Dual plasmonic gold nanostars for photoacoustic imaging and photothermal therapy.

AIM: To fabricate multimodal nanoconstruct that act as a single node for photoacoustic imaging (PAI) and photothermal therapy (PTT) in the fight against cancer. MATERIALS & METHODS: Dual plasmonic gold nanostars (DPGNS) were chemically synthesized by reducing gold precursor using ascorbic acid and silver ions as shape directing agent. PAI and PTT were performed using commonly available 1064 nm laser source on DPGNS embedded tumor xenografts on mice. RESULTS & CONCLUSION: Photoacoustic amplitude increase with longer wavelength source and with silica coating of DPGNS. The in vivo photothermal capability of DPGNS resulted in a significant decrease in the tumor cellular area. DPGNS exhibited potential for single node diagnosis and therapy with longer wavelength facilitating deeper imaging and therapy.

Functional imaging for regenerative medicine.

In vivo imaging is a platform technology with the power to put function in its natural structural context. With the drive to translate stem cell therapies into pre-clinical and clinical trials, early selection of the right imaging techniques is paramount to success. There are many instances in regenerative medicine where the biological, biochemical, and biomechanical mechanisms behind the proposed function of stem cell therapies can be elucidated by appropriate imaging. Imaging techniques can be divided according to whether labels are used and as to whether the imaging can be done in vivo. In vivo human imaging places additional restrictions on the imaging tools that can be used. Microscopies and nanoscopies, especially those requiring fluorescent markers, have made an extraordinary impact on discovery at the molecular and cellular level, but due to their very limited ability to focus in the scattering tissues encountered for in vivo applications they are largely confined to superficial imaging applications in research laboratories. Nanoscopy, which has tremendous benefits in resolution, is limited to the near-field (e.g. near-field scanning optical microscope (NSNOM)) or to very high light intensity (e.g. stimulated emission depletion (STED)) or to slow stochastic events (photo-activated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM)). In all cases, nanoscopy is limited to very superficial applications. Imaging depth may be increased using multiphoton or coherence gating tricks. Scattering dominates the limitation on imaging depth in most tissues and this can be mitigated by the application of optical clearing techniques that can impose mild (e.g. topical application of glycerol) or severe (e.g. CLARITY) changes to the tissue to be imaged. Progression of therapies through to clinical trials requires some thought as to the imaging and sensing modalities that should be used. Smoother progression is facilitated by the use of comparable imaging modalities throughout the discovery and trial phases, giving label-free techniques an advantage wherever they can be used, although this is seldom considered in the early stages. In this paper, we will explore the techniques that have found success in aiding discovery in stem cell therapies and try to predict the likely technologies best suited to translation and future directions.

Allogeneic murine mesenchymal stem cells: migration to inflamed joints in vivo and amelioration of collagen induced arthritis when transduced to express CTLA4Ig.

Despite the immunosuppressive, homing, and regenerative capabilities of mesenchymal stem cells (MSCs), their ability to migrate to arthritic joints and influence the course of arthritis in vivo remains poorly understood. The objective of this study was to determine if allogeneic MSCs migrate to inflamed joints in vivo and to determine if MSCs expressing the costimulation blocker cytotoxic T lymphocyte associated antigen-4 coupled to immunoglobulin-G (CTLA4Ig) could be used to ameliorate collagen induced arthritis (CIA). The migration of systemically delivered inbred mouse strain (FVB) MSCs to migrate to inflamed joints in CIA was studied using real-time quantitative polymerase chain reaction. Furthermore, the effect of BALB/c MSCs modified with an adenoviral vector to express CTLA4Ig, on T cell function in vitro and on CIA in vivo was assessed. After systemic delivery of FVB MSCs, eGFP DNA was detectable in the joints of mice with CIA confirming that some MSCs had reached to inflamed joints. BALB/c MSCs suppressed the secretion of both TNFα and IFNγ, and reduced the ratio of Th1:Th2 cytokine expression, by DBA/1 T cells in vitro irrespective of viral modification. The expression of CTLA4Ig did not augment this effect. Despite a worsening of disease scores after infusion of BALB/c MSCs in vivo, BALB/c MSCs expressing CTLA4Ig significantly delayed the onset of inflammatory arthritis in CIA. These data demonstrate that allogeneic MSCs can migrate to the inflamed joints of CIA in vivo and that genetically modified allogeneic MSCs may be considered for development of gene therapy strategies for inflammatory arthritis.

Genetic mismatch affects the immunosuppressive properties of mesenchymal stem cells in vitro and their ability to influence the course of collagen-induced arthritis.

INTRODUCTION: The immunological and homing properties of mesenchymal stem cells (MSCs) provide a potentially attractive treatment for arthritis. The objective of this study was to determine effects of genetic disparity on the immunosuppressive potential of MSCs in vitro and in vivo within collagen induced arthritis (CIA). METHODS: The ability of DBA/1, FVB and BALB/c MSC preparations to impact the cytokine release profile of CD3/CD28 stimulated DBA/1 T cells was assessed in vitro. The effect of systemically delivered MSCs on the progression of CIA and cytokine production was assessed in vivo. RESULTS: All MSC preparations suppressed the release of TNFα and augmented the secretion of IL-4 and IL-10 by stimulated DBA/1 T-cells. However, assessment of the ratio of IFNγ to IL-4 production indicated that the more genetically distant BALB/c MSCs had significantly less immunosuppressive capacity. Systemic delivery of BALB/c MSC resulted in an exacerbation of CIA disease score in vivo and a higher erosive disease burden. This was not seen after treatment with syngeneic or partially mismatched MSCs. An increase in serum levels of IL-1ß was observed up to 20 days post treatment with allogeneic MSCs. An initial elevation of IL-17 in these treatment groups persisted in those treated with fully mismatched BALB/c MSCs. Over the course of the study, there was a significant suppression of serum IL-17 levels in groups treated with syngeneic MSCs. CONCLUSIONS: These data demonstrate a significant difference in the immunosuppressive properties of syngeneic and allogeneic MSCs in vitro and in vivo, which needs to be appreciated when developing MSC based therapies for inflammatory arthritis.

