Reactivity of new adhesion molecules on lymphocytes from patients with chronic graft versus host disease

Reaction patterns of the 7th Human Leukocyte Differentiation Antigen Workshop blind panel adhesion molecules were studied on CD3/CD4, CD3/CD8, CD3/TCR gamma delta double positive T cells from peripheral blood of patients with chronic graft versus host disease (n = 8) and healthy controls (n = 4). Reactivity of 14 adhesion antibodies was tested by three-colour immunophenotyping. The mean proportion of CD3+ T cells (69 +/- 19%). CD3/CD8++ (31 +/- 13%) and CD3/TCR gamma delta++ (4 +/- 2%) T sub-populations of patients were comparable with the healthy controls. However, the mean percentage of CD3/CD4++ T cell subset in patients (14 +/- 12%) proved to be significantly decreased in comparison with the normal control value (34 +/- 16%) presumably due to secondary immunodeficiency. The workshop antibodies proved to be reactive with three T cell subsets expressing the examined antigens. Based on the results of the adhesion molecule workshop new CD categories have been introduced: CD156b as a transmembrane protein, CD167a as an epithelial tyrosin kinase receptor, CD168 as a receptor for hyaluronan mediated motility (RHAMM) and CD171 as a co-stimulatory adhesion molecule. There were significant differences in the expression of the CD167a and CD156b antigens on the CD3/CD4++ subset between the samples of patients compared with the controls characterizing the CD4+ T lymphocyte subpopulation in chronic graft versus host disease.     

MDSCs Mediate Angiogenesis and Predispose Canine Mammary Tumor Cells for Metastasis via IL-28/IL-28RA (IFN-λ) Signaling

Background

Myeloid-derived suppressor cells (MDSCs) function in immunosuppression and tumor development by induction of angiogenesis in a STAT3-dependent manner. Knowledge of MDSC biology is mainly limited to mice studies, and more clinical investigations using spontaneous tumor models are required. Here we performed in vitro experiments and clinical data analysis obtained from canine patients.

Methods

Using microarrays we examined changes in gene expression in canine mammary cancer cells due to their co-culture with MDSCs. Further, using Real-time rt-PCR, Western blot, IHC, siRNA, angiogenesis assay and migration/invasion tests we examined a role of the most important signaling pathway.

Results

In dogs with mammary cancer, the number of circulating MDSCs increases with tumor clinical stage. Microarray analysis revealed that MDSCs had significantly altered molecular pathways in tumor cells in vitro. Particularly important was the detected increased activation of IL-28/IL-28RA (IFN-λ) signaling. The highest expression of IL-28 was observed in stage III/IV mammary tumor-bearing dogs. IL-28 secreted by MDSCs stimulates STAT3 in tumor cells, which results in increased expression of angiogenic factors and subsequent induction of angiogenesis by endothelial cells, epithelial-mesenchymal transition (EMT) and increased migration of tumor cells in vitro. Knockdown of IL-28RA decreased angiogenesis, tumor cell invasion and migration.

Conclusions

We showed for the first time that MDSCs secrete IL-28 (IFN-λ), which promotes angiogenesis, EMT, invasion and migration of tumor cells. Thus, IL-28 may constitute an interesting target for further therapies. Moreover, the similarity in circulating MDSC levels at various tumor clinical stages between canine and human patients indicates canines as a good model for clinical trials of drugs targeting MDSCs.     

Mapping of nucleoside phosphorylase (Np-1) and esterase 10 (Es-10) on mouse chromosome 14

A method for detecting two alleles at Np-1 (nucleoside phosphorylase) and three alleles at Es-10 (esterase 10) from mouse blood by cellulose acetate electrophoresis is described. The allelic constitution at these loci for 44 inbred strains and stocks was determined. The location of Np-1 on chromosome 14 was established by backcross experiments in which alleles at Np-1 and Robertsonian translocations were segregating. Es-10 was shown to be linked to Np-1, and the following genetic map of Chr 14 was constructed: centromere-(8.9±4.0 cM)-[Np-1, Wc]-(10.2±1.9 cM)-Es-10-(15.5±3.7 cM)-s. The homologous human loci, NP and ES-D, are not linked.This work was supported by Contract E(11-1)-3267 with the Energy Research and Development Administration, by Contracts NO1-ES4-2156 and NO1-ES4-2159 with the National Institute of Environmental Health Sciences, and by Grants GM 19656 and GM 20919 from the National Institute of General Medical Sciences. D. A. K. was a participant in the 1975 Summer Program for College, Graduate, and Medical Students, which was supported, in part, by the Clark Foundation. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care.     

La grotte de Santa Ana (Cáceres, Espagne) et l'évolution technologique au Pléistocène dans la Péninsule ibérique

The excavations carried out in the cave of Santa Ana (Cáceres, Spain), cave of the karstic network of the “Calerizo” of Cáceres, enabled us to know the existence, in stratigraphy, of the three lito-techniques modes which characterize the industrial development of lower and middle Pleistocene in the Iberian Peninsula. On standby of new research, the results obtained until now permit us two work out a diachronic assumption of technical evolution. In the Iberian Peninsula, there are only two karstic systems where we can fallow this technological development; one is the “Sierra de Atapuerca” (Burgos) and the other is the “Calerizo de Cáceres” (Cáceres).     

