“Antitumor Activity of Novel Anti-MM Agents and Combinations, the Proteasome Inhibitor Bortezomib and Multikinase Inhibitor Sorafenib, Both Applied as Monotherapy and in Combination in NOD/SCID-IL2-Receptor-Gamma-chain−/− (NSG) Mice Using a Intratibial Tumor Dissemination approach”.

Abstract Abstract 4912 Since novel treatment options are needed in multiple myeloma (MM), novel anti-MM agents and combinations are eagerly pursued to further improve the prognosis for MM patients. For potentially novel therapeutic agents, functional in vivo models are highly valuable. We have established a cell line-based, disseminated MM model in NOD/SCID-IL2-receptor-gamma-chain−/− (NSG) mice. In our current analysis, the multikinase inhibitor sorafenib was validated alone and in combination with the well-established anti-MM agent bortezomib in 6 independent experiments. Optimized dose and schedule were determined as follows: 1. sorafenib (100mg/kg/d; d0-11) alone, 2. bortezomib (0.7mg/kg/day (d); d0,4,11) alone, 3. both in combination with the respective doses and schedules compared to 4. a control group. L363 cells were injected intratibialy into NSG mice and respective therapies were started 7 days after L363-injection (d0). Tumor growth was monitored with daily monitoring of MM-symptoms, flow-cytometry (FACS) and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean MM-cell-infiltration at the respective compartment of the test- compared to the control-group (optimal T/C in %). Furthermore, hollow bones of the injected mice were retrieved when mice were sacrificed, cells flushed out and MM cells purified by MACS microbeads. Total RNA was isolated from these cells and gene expression profiles analyzed using the HG-U133 Plus 2.0 array (Affymetrix) and the Expressionist software (Genedata AG, Basel). L363 engrafted reliably (take rate=100%) at the injection site and in distant organs, such as bone marrow (BM; 100%), spleen (38%) and rarely liver (8%); in the latter organs as previously reported. Control mice developed MM symptoms, such as hind limb pareses, weight loss and osteolyses. At the respective doses and schedules, the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was 4% with mono- and 11% with combined therapy. Primary tumor development was markedly reduced by sorafenib (optimal T/C of 11% on d11), as well as with bortezomib, albeit to a lesser extend (optimal T/C: 22% on d5). BM metastases were also significantly reduced by sorafenib with an optimal T/C value of 21% on d11. Bortezomib reduced BM infiltration to an optimal T/C value of 46% on d5 as compared to the control. Combined therapy of sorafenib and bortezomib showed most pronounced anti-tumor and anti-metastatic effects, inducing T/C values of 17% (primary tumor) and 7% (BM) on day 11, respectively. Table 1. Antitumor effect of Sorafenib and Bortezomib in mono- and combined therapy in the L363-xenograft model Compound Side effects Primary tumor Bone marrow Dose Mortality Max. median bwc1 FI2 tumor inhibition FI2 tumor inhibition [mg/kg/d] [n] [%] [%] [%] Sorafenib 100 0 / 5 96 11 21 Bortezomib 0.7 0 / 5 97 22 46 Soraf. / Bortez. 100 / 0.7 0 / 5 89 17 7 1 bwc=body weight changes 2 Tumor inhibition was calculated as the median % of MM cells determined by FI at respective compartments of the test vs. control group multiplied by 100 (optimal test/control (T/C) in %) L363 engraftment into NSG is a valuable in vivo MM model which exhibits high reproducibility, take- and metastases-rates and closely mimics the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor MM growth. Sorafenib showed promising results in our MM model, in particular in combination with bortezomib. Amongst others, a detailed characterization of the anti-tumor activity of both compounds will be provided by gene expression analysis of L363 cells isolated from untreated vs. treated mice. Further investigations to validate other innovative anti-MM agents as well as their combinations are currently also pursued. Disclosures No relevant conflicts of interest to declare.

Download Full-text

Antitumor Activity of Small-Molecule SYK Inhibitor R788 and Fludarabinin Mono- and Combined Therapy in a Human B-CLL Xenograft Model.

