78 INTERFERON-τ SECRETION OF CRYOPRESERVED BOVINE TROPHOBLASTIC VESICLES DERIVED FROM IN VIVO

2012 ◽  
Vol 24 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Continuous Culture ◽  
Culture Media ◽  
Calf Serum ◽  
Measure Concentration ◽  
Fetal Bovine ◽  
The Mean ◽  
Student’S T ◽  
Student’S T Test

The co-transfer of bovine trophoblastic vesicles (bTVs) prepared from in vivo recovered conceptuses is known to promote the successful implantation of embryos, which expected lower viability, through the effects of interferon-τ (IFN-τ) secreted by bTVs. We have reported that the pregnancy rate was improved for co-transferred embryos with frozen-thawed bTVs using the direct-transfer technique (Hashiyada et al. 2008, 41st SSR). However, the IFN-τ secretion level from cryopreserved bTVs is not well known. The objective of the present study was to measure concentration of IFN-τ released from frozen-thawed bTVs individually cultured in vitro. bTVs were prepared from elongating blastocysts 3 to 20 mm in length, following superstimulatory treatment and recovered on Day 16 post-AI, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of a 96-well plate using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 or 48 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum, 1.5 M ethylene glycol (EG) and 0.1 M sucrose or 1.8 M EG. After thawing, each bTVs was cultured for 2 days to compare IFN-τ secretion between the 2 cryoprotectants. Furthermore, transition of IFN-τ level was assessed in continuous culture until Day 10 (the day of thawing was defined as Day 0). The volume of culture medium was 100 μL well–1 day–1 until Day 2 and thereafter changed to 200 μL well–1 day–1 until termination. Exchange and collection of culture media were performed on Day 1, 2, 4, 6, 8 and 10. Collected culture media were stored at –30°C until use. IFN-τ was measured by RIA (Takahashi et al. 2005 Theriogenology 63, 1050–1060). Data were analysed by Student's t-test. Initial IFN-τ secretion from bTVs before cryopreservation did not differ between 24 and 48 h of culture period to form vesicles, 44.0 ± 2.9 (mean ± standard error of the mean, n = 64) and 52.8 ± 6.4 ng mL–1 (n = 27), respectively. IFN-τ secretion was no difference between the 1.5 M EG group and the 1.8 M EG group on Day 1 (41.2 ± 4.9 ng mL–1, n = 42 and 30.4 ± 2.2 ng mL–1, n = 31) and on Day 2 (38.0 ± 5.4 and 38.2 ± 4.5 ng mL–1), respectively. In the continuous culture group (n = 28), IFN-τ secretion tended to increase from Day 2 (25.2 ± 3.4 ng mL–1) to Day 4 (51.8 ± 12.3 ng mL–1) and 6 (55.4 ± 13.3 ng mL–1) (P < 0.05). However, this amount of IFN-τ on Day 6 significantly decreased on Day 8 (25.6 ± 2.7 ng mL–1; P < 0.05) and Day 10 (15.5 ± 2.2 ng mL–1; P < 0.01), gradually. These results indicate that cryopreserved bTVs could secrete IFN-τ at the same level as fresh bTVs on Day 4 to 6 after thawing and then these amounts of IFN-τ significantly decrease in vitro.

74 EFFECT OF SIZE AND CULTURE PERIOD OF FROZEN–THAWED BOVINE TROPHOBLASTIC VESICLES ON INTERFERON-τ SECRETION

2014 ◽  
Vol 26 (1) ◽  
pp. 151
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
D. Yamaguchi ◽  
K. Imai ◽  
M. Geshi
Keyword(s):  
Significant Positive Correlation ◽  
Culture Media ◽  
Fetal Bovine Serum ◽  
Calf Serum ◽  
Culture Period ◽  
Fetal Bovine ◽  
Student’S T ◽  
Student’S T Test

