Tritiated Thymidine Research Articles

Lymphoproliferative responses to dendritic cell presentation of sensitizing allergens in atopic children with multiple allergies

BackgroundPeripheral blood mononuclear cells (PBMCs) proliferate inconsistently, rendering current lymphoproliferation assays unreliable in diagnosis. ObjectiveTo investigate the utility and nature of proliferation responses in allergy by comparison of the standard lymphoproliferation with a new dendritic cell (DC) stimulated assay. MethodsMonocyte-derived DCs were pulsed with allergens and incubated with autologous T cells for 7 days. DC-stimulated and standard PBMC proliferation responses to 3 common dietary allergens in children with allergy and without atopy were measured by incorporation of tritiated thymidine and reduction of carboxyl fluorescein succinimidyl ester staining. ResultsThe DC presentation of sensitizing allergens induced significantly higher proliferative responses than PBMC stimulation (P = .04) and greater distinction between normal and allergic responses. DC-induced stimulation indices of children without sensitivity and those with allergy were significantly different with all 3 foods (P < .001). All children with allergy presented with peanut allergy and 12 of 14 (86%) β-lactoglobulin–pulsed DC preparations proliferated more than 3.3-fold above un-pulsed cells, but 8 of 18 children (44%) with ovalbumin egg allergy showed proliferation below this level. The stimulation index of DC tritiated thymidine incorporation correlated closely with carboxyl fluorescein succinimidyl ester reduction (P < .001). Sensitivity of detection of peanut, milk, or egg allergy was 100%, 85.7%, or 55.6% and specificity was 60%, 88.9%, or 86.7%, respectively. DC-stimulated T cells expressed increased levels of CD45 RO and CD25 and most produced interferon-γ. DC-stimulated proliferation correlated with total immunoglobulin E and peanut antigen-stimulated proliferation correlated with peanut specific immunoglobulin E (P = .03). ConclusionThe DC-induced lymphoproliferation had higher sensitivity, specificity, and reproducibility than the standard assay and caused increased memory and activated T-cell proliferation in children with food allergy.

A New View of Stem Cell Dynamics

Our understanding of the biology of hematopoietic stem cells is evolving beyond hierarchical models and markers of stem cell purification to a far more fluid model wherein degrees of multipotency of individual cells can change over time. This review is to outline these new concepts: The long-term repopulating hematopoietic stem cell is a proliferating cell, which is continually changing in phenotype. It cannot be purified by current surface epitope techniques. The cycling nature of the cell has been established by three separate approaches; separation of whole marrow into G0/G1 and S/G2/M fractions followed by evaluation of long-term in vivo repopulation in irradiated mice, tritiated thymidine suicide of marrow cells with repopulation assays, and in vivo BrdU studies to estimate flux through cycle. Other work has suggested that stem cell studies need to be considered within the context of transplant-based or normal baseline hematopoiesis; they are quite distinct and likely have differential influences governing stem cell self-renewal and differentiation. Hematopoietic stem cell functions are further modulated by interactions with a variety of extracellular vesicles and influenced by circadian rhythms and other variables. The true nature of the system remains to be defined, and such definition will elucidate the basic nature of stem cell biology and indicate specifics of clinical relevance.

Spermatogenesis in a neotropical marsupial species, Philander frenatus (Olfers, 1818)

Despite the singular morphology of the male genital system and the different reproductive strategies of marsupials, little emphasis has been given to the testis morphology and spermatogenic kinetics in this mammalian order. The present study aimed to investigate the testis function and the duration of spermatogenesis in the southeastern four-eyed opossum, Philander frenatus. Testes of six adult males were routinely processed for histological and stereological analyses. In order to determine the duration of spermatogenesis, intratesticular injections of tritiated thymidine were performed 1h, 13days and 21days before the sacrifice. Based on the development of the acrosomic system, ten stages of the seminiferous epithelium cycle were characterized. The mean body and testis weights for the P. frenatus were respectively 326±20g and 0.4±0.05g, providing a gonadosomatic index of 0.3±0.02%. The most advanced germ cell types labeled at 1h, 13days and 21days after thymidine injections were, respectively, preleptotene spermatocytes at stage IV, pachytene spermatocytes at stage IV and diplotene spermatocytes at stage IX. Based on the stages frequencies and the most advanced labeled germ cells, each spermatogenic cycle and the entire spermatogenic process lasted respectively 13.5±0.5 and 60.9±2.4days. When compared to the vast majority of eutherian mammals already investigated, these data indicate that the Philander frenatus presents a relatively long duration of spermatogenesis.

