Influence of culture conditions on the molecular signature of mesenchymal stem cells

Cell based therapies require cells capable of self renewal and differentiation, and a prerequisite is the ability to prepare an effective dose of ex vivo expanded cells for autologous transplants. The in vivo identification of a source of physiologically relevant cell types suitable for cell therapies is therefore an integral part of tissue engineering. Bone marrow is the most easily accessible source of mesenchymal stem cells (MSCs), and harbours two distinct populations of adult stem cells; namely hematopoietic stem cells (HSCs) and bone mesenchymal stem cells (BMSCs). Unlike HSCs, there are yet no rigorous criteria for characterizing BMSCs. Changing understanding about the pluripotency of BMSCs in recent studies has expanded their potential application; however, the underlying molecular pathways which impart the features distinctive to BMSCs remain elusive. Furthermore, the sparse in vivo distribution of these cells imposes a clear limitation to their in vitro study. Also, when BMSCs are cultured in vitro there is a loss of the in vivo microenvironment which results in a progressive decline in proliferation potential and multipotentiality. This is further exacerbated with increased passage number, characterized by the onset of senescence related changes. Accordingly, establishing protocols for generating large numbers of BMSCs without affecting their differentiation potential is necessary. The principal aims of this thesis were to identify potential molecular factors for characterizing BMSCs from osteoarthritic patients, and also to attempt to establish culture protocols favourable for generating large number of BMSCs, while at the same time retaining their proliferation and differentiation potential. Previously published studies concerning clonal cells have demonstrated that BMSCs are heterogeneous populations of cells at various stages of growth. Some cells are higher in the hierarchy and represent the progenitors, while other cells occupy a lower position in the hierarchy and are therefore more committed to a particular lineage. This feature of BMSCs was made evident by the work of Mareddy et al., which involved generating clonal populations of BMSCs from bone marrow of osteoarthritic patients, by a single cell clonal culture method. Proliferation potential and differentiation capabilities were used to group cells into fast growing and slow growing clones. The study presented here is a continuation of the work of Mareddy et al. and employed immunological and array based techniques to identify the primary molecular factors involved in regulating phenotypic characteristics exhibited by contrasting clonal populations. The subtractive immunization (SI) was used to generate novel antibodies against favourably expressed proteins in the fast growing clonal cell population. The difference between the clonal populations at the transcriptional level was determined using a Stem Cell RT2 Profiler TM PCR Array which focuses on stem cell pathway gene expression. Monoclonal antibodies (mAb) generated by SI were able to effectively highlight differentially expressed antigenic determinants, as was evident by Western blot analysis and confocal microscopy. Co-immunoprecipitation, followed by mass spectroscopy analysis, identified a favourably expressed protein as the cytoskeletal protein vimentin. The stem cell gene array highlighted genes that were highly upregulated in the fast growing clonal cell population. Based on their functions these genes were grouped into growth factors, cell fate determination and maintenance of embryonic and neural stem cell renewal. Furthermore, on a closer analysis it was established that the cytoskeletal protein vimentin and nine out of ten genes identified by gene array were associated with chondrogenesis or cartilage repair, consistent with the potential role played by BMSCs in defect repair and maintaining tissue homeostasis, by modulating the gene expression pattern to compensate for degenerated cartilage in osteoarthritic tissues. The gene array also presented transcripts for embryonic lineage markers such as FOXA2 and Sox2, both of which were significantly over expressed in fast growing clonal populations. A recent groundbreaking study by Yamanaka et al imparted embryonic stem cell (ESCs) -like characteristic to somatic cells in a process termed nuclear reprogramming, by the ectopic expression of the genes Sox2, cMyc and Oct4. The expression of embryonic lineage markers in adult stem cells may be a mechanism by which the favourable behaviour of fast growing clonal cells is determined and suggests a possible active phenomenon of spontaneous reprogramming in fast growing clonal cells. The expression pattern of these critical molecular markers could be indicative of the competence of BMSCs. For this reason, the expression pattern of Sox2, Oct4 and cMyc, at various passages in heterogeneous BMSCs population and tissue derived cells (osteoblasts and chondrocytes), was investigated by a real-time PCR and immunoflourescence staining. A strong nuclear staining was observed for Sox2, Oct4 and cMyc, which gradually weakened accompanied with cytoplasmic translocation after several passage. The mRNA and protein expression of Sox2, Oct4 and cMyc peaked at the third passage for osteoblasts, chondrocytes and third passage for BMSCs, and declined with each subsequent passage, indicating towards a possible mechanism of spontaneous reprogramming. This study proposes that the progressive decline in proliferation potential and multipotentiality associated with increased passaging of BMSCs in vitro might be a consequence of loss of these propluripotency factors. We therefore hypothesise that the expression of these master genes is not an intrinsic cell function, but rather an outcome of interaction of the cells with their microenvironment; this was evident by the fact that when removed from their in vivo microenvironment, BMSCs undergo a rapid loss of stemness after only a few passages. One of the most interesting aspects of this study was the integration of factors in the culture conditions, which to some extent, mimicked the in vivo microenvironmental niche of the BMSCs. A number of studies have successfully established that the cellular niche is not an inert tissue component but is of prime importance. The total sum of stimuli from the microenvironment underpins the complex interplay of regulatory mechanisms which control multiple functions in stem cells most importantly stem cell renewal. Therefore, well characterised factors which affect BMSCs characteristics, such as fibronectin (FN) coating, and morphogens such as FGF2 and BMP4, were incorporated into the cell culture conditions. The experimental set up was designed to provide insight into the expression pattern of the stem cell related transcription factors Sox2, cMyc and Oct4, in BMSCs with respect to passaging and changes in culture conditions. Induction of these pluripotency markers in somatic cells by retroviral transfection has been shown to confer pluripotency and an ESCs like state. Our study demonstrated that all treatments could transiently induce the expression of Sox2, cMyc and Oct4, and favourably affect the proliferation potential of BMSCs. The combined effect of these treatments was able to induce and retain the endogenous nuclear expression of stem cell transcription factors in BMSCs over an extended number of in vitro passages. Our results therefore suggest that the transient induction and manipulation of endogenous expression of transcription factors critical for stemness can be achieved by modulating the culture conditions; the benefit of which is to circumvent the need for genetic manipulations. In summary, this study has explored the role of BMSCs in the diseased state of osteoarthritis, by employing transcriptional profiling along with SI. In particular this study pioneered the use of primary cells for generating novel antibodies by SI. We established that somatic cells and BMSCs have a basal level of expression of pluripotency markers. Furthermore, our study indicates that intrinsic signalling mechanisms of BMSCs are intimately linked with extrinsic cues from the microenvironment and that these signals appear to be critical for retaining the expression of genes to maintain cell stemness in long term in vitro culture. This project provides a basis for developing an “artificial niche” required for reversion of commitment and maintenance of BMSC in their uncommitted homeostatic state.

