Decrease In Total Cell Number Research Articles

Hypoxia-Inducible Factor 1α Affects Yak Oocyte Maturation and Early Embryonic Development by Regulating Autophagy

In animal assisted reproductive technology, the production of high-quality oocytes is crucial. The yak, having lived in the Qinghai-Tibet Plateau for an extended period, has reproductive cells that are regulated by hypoxia-inducible factor 1α (HIF-1α). This study aimed to investigate the impact of HIF-1α on yak oocyte maturation and early embryonic development in vitro through the regulation of autophagy. The in vitro maturation process of yak oocytes involved the addition of the HIF-1α inducer DFOM and the inhibitor LW6 to examine their effects on yak oocyte maturation, early embryonic development, cell autophagy, cytochrome P450s (CYP450s) enzyme expression, and cumulus diffusion factors. The findings revealed that DFOM significantly upregulated the expression of HIF-1α, resulting in increased the cumulus diffusion area, elevated first polar body expulsion rate of oocytes, enhanced mitochondrial and actin levels, decreased ROS production, and reduced early apoptosis levels of oocytes. Moreover, DFOM promoted the expression of autophagy-related proteins, CYP450s enzymes, and cumulus diffusion factors, thereby enhancing oocyte maturation and early embryonic development. Conversely, LW6 exhibited opposite effects. The inhibition of autophagy levels with 3-MA during DFOM treatment yielded similar outcomes. Furthermore, reducing autophagy led to increased apoptosis levels at all stages of early embryonic development, as well as a significant decrease in total cell number and ICM/TE ratio of blastocysts. Studies have shown that during the in vitro maturation of yak oocytes, HIF-1α can affect the cumulus expansion area of oocytes by regulating autophagy, the first polar body excretion rate, mitochondrial level, actin level, ROS and early apoptosis level, the CYP450s enzyme, and the expression of cumulus expansion factors, thereby improving the in vitro maturation and early embryonic development of yak oocytes. These findings offer valuable insights into the reproductive regulation mechanism of yaks in hypoxic environments and suggest potential strategies for the advancement of yak assisted reproductive technology.

TEAD4 regulates KRT8 and YAP in preimplantation embryos in mice but not in cattle.

Lineage specification plays a vital role in preimplantation development. TEAD4 is an essential transcription factor for trophectoderm lineage specification in mice but not in cattle. Tead4, a critical transcription factor expressed during preimplantation development, is essential for the expression of trophectoderm-specific genes in mice. However, the functional mechanism of TEAD4 in mouse preimplantation development and its conservation across mammals remain unclear. Here, we report that Tead4 is a crucial transcription factor necessary for blastocyst formation in mice. Disruption of Tead4 through base editing results in developmental arrest at the morula stage. Additionally, RNA-seq analysis reveals dysregulation of 670 genes in Tead4 knockout embryos. As anticipated, Tead4 knockout led to a decrease in trophectoderm genes Cdx2 and Gata3. Intriguingly, we observed a reduction in Krt8, suggesting that Tead4 influences the integrity of the trophectoderm epithelium in mice. More importantly, we noted a dramatic decrease in nuclear Yap in outside cells for Tead4-deficient morula, indicating that Tead4 directly regulates Hippo signaling. In contrast, bovine embryos with TEAD4 depletion could still develop to blastocysts with normal expression of CDX2, GATA3, and SOX2, albeit with a decrease in total cell number and ICM cell number. In conclusion, we propose that Tead4 regulates mouse blastocyst formation via Krt8 and Yap, both of which are critical regulators of mouse preimplantation development.