Morphofunctional evaluation of the testis, duration of spermatogenesis and spermatogenic efficiency in the Japanese fancy mouse (Mus musculus molossinus).

Japanese fancy mouse, mini mouse or pet mouse are common names used to refer to strains of mice that present with different colour varieties and coat types. Although many genetic studies that involve spotting phenotype based on the coat have been performed in these mice, there are no reports of quantitative data in the literature regarding testis structure and spermatogenic efficiency. Hence, in this study we researched testis function and spermatogenesis in the adult Japanese fancy mouse. The following values of 68 ± 6 mg and 0.94 ± 0.1% were obtained as mean testis weight and gonadosomatic index, respectively. In comparison with other investigated mice strains, the fancy mouse Leydig cell individual size was much smaller, resulting in higher numbers of these cells per gram of testis. As found for laboratory mice strains, as a result of the development of the acrosomic system, 12 stages of the seminiferous epithelium cycle have been described in this study. The combined frequencies of pre-meiotic and post-meiotic stages were respectively 24% and 64% and very similar to the laboratory mice. The more differentiated germ cell types marked at 1 h or 9 days after tritiated thymidine administration were preleptotene/leptotene and pachytene spermatocytes at the same stage (VIII). The mean duration of one spermatogenic cycle was 8.8 ± 0.01 days and the total length of spermatogenesis lasted 37.8 ± 0.01 days (4.5 cycles). A high number of germ cell apoptosis was evident during meiosis, resulting in lower Sertoli cell and spermatogenic efficiencies, when compared with laboratory mice strains.

Quantification of epithelial cell proliferation, cell dynamics, and cell kinetics in vivo.

The measurement of cell proliferation in vivo is usually carried out by the examination of static measures. These comprise the mitotic index or labeling indices using incorporation of DNA synthesis markers such as bromodeoxyuridine or tritiated thymidine, or intrinsic markers, such as Ki67 and proliferative cell nuclear antigen (PCNA). But static measures only provide a 'snapshot' of cell proliferation. Rate measures, including double labeling methods and the metaphase arrest method, can actually measure cell production rates but they are far less utilized at present. Transit times and migration rates can also be measured using pulse and chase labeling or by following the transit of labeled cells through the tissue. Simple indices of cell division can easily be confounded by concomitant changes in the compartment size and many alleged markers of proliferation have serious shortcomings, as the markers may be involved in multiple aspects of cell regulation. The complexities of studying proliferation in vivo are illustrated here with a focus on the gastrointestinal tract. Some of these methods can help elucidate the role of the stem cells and their relationship to label retaining cells. WIREs Dev Biol 2017, 6:e274. doi: 10.1002/wdev.274 For further resources related to this article, please visit the WIREs website.

Synthesis, cytotoxicity and antifungal activity of 5-nitro-thiophene-thiosemicarbazones derivatives

In the present work, twelve N-substituted 2-(5-nitro-thiophene)-thiosemicarbazones derivatives (L1-12) were synthesized, characterized and their in vitro cytotoxic and antifungal activities were evaluated against Candida sp. and Cryptococcus neoformans. The probable mechanisms of action have been investigated by sorbitol and ergosterol assays. Additionally, ultrastructural study by Scanning Electron Microscopy was performed with the L10 compound. All compounds were obtained in good yield and their chemical structures were characterized on basis of their physico-chemical and Nuclear Magnetic Resonance - NMR, Spectrophotometric Absorption in the Infrared - IR and High-resolution Mass Spectrometry - HRMS data. The results showed that all strains were more sensitive to the compound L10 except Candida tropicalis URM 6551. On the other hand, the cytotoxicity assay by incorporation of tritiated thymidine showed moderate cytotoxic activity on L8 of the 50 μg/mLat which had the best MIC-cytotoxicity relationship. Concerning the study of the possible mechanism of action, the compounds were not able to bind to ergosterol in the membrane, do not act by inhibiting the synthesis of fungal cell wall (sorbitol assay). However, the Scanning Electron Microscopy - SEM analysis shows significant morphological changes in shape, size, number of cells and hyphae, and cell wall indicating a possible mechanism of action by inhibition of enzymes related to the ergosterol biosynthesis pathway. Our results demonstrate that N-substituted 2-(5-nitro-thiophene)-thiosemicarbazones derivatives are potential antifungal agents with activity associated with inhibition of enzymes related to biosynthesis of ergosterol.

