Characterisation of mesenchymal cells from osteophytes in osteoarthritis

Osteophytes form through the process of chondroid metamorphosis of fibrous tissue followed by endochondral ossification. Osteophytes have been found to consist of three different mesenchymal tissue regions including endochondral bone formation within cartilage residues, intra-membranous bone formation within fibrous tissue and bone formation within bone marrow spaces. All these features provide evidence of mesenchymal stem cells (MSC) involvement in osteophyte formation; nevertheless, it remains to be characterised. MSC from numerous mesenchymal tissues have been isolated but bone marrow remains the “ideal” due to the ease of ex vivo expansion and multilineage potential. However, the bone marrow stroma has a relatively low number of MSC, something that necessitates the need for long-term culture and extensive population doublings in order to obtain a sufficient number of cells for therapeutic applications. MSC in vitro have limited proliferative capacity and extensive passaging compromises differentiation potential. To overcome this barrier, tissue derived MSC are of strong interest for extensive study and characterisation, with a focus on their potential application in therapeutic tissue regeneration. To date, no MSC type cell has been isolated from osteophyte tissue, despite this tissue exhibiting all the hallmark features of a regenerative tissue. Therefore, this study aimed to isolate and characterise cells from osteophyte tissues in relation to their phenotype, differentiation potential, immuno-modulatory properties, proliferation, cellular ageing, longevity and chondrogenesis in in vitro defect model in comparison to patient matched bone marrow stromal cells (bMSC). Osteophyte derived cells were isolated from osteophyte tissue samples collected during knee replacement surgery. These cells were characterised by the expression of cell surface antigens, differentiation potential into mesenchymal lineages, growth kinetics and modulation of allo-immune responses. Multipotential stem cells were identified from all osteophyte samples namely osteophyte derived mesenchymal stem cells (oMSC). Extensively expanded cell cultures (passage 4 and 9 respectively) were used to confirm cytogenetic stability and study signs of cellular aging, telomere length and telomerase activity. Cultured cells at passage 4 were used to determine 84 pathway focused stem cell related gene expression profile. Micro mass pellets were cultured in chondrogenic differentiation media for 21 days for phenotypic and chondrogenic related gene expression. Secondly, cell pellets differentiated overnight were placed into articular cartilage defects and cultured for further 21 days in control medium and chondrogenic medium to study chondrogenesis and cell behaviour. The surface antigen expression of oMSC was consistent with that of mesenchymal stem cells, such as lacking the haematopoietic and common leukocyte markers (CD34, CD45) while expressing those related to adhesion (CD29, CD166, CD44) and stem cells (CD90, CD105, CD73). The proliferation capacity of oMSC in culture was superior to that of bMSC, and they readily differentiated into tissues of the mesenchymal lineages. oMSC also demonstrated the ability to suppress allogeneic T-cell proliferation, which was associated with the expression of tryptophan degrading enzyme indoleamine 2,3 dioxygenase (IDO). Cellular aging was more prominent in late passage bMSC than in oMSC. oMSC had longer telomere length in late passages compared with bMSC, although there was no significant difference in telomere lengths in the early passages in either cell type. Telomerase activity was detectable only in early passage oMSC and not in bMSC. In osteophyte tissues telomerase positive cells were found to be located peri vascularly and were Stro-1 positive. Eighty-four pathway-focused genes were investigated and only five genes (APC, CCND2, GJB2, NCAM and BMP2) were differentially expressed between bMSC and oMSC. Chondrogenically induced micro mass pellets of oMSC showed higher staining intensity for proteoglycans, aggrecan and collagen II. Differential expression of chondrogenic related genes showed up regulation of Aggrecan and Sox 9 in oMSC and collagen II in bMSC. The in vitro defect models of oMSC in control medium showed rounded and aggregated cells staining positively for proteoglycan and presence of some extracellular matrix. In contrast, defects with bMSC showed fragmentation and loss of cells, fibroblast-like cell morphology staining positively for proteoglycans. For defects maintained in chondrogenic medium, rounded, aggregated and proteoglycan positive cells were found in both oMSC and bMSC cultures. Extracellular matrix and cellular integration into newly formed matrix was evident only in oMSC defects. For analysis of chondrocyte hypertrophy, strong expression of type X collagen could be noticed in the pellet cultures and transplanted bMSC. In summary, this study demonstrated that osteophyte derived cells had similar properties to mesenchymal stem cells in the expression of antigen phenotype, differential potential and suppression of allo-immune response. Furthermore, when compared to bMSC, oMSC maintained a higher proliferative capacity due to a retained level of telomerase activity in vitro, which may account for the relatively longer telomeres delaying growth arrest by replicative senescence compared with bMSC. oMSC behaviour in defects supported chondrogenesis which implies that cells derived from regenerative tissue can be an alternative source of stem cells and have a potential clinical application for therapeutic stem cell based tissue regeneration.