The effect of discrete wavelengths of visible light on the developing murine embryo

PurposeA current focus of the IVF field is non-invasive imaging of the embryo to quantify developmental potential. Such approaches use varying wavelengths to gain maximum biological information. The impact of irradiating the developing embryo with discrete wavelengths of light is not fully understood. Here, we assess the impact of a range of wavelengths on the developing embryo.MethodsMurine preimplantation embryos were exposed daily to wavelengths within the blue, green, yellow, and red spectral bands and compared to an unexposed control group. Development to blastocyst, DNA damage, and cell number/allocation to blastocyst cell lineages were assessed. For the longer wavelengths (yellow and red), pregnancy/fetal outcomes and the abundance of intracellular lipid were investigated.ResultsSignificantly fewer embryos developed to the blastocyst stage when exposed to the yellow wavelength. Elevated DNA damage was observed within embryos exposed to blue, green, or red wavelengths. There was no effect on blastocyst cell number/lineage allocation for all wavelengths except red, where there was a significant decrease in total cell number. Pregnancy rate was significantly reduced when embryos were irradiated with the red wavelength. Weight at weaning was significantly higher when embryos were exposed to yellow or red wavelengths. Lipid abundance was significantly elevated following exposure to the yellow wavelength.ConclusionOur results demonstrate that the impact of light is wavelength-specific, with longer wavelengths also impacting the embryo. We also show that effects are energy-dependent. This data shows that damage is multifaceted and developmental rate alone may not fully reflect the impact of light exposure.

Alternariol exerts embryotoxic and immunotoxic effects on mouse blastocysts through ROS-mediated apoptotic processes.

Alternariol (AOH), a mycotoxin belonging to the genus Alternaria, has been shown to induce cytotoxicity, including apoptosis and cell cycle arrest, in several mammalian cell types. However, its effects on early-stage embryonic development require further investigation. Here, we have shown that AOH exerts embryotoxic effects on mouse blastocyst-stage embryos and long-term adverse effects on immunity in one-day-old newborn mice of the next generation. Significant apoptosis and decrease in total cell number, predominantly through loss of inner cell mass (ICM), and to a minor extent, trophectoderm (TE) cells, were observed in AOH-treated blastocysts. Moreover, AOH exerted detrimental effects on pre- and post-implantation embryo development potential and induced a decrease in fetal weight in in vitro development and embryo transfer assays. Injection of pregnant mice with AOH (1, 3 and 5mg/kg body weight/day) for 4days resulted in apoptosis of blastocyst-stage embryos and injurious effects on embryonic development from the zygote to blastocyst stage or embryo degradation and a further decrease in fetal weight. Furthermore, AOH exerted a long-term impact on the next generation, triggering a significant increase in total oxidative stress content and expression of genes encoding antioxidant proteins. Lower expression of CXCL1, IL-1β and IL-8 related to innate immunity was detected in liver tissue extracts obtained from one-day-old newborns of AOH-injected pregnant mice (5mg/kg body weight/day) relative to their non-treated counterparts. In addition, ROS served as an upstream regulator of AOH-triggered apoptotic processes and impairment of embryonic development. Our collective results highlight the potential of AOH as an embryotoxic and immunotoxic risk factor during embryo and infant development stages in mice.

The impact of oral exposure to low-dose tris(2-butoxyethyl) phosphate in allergic asthmatic mice.

Tris(2-butoxyethyl) phosphate (TBEP) is a major organophosphorus flame retardant and has been widely increasing as a substitute for brominated flame retardants. TBEP may have adverse effects on human health; however, its impact on immune and allergic responses remains largely uncharacterized. In this study, the effects of low-dose TBEP comparable with the level of actual human exposure to that of human tolerable daily intake on allergic asthmatic mice were explored. Five-week-old C3H/HeJSlc male mice consumed a diet containing approximately 0.02, 0.2 or 2 μg/kg/day TBEP and were intratracheally administrated ovalbumin (OVA) (1 μg/mouse every 2 weeks from 5 to 11 weeks of age). Exposure to 2 μg/kg/day TBEP with OVA tended to enhance allergic pulmonary inflammation and significantly elevated mRNA levels of interleukin-5, eotaxin-1 and estrogen receptor alpha (ERα) compared with OVA alone. In mediastinal lymph nodes (MLNs), TBEP (0.2 or 2 μg/kg/day) with OVA significantly increased in total cell number and promoted conventional dendritic cell activation than OVA alone; MLN cell proliferation by OVA restimulation was also enhanced in these groups. In the bone marrow (BM), TBEP (0.02 or 0.2 μg/kg/day) with OVA resulted in a net decrease in total cell number and fraction of CCR2+ Gr-1+ cells; the fraction of Gr-1+ cells increased. In conclusion, oral exposure to low-dose TBEP levels equivalent to tolerable daily intake may exacerbate allergic pulmonary inflammation by promoting a skewed T-helper 2 cell response, upregulation of ERα and dysregulation of both MLN and BM microenvironments.