Frozen–thawed bovine trophoblastic vesicles (bTV) derived in vivo could secrete interferon-τ (IFN-τ) at the same level as fresh bTV on Days 4 to 6 after thawing. However, amounts of IFN-τ decreased following continuous in vitro culture (Hashiyada et al. 2012 38th IETS). Co-transfer of frozen–thawed bTV improved pregnancy rate of embryos due to the effects of IFN-τ secreted by bTV (Hashiyada et al. 2008 41th SSR). However, the relation between bTV size and IFN-τ secretion level during culture has not been well documented. The objective of present study was to characterise the concentration of IFN-τ related bTV volume and culture period after thawing of cryopreserved bTV. The bTV were prepared from Day 16 elongating blastocysts recovered nonsurgically. The dissected trophoblastic fragment, 1 to 1.5 mm in width, was cultured using TCM-199 supplemented with 20% (vol/vol) fetal bovine serum and 0.1 mM β-mercaptoethanol. Formed vesicles after 24 h of culture were cryopreserved using D-PBS supplemented with 20% calf serum and 1.8 M ethylene glycol. After thawing, bTV were cultured individually with 100 μL/well/day until Day 2 (i.e. the day of thawing was defined as Day 0), and thereafter changed to 200 μL/well/day to termination at Day 10. Collection of culture media and measurement of bTV diameter were performed before cryopreservation and after thawing for every day. Interferon-τ in collected media was measured by radioimmunoassay. The estimated bTV volume was calculated based on the diameter. Data were analysed by Student's t-test. Nine fresh bTV before cryopreservation were used to assess the IFN-τ secretion for 24 h in relation to bTV volume. A significant positive correlation was observed between secreted IFN-τ (mean ± s.e.M, 19.9 ± 3.1 ng mL–1) and bTV volume (1.49 ± 0.6 mm3, r = 0.91; P < 0.01). Initial IFN-τ secretion from bTV cultured for 24 h after thawing was significantly decreased compared with that before cryopreservation (29.1 ± 2.1 ng mL–1 and 58.4 ± 4.8 ng mL–1; P < 0.01, n = 27). In continuous culture of bTV (n = 8), IFN-τ secretion increased gradually from Day 2 (23.1 ± 9.0 ng mL–1) to Day 4 (32.2 ± 8.4 ng mL–1), and then maintained this level until Day 7 (33.4 ± 14.9 ng mL–1). However, this amount of IFN-τ tended to decrease on Day 8 (24.8 ± 5.0 ng mL–1), 9 (16.5 ± 4.4 ng mL–1), and 10 (12.0 ± 1.7 ng mL–1). Interferon-τ secretion from bTV on Day 9 and 10 was lower than that on Day 3, 4, 5, 6, and 8, respectively (P < 0.05). Volume of bTV increased also from Day 2 (0.2 ± 0.1 mm3) to Day 5, 6 (0.8 ± 0.3 mm3) and 7 (0.7 ± 0.2 mm3). However, bTV volumes shrank drastically on Day 8 (0.3 ± 0.1 mm3), 9, and 10 (0.2 ± 0.1 mm3). In comparison with bTV during culture, volumes on Day 4, 5, and 7 were greater than those on Day 2 and 3, and volumes on Day 6 and 7 were greater than on Day 8, 9, and 10 (P < 0.05). These results indicate that the dynamics of IFN-τ secretion reflected the expansion or reduction of bTV in continued culture after thawing. Interferon-τ secretion might be related to bTV volume. Moreover, we reconfirmed that cryopreserved bTV highly express IFN-τ during 4 to 7 days after thawing.