Modulation of the Chlamydia trachomatis In vitro transcriptome response by the sex hormones estradiol and progesterone

BACKGROUND:Chlamydia trachomatis is a major cause of sexually transmitted disease in humans. Previous studies in both humans and animal models of chlamydial genital tract infection have suggested that the hormonal status of the genital tract epithelium at the time of exposure can influence the outcome of the chlamydial infection. We performed a whole genome transcriptional profiling study of C. trachomatis infection in ECC-1 cells under progesterone or estradiol treatment.RESULTS:Both hormone treatments caused a significant shift in the sub-set of genes expressed (25% of the transcriptome altered by more than 2-fold). Overall, estradiol treatment resulted in the down-regulation of 151 genes, including those associated with lipid and nucleotide metabolism. Of particular interest was the up-regulation in estradiol-supplemented cultures of six genes (omcB, trpB, cydA, cydB, pyk and yggV), which suggest a stress response similar to that reported previously in other models of chlamydial persistence. We also observed morphological changes consistent with a persistence response. By comparison, progesterone supplementation resulted in a general up-regulation of an energy utilising response.CONCLUSION:Our data shows for the first time, that the treatment of chlamydial host cells with key reproductive hormones such as progesterone and estradiol, results in significantly altered chlamydial gene expression profiles. It is likely that these chlamydial expression patterns are survival responses, evolved by the pathogen to enable it to overcome the host's innate immune response. The induction of chlamydial persistence is probably a key component of this survival response.

SNAREing immunity : the role of SNAREs in the immune system

The trafficking of molecules and membranes within cells is a prerequisite for all aspects of cellular immune functions, including the delivery and recycling of cell surface proteins, secretion of immune mediators, ingestion of pathogens and activation of lymphocytes. SNARE (soluble-N-ethylmaleimide-sensitive-factor accessory-protein receptor)-family members mediate membrane fusion during all steps of trafficking, and function in almost all aspects of innate and adaptive immune responses. Here, we provide an overview of the roles of SNAREs in immune cells, offering insight into one level at which precision and tight regulation are instilled on immune responses.

Differential expression of chemokine and matrix re-modelling genes is associated with contrasting schistosome-induced hepatopathology in murine models

The pathological outcomes of schistosomiasis are largely dependent on the molecular and cellular mechanisms of the host immune response. In this study, we investigated the contribution of variations in host gene expression to the contrasting hepatic pathology observed between two inbred mouse strains following Schistosoma japonicum infection. Whole genome microarray analysis was employed in conjunction with histological and immunohistochemical analysis to define and compare the hepatic gene expression profiles and cellular composition associated with the hepatopathology observed in S. japonicum-infected BALB/c and CBA mice. We show that the transcriptional profiles differ significantly between the two mouse strains with high statistical confidence. We identified specific genes correlating with the more severe pathology associated with CBA mice, as well as genes which may confer the milder degree of pathology associated with BALB/c mice. In BALB/c mice, neutrophil genes exhibited striking increases in expression, which coincided with the significantly greater accumulation of neutrophils at granulomatous regions seen in histological sections of hepatic tissue. In contrast, up-regulated expression of the eosinophil chemokine CCL24 in CBA mice paralleled the cellular influx of eosinophils to the hepatic granulomas. Additionally, there was greater down-regulation of genes involved in metabolic processes in CBA mice, reflecting the more pronounced hepatic damage in these mice. Profibrotic genes showed similar levels of expression in both mouse strains, as did genes associated with Th1 and Th2 responses. However, imbalances in expression of matrix metalloproteinases (e.g. MMP12, MMP13) and tissue inhibitors of metalloproteinases (TIMP1) may contribute to the contrasting pathology observed in the two strains. Overall, these results provide a more complete picture of the molecular and cellular mechanisms which govern the pathological outcome of hepatic schistosomiasis. This improved understanding of the immunopathogenesis in the murine model schistosomiasis provides the basis for a better appreciation of the complexities associated with chronic human schistosomiasis.