High-fat diet leads to elevated lipid accumulation and endoplasmic reticulum stress in oocytes, causing poor embryo development.

The present study was designed to investigate the effect of diet-induced obesity on endoplasmic reticulum (ER) stress in oocytes. Swiss albino mice (3 weeks old) were fed with a high-fat diet (HFD) for 8 weeks. Oocytes were assessed for lipid droplet accumulation, oxidative stress, ER stress and their developmental potential invitro. High lipid accumulation (P<0.01) and elevated intracellular levels of reactive oxygen species were observed in both germinal vesicle and MII oocytes of HFD-fed mice (P<0.05 and P<0.01 respectively compared with control). Further, expression of the ER stress markers X-box binding protein 1 (XBP1), glucose-regulated protein 78 (GRP78), activating transcription factor 4 (ATF4) and activating transcription factor 6 (ATF6) was significantly (P<0.001) higher in oocytes of the HFD than control group. Oocytes from HFD-fed mice exhibited poor fertilisation and blastocyst rates, a decrease in total cell number and high levels of DNA damage (P<0.01) compared with controls. In conclusion, diet-induced obesity resulted in elevated lipid levels and higher oxidative and ER stress in oocytes, which contributed to the compromised developmental potential of embryos.

Enniatin B1 exerts embryotoxic effects on mouse blastocysts and induces oxidative stress and immunotoxicity during embryo development.

Enniatins are mycotoxins of Fusarium fungi that naturally exist as mixtures of cyclic depsipeptides. Previous reports have documented hazardous effects of enniatins on cells, such as apoptosis. However, their effects on pre- and post-implantation embryonic development require further clarification. Here, we showed for the first time that enniatin B1 (ENN B1) exerts cytotoxic effects on mouse blastocyst-stage embryos and induces intracellular oxidative stress and immunotoxicity in mouse fetuses. Co-incubation of blastocysts with ENN B1 triggered significant apoptosis and led to a decrease in total cell number predominantly through loss of inner cell mass. In addition, ENN B1 appeared to exert hazardous effects on pre and postimplantation embryo development potential in an in vitro development assay. Treatment of blastocysts with 1-10 μM ENN B1 led to increased resorption of post-implantation embryos and decreased fetal weight in the embryo transfer assay in a dose-dependent manner. Importantly, in an in vivo model, intravenous injection with ENN B1 (1, 3, and 5 mg/kg body weight/d) for 4 days resulted in apoptosis of blastocyst-stage embryos and impairment of embryonic development from the zygote to blastocyst stage, subsequent degradation of embryos, and further decrease in fetal weight. Intravenous injection with 5 mg/kg body weight/d ENN B1 additionally induced a significant increase in total reactive oxygen species (ROS) content and transcription levels of genes encoding antioxidant proteins in mouse fetal liver. Moreover, ENN B1 triggered apoptosis through ROS generation and strategies to prevent apoptotic processes effectively rescued ENN B1-mediated hazardous effects on embryonic development. Transcription levels of CXCL1, IL-1β, and IL-8 related to innate immunity were downregulated after intravenous injection of ENN B1. These results collectively highlight the potential of ENN B1 to exert cytotoxic effects on embryos as well as oxidative stress and immunotoxicity during mouse embryo development.

Targeting Differentially Expressed UPR Mediators in Mucin‐rich Colorectal Cancers and Conventional Colorectal Adenocarcinomas

