Different correlation of Sphingosine-1-Phosphate receptor 1 with receptor activator of nuclear factor kappa B ligand and regulatory T cells in rat periapical lesions

Introduction Sphingosine-1-phosphate receptor 1 (S1P1) is crucial for regulation of immunity and bone metabolism. This study aimed to investigate the expression of S1P1 in rat periapical lesions and its relationship with receptor activator of nuclear factor kappa B ligand (RANKL) and regulatory T (Treg) cells. Methods Periapical lesions were induced by pulp exposure in the first lower molars of 55 Wistar rats. Thirty rats were killed on days 0, 7, 14, 21, 28, and 35, and their mandibles were harvested for x-ray imaging, micro–computed tomography scanning, histologic observation, immunohistochemistry, enzyme histochemistry, and double immunofluorescence analysis. The remaining 25 rats were killed on days 0, 14, 21, 28, and 35, and mandibles were harvested for flow cytometry. Results The volume and area of the periapical lesions increased from day 0 to day 21 and then remained comparably stable after day 28. S1P1-positive cells were observed in the inflammatory periapical regions; the number of S1P1-positive cells peaked at day 14 and then decreased from day 21 to day 35. The distribution of S1P1-positive cells was positively correlated with the dynamics of RANKL-positive cells but was negatively correlated with that of Treg cells. Conclusions S1P1 expression was differentially correlated with RANKL and Treg cell infiltration in the periapical lesions and is therefore a contributing factor to the pathogenesis of such lesions.

The chromosome 16q region associated with ankylosing spondylitis includes the candidate gene tumour necrosis factor receptor type 1-associated death domain (TRADD)

Objective: To replicate and refine the reported association of ankylosing spondylitis (AS) with two nonsynonymous single nucleotide polymorphisms (nsSNPs) on chromosome 16q22.1. Methods: Firstly, 730 independent UK patients with AS were genotyped for rs9939768 and rs6979 and allele frequencies were compared with 2879 previously typed historic disease controls. Secondly, the two data sets were combined in meta-analyses. Finally, 5 tagging SNPs, located between rs9939768 and rs6979, were analysed in 1604 cases and 1020 controls. Results: The association of rs6979 with AS was replicated, p=0.03, OR=1.14 (95% CI 1.01 to 1.28), and a trend for association with rs9939768 detected, p=0.06, OR=1.25 (95% CI 0.99 to 1.57). Meta-analyses revealed association of both SNPs with AS, p=0.0008, OR=1.31 (95% CI 1.12 to 1.54) and p=0.0009, OR=1.15 (95% CI 1.06 to 1.23) for rs9939768 and rs6979, respectively. New associations with rs9033 and rs868213 (p=0.00002, OR=1.23 (95% CI 1.12 to 1.36) and p=0.00002 OR=1.45 (95% CI 1.22 to 1.72), respectively, were identified. Conclusions: The region on chromosome 16 that has been replicated in the present work is interesting as the highly plausible candidate gene, tumour necrosis factor receptor type 1 (TNFR1)-associated death domain (TRADD), is located between rs9033 and rs868213. It will require additional work to identify the primary genetic association(s) with AS.

Evidence of genetic association between TNFRSF1A encoding the p55 tumour necrosis factor receptor, and ankylosing spondylitis in UK Caucasians

Objectives: To replicate the possible genetic association between ankylosing spondylitis (AS) and TNFRSF1A. Methods: TNFRSF1A was re-sequenced in 48 individuals with AS to identify novel polymorphisms. Nine single nucleotide polymorphisms (SNPs) in TNFRSF1A and 5 SNPs in the neighbouring gene SCNN1A were genotyped in 1604 UK Caucasian individuals with AS and 1019 matched controls. An extended study was implemented using additional genotype data on 8 of these SNPs from 1400 historical controls from the 1958 British Birth Cohort. A meta-analysis of previously published results was also undertaken. Results: One novel variant in intron 6 was identified but no new coding variants. No definite associations were seen in the initial study but in the extended study there were weak associations with rs4149576 (p=0.04) and rs4149577 (p=0.007). In the metaanalysis consistent, somewhat stronger associations were seen with rs4149577 (p=0.002) and rs4149578 (p=0.006). Conclusions: These studies confirm the weak genetic associations between AS and TNFRSF1A. In view of the previously reported associations of TNFRSF1A with AS, in Caucasians and Chinese, and the biological plausibility of this candidate gene, replication of this finding in well powered studies is clearly indicated.

Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Growth differentiation factor 9 signalling in the ovary

In the ovary, two new members of the large TGF-beta superfamily of growth factors were discovered in the 1990s. The oocyte was shown to express two closely related growth factors that were named growth differentiation factor 9 (GDF-9) and growth differentiation factor 9B (GDF-9B). Both of these proteins are required for normal ovarian follicle development although their individual significance varies between species. GDF-9 and GDF-9B mRNAs are expressed in the human oocytes from the primary follicle stage onwards. This thesis project was aimed to define the signalling mechanisms utilized by the oocyte secreted GDF-9. We used primary cultures of human granulosa luteal cells (hGL) as our cell model, and recombinant adenovirus-mediated gene transfer in manipulating the TGF-b family signalling cascade molecules in these cells. Overexpression of the constitutively active forms of the seven type I receptors, the activin receptor-like kinases 1-7 (ALK1-7), using recombinant adenoviruses caused a specific activation of either the Smad1 or Smad2 pathway proteins depending on the ALK used. Activation of both Smad1 and Smad2 proteins also stimulated the expression of dimeric inhibin B protein in hGL cells. Treatment with recombinant GDF-9 protein induced the specific activation of the Smad2 pathway and stimulated the expression of inhibin betaB subunit mRNA as well as inhibin B protein secretion in our cell model. Recombinant GDF-9 also activated the Smad3-responsive CAGA-luciferase reported construct, and the GDF-9 response in hGL cells was markedly potentiated upon the overexpression of Alk5 by adenoviral gene transduction. Alk5 overexpression also enhanced the GDF-9 induced inhibin B secretion by these cells. Similarly, in a mouse teratocarcinoma cell line P19, GDF-9 could activate the Smad2/3 pathway, and overexpression of ALK5 in COS7 cells rendered them responsive to GDF-9. Furthermore, transfection of rat granulosa cells with small interfering RNA for ALK5 or overexpression of the inhibitory Smad7 resulted in dose-dependent suppression of GDF-9 effects. In conclusion, this thesis shows that both Smad1 and Smad2 pathways are involved in controlling the regulation of inhibin B secretion. Therefore, in addition to endocrine control of inhibin production by the pituitary gonadotropins, also local paracrine factors within in the ovary, like the oocyte-derived growth factors, may contribute to controlling inhibin secretion. This thesis shows as well that like other TGF-beta family ligands, also GDF-9 signalling is mediated by the canonical type I and type II receptors with serine/threonine kinase activity, and the intracellular transcription factors, the Smads. Although GDF-9 binds to the BMP type II receptor, its downstream actions are specifically mediated by the type I receptor, ALK5, and the Smad2 and Smad3 proteins.

Activator Protein-1 and Epidermal Growth Factor Receptor Interplay : In Vivo & In Vitro Studies

Critical cellular decisions such as should the cell proliferate, migrate or differentiate, are regulated by stimulatory signals from the extracellular environment, like growth factors. These signals are transformed to cellular responses through their binding to specific receptors present at the surface of the recipient cell. The epidermal growth factor receptor (EGF-R/ErbB) pathway plays key roles in governing these signals to intracellular events and cell-to-cell communication. The EGF-R forms a signaling network that participates in the specification of cell fate and coordinates cell proliferation. Ligand binding triggers receptor dimerization leading to the recruitment of kinases and adaptor proteins. This step simultaneously initiates multiple signal transduction pathways, which result in activation of transcription factors and other target proteins, leading to cellular alterations. It is known that mutations of EGF-R or in the components of these pathways, such as Ras and Raf, are commonly involved in human cancer. The four best characterized signaling pathways induced by EGF-R are the mitogen-activated protein kinase cascades (MAPKs), the lipid kinase phosphatidylinositol 3 kinase (PI3K), a group of transcription factors called Signal Transducers and Activator of Transcription (STAT), and the phospholipase Cγ; (PLCγ) pathways. The activation of each cascade culminates in kinase translocation to the nucleus to stimulate various transcription factors including activator protein 1 (AP-1). AP-1 family proteins are basic leucine zipper (bZIP) transcription factors that are implicated in the regulation of a variety of cellular processes (proliferation and survival, growth, differentiation, apoptosis, cell migration, transformation). Therefore, the regulation of AP-1 activity is critical for the decision of cell fate and their deregulated expression is widely associated with many types of cancers, such as breast and prostate cancers. The aims of this study were to characterize the roles of EGF-R signaling during normal development and malignant growth in vitro and in vivo using different cell lines and tissue samples. We show here that EGF-R regulates cell proliferation but is also required for regulation of AP-1 target gene expression in fibroblasts in a MAP-kinase mediated manner. Furthermore, EGF-R signaling is essential for enterocyte proliferation and migration during intestinal maturation. EGF-R signaling network, especially PI3-K-Akt pathway mediated AP-1 activity is involved in cellular survival in response to ionizing radiation. Taken together, these results elucidate the connection of EGF-R and AP-1 in various cellular contexts and show their importance in the regulation of cellular behaviour presenting new treatment cues for intestinal perforations and cancer therapy.

