Mitogenic Concentrations Research Articles

Facile Macrocyclic Polyphenol Barrier Coatings for PDMS Microfluidic Devices

AbstractSoft lithography techniques using polydimethylsiloxane (PDMS) are a cornerstone of microfluidic microdevices and emerging technologies such as microphysiological systems (MPS). Most of these systems employ hydrophobic small molecules during either stem cell differentiation, drug screening, or organoid development. However, due to PDMS's structure and hydrophobicity, lipophilic molecules are strongly absorbed creating unpredictable concentrations of mitogens, drugs, differentiation factors, and analytes, which is a major limitation in its use for biological applications. In this study, several catechol‐functionalized calix[4]arene based macrocyclic polyphenols (MPPs) are synthesized and coated on PDMS through a dip‐coating or flow through process. One molecule, MPP5cone, synthesized from catechol and resorcinol in its cone isomer form, increases the hydrophilicity of PDMS and drastically reduces the absorption of a number of hydrophobic drug surrogates, while preserving high oxygen permeability, good cell viability and function. However, simple rules of molecular absorption based on Log P are not observed, suggesting screening barrier coatings for PDMS with single probes is not sufficient. The coating procedure is easily translated to microfluidic devices by infusion through channels with a pump, and therefore should find use in applications where molecular absorption into PDMS is a significant problem.

Changes in Nup62 content affect contact-induced differentiation of cultured myoblasts

Differentiation of cultured skeletal myoblasts is induced by extrinsic signals that include reduction in ambient mitogen concentration and increased cell density. Using an established murine myoblast cell line (C2C12), we have found that experimental reduction of the nucleoporin p62 (Nup62) content of myoblasts enhances differentiation in high-mitogen medium, while forced expression of Nup62 inhibits density-induced differentiation. In contrast, differentiation of myoblasts induced by low-mitogen medium was unaffected by ectopic Nup62 expression. Further analyses suggested that Nup62 content affects density-induced myoblast differentiation through a mechanism involving activation of p38 MAP kinase. Nuclear pore complex (NPC) composition, in particular changes in NUP62 content, may be altered during viral infection, differentiation, and in neoplastic growth. The results support a functional role for changes in Nup62 composition in NPCs and density-induced myogenic differentiation, and suggest a link between loss of Nup62 content and induction of an intracellular stress signaling pathways.

Khảo sát một số điều kiện nuôi cấy in vitro tế bào đơn nhân được chiết từ máu ngoại vi người

The purpose of this study is to optimize some conditions for human PBMCs in vitro culture. PBMCs were isolated within 4 hours from the EDTA – coagulated whole blood samples collected from the 10 healthy volunteers. 100 μL of a cell suspension (approximately 5x105 cells/mL)were added to each well in a 96 well microplate and incubated at 37°C, 95±5% humidity and 5% CO2 for 24 hrs. Then the isolation of PBMCs from 6 ml whole blood using 10 mL Leucosep® tube was optimized; cell viability using trypan blue dye was evaluated; the suitable solvents for the dissolution of formazan crystals in the MTT assay and the conditions of PBMCs in vitro culture such as PBMCs density per well, FBS concentration, time for PBMCs culture and PHA mitogen concentration for PBMCs proliferation were examined. The results of this study showed that the centrifuge speed and time for isolating PBMCs from 6 mL whole blood using Leucosep® tube was determined at 1200 xg for 20 minutes. After that, PBMCs viability was found above 95% using trypan blue and hemocytometer. The dissolution of formazan crystals in DMSO/NH3 was better than in either DMSO, isopropanol or isopropanol/HCl. Moreover, the optimal conditions for cell culture media containing 10% FBS, 1.5% PHA mitogen and the range of PBMCs density from 104 – 105 cells/well were determined. These results may be used for the further studies to assess the effects of medicinal plants or new drugs on in vitro PBMCs proliferation.