MMGL receptor plays an important role to macrophage activation during Trypanosoma cruzi infection

Abstract The C-type lectin receptor (MGL) is exclusively expressed on macrophages (MΦs) and dendritic cells (DCs), recognizes glycosylated antigens, particularly galactose (Gal) and N-acetylgalactosamine (GalNAc), that are present in tumors, fungi and viruses. However, little is known the role of MGL in the interaction with parasites. We evaluated the role of murine MGL (mMGL) in the activation and functionality of MΦs in Trypanosoma cruzi (T. cruzi) infection, a parasite that has molecules glycosylated rich in Gal and GalNAc in its membrane. Peritoneal MΦs from C57BL/6 WT and mMGL nockout (mMGL−/−) mice were infected in vitro with epimastigotes of T. cruz,i Qro strain (10000 parasites). The percentage of infected MΦs and the number of internalized parasites/MΦs was analyzed by microcopy. Cytokines TNF-α, IL-12 and IL-10 were determine in supernatants by ELISA. The trypanocidal capacity of MΦs was determined by the incorporation of tritiated thymidine ([3H] TdR). Expression of TLR-4, TLR-2 and mannose receptor (MR) in MΦs stimulated in vitro with T. cruzi antigen was analyzed by flow cytometry. mMGL−/− MΦs were higher infected with more parasites per MΦ and had a reduced capacity to eliminate the T. cruzi, these were associated to lower levels of TNF-α, TLR-4 and MR expression compared to WT MΦs. These results demonstrate that absence of mMGL favors susceptibility to infection with T. cruzi, suggesting a regulatory role of mMGL on the expression of other important receptors in the immune response to T. cruzi infection.

Differential regulation of surface receptor expression, proliferation, and apoptosis in HaCaT cells stimulated with interferon-γ, interleukin-4, tumor necrosis factor-α, or muramyl dipeptide.

Keratinocytes are routinely subjected to both internal and external stimulation. This study investigates the effects of interferon gamma, interleukin-4, tumor necrosis factor alpha, and the synthetic immunomodulator muramyl dipeptide on the human keratinocyte cell line, HaCaT. Following HaCaT stimulation with cytokines or muramyl dipeptide for different time periods, changes in the expression of different cell surface receptors, cell proliferation, and cell apoptosis were evaluated by flow cytometry, tritiated thymidine uptake, and annexin-V staining, respectively. A significant decrease in the expression of CD49d was found upon treatment with interleukin-4. Interferon gamma and tumor necrosis factor alpha increased the expression of intercellular adhesion molecule 1 and major histocompatibility complex class I, whereas major histocompatibility complex class II and CD1b were only upregulated by interferon gamma. Interferon gamma and tumor necrosis factor alpha had opposite effects regarding CD119 expression, with the former downregulating, while the latter upregulating its expression. Of the stimuli tested, only interferon gamma and tumor necrosis factor alpha significantly inhibited proliferation of HaCaT cells, yet only interferon gamma played a significant role in inducing HaCaT cell apoptosis. Our data demonstrate differential effects of the three tested cytokines on keratinocytes and reveal that the absence of HaCaT cell responses to muramyl dipeptide is associated with undetectable levels of its cytoplasmic receptor, nucleotide-binding oligomerization domain–containing protein 2.

Effects of β-adrenergic receptor drugs on embryonic ventricular cell proliferation and differentiation and their impact on donor cell transplantation.

