study, BL3 bovine leukaemia cells were exposed to LKT and assessed by molecular and flow cytometric techniques that measure different aspects of apoptotic cell death. The intoxicated cells demonstrated morphological, light scatter and Hoechst 33258 staining characteristics consistent with cells undergoing apoptosis. The cells also exhibited internucleosomal DNA fragmentation and poly (ADP-ribose) polymerase (PARP) cleavage, both indicators of apoptosis. LKT-treated cells bound annexin-V-FITC indicating that phosphatidylserine groups were translocated from the inner to the outer leaflet of the cell membrane. The effect of LKT on cells was dose dependent and inhibitable by incubation with anti-LKT monoclonal antibody. Finally, an early step for induction of apoptosis appears to be the binding of LKT to a β2 integrin since pre-incubating cells with anti-β2 integrin antibodies inhibited LKT-induced apoptosis. This study provides new insights into understanding the pathogenesis of bovine pasteurellosis and could lead to the development of both preventative and therapeutic strategies for disease management.     

Stimulation of Vgamma9/Vdelta2 T-lymphocyte proliferation by the isoprenoid precursor, (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate

(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate, a recently discovered intermediate in the deoxyxylulose phosphate pathway of isoprenoid biosynthesis, has been shown to act as a potent immunomodulator. In cultures of human peripheral blood mononuclear cells from eight non-related donors, the compound stimulated the proliferation of Vgamma9/Vdelta2 T lymphocytes with a median EC(50) of 70 pM when 10 U/ml of IL-2 was used as costimulant. Isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP) and some structural analogs of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate also stimulated Vgamma9/Vdelta2 T-cell proliferation, albeit at much higher concentrations. The Vgamma9/Vdelta2 T-cell proliferation is highly dependent on the seeding density used in culture. All phosphoantigens tested elicited the proliferation of two T-lymphocyte populations with different apparent ratios between the expression level of Vdelta2 and Vgamma9 chains.     

Altered melanoma cell surface glycosylation mediates organ specific adhesion and metastasis via lectin receptors on the lung vascular endothelium

Adhesive interactions between the molecules on cancer cells and the target organ are one of the key determinants of the organ specific metastasis. In this communication we show that b1,6 branched N-oligosaccharides which are expressed in a metastasis-dependent manner on B16-melanoma metastatic cell lines, participate in the adhesion process. We demonstrate that high metastatic cells show significantly increased translocation of one of the major carriers of these oligosaccharides, lysosome associated membrane protein (LAMP1), to the cell surface. LAMP1 on high metastatic cells, carry very high levels of these oligosaccharides, which are further substituted with poly N-acetyl lactosamine (polylacNAc), resulting in the expression of high density of very high affinity ligands for galectin-3 on the cell surface. We show that galectin-3 is expressed in highest amount in the lungs as compared to other representative organs. Blocking galectin-3 by pre-incubating the frozen sections of the lungs with 100 mM lactose, substantially inhibited the adhesion of high metastatic cells, while pre-incubation with sucrose had no effect. Finally, by in situ labeling and immunoprecipitation experiment, we demonstrated that the lung vascular endothelial cells express galectin-3 constitutively on their surface. Galectin-3 on the organ endothelium could thus serve as the first anchor for the circulating cancer cells, expressing high density of very high affinity ligands on their surface, and facilitate organ specific metastasis.     

Phagocytic signaling strategies: Fcγreceptor-mediated phagocytosis as a model system

Phagocytosis is a phylogenetically ancient process by which eukaryotic cells engulf insoluble substances whose size exceeds approximately 0.5 μ m. The engulfment process requires the concerted action of several fundamental cellular pathways and is governed by multiple transmembrane signaling events. Here we focus on phagocytosis mediated by a well-studied class of phagocytic receptors that recognize the Fc portion of IgG (Fc_γRs ).