Blood ◽

10.1182/blood.v114.22.2378.2378 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2378-2378

Author(s):

Julia Schüler ◽

Maike Buchner ◽

Heinz-Herbert Fiebig ◽

Hendrik Veelken ◽

Katja Zirlik

Keyword(s):

B Cells ◽

Antitumor Activity ◽

B Cell ◽

Combined Therapy ◽

Whole Body ◽

Control Group ◽

In Vivo Models ◽

Nsg Mice ◽

Syk Inhibitor

Abstract Abstract 2378 Poster Board II-355 B cell chronic lymphocytic leukemia (B-CLL), one of the most common leukemia in adults, is characterized by the accumulation of mature B cells expressing CD19 and CD5. Improved understanding of CLL has lead to new prognostic tools and therapeutic options. The protein kinase Syk as a key mediator of proximal B-cell receptor (BCR) signalling is constitutively phosphorylated in CLL B cells. For these novel therapeutics, functional in vivo models are highly valuable. We have established a cell line-based, disseminated B-CLL model in NOD/SCID-IL2-receptor-gamma-chain-/- (NSG) mice. In the current study, SYK inhibitor R788 (60mg/kg/day (d); applied daily) was evaluated in mono- and in combined therapy with Fludarabin (8mg/kg/day; applied 5 consecutive days in two cycles) as a well established compound in B-CLL treatment regimens in comparison with a control group. Equal parts of MEC1 cells were injected intravenously and into the peritoneal cavity of NSG mice and respective therapies were started 7 days after implantation. Tumor growth was monitored with flow-cytometry (FACS), daily monitoring of B-CLL symptoms and fluorescence-based in vivo imaging (FI). Tumor inhibition was calculated as the proportional reduction of mean B-CLL cell infiltration at the respective compartment of the test- compared to the control-group (in %). MEC1 cells engrafted predominantly in bone marrow (BM; take rate = 100%), but were as well detectable in spleen (33%) and peripheral blood (PB; 8%). At the respective doses and schedules the examined compounds were well tolerated in tumor-bearing mice. No acute toxicity could be observed and maximal body weight loss was below 15% in mono- and combined therapy. Tumor development was clearly reduced by R788 (optimal T/C of 48% on day 18), albeit to a lesser extend then standard anticancer agent Fludarabin (optimal T/C of 19% on day 18). Combined therapy induced no synergistic effects showing an optimal T/C value of 42% on day 19. Thus, MEC1 engraftment into NSG mice is a valuable in vivo model for B-CLL which exhibits high reproducibility and take-rates in relevant compartments closely mimicking the clinical situation. Collection of whole-body FI data proved to be a time- and animal-saving analysis that allows to closely monitor B-CLL growth. Further investigations will optimize the very promising antitumor activity of R788 and evaluate the potentially synergistic effect of R788 with additional well-established B-CLL therapeutics. Disclosures: No relevant conflicts of interest to declare.

Download Full-text

Anti-Obesity and Anti-Adipogenic Effects of Chitosan Oligosaccharide (GO2KA1) in SD Rats and in 3T3-L1 Preadipocytes Models

Molecules ◽

10.3390/molecules26020331 ◽

2021 ◽

Vol 26 (2) ◽

pp. 331

Author(s):

Jung-Yun Lee ◽

Tae Yang Kim ◽

Hanna Kang ◽

Jungbae Oh ◽

Joo Woong Park ◽

...

Keyword(s):

Gene Expression ◽

Body Weight ◽

Density Lipoprotein ◽

Treated Group ◽

Control Group ◽

Chitosan Oligosaccharide ◽

Sd Rats ◽

Adipogenic Gene

Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

Download Full-text

Effects of Vitrification on the Blastocyst Gene Expression Profile in a Porcine Model

International Journal of Molecular Sciences ◽

10.3390/ijms22031222 ◽

2021 ◽

Vol 22 (3) ◽

pp. 1222

Author(s):

Cristina Cuello ◽

Cristina A. Martinez ◽

Josep M. Cambra ◽

Inmaculada Parrilla ◽

Heriberto Rodriguez-Martinez ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Analysis ◽

