Recent developments in cell shipping methods.

As opposed to remarkable advances in the cell therapy industry, research reveal inexplicable difficulties associated with preserving and post-thawing cell death. Post cryopreservation apoptosis is a common occurrence that has attracted the attention of scientists to use apoptosis inhibitors. Transporting cells without compromising their survival and function is crucial for any experimental cell-based therapy. Preservation of cells allows the safe transportation of cells between distances and improves quality control testing in clinical and research applications. The vitality of transported cells is used to evaluate the efficacy of transportation strategies. For many decades, the conventional global methods of cell transfer were not only expensive but also challenging and had adverse effects. The first determination of some projects is optimizing cell survival after cryopreservation. The new generation of cryopreservation science wishes to find appropriate and alternative methods for cell transportation to ship viable cells at an ambient temperature without dry ice or in media-filled flasks. The diversity of cell therapies demands new cell shipping methodologies and cryoprotectants. In this review, we tried to summarize novel improved cryopreservation methods and alternatives to cryopreservation with safe and viable cell shipping at ambient temperature, including dry preservation, hypothermic preservation, gel-based methods, encapsulation methods, fibrin microbeads, and osmolyte solution compositions.

The Oral and Fecal Microbiota in a Canadian Cohort of Alzheimer's Disease.

BACKGROUND: Despite decades of research, our understanding of Alzheimer's disease (AD) etiology remains incomplete. In recent years, appreciation has grown for potential roles for the microbiota in shaping neurological health. OBJECTIVE: This study aimed to examine associations between the microbiota and AD in a human cross-sectional cohort. METHODS: Forty-five AD patients and 54 matched controls were recruited in Vancouver, Canada. Fecal and oral samples underwent 16S microbiota sequencing. A wide array of demographic and clinical data were collected. Differences between participant groups were assessed, and associations between microbes and clinical variables were examined within the AD population. RESULTS: The gut microbiota of AD patients displayed lower diversity relative to controls, although taxonomic differences were sparse. In contrast, the AD oral microbiota displayed higher diversity, with several taxonomic differences relative to controls, including a lower abundance of the families Streptococcaceae and Actinomycetaceae, and a higher abundance of Weeksellaceae, among others. The periodontitis-associated oral microbe Porphyromonas gingivalis was 5 times more prevalent among patients. No significant associations between gut or oral microbes and cognition were detected, but several correlations existed between microbes and mood disorders and BMI among patients, including a strong positive correlation between Alphaproteobacteria and depression score. CONCLUSION: The gut microbiota of AD patients was not overtly different from controls, although it displayed lower diversity, an overall marker of microbiota health. The oral microbiota did display marked differences. Cognition was not associated with a microbial signature, but other relevant AD factors including mood and BMI did demonstrate an association.

Low level of plasma fibulin-1 in patients with thyroid lesions: a case-control study and literature review.

Investigating novel biomarkers discriminating thyroid nodules is a matter of great importance for differential diagnosis. The current study was planned to investigate the diagnostic value of fibulin-1 in plasma specimens of patients with thyroid nodules. A literature review was also performed to gain an understanding of the existing research relevant to the main role of fibulin-1 in carcinogenesis. In this case-control study, the levels of plasma fibulin-1 were compared in 82 subjects including papillary thyroid cancer (PTC; n = 30), multinodular goiter (MNG; n = 30), and healthy subjects (n = 22) using enzyme-linked immunosorbent assay (ELISA). Fibulin-1 levels of patients with PTC and MNG were documented to be significantly lower than those of healthy subjects (PTC vs. Healthy; P = 0.000, MNG vs. Healthy; P = 0.000). No statistically significant differences were found between PTC and MNG groups when fibulin-1 levels were compared (P > 0.05). Low level of plasma fibulin-1 was associated with an increased risk of PTC tumorigenesis (odds ratio = 0.810; 95% CI: 0.704-0.933; P = 0.003). Further, fibulin-1 had an appropriate diagnostic value for detecting PTC patients with a sensitivity of 73.33%, and specificity of 100% at the cutoff value > 4.9 (ng/ml). According to the results of the present research which are tied well with previous studies, the abnormal downregulation of fibulin-1 may play a role in the PTC and MNG tumorigenesis. In addition, fibulin-1 probably promotes the development and progression of other human cancer; however, further studies are needed to improve current understandings.

The impact of pre-analytical variations on biochemical analytes stability: A systematic review.