Partial protection against chlamydial reproductive tract infection by a recombinant major outer membrane protein/CpG/cholera toxin intranasal vaccine in the guinea pig Chlamydia cavaie model

Chlamydia trachomatis is a major cause of sexually transmitted diseases worldwide. There currently is no vaccine to protect against chlamydial infection of the female reproductive tract. Vaccine development has predominantly involved using the murine model, however infection of female guinea pigs with Chlamydia caviae more closely resembles chlamydial infection of the human female reproductive tract, and presents a better model to assess potential human chlamydial vaccines. We immunised female guinea pigs intranasally with recombinant major outer membrane protein (r-MOMP) combined with CpG-10109 and cholera toxin adjuvants. Both systemic and mucosal immune responses were elicited in immunised animals. MOMP-specific IgG and IgA were present in the vaginal mucosae, and high levels of MOMP-specific IgG were detected in the serum of immunised animals. Antibodies from the vaginal mucosae were also shown to be capable of neutralising C. caviae in vitro. Following immunisation, animals were challenged intravaginally with a live C. caviae infection of 102 inclusion forming units. We observed a decrease in duration of infection and a significant (p<0.025) reduction in infection load in r-MOMP immunised animals, compared to animals immunised with adjuvant only. Importantly, we also observed a marked reduction in upper reproductive tract (URT) pathology in r-MOMP immunised animals. Intranasal immunisation of female guinea pigs with r-MOMP was able to provide partial protection against C. caviae infection, not only by reducing chlamydial burden but also URT pathology. This data demonstrates the value of using the guinea pig model to evaluate potential chlamydial vaccines for protection against infection and disease pathology caused by C. trachomatis in the female reproductive tract.