BackgroundDespite the improved 5‐year survival rate, colorectal cancer (CRC) is the second leading cause of cancer deaths in the United States and is sub‐classified according to mucin content as mucin‐rich CRC (MCRC, ≥50% mucin content) or conventional colorectal adenocarcinoma (CCRA, &lt;50% mucin content). Ten percent of CRCs are considered mucin‐rich and are reported to be less sensitive to chemotherapy treatment than CCRAs. The unfolded protein response (UPR) is an adaptive response activated when unfolded proteins accumulate in the endoplasmic reticulum (ER). When the UPR is incapable of dealing with an increased protein load, it leads to apoptosis. Despite the increased protein load and UPR activation, tumor cells are known to maintain protein homeostasis and avoid apoptosis.ObjectivesWe hypothesized that the higher protein load in MCRC cells, initiated by the high demand of producing mucins, induces greater ER stress and activation of the UPR. Here, we aimed to identify UPR markers uniquely upregulated in their expression in MCRC versus CCRA patient biopsies. We also utilized CRC cell lines with similar mucin and UPR profiles as the biopsies, to target differentially regulated UPR markers for inhibition as potential targets for CRC therapy.ResultsOur data showed that when compared to CCRA samples, MCRC biopsies express significantly higher mRNAs for several UPR markers such as IRE1α, XBP1, and CHOP, which significantly correlate (p&lt;0.0001, p&lt;0.0001, p=0.0003, respectively) with MUC6 expression. In contrast, CCRA samples expressed higher levels of several protein disulfide isomerases (PDIs) including PDIA1 (p=0.0001), PDIA3 (p&lt;0.0001), PDIA5 (p&lt;0.0001), and PDIA17 (p=0.0025), that are known to be the downstream of canonical transducers of the UPR. We also identified CCRA (Caco2) and MCRC (LS174T) cell lines with similar UPR profiles to the biopsies. Our data indicate that cells from the MCRC cell line were less responsive to LOC14, a PDI inhibitor, whereas the CCRA cell line, which expressed higher levels of several PDIs, were less proliferative and had a greater decrease in total cell numbers following 6 days of treatment with LOC14. Accordingly, treatment with LOC14 inhibited one of the PDIs, PDIA3 in the CCRA cell, but not the MCRC cell line.ConclusionsThese novel results suggest that CCRAs and MCRCs have different signatures of UPR markers and CCRAs may be more responsive to PDI inhibitors of the UPR than MCRCs. Further characterization of the UPR profiles of CCRAs and MCRCs could aid in the development of future anti‐cancer therapies and targeting of UPR transducers and their downstream mediators.Support or Funding InformationNIH R01 HL122383 and Department of Pathology and Laboratory Medicine (UVM) Startup Fund (VA)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Abstract MIP-047: INHIBITION OF POLY ADP–RIBOSE POLYMERASE (PARP) IN OVARIAN CANCER CELLS ENRICHES FOR CD133 AND CD117 POSITIVE POPULATIONS

Abstract Keywords: chemoresistance, cancer stem cells (CSC), DNA repair pathways, PARP inhibition INTRODUCTION: Ovarian cancer (OvCa) is the most lethal gynecologic malignancy as women commonly present with metastatic disease that ultimately develops chemotherapy resistance. The development of OvCa has been linked to alterations in DNA repair pathways and the poly (adenosine diphosphate [ADP]–ribose) polymerase (PARP) inhibitor olaparib has demonstrated antitumor activity and is an FDA approved monotherapy for the treatment of recurrent OvCa in patients with a germline BRCA mutations. Despite promising response rates, not all patients derive benefit, and the majority develop resistance to this therapy that is poorly understood. We hypothesized that ultimate resistance to olaparib is in part mediated by small populations of cancer stem cells (CSCs) that are unaffected by olaparib therapy and may promote tumor resurgence. METHODS: UWB1.289 (BRCA1 mutated), UWB1.289 (WT), OVCAR4 (WT), and OVCAR3 (WT) cell lines and primary cell lines derived from serous OvCa obtained with IRB approval were treated in vitro with 0.1 μM, 1 μM and 10 μM olaparib for 7 days. We assessed cell viability and CSC marker expression (CD133 and CD117) via flow cytometric analysis, and sphere forming capacity via extreme limiting dilution assay (ELDA). RESULTS: Consecutive treatment over 7 days with olaparib decreased the viability of the non–sorted tumor cell population relative to the vehicle control treated cells. Despite the decrease in total cell number, the relative frequency of CD133+ and CD117+ cells increased (all lines p &amp;lt;0.05) in response to treatment with olaparib. This shift was not observed in vehicle treated cells. The increase in CD133+ and CD117+ cell frequency in response to olaparib was not different in the BRCA1 mutant line when compared to the BRCA1 wild type lines. ELDA revealed an increase in the frequency of sphere formation following olaparib treatment relative to that observed with vehicle treated cells, though mutation status did not impact this frequency. CONCLUSIONS: These results suggest that olaparib may enrich for CSC populations that could contribute to resistance and recurrence. These data expose a potential resistance mechanism to olaparib therapy that is in part independent of BRCA mutation status. Citation Format: Chiara Bellio, Rosemary Foster, Whitfield B. Growdon Panagiotis Konstantinopoulos, and Bo R. Rueda. INHIBITION OF POLY ADP–RIBOSE POLYMERASE (PARP) IN OVARIAN CANCER CELLS ENRICHES FOR CD133 AND CD117 POSITIVE POPULATIONS [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr MIP-047.