OBJECTIVE: A common problem in clinical laboratories is maintaining the stability of analytes during pre-analytical processes. The aim of this study was to systematically summarize the results of a set of studies about the biochemical analytes stability. METHODS: A literature search was performed on the Advanced search field of PubMed using the keywords: "(stability) AND (analytes OR laboratory analytes OR laboratory tests OR biochemical analytes OR biochemical tests OR biochemical laboratory tests)." A total of 56 entries were obtained. After applying the selection criteria, 20 articles were included in the study. RESULTS: In the 20 included references, up to 123 different analytes were assessed. The 34 analytes in order of the most frequently studied analytes were evaluated: Alanine aminotransferase, aspartate aminotransferase, potassium, triglyceride, alkaline phosphatase, creatinine, total cholesterol, albumin, lactate dehydrogenase, sodium, calcium, γ-glutamyltransferase, total bilirubin, urea, creatine kinase, inorganic phosphate, total protein, uric acid, amylase, chloride, high-density lipoprotein, magnesium, glucose, C-reactive protein, bicarbonate, ferritin, iron, lipase, transferrin, cobalamin, cortisol, folate, free thyroxine, and thyroid-stimulating hormone. Stable test results could be varied between 2 hours and 1 week according to the type of samples and/or type of blood collection tubes on a basic classification set as refrigerated or room temperature. CONCLUSIONS: Biochemical analytes stability could be improved if the best pre-analytical approaches are used.

Versican localizes to the nucleus in proliferating mesenchymal cells.

OBJECTIVE: Versican is a versatile and highly interactive chondroitin sulfate proteoglycan that is found in the extracellular matrix (ECM) of many tissues and is a major component of developing and developed lesions in atherosclerotic vascular disease. In this paper, we present data to indicate that versican may have important intracellular functions in addition to its better known roles in the ECM. METHODS AND RESULTS: Rat aortic smooth muscle cells were fixed and immunostained for versican and images of fluorescently labeled cells were obtained by confocal microscopy. Intracellular versican was detected in the nucleus and cytosol of vascular smooth muscle cells. The use of a synthetic neutralizing peptide eliminated versican immunostaining, demonstrating the specificity of the antibody used in this study. Western blot of pure nuclear extracts confirmed the presence of versican in the nucleus, and multifluorescent immunostaining showed strong colocalization of versican and nucleolin, suggesting a nucleolar localization of versican in nondividing cells. In dividing valve interstitial cells, a strong signal for versican was observed in and around the condensed chromosomes during the various stages of mitosis. Multifluorescent immunostaining for versican and tubulin revealed versican aggregated at opposing poles of the mitotic spindle during metaphase. Knockdown of versican expression using siRNA disrupted the organization of the mitotic spindle and led to the formation of multipolar spindles during metaphase. CONCLUSIONS: Collectively, these data suggest an intracellular function for versican in vascular cells where it appears to play a role in mitotic spindle organization during cell division. These observations open a new avenue for studies of versican, suggesting even more diverse roles in vascular health and disease.

Versican V1 Overexpression Induces a Myofibroblast-Like Phenotype in Cultured Fibroblasts.

BACKGROUND: Versican, a chondroitin sulphate proteoglycan, is one of the key components of the provisional extracellular matrix expressed after injury. The current study evaluated the hypothesis that a versican-rich matrix alters the phenotype of cultured fibroblasts. METHODS AND RESULTS: The full-length cDNA for the V1 isoform of human versican was cloned and the recombinant proteoglycan was expressed in murine fibroblasts. Versican expression induced a marked change in fibroblast phenotype. Functionally, the versican-expressing fibroblasts proliferated faster and displayed enhanced cell adhesion, but migrated slower than control cells. These changes in cell function were associated with greater N-cadherin and integrin ß1 expression, along with increased FAK phosphorylation. The versican-expressing fibroblasts also displayed expression of smooth muscle α-actin, a marker of myofibroblast differentiation. Consistent with this observation, the versican fibroblasts displayed increased synthetic activity, as measured by collagen III mRNA expression, as well as a greater capacity to contract a collagen lattice. These changes appear to be mediated, at least in part, by an increase in active TGF-ß signaling in the versican expressing fibroblasts, and this was measured by phosphorylation and nuclear accumulation of SMAD2. CONCLUSIONS: Collectively, these data indicate versican expression induces a myofibroblast-like phenotype in cultured fibroblasts.

1,25 Dihydroxyvitamin D3 Inhibits TGFß1-Mediated Primary Human Cardiac Myofibroblast Activation.

AIMS: Epidemiological and interventional studies have suggested a protective role for vitamin D in cardiovascular disease, and basic research has implicated vitamin D as a potential inhibitor of fibrosis in a number of organ systems; yet little is known regarding direct effects of vitamin D on human cardiac cells. Given the critical role of fibrotic responses in end stage cardiac disease, we examined the effect of active vitamin D treatment on fibrotic responses in primary human adult ventricular cardiac fibroblasts (HCF-av), and investigated the relationship between circulating vitamin D (25(OH)D3) and cardiac fibrosis in human myocardial samples. METHODS AND RESULTS: Interstitial cardiac fibrosis in end stage HF was evaluated by image analysis of picrosirius red stained myocardial sections. Serum 25(OH)D3 levels were assayed using mass spectrometry. Commercially available HCF-av were treated with transforming growth factor (TGF)ß1 to induce activation, in the presence or absence of active vitamin D (1,25(OH)2D3). Functional responses of fibroblasts were analyzed by in vitro collagen gel contraction assay. 1,25(OH)2D3 treatment significantly inhibited TGFß1-mediated cell contraction, and confocal imaging demonstrated reduced stress fiber formation in the presence of 1,25(OH)2D3. Treatment with 1,25(OH)2D3 reduced alpha-smooth muscle actin expression to control levels and inhibited SMAD2 phosphorylation. CONCLUSIONS: Our results demonstrate that active vitamin D can prevent TGFß1-mediated biochemical and functional pro-fibrotic changes in human primary cardiac fibroblasts. An inverse relationship between vitamin D status and cardiac fibrosis in end stage heart failure was observed. Collectively, our data support an inhibitory role for vitamin D in cardiac fibrosis.