β-Adrenergic receptors (β-ARs) and catecholamines are present in rodents as early as embryonic day (E)10.5. However, it is not known whether β-AR signaling plays any role in the proliferation and differentiation of ventricular cells in the embryonic heart. Here, we characterized expression profiles of β-AR subtypes and established dose-response curves for the nonselective β-AR agonist isoproterenol (ISO) in the developing mouse ventricular cells. Furthermore, we investigated the effects of ISO on cell cycle activity and differentiation of cultured E11.5 ventricular cells. ISO treatment significantly reduced tritiated thymidine incorporation and cell proliferation rates in both cardiac progenitor cell and cardiomyocyte populations. The ISO-mediated effects on DNA synthesis could be abolished by cotreatment of E11.5 cultures with either metoprolol (a β1-AR antagonist) or ICI-118,551 (a β2-AR antagonist). In contrast, ISO-mediated effects on cell proliferation could be abolished only by metoprolol. Furthermore, ISO treatment significantly increased the percentage of differentiated cardiomyocytes compared with that in control cultures. Additional experiments revealed that β-AR stimulation leads to downregulation of Erk and Akt phosphorylation followed by significant decreases in cyclin D1 and cyclin-dependent kinase 4 levels in E11.5 ventricular cells. Consistent with in vitro results, we found that chronic stimulation of recipient mice with ISO after intracardiac cell transplantation significantly decreased graft size, whereas metoprolol protected grafts from the inhibitory effects of systemic catecholamines. Collectively, these results underscore the effects of β-AR signaling in cardiac development as well as graft expansion after cell transplantation.NEW & NOTEWORTHY β-Adrenergic receptor (β-AR) stimulation can decrease the proliferation of embryonic ventricular cells in vitro and reduce the graft size after intracardiac cell transplantation. In contrast, β1-AR antagonists can abrogate the antiproliferative effects mediated by β-AR stimulation and increase graft size. These results highlight potential interactions between adrenergic drugs and cell transplantation.

Reinvestigation of the T-Cell Inactivation Achieved for the Prevention of TA GV HD By 2500 Gy of Gamma Irradiation, a Comparison with Pathogen Reduction

▪Background: Transfusion Associated Graft vs. Host Disease (TA GVHD) results in high morbidity and mortality, caused by contaminating T-cells present in transfused blood products. Gamma irradiation (GI) of platelet components (PC) with a dose of 2500 cGy is the approved methodology to prevent TA GVHD in high-risk patients. That methodology has been established based on in vitro Limiting Dilution Assays (LDA) that quantify the inactivation of T-cells. The limited natural abundance of T-cells in blood products and the number of T-cells that can be reproducibly cultured in single wells have defined LDA assay sensitivity. LDA assays usually have a dynamic range of 4-5 log10. Even though that methodology has been used for decades with presumable success, cases of GVHD recognized as transfusion associated, have to be connected temporally and phenotypically to the blood product in use, in order to be characterized as such. That leaves open the possibility that some atypical cases of GVHD in blood product recipients may be miss-assigned and go unrecognized.An alternative methodology to prevent TA GVHD is the photochemical treatment of PC with amotosalen/UVA (PCT; INTERCEPTTM Blood System). PCT has replaced GI in Europe for pathoen inactivated PC for more than 10 years. In this study, we evaluated both GI and PCT on inactivation of T-cells using a highly sensitive approach that includes the use of higher numbers of PBMCs isolated by leukapheresis, and an increased number of cells cultured in a single well (107/well) by using larger wells and optimizing culture conditions.Methods: PBMCs harvested by leukapheresis from individual donors (AllCells, Alameda, CA) were spiked (106/mL) into identical units of human plasma and inactivated using either GI with 2500 cGy, PCT, or were retained as untreated control. For the LDA assay, PCT- or GI-treated cells isolated post treatment, were incubated in individual wells for 14 days in the presence of pooled allostimulator cells from 10 unrelated donors (5X106 treated with 7500 cGy) and growth stimulating factors (PHA and IL-2) under standard culture conditions. Proliferation was assessed by tritiated thymidine ([3H]TdR6.7 Ci/mmol) incorporation into PBMC, as well as by the observation of bright large clusters that were clearly observed at 40x magnification.Results: Initial experiments showed that 107PBMCs/well or 106PBMCs/well resulted in proliferation after GI at 2500 cGy, while 105 PBMCs/well did not. On the other hand, 107 PCT treated-PBMCs/well were found neither to proliferate, nor to incorporate [3H]TdRabove background. T-cell precursor frequency was measured for each donor by incubation of serial dilutions of viable PBMCs (50 - 1 /well; 12 wells per dilution) in the presence of 107 inactivated PBMCs. The experiment was repeated 10 times with PBMCs from different donors.For GI, [3H]TdR incorporation above background indicative of T-cell growth, as well as T-cell proliferating colonies were observed in 4 of 10 experiments when 106PBMCs/ well were cultured. No T-cell growth was detected by [3H]TdR incorporation when 105 GI PBMCs/well were cultured, while proliferating colonies were observed in 1 of 10 experiments. No T-cell proliferation was detected by either criterion, when 107 PCT-treated PBMCs/well were cultured.Conclusions: The accepted dose of 2500 cGy gamma irradiation for prevention of TA GVHD results in more than 4.2log10 but less than 6.2 log10 T-cell inactivation. However, it still allows the proliferation of T-cell clones from some donors. Treatment of T-cells with amotosalen/UVA results in complete inactivation of T-cells (>6.2 log10) with no break though proliferation detected.The data in this study have not been reviewed by the FDA. DisclosuresMerkel:Cerus Corporation: Other: fee for service with Cerus Corporation. Giclas:Cerus Corporation: Other: fee for service with Cerus Corporation. Gibula:Cerus Corporation: Other: fee for service with Cerus Corporation. Andersen:Cerus Corporation: Other: fee for service with Cerus Corporation. Knight:Cerus Corporation: Other: fee for service with Cerus Corporation. Lin:Cerus corp: Employment. Castro:Cerus Corporation: Employment. Green:Cerus Corporation: Employment. Stassinopoulos:Cerus Corporation: Employment.