Association of immunoproteasomes with the endoplasmic reticulum

Proteasomes are complex multisubunit proteases which play a critical role in intracellular proteolysis. Immunoproteasomes, which contain three c-interferon-inducible subunits, are a subset of proteasomes which have a specialized function in antigen processing for presentation by the MHC class I pathway. Two of the c-interferon inducible subunits, LMP2 and LMP7, are encoded within the MHC class II region adjacent to the two TAP (transporter associated with antigen presentation) genes. We have investigated the localization of immunoproteasomes using monoclonal antibodies to LMP2 and LMP7. Immunoproteasomes were strongly enriched around the endoplasmic reticulum as judged by double-immuno¯uorescence experiments with anticalreticulin antibodies, but were also present in the nucleus and throughout the cytosol. In contrast, proteasome subunit C2, which is present in all proteasomes, was found to be evenly distributed throughout the cytoplasm and in the nucleus, as was the delta subunit, which is replaced by LMP2 in immunoproteasomes. c-Interferon increased the level of immunoproteasomes, but had no effect on their distribution. Our results provide the ®rst direct evidence that immunoproteasomes are strongly enriched at the endoplasmic reticulum, where they may be located close to the TAP transporter to provide efficient transport of peptides into the lumen of the endoplasmic recticulum for association with MHC class I molecules.

β-glucan triggers spondylarthritis and Crohn's disease-like ileitis in SKG mice

Objective The spondylarthritides (SpA), including ankylosing spondylitis (AS), psoriatic arthritis (PsA), reactive arthritis, and arthritis associated with inflammatory bowel disease, cause chronic inflammation of the large peripheral and axial joints, eyes, skin, ileum, and colon. Genetic studies reveal common candidate genes for AS, PsA, and Crohn's disease, including IL23R, IL12B, STAT3, and CARD9, all of which are associated with interleukin-23 (IL-23) signaling downstream of the dectin 1 β-glucan receptor. In autoimmune-prone SKG mice with mutated ZAP-70, which attenuates T cell receptor signaling and increases the autoreactivity of T cells in the peripheral repertoire, IL-17–dependent inflammatory arthritis developed after dectin 1–mediated fungal infection. This study was undertaken to determine whether SKG mice injected with 1,3-β-glucan (curdlan) develop evidence of SpA, and the relationship of innate and adaptive autoimmunity to this process. Methods SKG mice and control BALB/c mice were injected once with curdlan or mannan. Arthritis was scored weekly, and organs were assessed for pathologic features. Anti–IL-23 monoclonal antibodies were injected into curdlan-treated SKG mice. CD4+ T cells were transferred from curdlan-treated mice to SCID mice, and sera were analyzed for autoantibodies. Results After systemic injection of curdlan, SKG mice developed enthesitis, wrist, ankle, and sacroiliac joint arthritis, dactylitis, plantar fasciitis, vertebral inflammation, ileitis resembling Crohn's disease, and unilateral uveitis. Mannan triggered spondylitis and arthritis. Arthritis and spondylitis were T cell– and IL-23–dependent and were transferable to SCID recipients with CD4+ T cells. SpA was associated with collagen- and proteoglycan-specific autoantibodies. Conclusion Our findings indicate that the SKG ZAP-70W163C mutation predisposes BALB/c mice to SpA, resulting from innate and adaptive autoimmunity, after systemic β-glucan or mannan exposure.