162 IMPROVED EMBRYO SURVIVAL AND QUALITY WITH EMCARE II

2006 ◽  
Vol 18 (2) ◽  
pp. 189
Author(s):  
A. Harvey ◽  
M. Lane ◽  
J. Thompson
Keyword(s):  
Calf Serum ◽  
Cell Number ◽  
Tunel Staining ◽  
Embryo Survival ◽  
Improved Outcomes ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Collection of embryos exposes them to a number of stresses, including light, air, and changes in temperature. Improvement of holding media to reduce the impact of handling stresses on the embryo during in vivo collection and transfer is therefore beneficial to ensure maintenance of viability following transfer. The aim of this study was to compare the effect of holding IVP-derived blastocysts at 25°C in Emcare I (ECMI, Emcare, Dallas, TX, USA) with those held in Emcare II (ECMII), a proprietry formulation designed to reduce in vitro-induced stress. In vitro-produced bovine embryos were generated using standard protocols. Blastocysts were randomly allocated to either ECMI or ECMII (ICPBio, Aukland, New Zealand) on Day 7 and were held at 25°C for a period of 24 h, after which they were cultured in Cook Bovine Blast (Cook Australia, Brisbane, Australia) supplemented with 10% fetal calf serum for 48 h. At 24 and 48 h, embryos were scored for hatching, and a cohort removed for TUNEL staining at each time point. Differences were analyzed by Student's t-test. At both 24- and 48-h culture, hatching rates tended to be higher for embryos held in ECMII than in ECMI (Table 1). The level of apoptosis at 48 h was reduced in blastocysts held in ECMII (P = 0.06). Moreover, the total cell number of hatched blastocysts at 48 h was significantly increased (1.5-fold) in those held in ECMII (P = 0.01). Results suggest that the formulation of ECMII improves the ability of IVP bovine blastocysts to re-expand and hatch following an imposed stress (25°C for 24 h). Furthermore, ECMII improves overall embryo quality through a reduction in the percentage of cells undergoing apoptosis as well as through increased cell numbers, evident 48 h following cessation of the stress. We suggest that Emcare II reduces the impact of (or increases the embryo's tolerance to and recovery from) an imposed stress, which, although severe in the present study, may provide improved outcomes following embryo transfer in field situations. Table 1. Hatching and apoptosis of blastocysts held at 25°C for 24 h in Emcare I or Emcare II This work was supported with funding by ICPBio (NZ).

179 EFFECT OF SERUM DURING CULTURE ON DAY 14 ELONGATED BOVINE EMBRYOS

2011 ◽  
Vol 23 (1) ◽  
pp. 191 ◽  
Author(s):  
J. Angulo ◽  
G. T. Gentry ◽  
R. A. Godke ◽  
K. R. Bondioli
Keyword(s):  
Gene Expression ◽  
Messenger Rna ◽  
Culture Media ◽  
Calf Serum ◽  
Blastocyst Development ◽  
Cleavage Rate ◽  
Expression Levels ◽  
The Mean

It has been reported that the addition of serum to embryo culture media alters gene expression and triggers the development of large offspring syndrome. The objectives of this study were to determine gene expression levels in embryos cultured in the absence or presence of 5% calf serum and in vivo-derived (IVD) embryos and to determine the effects of serum on the length of elongated embryos. Abattoir-derived oocytes were obtained from a commercial provider and fertilized at 24 h of maturation with semen from a bull previously used for IVF. At 18 h post-insemination (hpi), embryos were denuded and groups of 15 presumptive zygotes were cultured in 30-μL drops of modified SOF medium with amino acids and 6 mg mL–1 of BSA (mSOFaa). At 72 hpi, cleavage rate was assessed and embryos were randomly allocated into 2 treatments: mSOFaa without and with 5% calf serum. Embryos were then cultured to 168 hpi and blastocyst rates were assessed and recorded. Blastocysts (n = 5 to 10) from each treatment were transferred into synchronized recipients, and Day 14 embryos were recovered 7 days post-transfer. Embryos were photographed, measured, and immediately stored at –80°C in a minimal volume of PBS + 0.1% polyvinyl alcohol. Messenger RNA was isolated using a Dynabeads mRNA Direct Kit™ (Invitrogen, Carlsbad, CA), and reverse transcription was performed using an iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., CA). Quantitative PCR was performed to determine the transcript abundance for COX6A, IFNT1a, PLAC8, IGF2R, and GAPDH for each sample. The GAPDH was used as a reference gene, and gene expression was calculated as a ratio of expression levels between each gene of interest and GAPDH. Expression levels for each gene were determined from standard curves generated by serial dilutions of PCR amplicons starting with 0.4 pg/reaction. Blastocyst development rates were higher in embryos cultured with serum compared with the nonserum treatment (14.9 and 7.4% respectively; chi-square, P < 0.001). Lengths of elongated embryos from the serum (3395.3 ± 414.7 μm) and nonserum (2784 ± 741.8 μm) culture treatments differed from the IVD (6297.7 ± 677.2 μm) treatment (mean ± SE; ANOVA, P < 0.0052). There were no differences in the mean expression levels for COX6A, IFNT1a, PLAC8, and IGF2R across treatment groups, but in the serum treatment, 3 out 11 overexpressed IFNT1a, 4 out of 11 overexpressed IGF2R, and 2 out of 11 overexpressed PLAC8, defined as being 2 standard deviations above the mean of the IVD treatment for each respective gene. In the in vitro-produced nonserum and IVD treatments, overexpression by this definition was not observed. Although mean expression levels were not affected by culture with serum under these conditions, very high expression of IFNT1a, IGF2R, and PLAC8 was observed in some embryos cultured with serum, but not in embryos cultured without serum or IVD embryos.