Scintillation imaging of tritium radioactivity distribution during tritiated thymidine uptake by PC12 cells using a melt-on scintillator

A scintillation image of tritium fixed in a melt-on scintillator was obtained using a charged-coupled device (CCD) imager, and a linear relationship was observed between the intensity of the scintillation image and the radioactivity of tritium. In a [3H]thymidine uptake experiment, a linear correlation between the intensity of the CCD image and the dilution ratio of cells was confirmed. Scintillation imaging has the potential for use in direct observation of tritium radioactivity distribution.

Improved osteogenesis and upregulated immunogenicity in human placenta-derived mesenchymal stem cells primed with osteogenic induction medium.

BackgroundMesenchymal stem cells (MSCs) are widely used in cell-based therapy owing to their multilineage potential and low immunogenicity. However, low differentiation efficiency and unpredictable immunogenicity of allogeneic MSCs in vivo limit their success in therapeutic treatment. Herein, we evaluated the differentiation potential and immunogenicity of human placenta-derived MSCs manipulated with osteogenic priming and dedifferentiation process.MethodsMSCs from human placentas were subjected to osteogenic induction and then cultivated in osteogenic factor-free media; the obtained cell population was termed dedifferentiated mesenchymal stem cells (De-MSCs). De-MSCs were induced into osteo-, chondro- and adipo-differentiation in vitro. Cell proliferation was quantified by a Cell-Counting Kit-8 or tritiated thymidine ([3H]-TdR) incorporation. Meanwhile, the osteogenesis of De-MSCs in vivo was assayed by real-time PCR and histological staining. The expressions of stem cell markers and co-stimulatory molecules on De-MSCs and lymphocytes from primed BALB/c mouse with De-MSCs were determined by flow cytometry.ResultsDe-MSCs exhibited some properties similar to MSCs including multiple differentiation potential and hypoimmunogenicity. Upon re-osteogenic induction, De-MSCs exhibited higher differentiation capability than MSCs both in vitro and in vivo. Of note, De-MSCs had upregulated immunogenicity in association with their osteogenesis, reflected by the alternated expressions of co-stimulatory molecules on the surface and decreased suppression on T cell activation. Functionally, De-MSC-derived osteoblasts could prime lymphocytes of peripheral blood and spleen in BALB/c mice in vivo.ConclusionsThese data are of great significance for the potential application of De-MSCs as an alternative resource for regenerative medicine and tissue engineering. In order to avoid being rejected by the host during allogeneic De-MSC therapy, we suggest that immune intervention should be considered to boost the immune acceptance and integration because of the upregulated immunogenicity of De-MSCs with redifferentiation in clinical applications.