Effects of polychlorinated biphenyls (PCB) on California sea lion (Zalophus californianus) lymphocyte functions upon in vitro exposure

Polychorinated biphenyl (PCB) congeners are a cause for concern due to their persistence in the environment, their lipophilic properties that cause them to bio-accumulate in top predators, and their adverse effects on mammalian health. For example, the common urogenital carcinoma reported in California sea lions (Zalophus californianus) (CSL) is associated with high tissue levels of PCBs, but the mechanisms responsible for this association are unknown. This study investigated the effect of exposure to six PCB congeners and a congener mix at low and environmentally relevant concentrations on NK cell-like and T cell activity using in vitro assays on cryopreserved lymph node mononuclear cells isolated from dead CSL. Non dioxin-like congeners 153 and 180 increased lymphocyte proliferation at 5 and 10 ppm, while congener 138 decreased proliferation by up to 43% at 15 ppm. Dioxin-like PCBs 118 and 169 did not affect lymphocyte proliferation, while the effects of congener 105 depended on the mitogen concentration; these did not correlate with their predicted toxic equivalent factors. NK cell-like activity was affected only by the highest concentration of PCBs tested; it was increased by non-dioxin-like congeners 138 and 153, and decreased by dioxin-like congener 169. The PCB congener mix suggested that the effects of PCB congeners were not simply additive. Our results concur with effects of PCBs reported for other pinniped's lymphocytes and add further experimental support to the observation that dioxin-like PCBs are not the most toxic congeners for marine mammals, contrary to effects in other species. This is the first evidence of in vitro suppression of NK cell-like cytotoxicity by a dioxin-like congener in a pinniped. More importantly, the observed results suggest that PCBs can modulate the CSL immune system, increasing exposed individuals’ susceptibility to viral and oncogenic challenges.

Evaluating the Proliferation of Human PeripheralBlood Mononuclear Cells Using MTT Assay

Introduction: 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay is a safe, convenient, and low-cost technique with high applications for the measurement of cell proliferation rate in researches and clinical laboratories. Our aim was to evaluate the proliferation rate of human peripheral blood mononuclear cells (PBMCs) and production rate of Tumor necrosis factor alpha (TNF-α) by these cells after various mitogens stimulation in different situations. Methods: The MTT test was performed with various concentrations of mitogens including concanavalin A (ConA), lipopolysaccharide (LPS) and phytohemagglutinin (PHA) on the PBMCs. The cells were incubated for 24, 48, 72, and 96 hours in the culture medium and TNF-α cytokine assay was performed on the supernatant of the cultured splenocytes using the enzyme-linked immunosorbent assay (ELISA) method. Results: The optimal time and incubation of the PBMCs with the mixture of PHA-ConA were 5 μg/mL and 72 hours, respectively. The TNF-α level increased significantly after PHA-ConA and PHA stimulation. Conclusion: The results showed that the mixture of PHA-ConA (at the concentration of 5 μg/mL) can give rise to the optimal results on stimulation of the PBMcs using the MTT assay after 72 hours incubation.

Telomere shortening and immune activity in war veterans with posttraumatic stress disorder

There is increasing evidence that chronic stress accelerates telomere erosion in leukocytes/peripheral blood mononuclear cells (PBMCs). However, functional changes associated with telomere shortening are poorly understood. We hypothesized that war veterans with PTSD would have shorter telomeres in PBMCs and that these cells might exhibit changes in measures of immune reactivity such as proliferation, cytokine production and expression of regulators of immune responses. We measured relative telomere length and basal telomerase activity in PBMCs of 62 individuals (PTSD patients (N=30); age-matched healthy controls (N=17), elderly volunteers (N=15)). In parallel, we have assessed proliferation of activated T cells, interferon (IFN)-γ, interleukin (IL)-2, IL-4, tumor necrosis factor (TNF)-α and IL-6 cytokine production and expression of programmed death 1 (PD-1) receptor and its ligand PD-L1 on activated T cells. Middle-aged war veterans with current PTSD had shorter PBMC telomere length than their age-matched healthy controls while the elderly had the shortest telomeres. There was no difference in telomerase activity between PTSD patients and healthy controls while telomerase activity was significantly lower in the elderly. While the elderly group exhibited robust changes in immune activity such as increased production of proinflammatory cytokines (TNF-α, IL-6) and reduced proliferation of all T cells, the PTSD group showed reduced proliferative response of CD8(+) T cells to high concentrations of mitogen and reduced spontaneous production of IL-2 and IFN-γ. This study adds to the accumulating evidence that psychological trauma and chronic stress are associated with accelerated telomere attrition. However, changes in immune function associated with stress-related telomere shortening are not well understood. Although much less pronounced in PTSD patients than in elderly persons, reduced proliferative responses of T cells accompanied by shorter telomeres might be a sign of early immunosenescence. Together with reduced production of Th1 cytokines, observed immune changes may contribute to health risks associated with PTSD.