Intracellular and extracellular factors influencing the genotoxicity of nitric oxide and reactive oxygen species.

A number of factors affect cellular responses to nitric oxide (NO•) and reactive oxygen species (ROS), including their source, concentration, cumulative dose, target gene and biological milieu. This limits the extrapolation of data to in vivo pathological states in which NO• and ROS may be important. The present study investigated lethality and mutagenesis in the HPRT and TK1 genes of human lymphoblastoid TK6 cells exposed to NO• and ROS derived from two delivery methods: A reactor system and a Transwell™ co-culture. The delivery of NO• into the medium at controlled steady-state concentrations (given in µM/min) and the production of NO• and ROS by activated macrophages, resulted in a time-dependent decrease in total cell numbers, and an increase in mutation frequency (MF), compared with untreated controls. This increase in MF was effectively suppressed by N-methyl-L-arginine monoacetate. Single base substitutions were the most common type of spontaneous and NO• induced mutations in HPRT, followed by exon exclusions and small deletions in both delivery systems. Among the single base pair substitutions, an equal frequency of four types of single base substitutions were identified in TK6 cells exposed to NO• delivered by the reactor system, whereas G:C to T:A transversions and A:T to G:C transitions were more frequent in the co-culture system. Taken together, these results demonstrate that both the delivery method of NO• and ROS, and the target genes are determinants of observed cytotoxic and mutagenic responses, indicating that these parameters need to be considered in assessing the potential effects of NO• and ROS in vivo.

A new simple and reliable vitrification device based on Hollow Fiber Vitrification (HFV) method evaluated using IVP bovine embryos

A new vitrification device based on hollow fiber vitrification (HFV) was constructed using a glass capillary, which lead to simplified construction process and increased practicality of the device. The hollow fiber was attached to heat-pulled tip of the glass capillary using forceps. A protective sheath fitted on the capillary provided protection for the cellulose triacetate hollow fiber with loaded embryos and allowed safe storage in liquid nitrogen for long periods of time (2-12 month), transfer between tanks with liquid nitrogen and transportation within these tanks. No embryos were lost in the process. The device was tested using seven-dayold and eight-day-old IVP bovine blastocysts and expanded blastocysts as a model. Obtained survival (90% at 24 h post warming) and hatching rates (62% at 72 h post warming) of day 7 blastocysts and expanded blastocysts were comparable to those gained using various vitrification carriers. Vitrified embryos did not show an increase in the number of cells with damaged membrane or a decrease in total cell number per embryos in comparison to their non-vitrified counterparts. Day 7 and 8 expanded blastocysts did not differ significantly in terms of survival at 24 (97.01 vs. 97.50%) and 48 h post warming (95.52 vs. 95%), but showed significantly higher survival and hatching rates than day 7 and 8 blastocysts. These results indicated that high and repeatable survival rates can be obtained by selection of IVP bovine embryos at the developmental stage of expanded blastocyst for HFV. Further modification of the method may be required to achieve high and stable results with different developmental stages of IVP bovine embryo. The vitrification device presented in the current article may contribute to wider application of HFV method in livestock production.