JAK inhibitor JTE-052 regulates contact hypersensitivity by downmodulating T cell activation and differentiation

BackgroundUsing JAK inhibitors to inhibit cytokine signaling is presumed to be a possible means of treating skin inflammatory disorders such as contact dermatitis. ObjectiveTo clarify the action site of JAK inhibitors in skin inflammatory disorders. MethodsWe analyzed the mechanism of action of the JAK inhibitor JTE-052 using murine skin inflammation models, including contact hypersensitivity (CHS) and irritant contact dermatitis. Cells isolated from ear tissue or lymph node (LN) were analyzed by flow cytometry. The amounts of cytokines in the culture medium were measured by ELISA or bead array system. Proliferation of LN cells was evaluated by measurement of tritiated thymidine incorporation. ResultsOral administration of JTE-052 during both sensitization and elicitation phase attenuated CHS, but did not affect croton oil-induced irritant contact dermatitis. JTE-052 potently inhibited T cell proliferation and activation by antigen presentation in vitro, and attenuated skin inflammation in a sensitized-lymphocyte transfer model without suppressing T cell migration. JTE-052 did not affect hapten-induced cutaneous dendritic cell migration into draining lymph nodes or their costimulatory molecule expressions. ConclusionThe JAK inhibitor JTE-052 exerts an inhibitory effect on antigen-specific T cell activation and subsequent inflammation in acquired skin immunity, such as CHS.

Human immune-cell response towards diverse xenogeneic and allogeneic decellularized biomaterials

BackgroundImmunological knowledge on processed biological implants and mesh-prostheses is still mainly based on animal models, lacking information on the species-specific human immune response. We hypothesized that in contrast to human tissue even decellularized xenogenic specimens would lead to significant and tissue source dependent human immune reactions. MethodsSpecimens from processed allogenic and xenogenic pulmonary arteries, pericardium or dermis, were co-cultured with human peripheral blood mononuclear cells (PBMNC). Proliferative responses were measured in tritiated thymidine incorporation assays (n = 10). Stimulation indices (SI), calculated as counts-per-minute of co-cultured PBMNC divided by the cpm of basic cell proliferation, were compared. ResultsCompared to native porcine pulmonary artery tissue decellularization significantly reduced human PBMNC proliferation (mean SI: 48.7 vs. 18.0, p < 0.01), which was still higher compared to the human equivalent (SI: 0.7 vs. 1.7). Also the processed human dermal implant did not elicit immune response (SI: 1.5), whereas the decellularized and cross-linked porcine dermis lead to a significant human cell-proliferation (SI: 8.4, p < 0.01). Interestingly, both the processed human (SI: 15.2) and bovine pericardial patches (SI: 15.1) led to higher immune cell proliferation. ConclusionEven decellularized or cross-linked xenogenic cardiovascular and reconstructive biomaterials elicit increased human immune responses not seen in the majority of allogenic specimens tested.

Commentary: Blurring Borders: Innate Immunity with Adaptive Features.

Commentary: Blurring Borders: Innate Immunity with Adaptive Features.

Endocrine and social regulation of adult neurogenesis in songbirds.

The identification of pronounced seasonal changes in the volume of avian song control nuclei stimulated the discovery of adult neurogenesis in songbirds as well as renewed studies in mammals including humans. Neurogenesis in songbirds is modulated by testosterone and other factors such as photoperiod, singing activity and social environment. Adult neurogenesis has been widely studied by labeling, with tritiated thymidine or its analog BrdU, cells duplicating their DNA in anticipation of their last mitotic division and following their fate as new neurons. New methods based on endogenous markers of cell cycling or of various stages of neuronal life have allowed for additional progress. In particular immunocytochemical visualization of the microtubule-associated protein doublecortin has provided an integrated view of neuronal replacement in the song control nucleus HVC. Multiple questions remain however concerning the specific steps in the neuronal life cycle that are modulated by various factors and the underlying cellular mechanisms.

Cell Proliferation Analysis Using EdU Labeling in Whole Plant and Histological Samples of Arabidopsis.

The ability to analyze cell division in both spatial and temporal dimensions within an organism is a key requirement in developmental biology. Specialized cell types within individual organs, such as those within shoot and root apical meristems, have often been identified by differences in their rates of proliferation prior to the characterization of distinguishing molecular markers. Replication-dependent labeling of DNA is a widely used method for assaying cell proliferation. The earliest approaches used radioactive labeling with tritiated thymidine, which were later followed by immunodetection of bromodeoxyuridine (BrdU). A major advance in DNA labeling came with the use of 5-ethynyl-2'deoxyuridine (EdU) which has proven to have multiple advantages over BrdU. Here we describe the methodology for analyzing EdU labeling and retention in whole plants and histological sections of Arabidopsis.