Mesenchymal Stem Cell-mediated delivery of the sodium iodide symporter supports radionuclide imaging and treatment of breast cancer.

Mesenchymal Stem Cells (MSCs) migrate specifically to tumors in vivo, and coupled with their capacity to bypass immune surveillance, are attractive vehicles for tumor-targeted delivery of therapeutic agents. This study aimed to introduce MSC-mediated expression of the sodium iodide symporter (NIS) for imaging and therapy of breast cancer. Tumor bearing animals received an intravenous or intratumoral injection of NIS expressing MSCs (MSC-NIS), followed by (99m) Technetium pertechnetate imaging 3-14 days later using a BazookaSPECT γ-camera. Tissue was harvested for analysis of human NIS (hNIS) expression by relative quantitative-polymerase chain reaction. Therapy animals received an i.p. injection of (131) I or saline 14 days after injection of MSC-NIS, and tumor volume was monitored for 8 weeks. After injection of MSC-NIS, BazookaSPECT imaging revealed an image of animal intestines and chest area at day 3, along with a visible weak tumor image. By day 14, the tumor was visible with a significant reduction in radionuclide accumulation in nontarget tissue observed. hNIS gene expression was detected in the intestines, heart, lungs, and tumors at early time points but later depleted in nontarget tissues and persisted at the tumor site. Based on imaging/biodistribution data, animals received a therapeutic dose of (131) I 14 days after MSC-NIS injection. This resulted in a significant reduction in tumor growth (mean ± SEM, 236 ± 62 mm(3) vs. 665 ± 204 mm(3) in controls). The ability to track MSC migration and transgene expression noninvasively in real time before therapy is a major advantage to this strategy. This promising data supports the feasibility of this approach as a novel therapy for breast cancer.

Mesenchymal stem cells and osteoarthritis: remedy or accomplice?

Multipotent mesenchymal stromal or stem cells (MSCs) are likely to be agents of connective tissue homeostasis and repair. Because the hallmark of osteoarthritis (OA) is degeneration and failure to repair connective tissues it is compelling to think that these cells have a role to play in OA. Indeed, MSCs have been implicated in the pathogenesis of OA and, in turn, progression of the disease has been shown to be therapeutically modulated by MSCs. This review discusses current knowledge on the potential of both marrow- and local joint-derived MSCs in OA, the mode of action of the cells, and possible effects of the osteoarthritic niche on the function of MSCs. The use of stem cells for repair of isolated cartilage lesions and strategies for modulation of OA using local cell delivery are discussed as well as therapeutic options for the future to recruit and appropriately activate endogenous progenitors and/or locally systemically administered MSCs in the early stages of the disease. The use of gene therapy protocols, particularly as they pertain to modulation of inflammation associated with the osteoarthritic niche, offer an additional option in the treatment of this chronic disease. In summary, elucidation of the etiology of OA and development of technologies to detect early disease, allied to an increased understanding of the role MSCs in aging and OA, should lead to more targeted and efficacious treatments for this debilitating chronic disease in the future.

Membrane ERalpha-dependent activation of PKCalpha in endometrial cancer cells by estradiol.

The purpose of this study was to investigate the role of the oestrogen receptor subtypes ERalpha and ERbeta in mediating the non-genomic effects of 17-beta-estradiol (E(2)) in two human endometrial cancer cell lines (RL95-2 and HEC-1A) expressing different levels of these receptor subtypes. Western blotting analysis using phosphorylation site-specific antibodies showed that physiological concentrations of E(2) rapidly (<20 min) activated PKCalpha, but not PKCdelta in the RL95-2 cell line. E(2) had no effect on PKCalpha or PKCdelta activity in the HEC-1A cell line and suppressed basal levels of PKA activity in both cell lines. PKCalpha activation coincided with its membrane translocation. ERalpha was detected in the RL95-2 cell line by Western blotting and RT-PCR but not in the HEC-1A cells, which did express ERbeta. A selective ERalpha agonist PPT had the same effect as E(2) on PKCalpha activation in the RL95-2 cells, but the selective ERbeta agonist DPN had no such effect. A 46kDa variant of ERalpha increased in abundance in the cell membrane within 20 min of E(2) treatment suggesting that ERalpha mediated the E(2) non-genomic effects on PKCalpha through the formation of a membrane associated signalling complex.

Telomerase: is it the future diagnostic and prognostic tool in human cancer?

A number of methods exist to detect levels of telomerase activity and the presence of telomerase subunits in a variety of tissues. As telomerase activation seems to be an important step in tumorigenesis, accurate detection of the presence and activity of the enzyme and its subunits is vital. The original method of detecting telomerase activity was developed by Kim and coworkers in 1994, and was termed the telomeric repeat amplification protocol. This assay led to a staggering increase in the number of telomerase-associated publications in scientific journals (85 publications from 1974-1994, 5063 publications from 1994-2004). A number of methods have been described to detect telomeres and to measure their length, with the standard measurement of telomere length performed using a modification of the Southern blot protocol. RNA in situ hybridization can be performed to detect levels of the RNA component of telomerase, and standard in situ hybridization and immunohistochemistry can be applied to examine expression levels and localization of the catalytic subunit of the enzyme. Reverse transcriptase PCR has also been applied to assess expression levels of the telomerase components in various tissues. This review provides a synopsis of telomeres, telomerase, telomerase and cancer, and finally, methods for the detection of telomerase in cancer.