神経障害性疼痛モデル動物におけるトラニラストの鎮痛効果とテトラヒドロビオプテリン合成制御の役割

Trigeminal neuralgia is unilateral, lancinating, episodic pain that can be provoked by routine activities. Anticonvulsants, such as carbamazepine, are the drugs of choice; however, these possess side-effects. Microvascular decompression is the most effective surgical technique with a higher success rate, although occasionally causes adverse effects. The potential treatment for this type of pain remains unmet. Increased tetrahydrobiopterin (BH4) levels have been reported in association with axonal injury. This study aimed to evaluate the effect of tranilast on relieving neuropathic pain in animal models and analyze the changes in BH4 synthesis. Neuropathic pain was induced via infraorbital nerve constriction. Tranilast, carbamazepine, or saline was injected intraperitoneally to assess the rat’s post-intervention pain response. In the von Frey’s test, the tranilast and carbamazepine groups showed significant changes in the head withdrawal threshold in the ipsilateral whisker pad area. The motor coordination test showed no changes in the tranilast group, whereas the carbamazepine group showed decreased performance, indicating impaired motor coordination. Trigeminal ganglion tissues were used for the PCR array analysis of genes that regulate the BH4 pathway. Downregulation of the sepiapterin reductase (Spr) and aldoketo reductase (Akr) genes after tranilast injection was observed compared to the pain model. These findings suggest that tranilast effectively treats neuropathic pain

Mesenchymal Stem Cell Therapy for Osteoarthritis: The Critical Role of the Cell Secretome

Osteoarthritis (OA) is an inflammatory condition still lacking effective treatments. Mesenchymal stem/stromal cells (MSCs) have been successfully employed in pre-clinical models aiming to resurface the degenerated cartilage. In early-phase clinical trials, intra-articular (IA) administration of MSCs leads to pain reduction and cartilage protection or healing. However, the consistent lack of engraftment indicates that the observed effect is delivered through a “hit-and-run” mechanism, by a temporal release of paracrine molecules. MSCs express a variety of chemokines and cytokines that aid in repair of degraded tissue, restoration of normal tissue metabolism and, most importantly, counteracting inflammation. Secretion of therapeutic factors is increased upon licensing by inflammatory signals or apoptosis, induced by the host immune system. Trophic effectors are released as soluble molecules or carried by extracellular vesicles (ECVs). This review provides an overview of the functions and mechanisms of MSC-secreted molecules found to be upregulated in models of OA, whether using in vitro or in vivo models

Feasibility of mesenchmyal stem cells as modulators of inflammation and as a cellular model to study cartilage damage in osteoarthritis

Osteoarthritis (OA) is the most common degenerative condition affecting whole joints and causing pain and cartilage degeneration, particularly in the elderly population. Inflammation of the synovium is now recognised as an important clinical feature initiating and promoting disease progression. Activated macrophages and T lymphocytes infiltrating the OA synovial lining mediate inflammatory responses such as production of pro-inflammatory cytokines, which can induce destructive processes in the cartilage. In addition to this danger signals like alarmins, released as an immune response, further lead to production of soluble mediators that could accelerate cartilage matrix degradation resulting in altered chondrocyte behaviour and hypertrophy. Mesenchymal stem cells (MSCs) have been considered as an attractive option for OA cell therapy. However, inflammation induced catabolic factors are also known to negatively impact cartilage engineering strategies, perhaps inhibiting the use of therapeutic cells for the treatment of OA. The work presented in this thesis sought to investigate the use of engineered MSCs as cellular mediators of anti-inflammation via viral interleukin-10 (vIL10) expression and also the potential of an in vitro model using MSCs to study inflammation-driven cartilage damage in OA. The tetracycline system (Tet) was used to modify mouse mesenchymal stem cells (mMSCs) to over-express vIL10 via adenoviral transduction. Doxycycline acted as a pharmacological switch to control the Tet system and successfully demonstrated efficient and tightly controlled vIL10 production by mMSCs. Engineered vIL10 MSCs proved to be immunosuppressive on activated macrophages and splenocytes in vitro via juxtacrine and paracrine signalling. These finding suggest that the Tet system of inducible vIL10 expression by MSCs may serve as a feasible strategy to enhance MSC-mediated immune regulation that can be translated towards attenuation of inflammation in OA. Furthermore, a three-dimensional in vitro cartilage model to study the effects of inflammation-triggered chondrocyte alterations in OA was developed. Activation of the pathogen recognising receptor NLRP3 inflammasome pathway in the presence of the S100A8/A9 danger ligand signal, in alginate encapsulated articular chondrocyte progenitors (ACPs) and human MSCs (hMSCs) demonstrated anti-chondrogenic effects. This novel model interrogating this ligand receptor interaction could offer a new direction to control/prohibit the release of catabolic factors associated with early inflammatory responses, thereby improving MSC and/or chondroprogenitor-based cartilage engineering strategies. Overall, this thesis showcased the viability of MSCs as potential modulators of inflammation and a possible model to generate novel cartilage regeneration strategies.2022-04-1