Differential expression and distribution of syndecan-1 and -2 in periodontal wound healing of the rat

Cell-surface proteoglycans participate in several biological functions including interactions with adhesion molecules, growth factors and a variety of other effector molecules. Accordingly, these molecules play a central role in various aspects of cell–cell and cell–matrix interactions. To investigate the expression and distribution of the cell surface proteoglycans, syndecan-1 and -2, during periodontal wound healing, immunohistochemical analyses were carried out using monoclonal antibodies against syndecan-1, or -2 core proteins. Both syndecan-1 and -2 were expressed and distributed differentially at various stages of early inflammatory cell infiltration, granulation tissue formation, and tissue remodeling in periodontal wound healing. Expression of syndecan-1 was noted in inflammatory cells within and around the fibrin clots during the earliest stages of inflammatory cell infiltration. During granulation tissue formation it was noted in fibroblast-like cells and newly formed blood vessels. Syndecan-1 was not seen in newly formed bone or cementum matrix at any of the time periods studied. Syndecan-1 expression was generally less during the late stages of wound healing but was markedly expressed in cells that were close to the repairing junctional epithelium. In contrast, syndecan-2 expression and distribution was not evident at the early stages of inflammatory cell infiltration. During the formation of granulation tissue and subsequent tissue remodeling, syndecan-2 was expressed extracellularly in the newly formed fibrils which were oriented toward the root surface. Syndecan-2 was found to be significantly expressed on cells that were close to the root surface and within the matrix of repaired cementum covering root dentin as well as at the alveolar bone edge. These findings indicate that syndecan-1 and -2 may have distinctive functions during wound healing of the periodontium. The appearance of syndecan-1 may involve both cell–cell and cell–matrix interactions, while syndecan-2 showed a predilection to associate with cell–matrix interactions during hard tissue formation.

An introduction to biologics

An introduction to biologics 23.1 Introduction: Principles of biologics and their use as medicines 23.2 Protein biologics used as drugs 23.2.1 Proteins that function through enzymatic or regulatory activity. 23.2.1.1 Biologics as replacement of a deficient or abnormal protein. 23.2.1.2 Proteins that augment an existing biological process. 23.2.1.3 Proteins that provide a novel function or activity. 23.2.2. Proteins that function through specific targeting activity. 23.2.2.1. Monoclonal antibody nomenclature. 23.2.2.2. Naked monoclonal antibodies. 23.2.2.3. Conjugated monoclonal antibodies. 23.2.3. Recombinant protein vaccines.

An introduction to anticancer drugs

An introduction to anticancer drugs 24.1 Introduction 24.2 The rationale behind anticancer drug therapy 24.3 Drugs used in cancer 24.3.1 Alkylating agents 24.3.2 Cytotoxic antibiotics 24.3.3 Antimetabolites 24.3.4 Microtubule inhibitors 24.3.5 Monoclonal antibodies 24.3.6 Steroid hormones and their antagonists 24.3.7 Other treatments

Expression of HPV-11 L1 protein in transgenic Arabidopsis thaliana and Nicotiana tabacum

Background We have investigated the possibility and feasibility of producing the HPV-11 L1 major capsid protein in transgenic Arabidopsis thaliana ecotype Columbia and Nicotiana tabacum cv. Xanthi as potential sources for an inexpensive subunit vaccine. Results Transformation of plants was only achieved with the HPV-11 L1 gene with the C-terminal nuclear localization signal (NLS-) encoding region removed, and not with the full-length gene. The HPV-11 L1 NLS- gene was stably integrated and inherited through several generations of transgenic plants. Plant-derived HPV-11 L1 protein was capable of assembling into virus-like particles (VLPs), although resulting particles displayed a pleomorphic phenotype. Neutralising monoclonal antibodies binding both surface-linear and conformation-specific epitopes bound the A. thaliana-derived particles and - to a lesser degree - the N. tabacum-derived particles, suggesting that plant-derived and insect cell-derived VLPs displayed similar antigenic properties. Yields of up to 12 μg/g of HPV-11 L1 NLS- protein were harvested from transgenic A. thaliana plants, and 2 μg/g from N. tabacum plants - a significant increase over previous efforts. Immunization of New Zealand white rabbits with ∼50 μg of plant-derived HPV-11 L1 NLS- protein induced an antibody response that predominantly recognized insect cell-produced HPV-11 L1 NLS- and not NLS+ VLPs. Evaluation of the same sera concluded that none of them were able to neutralise pseudovirion in vitro. Conclusion We expressed the wild-type HPV-11 L1 NLS- gene in two different plant species and increased yields of HPV-11 L1 protein by between 500 and 1000-fold compared to previous reports. Inoculation of rabbits with extracts from both plant types resulted in a weak immune response, and antisera neither reacted with native HPV-11 L1 VLPs, nor did they neutralise HPV-11 pseudovirion infectivity. This has important and potentially negative implications for the production of HPV-11 vaccines in plants. © 2007 Kohl et al; licensee BioMed Central Ltd.