140 INTERFERON-τ SECRETION BY IN VIVO DERIVED BOVINE TROPHOBLASTIC VESICLES

2010 ◽  
Vol 22 (1) ◽  
pp. 229
Author(s):  
Y. Hashiyada ◽  
H. Takahashi ◽  
M. Geshi
Keyword(s):  
Culture Medium ◽  
Culture Media ◽  
Spherical Shape ◽  
Morphological Observation ◽  
Measure Concentration ◽  
Fetal Bovine ◽  
Implantation Period

In ruminants, interferon-τ (IFN-τ) is a major pregnancy factor, secreted by the embryonic trophoblast cells during the pre-implantation period, being important for the maternal-fetal recognition. The co-transfer of bovine trophoblastic vesicles (bTVs) derived from in vivo recovered conceptuses is known to promote the successful implantation of embryos with expected lower viability, such as in vitro handled embryos, through the effects of IFN-τ secreted by bTVs. We have also reported that the pregnancy rate was improved using this technique in early pregnancy phase (Hashiyada et al. 2005 J. Reprod. Dev. 51, 749-756). However, the IFN-τ secretion level from bTVs has not been well known. Therefore, the objective of the present study was to measure concentration of IFN-τ released from individually cultured bTVs in vitro. Furthermore, we also investigated the transition of IFN-τ level in continuous culture of bTVs. Blastocysts were produced by artificial insemination of Japanese black cows following superstimulatory treatment and were recovered on 16 or 18 days post-estrus. Sixty-eight bTVs were prepared from 23 elongating blastocysts, 3 to 20 mm in length, by dissection using a surgical blade. Each trophoblastic fragment, 1 to 1.5 mm in width, was cultured in a well of 96-well plates using TCM-199 supplemented with 20% (v/v) fetal bovine serum and 0.1 mM β-mercaptoethanol at 38.5°C in a humidified atmosphere of 5% CO2 in air. After 24 h of culture, fragments of unformed vesicles were re-cultured for an additional 24 h; 10 bTVs from this group were continuously cultured until Day 24 (the day of insemination was defined as Day 0). The volume of culture medium was 100 μL/well/day until Day 2 and thereafter changed to 200 μL/well/2 days to terminate. The viability of bTVs was assessed based on maintained spherical shape of vesicle, morphologically. Exchange and collection of culture media, morphological observation of bTVs were performed on Days 1, 2, 4, 8, 10, 12, 14, 16, 18, 20, 22, and 24. Culture fluids were stored at -30°C. IFN-τ was measured by RIA (Takahashi H et al. 2005 Theriogenology 63, 1050-1060). Data were analyzed by Student’s τ-test Initial IFN-τ secretion did not differ between groups that had formed and unformed vesicles on Day 1, 89.8 ± 7.1 (mean ± SEM, n = 41) and 76.6 ± 7.2 ng mL-1 (n = 27), respectively. On Day 2, in the unformed group, all of the fragments had made vesicles and the IFN-τ increased to 99.4 ± 11.8 ng mL-1. In the extended culture group (n = 10), IFN-τ secretion tended to increase from Day 2 (66.9 ± 14.2 ng mL-1) to Day 8 (166.0 ± 46.7 ng mL-1) (P = 0.06). However, this large amount of IFN-τ on Day 8 significantly decreased from Day 10 (32 ± 4.9 ng mL-1, P < 0.05) to Day 24 (9.2 ± 1.0 ng mL-1, P < 0.05) gradually. The survival rate of these bTVs decreased to 90% (9/10) on Day 10 and then to 60% (6/10) during Days 18 to 22. These results indicate that bTVs cultured for a long term in vitro might decrease IFN-τ secretion.

scholarly journals Inhibition of Demineralization at Restoration Margins of Z100 and Tetric EvoCeram Bulk Fill in Dentin and Enamel