Feasibility of mesenchmyal stem cells as modulators of inflammation and as a cellular model to study cartilage damage in osteoarthritis

Osteoarthritis (OA) is the most common degenerative condition affecting whole joints and causing pain and cartilage degeneration, particularly in the elderly population. Inflammation of the synovium is now recognised as an important clinical feature initiating and promoting disease progression. Activated macrophages and T lymphocytes infiltrating the OA synovial lining mediate inflammatory responses such as production of pro-inflammatory cytokines, which can induce destructive processes in the cartilage. In addition to this danger signals like alarmins, released as an immune response, further lead to production of soluble mediators that could accelerate cartilage matrix degradation resulting in altered chondrocyte behaviour and hypertrophy. Mesenchymal stem cells (MSCs) have been considered as an attractive option for OA cell therapy. However, inflammation induced catabolic factors are also known to negatively impact cartilage engineering strategies, perhaps inhibiting the use of therapeutic cells for the treatment of OA. The work presented in this thesis sought to investigate the use of engineered MSCs as cellular mediators of anti-inflammation via viral interleukin-10 (vIL10) expression and also the potential of an in vitro model using MSCs to study inflammation-driven cartilage damage in OA. The tetracycline system (Tet) was used to modify mouse mesenchymal stem cells (mMSCs) to over-express vIL10 via adenoviral transduction. Doxycycline acted as a pharmacological switch to control the Tet system and successfully demonstrated efficient and tightly controlled vIL10 production by mMSCs. Engineered vIL10 MSCs proved to be immunosuppressive on activated macrophages and splenocytes in vitro via juxtacrine and paracrine signalling. These finding suggest that the Tet system of inducible vIL10 expression by MSCs may serve as a feasible strategy to enhance MSC-mediated immune regulation that can be translated towards attenuation of inflammation in OA. Furthermore, a three-dimensional in vitro cartilage model to study the effects of inflammation-triggered chondrocyte alterations in OA was developed. Activation of the pathogen recognising receptor NLRP3 inflammasome pathway in the presence of the S100A8/A9 danger ligand signal, in alginate encapsulated articular chondrocyte progenitors (ACPs) and human MSCs (hMSCs) demonstrated anti-chondrogenic effects. This novel model interrogating this ligand receptor interaction could offer a new direction to control/prohibit the release of catabolic factors associated with early inflammatory responses, thereby improving MSC and/or chondroprogenitor-based cartilage engineering strategies. Overall, this thesis showcased the viability of MSCs as potential modulators of inflammation and a possible model to generate novel cartilage regeneration strategies.2022-04-1

Evaluation of Genotoxicity of Ethyl Methanesulfonate (EMS) Using Human Lymphocytes

ABSTRACT Among the class of ethylating agents known, ethyl methanesulfonate (EMS) is a potential carcinogen and a mutagen. The objective of the present study is to determine the genotoxic potential of ethyl methanesulfonate in human peripheral blood lymphocytes using the gold-standard chromosomal aberration assay. Peripheral blood, collected from a healthy donor was exposed to various concentrations (0.5M, 0.05M and 0.005M) of EMS for 24 hours and 48 hours. Following exposure, the analysis revealed the presence of different types of unstable aberrations like chromatid breaks, gaps and radials. Chromosomal aberration frequency was calculated for EMStreated cells and untreated (control sample) cells. The results were dose-dependent. From the dose-response curve it was evident that cells exposed for 24hours showed more damage compared with those exposed for 48hours. This could probably be attributed to DNA repair mechanism in the subsequent rounds of replication. This finding was consistently observed in all the three doses. Yet another important finding was that, there was no aberration documented in the sample treated with 100µl of 0.005M EMS. This particular observation therefore, helped to define the threshold molar dose below which no genotoxic effect was documented

Interplay of Inflammatory Mediators with Epigenetics and Cartilage Modifications in Osteoarthritis