Third International Conference on plant-based vaccines and antibodies

This relatively new biennial meeting - the first was in Prague in 2005 - was chaired by Julian Ma (Guy's Hospital, London, UK), with Mario Pezzotti (University of Verona, Italy) as local organizer, and attracted approximately 180 delegates from 25 countries. The theme was 'Plant Expression Systems for Recombinant Pharmacologics': there were 46 talks gathered into two plenaries, 12 themed sessions and 72 posters. Topics covered included publicly funded and commercial developments, innovation, regulation and commercialization, competition with conventional technology, manufacture and new products. © 2009 Expert Reviews Ltd.

More men than women make mucosal IgA antibodies to human papillomavirus type 16 (HPV-16) and HPV-18: A study of oral HPV and oral HPV antibodies in a normal healthy population

Background: We have previously shown the high prevalence of oral anti-human papillomavirus type 16 (HPV-16) antibodies in women with HPV-associated cervical neoplasia. It was postulated that the HPV antibodies were initiated after HPV antigenic stimulation at the cervix via the common mucosal immune system. The present study aimed to further evaluate the effectiveness of oral fluid testing for detecting the mucosal humoral response to HPV infection and to advance our limited understanding of the immune response to HPV. Methods: The prevalence of oral HPV infection and oral antibodies to HPV types 16, 18 and 11 was determined in a normal, healthy population of children, adolescents and adults, both male and female, attending a dental clinic. HPV types in buccal cells were determined by DNA sequencing. Oral fluid was collected from the gingival crevice of the mouth by the OraSure method. HPV-16, HPV-18 and HPV-11 antibodies in oral fluid were detected by virus-like particle-based enzyme-linked immunosorbent assay. As a reference group 44 women with cervical neoplasia were included in the study. Results: Oral HPV infection was h ighest in children (9/114, 7.9%), followed by adolescents (4/78, 5.1%), and lowest in normal adults (4/116, 3.5%). The predominant HPV type found was HPV-13 (7/22, 31.8%) followed by HPV-32 (5/22, 22.7%). The prevalence of oral antibodies to HPV-16, HPV-18 and HPV-11 was low in children and increased substantially in adolescents and normal adults. Oral HPV-16 IgA was significantly more prevalent in women with cervical neoplasia (30/44, 68.2%) than the women from the dental clinic (18/69, 26.1% P = 0.0001). Significantly more adult men than women displayed oral HPV-16 IgA (30/47 compared with 18/69, OR 5.0, 95% CI 2.09-12.1, P < 0.001) and HPV-18 IgA (17/47 compared with 13/69, OR 2.4, 95% CI 0.97-6.2, P = 0.04). Conclusion: The increased prevalence of oral HPV antibodies in adolescent individuals compared with children was attributed to the onset of sexual activity. The increased prevalence of oral anti-HPV IgA in men compared with women was noteworthy considering reportedly fewer men than women make serum antibodies, and warrants further investigation. © 2006 Marais et al; licensee BioMed Central Ltd.

An investigation into the use of human papillomavirus type 16 virus-like particles as a delivery vector system for foreign proteins: N- and C-terminal fusion of GFP to the L1 and L2 capsid proteins

Development of vaccine strategies against human papillomavirus (HPV), which causes cervical cancer, is a priority. We investigated the use of virus-like particles (VLPs) of the most prevalent type, HPV-16, as carriers of foreign proteins. Green fluorescent protein (GFP) was fused to the N or C terminus of both L1 and L2, with L2 chimeras being co-expressed with native L1. Purified chimaeric VLPs were comparable in size (∼55 nm) to native HPV VLPs. Conformation-specific monoclonal antibodies (Mabs) bound to the VLPs, thereby indicating that they possibly retain their antigenicity. In addition, all of the VLPs encapsidated DNA in the range of 6-8 kb. © 2007 Springer-Verlag.