α‐Hemolysin enhances Staphylococcus aureus internalization and survival within mast cells by modulating the expression of β1 integrin

Mast cells (MCs) are important sentinels of the host defence against invading pathogens. We previously reported that Staphylococcus aureus evaded the extracellular antimicrobial activities of MCs by promoting its internalization within these cells via β1 integrins. Here, we investigated the molecular mechanisms governing this process. We found that S. aureus responded to the antimicrobial mediators released by MCs by up‐regulating the expression of α‐hemolysin (Hla), fibronectin‐binding protein A and several regulatory systems. We also found that S. aureus induced the up‐regulation of β1 integrin expression on MCs and that this effect was mediated by Hla‐ADAM10 (a disintegrin and metalloproteinase 10) interaction. Thus, deletion of Hla or inhibition of Hla‐ADAM10 interaction significantly impaired S. aureus internalization within MCs. Furthermore, purified Hla but not the inactive Hla_H35L induced up‐regulation of β1 integrin expression in MCs in a dose‐dependent manner. Our data support a model in which S. aureus counter‐reacts the extracellular microbicidal mechanisms of MCs by increasing expression of fibronectin‐binding proteins and by inducing Hla‐ADAM10‐mediated up‐regulation of β1 integrin in MCs. The up‐regulation of bacterial fibronectin‐binding proteins, concomitantly with the increased expression of its receptor β1 integrin on the MCs, resulted in enhanced S. aureus internalization through the binding of fibronectin‐binding proteins to integrin β1 via fibronectin.     

Location of phosphorylase kinase (Phk) in the mouse X chromosome

The phosphorylase kinase deficiency (Phk) locus has been located in the mouse X chromosome, the order of genes being centromere-Bn-Phk-Ta-jp. Since the Phk locus of the mouse may be identical to the locus responsible for the X-linked phosphorylase kinase deficiency trait of man, and there may be a high degree of gene-order homology in the X chromosome of all mammals, the location of Phk in the mouse reported here may aid in locating the phosphorylase kinase gene on the X chromosome of man.This research was supported by grants AM 13359 (to F.H.) and AM 14461 (to D.L.C.) from the National Institute of Arthritis and Metabolic Diseases, and by an allocation (to E.M.E.) from NIH General Research Support Grant RR-05545 from the Division of Research Resources to The Jackson Laboratory. F.H. is a recipient of a Research Career Development Award (AM 46 421) of the National Institute of Arthritis and Metabolic Diseases.     

Time-Dependent Progression of Demyelination and Axonal Pathology in MP4-Induced Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.     

Intensified antineoplastic effect by combining an HDAC‐inhibitor,an mTOR‐inhibitor and low dosed interferon alpha in prostate cancer cells

A significant proportion of men diagnosed with prostate cancer (PCa) eventually develop metastatic disease, which progresses to castration resistance, despite initial response to androgen deprivation. As anticancer therapy has become increasingly effective, acquired drug resistance has emerged, limiting efficacy. Combination treatment, utilizing different drug classes, exemplifies a possible strategy to foil resistance development. The effects of the triple application of the histone deacetylase (HDAC) inhibitor valproic acid (VPA), the mammalian target of rapamycin inhibitor everolimus and low dosed interferon alpha (IFNα) on PCa cell growth and dissemination capacity were investigated. For that purpose, the human PCa cell lines, PC‐3, DU‐145 and LNCaP were treated with the combined regimen or separate single agents. Cell growth was investigated by the MTT dye reduction assay. Flow cytometry served to analyse cell cycle progression. Adhesion to vascular endothelium or immobilized collagen, fibronectin and laminin was quantified. Migration and invasion characteristics were determined by the modified Boyden chamber assay. Integrin α and β subtypes were investigated by flow cytometry, western blotting and RT‐PCR. Integrin related signalling, Epidermal Growth Factor Receptor (EGFr), Akt, p70S6kinase and extracellular signal‐regulated kinases (ERK)1/2 activation were also assessed. The triple application of VPA, everolimus and low dosed IFNα blocked tumour cell growth and dissemination significantly better than any agent alone. Antitumour effects were associated with pronounced alteration in the cell cycle machinery, intracellular signalling and integrin expression profile. Combining VPA, everolimus and low dosed IFNα might be a promising option to counteract resistance development and improve outcome in PCa patients.     

Sequence and domain arrangements influence mechanical properties of elastin‐like polymeric elastomers

Elastin is the polymeric, extracellular matrix protein that provides properties of extensibility and elastic recoil to large arteries, lung parenchyma, and other tissues. Elastin assembles by crosslinking through lysine residues of its monomeric precursor, tropoelastin. Tropoelastin, as well as polypeptides based on tropoelastin sequences, undergo a process of self‐assembly that aligns lysine residues for crosslinking. As a result, both the full‐length monomer as well as elastin‐like polypeptides (ELPs) can be made into biomaterials whose properties resemble those of native polymeric elastin. Using both full‐length human tropoelastin (hTE) as well as ELPs, we and others have previously reported on the influence of sequence and domain arrangements on self‐assembly properties. Here we investigate the role of domain sequence and organization on the tensile mechanical properties of crosslinked biomaterials fabricated from ELP variants. In general, substitutions in ELPs involving similiar domain types (hydrophobic or crosslinking) had little effect on mechanical properties. However, modifications altering either the structure or the characteristic sequence style of these domains had significant effects on such properties. In addition, using a series of deletion and replacement constructs for full‐length hTE, we provide new insights into the role of conserved domains of tropoelastin in determining mechanical properties. © 2012 Wiley Periodicals, Inc. Biopolymers 99: 392–407, 2013.