An ERK-p38 subnetwork coordinates host cell apoptosis and necrosis during coxsackievirus B3 infection.

The host response to a virus is determined by intracellular signaling pathways that are modified during infection. These pathways converge as networks and produce interdependent phenotypes, making it difficult to link virus-induced signals and responses at a systems level. Coxsackievirus B3 (CVB3) infection induces death of cardiomyocytes, causing tissue damage and virus dissemination, through incompletely characterized host cell signaling networks. We built a statistical model that quantitatively predicts cardiomyocyte responses from time-dependent measurements of phosphorylation events modified by CVB3. Model analysis revealed that CVB3-stimulated cytotoxicity involves tight coupling between the host ERK and p38 MAPK pathways, which are generally thought to control distinct cellular responses. The kinase ERK5 requires p38 kinase activity and inhibits apoptosis caused by CVB3 infection. By contrast, p38 indirectly promotes apoptosis via ERK1/2 inhibition but directly causes CVB3-induced necrosis. Thus, the cellular events governing pathogenesis are revealed when virus-host programs are monitored systematically and deconvolved mathematically.

Versican and CD44 in in vitro valvular interstitial cell injury and repair.

BACKGROUND: Versican is one of the key components of the extracellular matrix (ECM) that is expressed during injury, inflammatory, and repair processes. The current study evaluated the relationship between versican and the membrane receptor CD44 during in vitro valvular interstitial cell (VIC) injury and repair. METHODS: Subconfluent, confluent, and wounded cultures of human VICs were fixed and immunostained to detect versican and the membrane receptor CD44. To examine the relationship between versican and CD44, a blocking antibody to CD44 was added to cultured VICs, and in vitro wound repair along with pericellular versican organization and stress fiber formation were examined. RESULTS: Immunohistochemistry demonstrated that versican is prominent intracellularly, as well as extracellularly, in actively proliferating VICs. In contrast, versican was only localized to fibrils in the extracellular space in between cells in confluent (quiescent) cultures. Following wounding, versican expression was up-regulated, and it was secreted as ECM at the trailing edge of migrating cells. The staining for CD44 was similarly localized to the trailing edge of migrating VICs in wounded cultures. Treatment of VICs with a CD44-blocking antibody disrupted the organization of versican in the pericellular matrix and inhibited stress fiber formation in these cells. Functionally, blocking CD44 significantly inhibited VIC-mediated contraction of type I collagen gels (35.7%±0.7% vs. 23.3%±1.4% of initial gel area, P<.01). CONCLUSIONS: Versican is a key component of the provisional wound repair ECM that is expressed following injury to VICs. The receptor CD44 plays an important role in organizing the provisional ECM. SUMMARY: Our data suggests VICs synthesize and secrete versican following injury. These cells also up-regulate CD44, a receptor that binds versican. Blocking CD44 disrupted the organization of versican and inhibited stress fiber formation. Functionally, blocking CD44 inhibited cell-mediated contraction of a collagen matrix. Collectively, these data suggest versican expression and organization are important to valve cell injury and repair.

Coxsackievirus B3 activates nuclear factor kappa B transcription factor via a phosphatidylinositol-3 kinase/protein kinase B-dependent pathway to improve host cell viability.

Coxsackievirus B3 (CVB3) is the most common viral infectant of heart muscle. CVB3 directly injures cardiomyocytes. We have previously reported on a regulatory role for the phosphatidylinositol-3 kinase (PI3K)/protein kinase B (Akt) pathway during CVB3 infection. Yet, the mechanism underlying this regulatory role has not been elucidated. The PI3K/Akt pathway is involved in various cellular processes and exerts its function through the activation of several downstream effectors. Among them, nuclear factor kappa B (NFkappaB) transcription factor is involved in inflammation, survival and apoptosis. In this study, we investigated the role of NFkappaB as a potential downstream mediator of signals through the PI3K/Akt cascade, in regulating CVB3-induced cellular injury. We report that CVB3 infection induces the translocation of NFkappaB into the nucleus of infected cells. Inhibition of the PI3K/Akt pathway markedly decreases virus-induced NFkappaB activation. Further, NFkappaB inhibition significantly suppresses host viability, suggesting a pro-survival role for NFkappaB. Short-term treatment of cells with tumour necrosis factor-alpha (TNF-alpha), a potent activator of NFkappaB, promotes host cell viability without affecting virus replication. However, a prolonged treatment has a detrimental effect on cells, indicating the existence of a delicate balance between the anti- and pro-apoptotic roles of TNF-alpha in the setting of CVB3 infection.

Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter.

Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.