Report on the AIDS Vaccine 2008 Conference

The "AIDS Vaccine 2008" Conference was held in Cape Town, South Africa (October 13 to 16, 2008) and organized, under the aegis of the Global HIV Vaccine Enterprise, by Dr. Lynn Morris (Chair of the Conference) National Institute of Communicable Diseases; Dr. Koleka Mlisana from CAPRISA, University KwaZulu-Natal, Durban, Dr. Glenda Gray from Perinatal HIV Research Unit, University Witwatersrand, Johannesburg and Dr. Carolyn Williamson from Institute of Infectious Diseses. and Molecular Medicine, UCT, Cape Town (Co-Chairs of the Conference). Since the first AIDS Vaccine conference, organized in Paris in 2000, this was the first time it was held outside of the U.S. and Europe, and involved nearly 1,000 participants. Besides three Plenary Sessions with ten state-of-the-art plenary lectures and one Keynote Lecture given by Dr. A.S. Fauci (Director of NIAID, NIH, USA), the Conference was organized in nine oral sessions, four poster discussion groups covering a wide spectrum of scientific information relating to HIV vaccine research and development. Moreover three Symposia, two Special Sessions, one Roundtable as well as two Debates were held, the latter focusing on current controversial topics. The conference opening was memorable for a number of reasons: among these was the presence of South Africa's new Minister of Health, Barbara Hogan who, in her first speech in a major forum as a senior member of the SA Government, affirmed that HIV causes AIDS, and that the search for a vaccine is of paramount importance to SA and the rest of the world. A scientific summary of the Conference is reported in the present article, divided into four major topics: (1) vaccine concepts and design; (2) T-cell immunology and innate immunity; (3) B-cell immunology, neutralizing antibodies and mucosal immunology; and (4) clinical trials. © 2009 Landes Bioscience.

Multifocal urothelial cancers with the mutator phenotype are of monoclonal origin and require panurothelial treatment for tumor clearance

Purpose: UC is a disease of the entire urothelium, characterized by multiplicity and multifocality. The clonal relationship among multiple UCs has implications regarding adjuvant chemotherapy. It has been investigated in studies of chromosomal alteration and single gene mutation. However, these genetic changes can occur in unrelated tumors under similar carcinogenic selection pressures. Tumors with high MSI have numerous DNA mutations, of which many provide no selection benefit. While these tumors represent an ideal model for studying UC clonality, their low frequency has prevented their previous investigation. Materials and Methods: We investigated 32 upper and lower urinary tract UCs with high MSI and 4 nonUC primary cancers in 9 patients. We used the high frequency and specificity of individual DNA mutations in these tumors (MSI at 17 loci) and the early timing of epigenetic events (methylation of 7 gene promoters) to investigate tumor clonality. Results: Molecular alterations varied among tumors from different primary organs but they appeared related in the UCs of all 9 patients. While 7 patients had a high degree of concordance among UCs, in 2 the UCs shared only a few similar alterations. Genetic and epigenetic abnormalities were frequently found in normal urothelial samples. Conclusions: Multiple UCs in each patient appeared to arise from a single clone. The molecular order of tumor development varied from the timing of clinical presentation and suggested that residual malignant cells persist in the urinary tract despite apparent curative surgery. These cells lead to subsequent tumor relapse and new methods are required to detect and eradicate them.

Acetylcholine receptor antibody in the diagnosis and management of myasthenia gravis

The relationship of acetylcholine receptor (AchR) antibodies to disease activity in myasthenia gravis (MG) is controversial. Some authors claim a direct correlation with disease activity and treatment, in particular plasmapheresis therapy, whereas others have commented on the poor overall correlation of antibody levels with clinical state. Antibody levels were examined in a population of MG patients and correlated with disease activity and response to treatment. Antibodies to skeletal muscle AchR were found in most patients with generalised MG (24/25) and in about half of the patients with purely ocular MG (6/10) and in neither of 2 patients with congenital MG. There was scant correlation with disease activity or response to treatment. It is concluded that the assay is more useful for diagnosis than for management of MG.