Cyclic tensile stress of human annulus fibrosus cells induces MAPK activation: involvement in proinflammatory gene expression

To study the role of mitogen-activated protein kinases (MAPKs) in human annulus fibrosus (AF) cells subjected to cyclic tensile stress (CTS). An invitro system for CTS studies was established using AF cultures on fibronectin-coated silicone dishes. MAPK phosphorylation was studied by western analysis, while gene expression was followed by qRT-PCR. DNA synthesis was assessed by both tritiated thymidine incorporation and flow cytometry, and collagen synthesis using tritiated proline incorporation and the protease-free collagenase method. All three MAPKs studied, i.e., ERK, SAPK/JNK, and p38 were found to be phosphorylated immediately after CTS application within physiological range. A second wave of phosphorylation appeared at later time points. MAPK activation was elevated at higher CTS magnitudes, but independent of the frequency. CTS did not stimulate DNA synthesis neither extracellular matrix turnover, but it stimulated the proinflammatory genes, COX-2, IL-6, and IL-8. This stimulation was more intense at the highest magnitude (8%) testedand at the median frequency (1Hz) and time interval (12h). Blocking of ERK, SAPK/JNK, and p38 MAPK inhibited the CTS-induced stimulation of COX-2 and IL-8, while IL-6 expression was mediated only by SAPK/JNK and p38 MAPK. We have described for the first time the activation of MAPKs in human AF cells in response to CTSand showed that it drives an inflammatory reaction. These observations shed light on the mechanisms of intervertebral disc (IVD) cell responses to mechanical stress, contributing to the understanding of disc pathophysiologyand possibly to the design of novel therapeutic interventions.

Role for Megakaryocyte Phosphatidylinositol Transfer Proteins-Alpha and -Beta in TGF Beta-Mediated Regulation of Hematopoietic Homeostasis