Abrogation of contaminating RNA activity in HIV-1 Gag VLPs

Background: HIV-1 Gag virus like particles (VLPs) used as candidate vaccines are regarded as inert particles as they contain no replicative nucleic acid, although they do encapsidate cellular RNAs. During HIV-1 Gag VLP production in baculovirus-based expression systems, VLPs incorporate the baculovirus Gp64 envelope glycoprotein, which facilitates their entry into mammalian cells. This suggests that HIV-1 Gag VLPs produced using this system facilitate uptake and subsequent expression of encapsidated RNA in mammalian cells - an unfavourable characteristic for a vaccine. Methods. HIV-1 Gag VLPs encapsidating reporter chloramphenicol acetyl transferase (CAT) RNA, were made in insect cells using the baculovirus expression system. The presence of Gp64 on the VLPs was verified by western blotting and RT-PCR used to detect and quantitate encapsidated CAT RNA. VLP samples were heated to inactivate CAT RNA. Unheated and heated VLPs incubated with selected mammalian cell lines and cell lysates tested for the presence of CAT protein by ELISA. Mice were inoculated with heated and unheated VLPs using a DNA prime VLP boost regimen. Results: HIV-1 Gag VLPs produced had significantly high levels of Gp64 (∼1650 Gp64 molecules/VLP) on their surfaces. The amount of encapsidated CAT RNA/g Gag VLPs ranged between 0.1 to 7 ng. CAT protein was detected in 3 of the 4 mammalian cell lines incubated with VLPs. Incubation with heated VLPs resulted in BHK-21 and HeLa cell lysates showing reduced CAT protein levels compared with unheated VLPs and HEK-293 cells. Mice inoculated with a DNA prime VLP boost regimen developed Gag CD8 and CD4 T cell responses to GagCAT VLPs which also boosted a primary DNA response. Heating VLPs did not abrogate these immune responses but enhanced the Gag CD4 T cell responses by two-fold. Conclusions: Baculovirus-produced HIV-1 Gag VLPs encapsidating CAT RNA were taken up by selected mammalian cell lines. The presence of CAT protein indicates that encapsidated RNA was expressed in the mammalian cells. Heat-treatment of the VLPs altered the ability of protein to be expressed in some cell lines tested but did not affect the ability of the VLPs to stimulate an immune response when inoculated into mice. © 2011 Valley-Omar et al; licensee BioMed Central Ltd.

Continuum mechanics modelling of microfilament networks with different architectures based on molecular investigation of single F-actin

The actin microfilament plays a critical role in many cellular processes including embryonic development, wound healing, immune response, and tissue development. It is commonly organized in the form of networks whose mechanical properties change with changes in their architecture due to cell evolution processes. This paper presents a new nonlinear continuum mechanics model of single filamentous actin (F-actin) that is based on nanoscale molecular simulations. Following this continuum model of the single F-actin, mechanical properties of differently architected lamellipodia are studied. The results provide insight that can contribute to the understanding of the cell edge motions of living cells.

Molecular characterisation and expression analysis of interferon gamma in response to natural chlamydia infection in the koala, phascolarctos cinereus

Interferon gamma (IFNγ) is a key Th1 cytokine, with a principal role in the immune response against intracellular organisms such as Chlamydia. Along with being responsible for significant morbidity in human populations, Chlamydia is also responsible for wide spread infection and disease in many animal hosts, with reports that many Australian koala subpopulations are endemically infected. An understanding of the role played by IFNγ in koala chlamydial diseases is important for the establishment of better prophylactic and therapeutic approaches against chlamydial infection in this host. A limited number of IFNγ sequences have been published from marsupials and no immune reagents to measure expression have been developed. Through preliminary analysis of the koala transcriptome, we have identified the full coding sequence of the koala IFNγ gene. Transcripts were identified in spleen and lymph node tissue samples. Phylogenetic analysis demonstrated that koala IFNγ is closely related to other marsupial IFNγ sequences and more distantly related to eutherian mammals. To begin to characterise the role of this important cytokine in the koala's response to chlamydial infection, we developed a quantitative real time PCR assay and applied it to a small cohort of koalas with and without active chlamydial disease, revealing significant differences in expression patterns between the groups. Description of the IFNγ sequence from the koala will not only assist in understanding this species' response to its most important pathogen but will also provide further insight into the evolution of the marsupial immune system

Packed red blood cells suppress myeloid dendritic cell and monocyte inflammatory responses in a whole blood model of transfusion