Microglia change from a reactive to an age-like phenotype with the time in culture

Age-related neurodegenerative diseases have been associated with chronic neuroinflammation and microglia activation. However, cumulative evidence supports that inflammation only occurs at an early stage once microglia change the endogenous characteristics with aging and switch to irresponsive/senescent and dystrophic phenotypes with disease progression. Thus, it will be important to have the means to assess the role of reactive and aged microglia when studying advanced brain neurodegeneration processes and age-associated related disorders. Yet, most studies are done with microglia from neonates since there are no adequate means to isolate degenerating microglia for experimentation. Indeed, only a few studies report microglia isolation from aged animals, using either short-term cultures or high concentrations of mitogens in the medium, which trigger microglia reactivity. The purpose of this study was to develop an experimental process to naturally age microglia after isolation from neonatal mice and to characterize the cultured cells at 2 days in vitro (DIV), 10 DIV, and 16 DIV. We found that 2 DIV (young) microglia had predominant amoeboid morphology and markers of stressed/reactive phenotype. In contrast, 16 DIV (aged) microglia evidenced ramified morphology and increased matrix metalloproteinase (MMP)-2 activation, as well as reduced MMP-9, glutamate release and nuclear factor kappa-B activation, in parallel with decreased expression of Toll-like receptor (TLR)-2 and TLR-4, capacity to migrate and phagocytose. These findings together with the reduced expression of microRNA (miR)-124, and miR-155, decreased autophagy, enhanced senescence associated beta-galactosidase activity and elevated miR-146a expression, are suggestive that 16 DIV cells mainly correspond to irresponsive/senescent microglia. Data indicate that the model represent an opportunity to understand and control microglial aging, as well as to explore strategies to recover microglia surveillance function.

Polyfunctionality of naive T cells is enhanced in older age (CCR3P.223)

Abstract Reduced number of naïve T cells along with an accumulation of differentiated cell types in aging has been described but little is known about the polyfunctionality of T cells. In this study we compared the individual cytokine and polyfunctional profiles of T cell subsets, including newly described stem cell like memory T cells (TSCM) in response to stimulation with submaximal concentration of non-specific mitogen in older (median age 80, n=23) versus younger (median age 27; n=23) adults using multicolor flow cytometry. Secretion of IFN-γ, MIP-1α, TNF-α, perforin, and IL-2 were measured. Polyfunctionality was determined using FlowJo and SPICE (NIAID). Aging was associated with a marked decline in CD8+ naïve cells (11% vs. 47 %; p<0.0001) and an expansion in memory cell types including central memory (p<0.05), effector memory and effectors (p<0.001 for both) compared to younger subjects. There was also a decline in CD4+ naïve cells with age (33% vs. 45%; p=0.02). There were no differences in frequencies or cytokine profiles of TSCM between groups. CD8+ naïve cells in the older group had increased secretion of all cytokines measured compared to the younger subjects and exhibited greater polyfunctionality (p=0.03). CD4+ naïve cells among the older group also had greater polyfunctionality with a TNF-α and IL-2 dominance (p=0.001). The decline in naïve T cells in aging, rather than their polyfunctional potential maybe contributing to immunosenescence.

In vitro modulation of MMP-2 and MMP-9 in pediatric human sarcoma cell lines by cytokines, inducers and inhibitors

The highly aggressive pediatric sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and MMP-9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effects of cytokines, mitogens, inducers and inhibitors on MMP-2 and -9 expression in osteosarcoma (U2OS) and rhabdomyosarcoma (RD). The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. These cell lines were cultured in their respective media to near confluence and the cells were washed with PBS and incubated in serum-free medium with various concentrations of several cytokines, mitogens and inhibitors. After 24 h the media were removed and analyzed for MMP-2 and -9 by gelatinase zymography and quantitated by densitometry. Osteosarcoma and rhabdomyosarcoma showed bands corresponding to MMP-2 and -9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. Tumor necrosis factor-α, interleukin-1β and LPS enhanced osteosarcoma U2OS MMP-9 secretion but had no effect on MMP-2 secretion. Tumor necrosis factor-α stimulated rhabdomyosarcoma MMP-2 expression, but had no effect on MMP-9 secretion. Doxycycline, epigallocatechin gallate, nutrient mixture (NM), actinomycin-D, cyclohex-amide, retinoic acid and dexamethasone inhibited MMP-2 and -9 in U2OS osteosarcoma cells. PMA-treated RD cells showed dose-response inhibition of MMP-9 by doxycycline and epigallocatechin gallate and both MMPs by NM. Dexamethasone and actinomycin-D showed inhibition of MMP-2 secretion of RD cells. Our results show that cytokines, mitogens and inducers show variable upregulation of U2OS osteosarcoma and RD rhabdomyosarcoma MMP-2 and -9 secretion, and inhibitors demonstrate downregulation under stimulatory conditions, suggesting the application of these agents for the development of effective therapies in pediatric sarcomas.