Control of the red tide dinoflagellate Cochlodinium polykrikoides by ozone in seawater

The inactivation of C. polykrikoides, a red tide dinoflagellate, by ozonation was investigated in seawater by monitoring numbers of viable and total cells. Parameters affecting the inactivation efficacy of C. polykrikoides such as the ozone dose, initial cell concentration, pH, and temperature were examined. The viable cell number rapidly decreased in the initial stage of the reaction (mostly in 1–2 min), whereas the decrease in total cell number was relatively slow and steady. Increasing ozone dose and decreasing initial cell concentration increased the inactivation efficacy of C. polykrikoides, while increasing pH and temperature decreased the cell inactivation efficacy. The addition of humic acid (a promoter for the ozone decomposition) inhibited the inactivation of C. polykrikoides, whereas bicarbonate ion (an inhibitor for the ozone decomposition) accelerated the C. polykrikoides inactivation. Observations regarding the effects of pH, temperature, humic acid, and bicarbonate ion collectively indicate that the inactivation of C. polykrikoides by ozonation is mainly attributed to oxidative cell damages by molecular ozone, rather than by hydroxyl radical, produced during the ozone decomposition. At high ozone dose (e.g., 5 mg/L), hypobromous acid formed by the reaction of bromide with ozone may partially contribute to cell inactivation. The use of ozone of less than 1 mg/L produced 0.75–2.03 μg/L bromate.

Oxidative stresses-mediated apoptotic effects of ginsenoside Rb1 on pre- and post-implantation mouse embryosin vitroandin vivo

Ginsenoside Rb1, the major saponin component of ginseng root, has a wide range of therapeutic application. Previous studies have established that ginsenoside Rb1 inhibits the cell cycle and induces apoptosis. However, its side-effects, particularly those on embryonic development, have not been well characterized to date. In the current study, we examined whether ginsenoside Rb1 exerts a cytotoxic effect on mouse embryos at the blastocyst stage, and affects subsequent embryonic development in vitro and in vivo. Blastocysts treated with 25-100 μgmL-1 ginsenoside Rb1 exhibited significantly increased apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with ginsenoside Rb1 was lower than that of their control counterparts. Moreover, in vitro treatment with 25-100 μgmL-1 ginsenoside Rb1 was associated with increased resorption of post-implantation embryos and decreased fetal weight. In an in vivo model, intravenous injection with ginsenoside Rb1 (1, 3, 5 mgkg-1 body weight/day) for 4 days resulted in apoptosis of blastocyst stage embryos and early embryonic developmental injury. In addition, ginsenoside Rb1 appeared to induce injury in mouse blastocysts through oxidative stresses-triggered intrinsic apoptotic signaling processes to impair sequent embryonic development. The collective results strongly indicate that ginsenoside Rb1 induces apoptosis and retards early pre- and post-implantation development of mouse embryos, both in vitro and in vivo. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 1990-2003, 2017.

Abrogation of collagen-induced arthritis by a peptidyl arginine deiminase inhibitor is associated with modulation of T cell-mediated immune responses.

Proteins containing citrulline, a post-translational modification of arginine, are generated by peptidyl arginine deiminases (PAD). Citrullinated proteins have pro-inflammatory effects in both innate and adaptive immune responses. Here, we examine the therapeutic effects in collagen-induced arthritis of the second generation PAD inhibitor, BB-Cl-amidine. Treatment after disease onset resulted in the reversal of clinical and histological changes of arthritis, associated with a marked reduction in citrullinated proteins in lymph nodes. There was little overall change in antibodies to collagen or antibodies to citrullinated peptides, but a shift from pro-inflammatory Th1 and Th17-type responses to pro-resolution Th2-type responses was demonstrated by serum cytokines and antibody subtypes. In lymph node cells from the arthritic mice treated with BB-Cl-amidine, there was a decrease in total cell numbers but an increase in the proportion of Th2 cells. BB-Cl-amidine had a pro-apoptotic effect on all Th subsets in vitro with Th17 cells appearing to be the most sensitive. We suggest that these immunoregulatory effects of PAD inhibition in CIA are complex, but primarily mediated by transcriptional regulation. We suggest that targeting PADs is a promising strategy for the treatment of chronic inflammatory disease.