Tumour necrosis factor receptor-associated periodic syndrome (TRAPS) : Identification of novel TNFRSF1A mutations and intracellular signalling defects

The systemic autoinflammatory disorders are a group of rare diseases characterized by periodically recurring episodes of acute inflammation and a rise in serum acute phase proteins, but with no signs of autoimmunity. At present eight hereditary syndromes are categorized as autoinflammatory, although the definition has also occasionally been extended to other inflammatory disorders, such as Crohn s disease. One of the autoinflammatory disorders is the autosomally dominantly inherited tumour necrosis factor receptor-associated periodic syndrome (TRAPS), which is caused by mutations in the gene encoding the tumour necrosis factor type 1 receptor (TNFRSF1A). In patients of Nordic descent, cases of TRAPS and of three other hereditary fevers, hyperimmunoglobulinemia D with periodic fever syndrome (HIDS), chronic infantile neurologic, cutaneous and articular syndrome (CINCA) and familial cold autoinflammatory syndrome (FCAS), have been reported, TRAPS being the most common of the four. Clinical characteristics of TRAPS are recurrent attacks of high spiking fever, associated with inflammation of serosal membranes and joints, myalgia, migratory rash and conjunctivitis or periorbital cellulitis. Systemic AA amyloidosis may occur as a sequel of the systemic inflammation. The aim of this study was to investigate the genetic background of hereditary periodically occurring fever syndromes in Finnish patients, to explore the reliability of determining serum concentrations of soluble TNFRSF1A and metalloproteinase-induced TNFRSF1A shedding as helpful tools in differential diagnostics, as well as to study intracellular NF-κB signalling in an attempt to widen the knowledge of the pathomechanisms underlying TRAPS. Genomic sequencing revealed two novel TNFRSF1A mutations, F112I and C73R, in two Finnish families. F112I was the first TNFRSF1A mutation to be reported in the third extracellular cysteine-rich domain of the gene and C73R was the third novel mutation to be reported in a Finnish family, with only one other TNFRSF1A mutation having been reported in the Nordic countries. We also presented a differential diagnostic problem in a TRAPS patient, emphasizing for the clinician the importance of differential diagnostic vigiliance in dealing with rare hereditary disorders. The underlying genetic disease of the patient both served as a misleading factor, which possibly postponed arrival at the correct diagnosis, but may also have predisposed to the pathologic condition, which led to a critical state of the patient. Using a method of flow cytometric analysis modified for the use on fresh whole blood, we studied intracellular signalling pathways in three Finnish TRAPS families with the F112I, C73R and the previously reported C88Y mutations. Evaluation of TNF-induced phosphorylation of NF-κB and p38, revealed low phosphorylation profiles in nine out of ten TRAPS patients in comparison to healthy control subjects. This study shows that TRAPS is a diagnostic possibility in patients of Nordic descent, with symptoms of periodically recurring fever and inflammation of the serosa and joints. In particular in the case of a family history of febrile episodes, the possibility of TRAPS should be considered, if an etiology of autoimmune or infectious nature is excluded. The discovery of three different mutations in a population as small as the Finnish, reinforces the notion that the extracellular domain of TNFRSF1A is prone to be mutated at the entire stretch of its cysteine-rich domains and not only at a limited number of sites, suggesting the absence of a founder effect in TRAPS. This study also demonstrates the challenges of clinical work in differentiating the symptoms of rare genetic disorders from those of other pathologic conditions and presents the possibility of an autoinflammatory disorder as being the underlying cause of severe clinical complications. Furthermore, functional studies of fresh blood leukocytes show that TRAPS is often associated with a low NF-κB and p38 phosphorylation profile, although low phosphorylation levels are not a requirement for the development of TRAPS. The aberrant signalling would suggest that the hyperinflammatory phenotype of TRAPS is the result of compensatory NF-κB-mediated regulatory mechanisms triggered by a deficiency of the innate immune response.