In vitro modulation of MMP-2 and MMP-9 in adult human sarcoma cell lines by cytokines, inducers and inhibitors

The highly aggressive adult sarcomas are characterized by high levels of matrix metalloproteinase (MMP)-2 and -9, which play crucial roles in tumor invasion and metastasis by degradation of the extracellular membrane leading to cancer cell spread to distal organs. We examined the effect of cytokines, mitogens, inducers and inhibitors on MMP-2 and MMP-9 secretion in chondrosarcoma (SW-1353), fibrosarcoma (HT-1080), liposarcoma (SW-872) and synovial sarcoma (SW-982) cell lines. The selected compounds included natural cytokines and growth factors, as well as chemical compounds applied in therapy of sarcoma and natural compounds that have demonstrated anticancer therapeutic potential. MMP-2 and MMP-9 secretions were analyzed by gelatinase zymography following 24-h exposure to the tested agents and quantitated by densitometry. Fibrosarcoma, chondrosarcoma, liposarcoma and synovial sarcoma showed bands corresponding to MMP-2 and MMP-9 with dose-dependent enhancement of MMP-9 with phorbol 12-myristate 13-acetate (PMA) treatment. In chondrosarcoma cells, tumor necrosis factor (TNF)-α had a stimulatory effect on MMP-9 and insignificant effect on MMP-2 and interleukin (IL)-1β stimulated MMP-9 and MMP-2. In fibrosarcoma and liposarcoma cells, TNF-α had a profound stimulatory effect on MMP-9, but no effect on MMP-2 and in synovial sarcoma an inhibitory effect on MMP-2 and no effect on MMP-9. IL-1β had a slight inhibitory effect on fibrosarcoma, liposarcoma and synovial sarcoma MMP-2 and MMP-9 except for MMP-9 in synovial sarcoma which showed slight stimulation. Lipopolysaccharide (LPS) stimulated expression of MMP-2 in fibrosarcoma and chondrosarcoma while inhibited it in liposarcoma. Doxycycline, epigallocatechin gallate and the nutrient mixture inhibited MMP-2 and MMP-9 in all cell lines. Actinomycin-D, cyclohexamide, retinoic acid, and dexamethasone inhibited MMP-2 and -9 in chondrosarcoma and fibrosarcoma cells. Our results show that cytokines, mitogens, inducers and inhibitors have an up or down regulatory effect on MMP-2 and MMP-9 expression in adult sarcoma cell lines, suggesting these agents may be effective strategies to treat these cancers.

VEGF-A165 Potently Induces Human Blood–Nerve Barrier Endothelial Cell Proliferation, Angiogenesis, and Wound Healing In Vitro

Several mitogens such as vascular endothelial growth factor (VEGF) have been implicated in mammalian vascular proliferation and repair. However, the molecular mediators of human blood-nerve barrier (BNB) development and specialization are unknown. Primary human endoneurial endothelial cells (pHEndECs) were expanded in vitro and specific mitogen receptors detected by western blot. pHEndECs were cultured with basal medium containing different mitogen concentrations with or without heparin. Non-radioactive cell proliferation, Matrigel(™)-induced angiogenesis and sterile micropipette injury wound healing assays were performed. Proliferation rates, number and total length of induced microvessels, and rate of endothelial cell monolayer wound healing were determined and compared to basal conditions. VEGF-A165 in the presence of heparin, was the most potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. 1.31 nM VEGF-A165 induced ~110 % increase in cell proliferation relative to basal conditions (∼51 % without heparin). 2.62 pM VEGF-A165 induced a three-fold increase in mean number of microvessels and 3.9-fold increase in total capillary length/field relative to basal conditions. In addition, 0.26 nM VEGF-A165 induced ∼1.3-fold increased average rate of endothelial wound healing 4-18 h after endothelial monolayer injury, mediated by increased cell migration. VEGF-A165 was the only mitogen capable of complete wound closure, occurring within 30 h following injury via increased cell proliferation. This study demonstrates that VEGF-A165, in the presence of heparin, is a potent inducer of pHEndEC proliferation, angiogenesis, and wound healing in vitro. VEGF-A165 may be an important mitogen necessary for human BNB development and recovery in response to peripheral nerve injury.