A commensal bacteria-derived lipid, significantly decreased in the serum of multiple sclerosis patients, causes attenuation of experimental autoimmune encephalomyelitis (HUM3P.269)

Abstract The role of the microbiome in multiple sclerosis (MS) remains unknown. We have identified a novel commensal bacteria-produced lipodipeptide, Lipid 654 (L654), which functions as a toll-like receptor 2 agonist. We reported that L654 can be recovered in healthy human serum and that levels are significantly lower in patients with MS. The purpose of this study was to understand the functional relevance of the decreased serum L654 level in MS by determining: 1) if L654 attenuates clinical disease in a transfer model of EAE, and 2) the mechanism by which L654 mediates this attenuation. L654 administration significantly attenuated clinical disease severity in an adoptive transfer model of EAE in SJL/J mice (p &amp;lt; 0.0001). Cellular analysis of mononuclear cells in the spinal cord at day 18 showed a significant decrease in total cell number (p = 0.02) as well as a decrease in percentage of IL-17 producing, IFNγ producing and IL-17/IFNγ double-producing CD4+ T cells in mice administered L654. At day 11, we found a decrease in total mononuclear cell number and an increase in percentage of CD39+ Tregs and IL-10 producing CD11b+ cells in mice administered L654. These results suggest that L654, normally present in human serum but at significantly lower levels in MS serum, has the ability to mediate an immunoregulatory and disease inhibitory effect on CNS autoimmunity. L654 may thus have a role both in the abnormal immunoregulation in MS and as a potential therapeutic in this disease.

Cytotoxic Effects of Dillapiole on Embryonic Development of Mouse Blastocysts in Vitro and in Vivo

We examined the cytotoxic effects of dillapiole, a phenylpropanoid with antileishmanial, anti-inflammatory, antifungal, and acaricidal activities, on the blastocyst stage of mouse embryos, subsequent embryonic attachment and outgrowth in vitro, and in vivo implantation via embryo transfer. Blastocysts treated with 2.5–10 μM dillapiole exhibited a significant increase in apoptosis and corresponding decrease in total cell number. Notably, the implantation success rates of blastocysts pretreated with dillapiole were lower than those of their control counterparts. Moreover, in vitro treatment with 2.5–10 μM dillapiole was associated with increased resorption of post-implantation embryos and decreased fetal weight. Our results collectively indicate that dillapiole induces apoptosis and retards early post-implantation development, both in vitro and in vivo. However, the extent to which this organic compound exerts teratogenic effects on early human development is not known at present. Further studies are required to establish effective protection strategies against the cytotoxic effects of dillapiole.

Histone H2A deubiquitination by 2A-DUB/Mysm1 regulates critical checkpoints in early T cell development (HEM4P.226)

Abstract Developmental processes rely on collaboration of epigenetic modifications and sequence-specific transcription factors for timely activation and silencing of lineage-specific genes. Because histone H2A deubiquitination by 2A-DUB/Mysm1 was recently shown to affect hematopoiesis, in this investigation, we used 2A-DUB-deficient mice to systematically characterize the function of this epigenetic regulator in early T cell development. Thymi of 2A-DUB-/- mice were severely hypoplastic with &amp;gt;10-fold decrease in total cell numbers and increased fraction of apoptotic thymocytes compared with wild-type littermates. Diminished cell numbers resulted from a reduction of early thymocyte progenitors (ETPs) in context with defective HSCs, a partial block at the double-negative (DN)1-DN2 transition and increased apoptosis of double-positive (DP) thymocytes. In qPCR and IF analyses, we identified p19ARF and other genes involved in early T-cell development and protection from apoptosis as potential 2A-DUB target gene. ChIP analyses were conducted to explore binding of 2A-DUB to the Ink4a/Arf promoter in thymocytes. In addition, genetic approaches are used to validate the interaction of 2A-DUB/Mysm1 with known regulators of apoptosis and T cell development. We conclude that 2A-DUB/Mysm1 is critical for early T cell development via regulation of lineage-specific genes and that insufficient histone H2A deubiquitination induces apoptotic programs in HSCs, thymocytes and potentially other cell types.