2019 ◽  
Vol 6 (2) ◽  
pp. 36
Author(s):  
Cristina Leon-Pineda ◽  
Kevin Donly
Keyword(s):  
Light Microscopy ◽  
Polarized Light ◽  
In Vitro Study ◽  
The Other ◽  
Solution Ph ◽  
Class V ◽  
The Mean ◽  
Student’S T ◽  
Student’S T Test

Recurrent caries is still considered the main reason restorations need to be replaced. There are different materials available now that promise to reduce the possibility of recurrent caries by releasing fluoride and inhibiting restoration marginal caries. The purpose of this in vitro study was to evaluate the demineralization inhibition potential of a non-fluoride-releasing resin (Z100TM 3M, St. Paul, MN, USA) and a glass containing resin-based composite (Tetric EvoCeram Bulk Fill, Ivoclar/Vivadent AG, Schaan, Liechtenstein), which contains fluoride. Class V preparations were placed on 22 premolars; the gingival margin was below the cementoenamel junction and the occlusal margin was placed above the cemento-enamel junction. Ten teeth were randomly selected to be restored with Z100 while the other 10 were restored with Tetric EvoCeram Bulk Fill. Both groups were restored following manufacturer’s instructions. All teeth had an acid resistant varnish placed within one millimeter of the preparation margins. Both groups were placed in artificial caries challenge solution (pH 4.4). At the end of the 4 days; 100 µm buccolingual sections were obtained for each tooth; these were photographed under polarized light microscopy and the demineralized areas adjacent to the restorations were measured and quantified. The mean (±S.D.) area (µm2) of demineralization from the occlusal margin (enamel) and dentin margin were: Z100 2781.889 ± 1045.213; 3960.455 ± 705.964 and for Tetric EvoCeram Bulk Fill 1541.545 ± 1167.027; 3027.600 ± 512.078. Student’s t-test indicated that there was significantly less enamel and dentin demineralization adjacent to Tetric EvoCeram Bulk Fill compared to Z100; there was significantly less demineralization in enamel compared to dentin in both Tetric EvoCeral Bulk Fill and Z100. Tetric EvoCeram Bulk Fill performed better inhibiting demineralization at restoration margins when compared to Z100 and provided better demineralization inhibition in enamel than cementum/dentin.

Oxygen concentration and protein source affect the development of preimplantation goat embryos in vitro

1991 ◽  
Vol 3 (5) ◽  
pp. 601 ◽  
Author(s):  
PA Batt ◽  
DK Gardner ◽  
AW Cameron
Keyword(s):  
Bovine Serum Albumin ◽  
Serum Albumin ◽  
Oxygen Concentration ◽  
Bovine Serum ◽  
Calf Serum ◽  
Cell Number ◽  
The Mean ◽  
Number Of Cells

The effect of oxygen concentration and the source of protein in culture medium on the development of 2- to 4-cell goat embryos in vitro was investigated. Embryos were collected from superovulated Angora-Cashmere-cross goats 48 h after ovulation and cultured for 6 days in synthetic oviduct fluid (SOF) medium under one of two oxygen concentrations (20% or 7%) and in the presence of one of five protein sources; Miles bovine serum albumin (Miles BSA), Commonwealth Serum Laboratory bovine serum albumin (CSL BSA), goat serum (GS), fetal calf serum (FCS) and human serum (HS). In the presence of 20% oxygen the percentage of embryos reaching the expanded and/or hatched blastocyst stage in SOF medium containing Miles BSA was 29%, with a mean cell number per embryo of 28.1 +/- 6.0 (+/- s.e.m.). Use of an oxygen concentration of 7% significantly increased the percentage of embryos reaching this stage (80%, P less than 0.01) and the mean number of cells per embryo (65.3 +/- 8.2, P less than 0.01). The mean number of cells of the early-cleavage-stage embryos was significantly lower when the medium contained CSL BSA, GS or FCS (42.7 +/- 5.6, 29.0 +/- 6.1 and 21.3 +/- 3.2, respectively) than with Miles BSA (92.8 +/- 6.4) or HS (104.8 +/- 17.2) (P less than 0.01). Under 7% oxygen and with Miles BSA or HS, embryos were morphologically comparable to those developed in vivo, but the mean cell numbers in vitro were only approximately half those obtained in vivo.