Assessment of cell mediated immune response in rabbits immunized with affinity purified 35kDa midgut antigen of Rhipicephalus haemaphysaloides ticks.

Cell mediated immune response to immunoaffinity chromatography purified midgut antigen of Rhipicephalus haemaphysaloides ticks in rabbits was studied by using lymphocyte transformation test. This test was carried out by using 5-bromo-2'-deoxy-uridine kit method. The blastogenic response of peripheral blood lymphocytes of normal rabbit to different concentrations of antigen and mitogen (Con A) showed that 2μg of antigen and 2μg of mitogen gave maximum stimulation index. The antigen specific responsiveness of immunized rabbits with affinity purified 35kDa midgut antigen was highly significant (P≤0.01) compared to mitogen. The maximum lymphocyte stimulation index (LSI) of 2.47 was observed on 49 day post immunization in immunized group. The lymphocytes separated from control animal cultured in RPMI1640 medium with 2μg of antigen and 2μg of mitogen (Con A) were never stimulated and their LSI values were below 2.0.

The Role of Syndecan-1 in Cellular Signaling and its Effects on Heparan Sulfate Biosynthesis in Mesenchymal Tumors

Proteoglycans (PGs) and in particular the syndecans are involved in the differentiation process across the epithelial-mesenchymal axis, principally through their ability to bind growth factors and modulate their downstream signaling. Malignant tumors have individual proteoglycan profiles, which are closely associated with their differentiation and biological behavior, mesenchymal tumors showing a different profile from that of epithelial tumors. Syndecan-1 is the main syndecan of epithelial malignancies, whereas in sarcomas its expression level is generally low, in accordance with their mesenchymal phenotype and highly malignant behavior. This proteoglycan is often overexpressed in adenocarcinoma cells, whereas mesothelioma and fibrosarcoma cells express syndecan-2 and syndecan-4 more abundantly. Increased expression of syndecan-1 in mesenchymal tumors changes the tumor cell morphology to an epithelioid direction whereas downregulation results in a change in shape from polygonal to spindle-like morphology. Although syndecan-1 plays major roles on the cell-surface, there are also intracellular functions, which are not very well studied. On the functional level, syndecan-1 affects mesenchymal tumor cell proliferation, adhesion, migration and motility, and the effect varies with the different domains of the core protein. Syndecan-1 may exert stimulatory or inhibitory effects, depending on the concentration of various mitogens, enzymes, and signaling molecules, the ratio between the shed and membrane-associated syndecan-1 and histological grade of the tumour. Growth factor signaling seems to be delicately controlled by regulatory loops involving the syndecan expression levels and their sulfation patterns. Overexpression of syndecan-1 modulates the biosynthesis and sulfation of heparan sulfate and it also affects the expression of other PGs. On transcriptomic level, syndecan-1 modulation results in profound effects on genes involved in regulation of cell growth

Identification and quantification of concentration-dependent biomarkers in MCF-7/BOS cells exposed to 17β-estradiol by 2-D DIGE and label-free proteomics

This paper reports the identification of biomarkers resulting from the exposure of MCF-7/BOS cells to 17β-estradiol (E(2)). The biomarkers were identified using 2 independent and complementary techniques, 2-D DIGE/MALDI-TOF peptide mass fingerprint, and 2-D UPLC-ESI MS/MS. They were identified from the cytosolic fractions of cells treated for 24h with mitogenic concentrations of 1, 30 and 500 pM of 17β-estradiol. Five biomarkers were up-regulated proteins, namely HSP 74, EF2, FKBP4, EF1 and GDIB and one was a down-regulated protein, namely K2C8. Three of these proteins, EF2, FKBP4 and K2C8 are implicated in a network centered on the estrogen receptors ESR1 and ESR2 as well as on AKT1. After the discovery phase, three biomarkers were selected to test the presence of estrogens using selected reaction monitoring (SRM). They were monitored using SRM after incubation of MCF-7/BOS in the presence of E(2) for confirmation or selected xenoestrogens. Daidzein, coumestrol and enterolactone induced an up-regulation of EF2 and FKPB4 proteins, while tamoxifen and resveratrol induced a down-regulation. The exposure of all phytoestrogens induced the down-regulation of K2C8. These markers form a preliminary molecular signature that can be used when testing the estrogenic activity of xenobiotics, either pure or in mixtures.