Purpose/Objective: The basis for poor outcomes in some patients post transfusion remains largely unknown. Despite leukodepletion, there is still evidence of immunomodulatory effects of transfusion that require further study. In addition, there is evidence that the age of blood components transfused significantly affects patient outcomes. Myeloid dendritic cell (DC) and monocyte immune function were studied utilising an in vitro whole blood model of transfusion. Materials and methods: Freshly collected (‘recipient’) whole blood was cultured with ABO compatible leukodepleted PRBC at 25% blood replacement-volume (6hrs). PRBC were assayed at [Day (D) 2, 14, 28and 42 (date-of expiry)]. In parallel, LPS or Zymosan (Zy) were added to mimic infection. Recipients were maintained for the duration of the time course (2 recipients, 4 PRBC units, n = 8).Recipient DC and monocyte intracellular cytokines and chemokines (IL-6, IL-10, IL-12,TNF-a, IL-1a, IL-8, IP-10, MIP-1a, MIP-1b, MCP-1) were measured using flow cytometry. Changes in immune response were calculated by comparison to a parallel no transfusion control (Wilcoxin matched pairs). Influence of storage age was calculated using ANOVA. Results: Significant suppression of DC and monocyte inflammatory responses were evident. DC and monocyte production of IL-1a was reduced following exposure to PRBC regardless of storage age (P < 0.05 at all time points). Storage independent PRBC mediated suppression of DC and monocyte IL-1a was also evident in cultures costimulated with Zy. In cultures co-stimulated with either LPS or Zy, significant suppression of DC and monocyte TNF-a and IL-6 was also evident. PRBC storage attenuated monocyte TNF-a production when co-cultured with LPS (P < 0.01 ANOVA). DC and monocyte production of MIP-1a was significantly reduced following exposure to PRBC (DC: P < 0.05 at D2, 28, 42; Monocyte P < 0.05 all time points). In cultures co-stimulated with LPS and zymosan, a similar suppression of MIP-1a production was also evident, and production of both DC and monocyte MIP-1b and IP-10 were also significantly reduced. Conclusions: The complexity of the transfusion context was reflected in the whole blood approach utilised. Significant suppression of these key DC and monocyte immune responses may contribute to patient outcomes, such as increased risk of infection and longer hospital stay, following blood transfusion.

The role of inflammation in the pathogenesis of non-small cell lung cancer

The link between chronic immune activation and tumorigenesis is well established. Compelling evidence has accumulated that histologic assessment of infiltration patterns of different host immune response components in non-small cell lung cancer specimens helps identify different prognostic patient subgroups. This review provides an overview of recent insights gained in the understanding of the role played by chronic inflammation in lung carcinogenesis. The usefulness of quantification of different populations of lymphocytes, natural killer cells, macrophages, and mast cells within the tumor microenvironment in non-small cell lung cancer is also discussed. In particular, the importance of assessment of inflammatory cell microlocalization within both the tumor islet and surrounding stromal components is emphasized. Copyright © 2010 by the International Association for the Study of Lung Cancer.

Epidermal growth factor receptors and cyclooxygenase-2 in the pathogenesis of non-small cell lung cancer : potential targets for chemoprevention and systemic therapy

The epidermal growth factor receptor (EGFR) is part of a family of plasma membrane receptor tyrosine kinases that control many important cellular functions, from growth and proliferation to cell death. Cyclooxygenase (COX)-2 is an enzyme which catalyses the conversion of arachidonic acid to prostagladins and thromboxane. It is induced by various inflammatory stimuli, including the pro-inflammatory cytokines, Interleukin (IL)-1β, Tumour Necrosis Factor (TNF)-α and IL-2. Both EGFR and COX-2 are over-expressed in non-small cell lung cancer (NSCLC) and have been implicated in the early stages of tumourigenesis. This paper considers their roles in the development and progression of lung cancer, their potential interactions, and reviews the recent progress in cancer therapies that are directed toward these targets. An increasing body of evidence suggests that selective inhibitors of both EGFR and COX-2 are potential therapeutic agents for the treatment of NSCLC, in the adjuvant, metastatic and chemopreventative settings. © 2002 Elsevier Science Ireland Ltd. All rights reserved.