Osteoarthritis (OA), a degenerative disease of diarthrodial joints, is influenced by mechanical and inflammatory factors with aging, obesity, chronic injuries, and secondary diseases thought to be major factors driving the process of articular cartilage degeneration. Chondrocytes, the cellular component of cartilage, reside in an avascular environment and normally have limited potential to replicate. However, extrinsic factors such as injury to the joint or intrinsic alterations to the chondrocytes themselves can lead to an altered phenotype and development of OA. Synovial inflammation is also a pivotal element of the osteoarthritic, degenerative process: influx of pro-inflammatory cytokines and production of matrix metalloproteinases accelerate advanced cellular processes such as synovitis and cartilage damage. As well as a genetic input, recent data have highlighted epigenetic factors as contributing to disease. Studies conducted over the last decade have focused on three key aspects in OA; inflammation and the immune response, genome-wide association studies that have identified important genes undergoing epigenetic modifications, and finally how chondrocytes transform in their function during development and disease. Data highlighted here have identified critical inflammatory genes involved in OA and how these factors impact chondrocyte hypertrophy in the disease. This review also addresses key inflammatory factors in synovial inflammation, epigenetics, and chondrocyte fate, and how agents that inhibit epigenetic mechanisms like DNA methylation and histone modifications could aid in development of long-term treatment strategies for the disease

Status of TMPRSS2–ERG fusion in prostate cancer patients from India: correlation with clinico-pathological details and TMPRSS2 Met160Val polymorphism

Background: Prostate cancer (PCa) shows considerable clinical heterogeneity that has been primarily attributed to variable molecular alterations. TMPRSS2–ERG fusion is one such molecular subtype that has been associated with predominantly poor prognosis. More recently, a single nucleotide polymorphism (SNP) in the TMPRSS2 gene rs12329760 C>T (Met160Val) has been shown to positively correlate with the fusion status and also to be associated with increased risk for PCa. The aim of the present study is to determine the frequency of TMPRSS2–ERG fusion and association of rs12329760 in Indian PCa patients with fusion status. Methods: TMPRSS2–ERG fusion by fluorescence in situ hybridization was determined in 102 of 150 PCa biopsy-proven cases. Genotyping for rs12329760 was performed on the entire cohort of 150 cases by Sanger sequencing. Results: TMPRSS2–ERG fusion was seen in 27 of 102 (26%) cases. Fusion-positive patterns in this study showed fusion by translocation in nine of 27 cases (33.5%), by deletion in six of 27 (22%) cases, and by insertion in 12 of 27 cases (44.5%). No association of the fusion status with Gleason Score, pattern, or perineural invasion was seen. The TMPRSS2 SNP rs12329760 ‘T’ allele was prevalent with a frequency of 0.27 in the PCa patients. The SNP was significantly associated with fusion [odds ratio (OR) = 2.176, 95% confidence interval (CI) = 1.012–4.684, P = 0.04], more specifically fusion by deletion (P = 0.04). Conclusion: The results provided here determine the frequency of TMPRSS2–ERG fusions (26%) in a fairly large cohort of Indian PCa cases and also the association of rs12329760 SNP with TMPRSS2–ERG fusion. No association with other clinico-pathological features was observed. Future studies with clinical outcomes are warranted in this population. Keywords: Fluorescence in situ hybridization, Indians, Prostate cancer, rs12329760, Single nucleotide polymorphism, TMPRSS2–ER

International Nosocomial Infection Control Consortiu (INICC) report, data summary of 43 countries for 2007-2012. Device-associated module

We report the results of an International Nosocomial Infection Control Consortium (INICC) surveillance study from January 2007-December 2012 in 503 intensive care units (ICUs) in Latin America, Asia, Africa, and Europe. During the 6-year study using the Centers for Disease Control and Prevention's (CDC) U.S. National Healthcare Safety Network (NHSN) definitions for device-associated health care–associated infection (DA-HAI), we collected prospective data from 605,310 patients hospitalized in the INICC's ICUs for an aggregate of 3,338,396 days. Although device utilization in the INICC's ICUs was similar to that reported from ICUs in the U.S. in the CDC's NHSN, rates of device-associated nosocomial infection were higher in the ICUs of the INICC hospitals: the pooled rate of central line–associated bloodstream infection in the INICC's ICUs, 4.9 per 1,000 central line days, is nearly 5-fold higher than the 0.9 per 1,000 central line days reported from comparable U.S. ICUs. The overall rate of ventilator-associated pneumonia was also higher (16.8 vs 1.1 per 1,000 ventilator days) as was the rate of catheter-associated urinary tract infection (5.5 vs 1.3 per 1,000 catheter days). Frequencies of resistance of Pseudomonas isolates to amikacin (42.8% vs 10%) and imipenem (42.4% vs 26.1%) and Klebsiella pneumoniae isolates to ceftazidime (71.2% vs 28.8%) and imipenem (19.6% vs 12.8%) were also higher in the INICC's ICUs compared with the ICUs of the CDC's NHSN