First-Line afatinib versus chemotherapy in patients with non–small cell lung cancer and common epidermal growth factor receptor gene mutations and brain metastases

Introduction Metastatic spread to the brain is common in patients with non–small cell lung cancer (NSCLC), but these patients are generally excluded from prospective clinical trials. The studies, phase III study of afatinib or cisplatin plus pemetrexed in patients with metastatic lung adenocarcinoma with EGFR mutations (LUX-Lung 3) and a randomized, open-label, phase III study of BIBW 2992 versus chemotherapy as first-line treatment for patients with stage IIIB or IV adenocarcinoma of the lung harbouring an EGFR activating mutation (LUX-Lung 6) investigated first-line afatinib versus platinum-based chemotherapy in epidermal growth factor receptor gene (EGFR) mutation-positive patients with NSCLC and included patients with brain metastases; prespecified subgroup analyses are assessed in this article. Methods For both LUX-Lung 3 and LUX-Lung 6, prespecified subgroup analyses of progression-free survival (PFS), overall survival, and objective response rate were undertaken in patients with asymptomatic brain metastases at baseline (n = 35 and n = 46, respectively). Post hoc analyses of clinical outcomes was undertaken in the combined data set (n = 81). Results In both studies, there was a trend toward improved PFS with afatinib versus chemotherapy in patients with brain metastases (LUX-Lung 3: 11.1 versus 5.4 months, hazard ratio [HR] = 0.54, p = 0.1378; LUX-Lung 6: 8.2 versus 4.7 months, HR = 0.47, p = 0.1060). The magnitude of PFS improvement with afatinib was similar to that observed in patients without brain metastases. In combined analysis, PFS was significantly improved with afatinib versus with chemotherapy in patients with brain metastases (8.2 versus 5.4 months; HR, 0.50; p = 0.0297). Afatinib significantly improved the objective response rate versus chemotherapy in patients with brain metastases. Safety findings were consistent with previous reports. Conclusions These findings lend support to the clinical activity of afatinib in EGFR mutation–positive patients with NSCLC and asymptomatic brain metastases.

Targeting the fibroblast growth factor receptor family in cancer

Fibroblast growth factors (FGFs) regulate a plethora of biological functions, in both the embryonic and adult stages of development, binding their cognate receptors and thus activating a variety of downstream signalling pathways. Deregulation of the FGF/FGFR signalling axis, observed in multifarious tumor types including squamous non-small cell lung cancer, occurs through genomic FGFR alterations that drive ligand-independent receptor signalling or alterations that support ligand-dependent activation. Mutations are not restricted to the tyrosine kinase domain and aberrations appear to be tumor type dependent. As well as its complementarity and synergy with VEGF of particular interest is the interplay between FGFR and EGFR and the ability of these pathways to offer a compensatory signalling escape mechanism when either is inhibited. Hence there exists a rationale for a combinatorial approach to inhibition of these dysregulated pathways to reverse drug resistance. To date, several multi-target tyrosine kinase inhibitors as well as FGFR specific tyrosine kinase inhibitors (TKIs), monoclonal antibodies and FGF ligand traps have been developed. Promising preclinical data has resulted in several drugs entering clinical trials. This review explores aberrant FGFR and its potential as a therapeutic target in solid tumors.

Antibodies against the extracellular domain of human Notch1 receptor reveal the critical role of epidermal-growth-factor-like repeats 25-26 in ligand binding and receptor activation