Progesterone activates multiple innate immune pathways inChlamydia trachomatis-Infected endocervical cells

Problem Susceptibility to Chlamydia trachomatis infection is increased by oral con- traceptives and modulated by sex hormones. We therefore sought to determine the effects of female sex hormones on the innate immune response to C. trachomatis infection. Method of study ECC-1 endometrial cells, pre-treated with oestradiol or progesterone, were infected with C. trachomatis and the host transcriptome analysed by Illumina Sentrix HumanRef-8 microarray. Primary endocervical epithe- lial cells, prepared at either the proliferative or secretory phase of the menstrual cycle, were infected with C. trachomatis and cytokine gene expression determined by quantitative RT-PCR analysis. Results Chlamydia trachomatis yield from progesterone-primed ECC-1 cells was significantly reduced compared with oestradiol-treated cells. Genes upregulated in progesterone-treated and Chlamydia-infected cells only included multiple CC and CXC chemokines, IL-17C, IL-29, IL-32, TNF-a, DEFB4B, LCN2, S100A7-9, ITGAM, NOD2, JAK1, IL-6ST, type I and II interferon receptors, numerous interferon-stimulated genes and STAT6. CXCL10, CXCL11, CX3CL1 and IL-17C, which were also upregu- lated in infected secretory-stage primary cells, and there was a trend towards higher levels of immune mediators in infected secretory-phase compared with proliferative-phase cells. Conclusion Progesterone treatment primes multiple innate immune pathways in hormone-responsive epithelial cells that could potentially increase resis- tance to chlamydial infection.

Innate transcriptional networks activated in bladder in response to uropathogenic Escherichia coli drive diverse biological pathways and rapid synthesis of IL-10 for defense against bacterial urinary tract infection

Early transcriptional activation events that occur in bladder immediately following bacterial urinary tract infection (UTI) are not well defined. In this study, we describe the whole bladder transcriptome of uropathogenic Escherichia coli (UPEC) cystitis in mice using genome-wide expression profiling to define the transcriptome of innate immune activation stemming from UPEC colonization of the bladder. Bladder RNA from female C57BL/6 mice, analyzed using 1.0 ST-Affymetrix microarrays, revealed extensive activation of diverse sets of innate immune response genes, including those that encode multiple IL-family members, receptors, metabolic regulators, MAPK activators, and lymphocyte signaling molecules. These were among 1564 genes differentially regulated at 2 h postinfection, highlighting a rapid and broad innate immune response to bladder colonization. Integrative systems-level analyses using InnateDB (http://www.innatedb.com) bioinformatics and ingenuity pathway analysis identified multiple distinct biological pathways in the bladder transcriptome with extensive involvement of lymphocyte signaling, cell cycle alterations, cytoskeletal, and metabolic changes. A key regulator of IL activity identified in the transcriptome was IL-10, which was analyzed functionally to reveal marked exacerbation of cystitis in IL-10–deficient mice. Studies of clinical UTI revealed significantly elevated urinary IL-10 in patients with UPEC cystitis, indicating a role for IL-10 in the innate response to human UTI. The whole bladder transcriptome presented in this work provides new insight into the diversity of innate factors that determine UTI on a genome-wide scale and will be valuable for further data mining. Identification of protective roles for other elements in the transcriptome will provide critical new insight into the complex cascade of events that underpin UTI.

The Escherichia coli O157:H7 EhaB autotransporter protein binds to laminin and collagen I and induces a serum IgA response in O157:H7 challenged cattle

Enterohaemorrhagic Escherichia coli (EHEC) are a subgroup of Shiga toxin-producing E. coli that cause gastrointestinal disease with the potential for life-threatening sequelae. Cattle serve as the natural reservoir for EHEC and outbreaks occur sporadically as a result of contaminated beef and other farming products. While certain EHEC virulence mechanisms have been extensively studied, the factors that mediate host colonization are poorly defined. Previously, we identified four proteins (EhaA,B,C,D) from the prototypic EHEC strain EDL933 that belong to the autotransporter (AT) family. Here we characterize the EhaB AT protein. EhaB was shown to be located at the cell surface and overexpression in E. coli K-12 resulted in significant biofilm formation under continuous flow conditions. Overexpression of EhaB in E. coli K12 and EDL933 backgrounds also promoted adhesion to the extracellular matrix proteins collagen I and laminin. An EhaB-specific antibody revealed that EhaB is expressed in E. coli EDL933 following in vitro growth. EhaB also cross-reacted with serum IgA from cattle challenged with E. coli O157:H7, indicating that EhaB is expressed in vivo and elicits a host IgA immune response.