Over Expression of MN1 Confers Resistance to Chemotherapy In Vitro and In Vivo

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 969-969
Author(s):  
Timothy Pardee ◽  
Teresa Mascenik ◽  
Britt H. Bolemon ◽  
Guerry J Cook
Keyword(s):  
Bone Marrow ◽  
The United States ◽  
Genetic Alterations ◽  
T Test ◽  
Over Expression ◽  
Student’S T ◽  
Worse Prognosis ◽  
Student’S T Test

Abstract Abstract 969 Acute myeloid leukemia (AML) is an accumulation of immature myeloid precursors that leads to progressive marrow failure and death. This disease affects approximately 12,000 people per year in the United States, causing 9,000 deaths. Despite decades of active research the overall 5 year survival remains a dismal 30–40%. The backbone of initial therapy for the last 30 years is combination chemotherapy containing cytarabine (Ara-C) and an anthracycline. Resistance to these therapies is a major problem and most patients diagnosed with AML will ultimately die from resistant disease. AML is characterized by heterogeneous genetic alterations that can be used to delineate prognosis. Using standard karyotyping techniques patients can be divided into good, intermediate and poor prognostic categories. There is a clear link between these chromosomal aberrations and response to chemotherapy as complete remission rates are significantly different between groups. Patients with no detectable cytogenetic abnormality fall into an intermediate prognostic group with a very heterogeneous outcome. Recent work has begun to uncover submicroscopic genetic alterations that effect prognosis for these patients. These alterations can be mutations, over or under expression of a particular gene. The MN1 gene encodes a transcription co-factor first identified by its involvement in a balanced translocation in a patient with a meningioma. Since its initial description it has been found over-expressed in multiple AML patient samples. There are several reports that over-expression of MN1 confers a worse prognosis in AML. High MN1 expressers were less likely to achieve a remission and had a lower 3 year survival rate. Additionally, over expression of MN1 in murine bone marrow leads to AML in transplanted recipients and predicts for resistance to ATRA in elderly AML patients. However, the effect of MN1 over expression on response to standard chemotherapy is currently unknown. To answer this question we used a murine model of AML driven by MLL-ENL. AML blasts were infected with retroviral vectors that contained MN1 and a GFP reporter. Partially infected blast populations were then exposed to various concentrations of either Ara-C or doxorubicin and the ratio of GFP positive and negative cells was compared to untreated controls. When blasts were exposed to 150 nM Ara-C the GFP+ percentage went from 21.10 (+/− 0.5302) in the control samples to 35.68 (+/−1.230) in the treated samples. This result was even more profound when cells were treated with 15 ng/ml doxorubicin where the percentage went from 21.10 (+/− 0.5302) to 80.27 (+/−1.615). Both results were highly statistically significant by two tailed student's t test with p values of 0.004 and <0.0001 respectively. Consistent results were obtained in multiple different infections and with separately derived MLL-ENL lines. These data demonstrate that blasts expressing MN1 had an advantage when exposed to either Ara-C or doxorubicin although the effect was far more pronounced with doxorubicin exposure. MN1 expressing blasts were also resistant to the combination of Ara-C and doxorubicin. In order to determine if MN1 conferred resistance to Ara-C and doxorubicin in vivo we injected sublethally irradiated, Ly5.1+ C57Bl6 recipients with a partially infected population of blasts. Ly5.1+ animals do not express the Ly5.2 allele; thus, staining cells for Ly5.2 allows differentiation of leukemic cells from endogenous marrow. Eight days after injection of blasts animals were treated with 100 mg/kg Ara-C plus 3 mg/kg doxorubicin daily for 5 days or observed. On day 6 animals were sacrificed and bone marrow from bilateral femurs was harvested, stained for Ly5.2 and analyzed by flow cytometery. Animals treated with Ara-C plus doxorubicin had 90.58% (+/−0.6638) Ly5.2+, GFP+ blasts compared to 55.38% (+/−5.245) in control animals. This result was highly statistically significant with a p value of <0.0001 by two tailed student's t test. This observation was reproducible in a separately derived MLL-ENL driven cell line. These data suggest that over expression of MN1 in this murine AML model confers resistance to both Ara-C and doxorubicin in vitro and in vivo and provides a biological explanation for the clinical observation that it confers a worse prognosis. The mechanisms involved in this resistance are currently under study. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Evaluation of the Safety and Usefulness of Citrus Jabara Fruit Peel Powder Cream for Patients with Atopic Dermatitis