Epigallocatechin gallate induces embryonic toxicity in mouse blastocysts through apoptosis

Catechins, a family of polyphenols found in tea, evoke various responses, including cell death. However, the side effects of these compounds, particularly those on embryonic development, have not been characterized in detail. A previous study by our group showed that (−)-epigallocatechin-3-gallate (EGCG), a catechin highly abundant in green tea, induces different cell-death modes in MCF-7 cells, depending on the treatment dosage. In the current study, we examined the effects of EGCG on mouse embryos at the blastocyst stage, subsequent embryonic attachment and outgrowth in vitro and in vivo implantation by embryo transfer. Blastocysts treated with 25–50 μM of EGCG exhibited a significant increase in apoptosis and a corresponding decrease in total cell number. Notably, the implantation success rate of blastocysts pretreated with EGCG was lower than that of their control counterparts. Moreover, in vitro treatment with 25–50 μM of EGCG led to increased resorption of postimplantation embryos and decreased fetal weight. EGCG appeared to induce injury in mouse blastocysts through intrinsic apoptotic signaling processes to impair sequent embryonic development. These results collectively highlight the potential of EGCG to induce embryonic cytotoxicity.

Oxidation of Microcystis aeruginosa and Anabaena flos-aquae by ozone: Impacts on cell integrity and chlorination by-product formation

Pre-ozonation of cyanobacterial (CB) cells in raw water and inter-ozonation of settled water can cause CB cell damage. However, there is limited information about the level of lysis or changes in cell properties after ozonation, release of intracellular compounds and their contribution to the formation of disinfection by-products (DBPs). This study aims to: (1) assess the extent of the pre-ozonation effects on CB cell properties; (2) determine the CT (ozone concentration × detention time) values required for complete loss of cell viability; and (3) study the DBPs formation associated with the pre-ozonation of cyanobacterial cells in laboratorial suspensions. To these ends, both Microcystis aeruginosa and Anabaena flos-aquae suspensions were prepared at concentrations of 250,000 cells mL−1 and 1,500,000 cells mL−1 and were subjected to ozone dosages of 0.5, 2.0 and 4.0 mg L−1 at pH 6 and pH 8. A quick and complete loss of viability was achieved for both CB species after exposure (CT) to ozone of <0.2 mg min L−1, although no significant decrease in total cell numbers was observed. Maximum dissolved organic carbon (DOC) releases of 0.96 mg L−1 and 1.63 mg L−1 were measured after ozonation of 250,000 cells mL−1 of M. aeruginosa and A. flos-aquae, respectively. DOC release was found to be pH and ozone dose dependent. Ozonation of CB cells increased formation of trihalomethanes (THM) and haloacetic acids (HAA), mainly for suspensions of A. flos-aquae at pH 8 (by 174% and 65% for THM and HAA respectively). Utilities considering using ozone for oxidising CB cells should weigh out the benefit of CB control with the potential increased formation of chlorinated DBPs.

CD4+ T Cell–Depleted Lymphocyte Infusion Impairs Neither the Recovery of Recipient Thymus nor the Development of Transplanted Thymus

Thymus transplantation, in conjunction with bone marrow transplantation (BMT), has been attracting attention for the treatment of various diseases. Recently, donor lymphocyte infusion (DLI) has been used as a helpful tool for establishing donor chimerism and preventing a relapse of leukemia/lymphoma. However, the effects of DLI on transplanted and recipient thymuses have not been explored. We therefore performed DLI in the intrabone marrow-BMT + thymus transplantation setting. We have found that DLI leads to derangements in both recipient thymuses and transplanted thymuses; by 2 wk after BMT, we saw a decrease in total cell number, a lower percentage of CD4(+)CD8(+) cells, and the obliteration of the thymic corticomedullary junction. Four weeks later, the thymic impairment became more serious. However, when we depleted the CD4(+) T cells (CD4(-)-DLI), the recipient thymic recovery and transplanted thymic development were significantly restored by the treatment. In addition, there were much greater levels of TNF-α and Fas ligand, and a lower percentage of regulatory T cells in the DLI group than in the CD4(-)-DLI group. These findings indicate that inflammation induced by DLI, especially by CD4(+) T cells, plays a crucial role in the thymic impairment.