Network Analysis of Epidermal Growth Factor Signaling Using Integrated Genomic, Proteomic and Phosphorylation Data

To understand how integration of multiple data types can help decipher cellular responses at the systems level, we analyzed the mitogenic response of human mammary epithelial cells to epidermal growth factor (EGF) using whole genome microarrays, mass spectrometry-based proteomics and large-scale western blots with over 1000 antibodies. A time course analysis revealed significant differences in the expression of 3172 genes and 596 proteins, including protein phosphorylation changes measured by western blot. Integration of these disparate data types showed that each contributed qualitatively different components to the observed cell response to EGF and that varying degrees of concordance in gene expression and protein abundance measurements could be linked to specific biological processes. Networks inferred from individual data types were relatively limited, whereas networks derived from the integrated data recapitulated the known major cellular responses to EGF and exhibited more highly connected signaling nodes than networks derived from any individual dataset. While cell cycle regulatory pathways were altered as anticipated, we found the most robust response to mitogenic concentrations of EGF was induction of matrix metalloprotease cascades, highlighting the importance of the EGFR system as a regulator of the extracellular environment. These results demonstrate the value of integrating multiple levels of biological information to more accurately reconstruct networks of cellular response.

Contents and Formulations of Platelet-Rich Plasma

Multiple studies have demonstrated a role for platelet-rich plasma (PRP) in accelerating and facilitating improved response to injury. The cellular response to injury progresses through 4 general stages: hemostasis, inflammation, proliferation, and finally remodeling. Each phase is characterized by enhanced cellular or molecular activity, all of which involve platelets. Blood plasma and platelets are responsible for hemostasis, whereas leukocytes and activated platelets mediate inflammation, and growth factors derived from platelet α-granules influence tissue regeneration. Specifically, the leukocyte content of PRP is thought to influence the inflammatory phase, whereas angiogenic and mitogenic growth factor concentrations are believed to aid tissue regeneration. Both the precise composition of PRP and its formulation affect the cellular environment in which it is placed and determine its overall effect on tissue repair.

GDNF restores human blood–nerve barrier function via RET tyrosine kinase-mediated cytoskeletal reorganization

Endoneurial microvessels and the perineurium are responsible for maintaining homeostasis in peripheral nerves. Endoneurial endothelial cells form the blood–nerve barrier (BNB). The molecular pathways responsible for endoneurial microvascular barrier formation in humans are not fully understood. We tested the effect of different mitogens on the transendothelial electrical resistance (TEER) of confluent primary human endoneurial endothelial cell (pHEndEC) cultures following serum withdrawal (mimicking diffuse endothelial injury) in vitro. We show that glial-derived neurotrophic factor (GDNF, 1ng/mL) sufficiently induced a maximal 114.2% recovery in TEER over basal conditions 48h after serum withdrawal. Solute permeability to high molecular weight dextran was reduced by 52.4% following GDNF treatment. GDNF-mediated increase in TEER was dependent on RET tyrosine-kinase signaling pathways and mildly enhanced by cyclic adenosine monophosphate in combination with maximal concentrations of multiple redundant mitogens. There was no significant increase in adherens or tight junction proteins β-catenin, VE-Cadherin, zona occludens-1 and occludin following GDNF treatment. GDNF induced a small increase in total claudin-5 protein expression without significant increase in messenger RNA or modulation in tyrosine phosphorylation following serum withdrawal. Indirect immunocytochemistry revealed membrane relocation of longitudinal F-actin cytoskeletal filaments in pHEndECs following GDNF treatment, resulting in more continuous intercellular contacts that formed adherens and tight junctions. Together, these results demonstrate a sufficient role for GDNF in human BNB recovery following serum withdrawal in vitro, facilitated primarily by endothelial cell cytoskeletal reorganization. These observations provide insights into the regulation of human BNB function during recovery from peripheral nerve injury.