2020 ◽  
Vol 4 (1) ◽  
Author(s):  
Yutaka Inaba ◽  
Fukumi Furukawa ◽  
Seisho Azuma ◽  
Kimiye Baba ◽  
Masahiko Taniguchi ◽  
...  
Keyword(s):  
Atopic Dermatitis ◽  
Oral Intake ◽  
Skin Lesions ◽  
Fruit Peel ◽  
Kii Peninsula ◽  
The Mean ◽  
Student’S T ◽  
The Relationship ◽  
Student’S T Test

Citrus jabara (CJ) is a rare citrus that originally grew wild only in the southern area of the Kii peninsula in Japan. In the relationship between CJ and atopic dermatitis (AD), improvement of AD by oral intake of CJ fruit juice was reported in AD model mice. Our previous study also showed anti-inflammatory potentials of CJ fruit peels in vitro. In this study, the applicability of CJ fruit peel powder (CJ powder) for topical application in patients with AD was investigated. After confirming both the safety of CJ powder in preclinical studies and the safety of 5% CJ powder cream in healthy volunteers, the safety and usefulness of 5% CJ powder cream were evaluated in 20 patients with AD. Evaluation of 5% CJ powder cream in patients with AD for 4 weeks showed improvement in the mean severity score of the affected area (from 3.0 to 2.0, p=0.001 by Student’s t test), improvement in skin lesions (11 of 20 participants), usefulness (16 of 20 participants), and safety (16 of 20 participants). Although aggravation of symptoms on application areas were observed on 4 participants, their aggravation were systemic, resulting from causes other than tested cream. These results suggested that 5% CJ powder cream is useful and safe for patients with AD.

Some aspects of metal oxide nanoparticles toxicity assessment on cell cultures as exemplified by NiO and Mn3O4

2017 ◽  
pp. 35-43 ◽  
Author(s):  
I. А. Minigalieva ◽  
T. V. Bushueva ◽  
V. G. Panov ◽  
A. N. Varaksin ◽  
V. Ya. Shur ◽  
...  
Keyword(s):  
Culture Media ◽  
Cell Damage ◽  
Metal Oxide Nanoparticles ◽  
Animal Experiments ◽  
Toxicity Assessment ◽  
In Vitro Tests ◽  
Fetal Bovine ◽  
Nio Nps

Comparative and combined damaging actions of NiO and Mn3O4 anoparticles were estimated on cultures of different established human cell lines. It was found out that the addition of the fetal bovine serum (FBS) to the culture media ,used in the investigation, renders NiO-NPs and, to even a greater extent, Mn3O4-NPs exponentially soluble while without FBS their dissolution was extremely low. Along with it, sedimentation of those MeO-NPs caused by their aggregation noticeably slowed down in the presence of the same FBS. The dependence of cell damage on the MeO-NPs concentration was found out, at a higher cytotoxicity of Mn3O4-NP as compared to NiO-NP. Thus, comparative assessment of NPs non-specific toxicity previously obtained in animal experiments was reproduced in the «in vitro» tests. However, with respect to manganese-specific brain damage «in vivo» discovered previously in sub-chronic intoxication with the same MeO-NPs, the present «in vitro» experiment on neurons only showed a certain enhancing effect of Mn3O4-NP on the action of NiO-NP, but the role of NiO-NP in the combination prevailed.

In Vitro and in Vivo Targeting of Chronic Lymphocytic Leukemia Using CX-4945, a Clinical-Stage CK2-Specific Inhibitor.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2897-2897 ◽  
Author(s):  
Leila R. Martins ◽  
Paulo Lúcio ◽  
Alice Melao ◽  
Bruno A. Cardoso ◽  
Ryan Stansfield ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Chronic Lymphocytic Leukemia ◽  
Cell Survival ◽  
Leukemia Cell ◽  
Lymphocytic Leukemia ◽  
Student’S T ◽  
The Impact ◽  
Student’S T Test