The Notch signalling pathway is implicated in a wide variety of cellular processes throughout metazoan development. Although the downstream mechanism of Notch signalling has been extensively studied, the details of its ligand-mediated receptor activation are not clearly understood. Although the role of Notch ELRs EGF (epidermal growth factor)-like-repeats] 11-12 in ligand binding is known, recent studies have suggested interactions within different ELRs of the Notch receptor whose significance remains to be understood. Here, we report critical inter-domain interactions between human Notch1 ELRs 21-30 and the ELRs 11-15 that are modulated by calcium. Surface plasmon resonance analysis revealed that the interaction between ELRs 21-30 and ELRs 11-15 is similar to 10-fold stronger than that between ELRs 11-15 and the ligands. Although there was no interaction between Notch 1 ELRs 21-30 and the ligands in vitro, addition of pre-clustered Jagged1Fc resulted in the dissociation of the preformed complex between ELRs 21-30 and 11-15, suggesting that inter-domain interactions compete for ligand binding. Furthermore, the antibodies against ELRs 21-30 inhibited ligand binding to the full-length Notch1 and subsequent receptor activation, with the antibodies against ELRs 25-26 being the most effective. These results suggest that the ELRs 25-26 represent a cryptic ligand-binding site which becomes exposed only upon the presence of the ligand. Thus, using specific antibodies against various domains of the Notch1 receptor, we demonstrate that, although ELRs 11-12 are the principal ligand-binding site, the ELRs 25-26 serve as a secondary binding site and play an important role in receptor activation.

Regulation of protumorigenic pathways by Insulin like growth factor binding protein2 and its association along with beta-catenin in breast cancer lymph node metastasis

Background: Insulin like growth factor binding proteins modulate the mitogenic and pro survival effects of IGF. Elevated expression of IGFBP2 is associated with progression of tumors that include prostate, ovarian, glioma among others. Though implicated in the progression of breast cancer, the molecular mechanisms involved in IGFBP2 actions are not well defined. This study investigates the molecular targets and biological pathways targeted by IGFBP2 in breast cancer. Methods: Transcriptome analysis of breast tumor cells (BT474) with stable knockdown of IGFBP2 and breast tumors having differential expression of IGFBP2 by immunohistochemistry was performed using microarray. Differential gene expression was established using R-Bioconductor package. For validation, gene expression was determined by qPCR. Inhibitors of IGF1R and integrin pathway were utilized to study the mechanism of regulation of beta-catenin. Immunohistochemical and immunocytochemical staining was performed on breast tumors and experimental cells, respectively for beta-catenin and IGFBP2 expression. Results: Knockdown of IGFBP2 resulted in differential expression of 2067 up regulated and 2002 down regulated genes in breast cancer cells. Down regulated genes principally belong to cell cycle, DNA replication, repair, p53 signaling, oxidative phosphorylation, Wnt signaling. Whole genome expression analysis of breast tumors with or without IGFBP2 expression indicated changes in genes belonging to Focal adhesion, Map kinase and Wnt signaling pathways. Interestingly, IGFBP2 knockdown clones showed reduced expression of beta-catenin compared to control cells which was restored upon IGFBP2 re-expression. The regulation of beta-catenin by IGFBP2 was found to be IGF1R and integrin pathway dependent. Furthermore, IGFBP2 and beta-catenin are co-ordinately overexpressed in breast tumors and correlate with lymph node metastasis. Conclusion: This study highlights regulation of beta-catenin by IGFBP2 in breast cancer cells and most importantly, combined expression of IGFBP2 and beta-catenin is associated with lymph node metastasis of breast tumors.

Co-liposomes comprising a lipidated multivalent RGD-peptide and a cationic gemini cholesterol induce selective gene transfection in alpha v beta 3 and alpha v beta 5 integrin receptor-rich cancer cells

The alpha v beta 3 and alpha v beta 5 integrins, transmembrane glycoprotein receptors, are over-expressed in numerous tumors and in endothelial cells that constitute tumor blood vessels. As this protein selectively binds to the Arg-Gly-Asp (RGD) sequence containing peptides, it is an attractive way to target tumors. Herein we have developed novel formulations for integrin mediated selective gene delivery. These formulations are composed of a novel palmitoylated tetrameric RGD containing scaffold (named RAFT-RGD), cationic gemini cholesterol (GL5) and a natural helper lipid 1,2-dioleoyl-L-alpha-glycero-3-phosphatidylethanolamine (DOPE). We have optimized a co-liposomal formulation to introduce the multivalent RGD-containing macromolecule in GL5: DOPE (GL5D) mixture to produce GL5D-RGD. We have unambiguously shown the selectivity of these formulations towards cancer cells that over express alpha v beta 3 and alpha v beta 5 integrins. Two reporter plasmids, pEGFP-C3 and PGL-3, were employed for the transfection experiments and it was shown that GL5D-RGD Liposomes increased exclusively the transfection in alpha v beta 3 and alpha v beta 5 overexpressing HeLa cells.