Engagement of IL-1 receptor accessory protein (IL-1RAcP) with the monoclonal antibody AY19 provides co-activating signals and prolongs the CD2-induced proliferation of peripheral blood lymphocytes

IL-1 receptor accessory protein (IL-1RAcP) is the second subunit required to form a functional receptor complex for IL-1α and β, IL-1F6, IL-1F8, IL1-F9 and IL-33. While it does not directly interact with the cytokines, IL-1RAcP is necessary to mediate signal transduction. We previously reported a monoclonal antibody with an unknown specificity, termed AY19, that was capable to induce a significant increase in the size of CFU-GM colonies when added to cultures of human cord blood CD34+ hematopoietic progenitors. Here we demonstrate that AY19 mAb recognizes IL1-RAcP. We show that this adaptor molecule is significantly present on peripheral blood monocytes and lymphocytes including CD4+ and CD8+ T lymphocytes, B and NK cells. Interestingly, its expression is found increased on CD127lowCD4+CD25high T cells when compared to CD127lowCD4+CD25− T cell subset, suggesting that the level of IL-1RAcP membrane expression could allow to distinguish within CD127lowCD4+ T lymphocytes the CD25high T regulatory subset from conventional CD25− T lymphocytes. Functional studies reveal that addition of AY19 mAb enhances the proliferation of peripheral blood mononuclear cells (PBMC) obtained with mitogenic concentrations of PMA. Interestingly, we found that although AY19 mAb does not increase the optimal PBMC proliferation induced by a mitogenic pair of anti-CD2 mAbs it prolongs their time of proliferation. Thus, these results indicate that the anti-IL-1RAcP mAb AY19 exhibits unique functional properties by triggering co-stimulatory signals in lymphocytes.

Simple method for culture of peripheral blood lymphocytes of Testudinidae

We developed and optimized a simple, efficient and inexpensive method for in vitro culture of peripheral blood lymphocytes from the Brazilian tortoise Chelonoidis carbonaria (Testudinidae), testing various parameters, including culture medium, mitogen concentration, mitotic index, culture volume, incubation time, and mitotic arrest. Peripheral blood samples were obtained from the costal vein of four couples. The conditions that gave a good mitotic index were lymphocytes cultured at 37°C in minimum essential medium (7.5 mL), with phytohemagglutinin as a mitogen (0.375 mL), plus streptomycin/penicillin (0.1 mL), and an incubation period of 72 h. Mitotic arrest was induced by 2-h exposure to colchicine (0.1 mL), 70 h after establishing the culture. After mitotic arrest, the cells were hypotonized with 0.075 M KCl for 2 h and fixed with methanol/acetic acid (3:1). The non-banded mitotic chromosomes were visualized by Giemsa staining. The diploid chromosome number of C. carbonaria was found to be 52 in females and males, and sex chromosomes were not observed. We were able to culture peripheral blood lymphocytes of a Brazilian tortoise in vitro, for the preparation of mitotic chromosomes.

Mathematical modeling of preadipocyte fate determination

White adipose tissue is the major energy storage depot for neutral lipids and is also a key endocrine regulator of a host of homeostatic activities, including metabolism, feeding behaviors, cardiovascular functions and reproduction. Abnormal fat accretion in the setting of obesity can lead to insulin resistance and type 2 diabetes, and has been linked to some cancers and arteriosclerosis. Thus, a thorough appreciation of the intricate signaling events that must take place as quiescent adipocyte precursors are recruited into the proliferating cell population that then must ‘decide’ to differentiate into fully functional fat cells is critical to our understanding of diseases related to excess adipogenesis. We are beginning to gain insights into the molecular regulators of adipocyte differentiation. A significant advance would be to construct mathematical modeling tools that would assist cell biologists in predicting how environmental and/or intrinsic inputs could influence preadipocyte fate decision making. We have developed a model of how preadipocytes may use the dynamic interplay of two transcription factors, nuclear factor- κ B ( NF ‐ κ B ) and peroxisome proliferator-activated receptor- γ (PPAR- γ ) in early proliferation and differentiation events in vitro. Critical to the model is the feedback signaling between NF ‐ κ B and its inhibitor, I κ B . The model is based on differential equations that describe the interactions of stimuli for NF ‐ κ B activation and mitogen concentrations and allows one to make predictions about how mouse 3T3-L1 preadipocytes choose between proliferation, differentiation or quiescence. Those predictions are supported by experiments on mouse 3T3-L1 cells.