Abstract Abstract 2897 Specific inhibition of signaling elements essential for chronic lymphocytic leukemia (CLL) cell survival offers great promise for the design of improved therapies against this still incurable malignancy. The serine/threonine protein kinase CK2 is frequently upregulated in cancer, and mounting evidence implicates CK2 in tumorigenesis. Here, we evaluated whether CK2 is a valid target for therapeutic intervention in CLL, by testing the efficacy of CX-4945, a potent and highly specific orally available ATP-competitive inhibitor of CK2 that is undergoing phase I clinical trials for solid tumors and multiple myeloma. We previously showed that CK2 phosphorylates and thereby inactivates PTEN in primary T-ALL and CLL cells, leading to the hyperactivation of PI3K signaling pathway, and consequently promoting leukemia cell survival (Silva et al, JCI 2008; Martins et al, Blood 2010). Therefore, we first analyzed the impact of CX-4945 on PTEN phosphorylation and PI3K pathway activation. Incubation of CLL cells with 20 μM CX-4945 for 2h resulted in striking downregulation of PTEN phosphorylation, indicative of increased PTEN activity, and a concomitant decrease in the activity of PI3K downstream targets Akt and PKC, as determined by Akt (S473), PKCβ (S660) and PKCδ (T550) phosphorylation in both MO1043 and primary CLL cells collected and isolated to >90% purity from the peripheral blood of untreated patients. Importantly, we confirmed that Akt phosphorylation on the CK2 direct target site (S129) was also inhibited by CX-4945. Next, we evaluated the functional impact of the CK2 inhibitor on CLL cell viability. Primary CLL cells (n=11) were cultured with 10 and 20 μM CX-4945. Both drug concentrations exerted clear pro-apoptotic effects in all cases (P<0.0001 for each dose, 2-tailed paired Student's t test), as determined by Annexin V-APC/7-AAD staining. Moreover, the effect of CX-4945 was time- and dose-dependent in 4 out of 4 cases that were more thoroughly analyzed. Similar results were obtained using MEC1, MEC2, WaC3CD5, JVM3 and MO1043 cell lines whose IC50 ranged between 3.1 and 5.8μM. Notably, although co-culture with OP9 stromal cells promoted primary leukemia cell survival, it did not prevent CX-4945-mediated apoptosis of CLL cells. Most importantly, CX-4945 induced a stronger decrease in the viability of CLL cells from patients with higher percentage of malignant cells in the blood (R2=0.4176, P=0.0317, n=11, Pearson correlation), Binet stage B/C (P=0.0424, n=10, 2-tailed unpaired Student's t test) or higher plasma β2 microglobulin levels (P=0.0239, n=9). Furthermore, CLL cells with a higher proliferation rate (LDT < 12 months) were also more sensitive to CX-4945 (P=0.0007, n=11). In accordance, the need for treatment positively correlated with the sensitivity to CX-4945 (R2=0.4504, P = 0.0238). These observations suggest that treatment with CK2 inhibitors may be especially beneficial to patients with more advanced or aggressive disease. The promising results obtained in vitro prompted us to assess the impact of CX-4945 on CLL tumor development in vivo. We implanted MO1043 CLL cells subcutaneously into Swiss nude mice. At day 3, all animals presented palpable tumor masses of approximately 150 mm3, and were randomly assigned into 4 groups (n=6 per group) to receive either CX-4945 alone (75mg/kg, bid, p.o.), fludarabine alone (34mg/kg, i.p., 5 days + 2 days rest, every week), the combination of both drugs, or vehicle control. A significant delay in tumor growth was observed in all of the treatment groups when compared to the control group (P<0.0001, 2-way ANOVA). Notably, CX-4945 was as effective as fludarabine when used as a single agent, and the combination of the two drugs was significantly more effective than fludarabine alone (P=0.0375). All treatments were well tolerated as evidenced by the maintenance of body weight and the inexistence of signs of overt toxicity. Overall, our data indicate that pharmacological inhibition of CK2 is a promising therapeutic strategy in CLL that may be of special benefit to patients with aggressive and advanced stage disease. Moreover, our studies pave the way to the development of clinical trials using CX-4945 or other CK2 antagonists to manage CLL. Disclosures: Stansfield: Cylene Pharmaceuticals Inc.: Employment. Drygin:Cylene Pharmaceuticals Inc.: Employment.

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