There are still unknowns regarding homeostatic regulation of hematopoietic stem (HSC) and progenitor (HPC) cells. Deciphering these processes are important for understanding and treating hematopoietic diseases. Phosphatidylinositol is a rare membrane structure lipid, but is critical for cellular signaling upon phosphorylation by lipid kinases to generate phosphoinositide. While phosphoinositide pathways contribute to events linked to the cytoskeleton, little is known of these pathways in regulating hematopoiesis. Critical to this pathway are phosphatidylinositol transfer proteins (PITPs) that in vitro enhance transfer of aqueous insoluble phosphatidylinositol from one membrane to another. Class I PITP proteins PITP α and β are highly conserved, small, and ubiquitously expressed in mammalian cells. To test the hypothesis that phosphatidylinositol signaling contributes to hematopoiesis, we generated conditional knock out mice that lack either PITPα single isoform (PITPαfl/fl PF4Cre+) or both PITPα and PITPβ (PITPαfl/fl βfl/fl PF4Cre+) specifically in their platelets and megakaryocytes, and observed a bone marrow (BM) HSC/HPC phenotype. BM from these mice and their littermate controls were evaluated for absolute numbers of nucleated cells, HSC, and HPC. Cells were analyzed by rigorous phenotyping for long-term (LT)-HSC, short-term (ST)-HSC, multipotential (MPP), common myeloid (CMP), and granulocyte macrophage (GMP) progenitors. They were also assessed for functional HPC by colony assays in vitro for multi-cytokine (Epo, GM-CSF, IL-3, SCF, hemin) stimulated granulocyte macrophage (CFU-GM), erythroid (BFU-E), and multipotential (CFU-GEMM) progenitors, and for their cycling status using a high specific activity tritiated thymidine kill assay. PITPα-/-, and to a greater extent PITPα/β-/-, progenitor cells demonstrated significant decreases in LT-HSC and ST-HSC per femur. While there were no significant changes in numbers of MPP, CMP, and GMP in the PITPα and PITPα/β-/- BM compared to controls, there were significant decreases of approximately 50% in numbers of CFU-GM, BFU-E, and CFU-GEMM per femur. PITC-/- HPC were in a slow or non-cycling state compared to the rapid cell cycle (40-57% in S-phase) of control HPC. Thus PITPα-/- and PITPα/β -/- BM cells were associated with decreased HSC and functional HPC numbers. To evaluate mechanisms for this phenotype, we focused on BM megakaryocytes, as they have been implicated in microenvironmental regulation of hematopoiesis, and PITPα and PITPα/β activities are associated with megakaryocyte/platelet function. BM derived TPO-culture expanded megakaryocytes were allowed to condition medium for 48 hours, and conditioned medium (CM) from PITPα-/-, PITPα/β-/-, and control BM megakaryocytes were assayed for effects on colony formation by multicytokine stimulated BM cells derived from normal mice. CM from PITPα-/- and PITPα/β-/- megakaryocytes, but not from control mice, significantly suppressed colony formation by CFU-GM, BFU-E and CFU-GEMM (by ~50%). Limiting dilution analysis of the CM demonstrated that PITPα/β-/- cells had more potent suppressor activity than PITPα-/- cells. Bioplex analysis of the CM from PITPα -/- and PITPα/β -/- megakaryocytes demonstrated significantly higher levels of cytokines/chemokines with known myelosuppressive activities (including: TNF-α, VEGF, LIF, IP-10, ENA-78, MDC, MIG, and MIP-1α). However, ELISA analysis of TGF-β1, demonstrated minimal protein in BM flushes from control mice, but large amounts of TGF-β (>350 pg/ml) in BM flushes from the PITPα/β -/- mice. CM from PITPα and α/β-/- megakaryocytes also contained highly elevated TGF-β protein. Thus, we hypothesized that the effect of PITP -/- on the suppression of HPC colony formation was mediated by TGF-β. The myelosuppressive CM derived from PITPα and PITPα/β -/- megakaryocytes was completely neutralized by a monoclonal TGF-β antibody. This demonstrates that PITPα and PITP α/β-/- megakaryocytes produce elevated TGF-β that at least in part, and possibly in synergy with other myelosuppressive cytokines/chemokines, decreases numbers of HSC and functional HPC. Our studies demonstrate a link between PITPα and α/β and TGF-β levels with significant effects on HSCs and HPCs, thus demonstrating involvement of the phosphoinositide pathway in homeostatic regulation of hematopoiesis. DisclosuresNo relevant conflicts of interest to declare.

Systematic differences in time of cerebellar-neuron origin derived from bromodeoxyuridine immunoperoxidase staining protocols and tritiated thymidine autoradiography: A comparative study

As exogenous markers of DNA synthesis, 5-bromo-2′-deoxyuridine (BrdU) and tritiated thymidine ([3H]TdR) have revolutionized our ability to identify proliferating neuroblasts and follow their fate during the development of the central nervous system. The effect of the incorporation of these molecules into DNA on cell proliferation, migration and differentiation is frequently neglected (Duque and Rakic, 2011. J. Neurosci. 31, 15205–15217). By a progressively delayed cumulative labeling method, the current paper analyzes the development of the cerebellum in mice exposed to either BrdU or [3H]TdR as embryos and collected at postnatal day 90. We observed that, in comparison to the saline group, several parameters of the cerebellum such as length of the cerebellar cortex, the area of the molecular layer, Purkinje cell (PCs) number, the areas of the cerebellar nuclei, and the number of the deep cerebellar nuclei (DCN) neurons were lower in the BrdU injected group. No consequence of [3H]TdR administration was observed. On the other hand, we also studied whether immunohistochemical methods, including BrdU antibodies from different vendors (Sigma and Dako), partial DNA denaturation procedures and trypsin pretreatments, alter the neurogenetic timetables of PC and DCN neurons that resulted from analysis of these tissue specimens. Our analysis revealed that the generative programs of these macroneurons were unrelated to differences in the sensibility of BrdU antibodies but were dependent on the partial denaturation of DNA and trypsin digestion protocols. Finally, we also compare the generation and spatial distribution of PC and DCN neurons in mice exposed to either BrdU or [3H]TdR to assess whether the results obtained by these two markers are quantitatively similar. The data presented here show that systematic differences exist in the pattern of neurogenesis and the spatial location of cerebellar neurons between mice injected with BrdU or [3H]TdR. These findings have implications for the interpretation of results obtained by both exogenous makers as an index of the production, migration and settling of neurons in the developing central nervous system.