Survival Rates ◽

Control Group ◽

P Value ◽

Transcriptome Profile ◽

Embryo Viability ◽

The Impact

This study was designed to investigate the impact of vitrification on the transcriptome profile of blastocysts using a porcine (Sus scrofa) model and a microarray approach. Blastocysts were collected from weaned sows (n = 13). A total of 60 blastocysts were vitrified (treatment group). After warming, vitrified embryos were cultured in vitro for 24 h. Non-vitrified blastocysts (n = 40) were used as controls. After the in vitro culture period, the embryo viability was morphologically assessed. A total of 30 viable embryos per group (three pools of 10 from 4 different donors each) were subjected to gene expression analysis. A fold change cut-off of ±1.5 and a restrictive threshold at p-value < 0.05 were used to distinguish differentially expressed genes (DEGs). The survival rates of vitrified/warmed blastocysts were similar to those of the control (nearly 100%, n.s.). A total of 205 (112 upregulated and 93 downregulated) were identified in the vitrified blastocysts compared to the control group. The vitrification/warming impact was moderate, and it was mainly related to the pathways of cell cycle, cellular senescence, gap junction, and signaling for TFGβ, p53, Fox, and MAPK. In conclusion, vitrification modified the transcriptome of in vivo-derived porcine blastocysts, resulting in minor gene expression changes.

Download Full-text

Pre-Clinical Efficacy of Combined Therapy with LSD1 Antagonist SP-2509 and Pan-Histone Deacetylase Inhibitor Against AML Blast Progenitor Cells

Blood ◽

10.1182/blood.v120.21.868.868 ◽

2012 ◽

Vol 120 (21) ◽

pp. 868-868 ◽

Cited By ~ 2

Author(s):

Warren Fiskus ◽

Sunil Sharma ◽

Sunil Abhyankar ◽

Joseph McGuirk ◽

David J. Bearss ◽

...

Keyword(s):

Progenitor Cells ◽

Histone Deacetylases ◽

Combined Therapy ◽

Colony Growth ◽

Clinical Activity ◽

Gene Promoters ◽

Chromatin Mark ◽

Tail Vein ◽

Nsg Mice

Abstract Abstract 868 LSD1 (KDM1A) is an FAD-dependent histone demethylase, with homology to amine oxidases. LSD1 demethylates di- and mono-methylated lysine (K) 4 on histone H3, reducing the permissive H3K4Me3 chromatin mark for gene expression. LSD1 forms a complex with the histone deacetylases (HDAC) 1 and 2 and with the co-repressor CoREST, which stimulates the activity of LSD1 toward nucleosomes. While high LSD1 expression may be an effector of blocked differentiation and confers poor prognosis in AML, LSD1 inhibition induces the expression of myeloid–differentiation associated genes and attenuates growth of AML blast progenitor cells (BPCs). Recently, LSD1 was shown to sustain the in vivo leukemogenic potential of MLL-AF9 expressing leukemia stem cells. Also, co-treatment with the LSD1 inhibitor tranylcypromine (TCP) and all-trans retinoic acid (ATRA) was shown to diminish the engraftment of primary AML BPCs in vivo in NOD-SCID-γIL-2 receptor deficient (NSG) mice. Previous studies have shown that HDAC inhibitors attenuate the levels of LSD1 through Sp1 inhibition. SP-2509 is a potent and selective FAD-binding pocket, non-MAOA and MAOB, inhibitor with an IC50 of 13 nM for LSD1. In the present studies, we determined the chromatin effects and anti-AML efficacy of SP-2509 alone and in combination with the pan-HDAC inhibitor panobinostat (PS) (Novartis Pharmaceuticals) in cultured (HL-60, OCI-AML3, MV4-11, MOLM13, THP1 and SKM1 cells) and primary human AML BPCs. Treatment with SP-2509 (250 to 1000 nM) dose-dependently increased the levels of H3K4Me2 & Me3 chromatin mark, and chromatin immunoprecipitation followed by QPCR analyses showed an increase in the H3K4Me3 mark on the gene promoters of KLF4, HMOX1, p57 and p21 in AML BPCs. SP-2509 treatment attenuated the binding of LSD1 with CoREST, accompanied with increased levels of p16, p21 and p27 in AML BPCs. Consistent with this, treatment with SP-2509 inhibited the suspension and colony growth of AML BPCs regardless of whether they expressed MLL fusion oncoproteins. Knockdown of LSD1 by shRNA also inhibited the suspension and colony growth of AML blast progenitor cells. SP-2509 also induced C/EBPα expression and features of morphologic differentiation in the cultured and primary AML BPCs. Following tail vein infusion and establishment of AML by OCI-AML3 or MOLM13 cells in NOD/SCID mice, treatment with SP-2509 (25 mg/kg b.i.w. via IP injection) for three weeks demonstrated improved survival of the mice compared to the vehicle control treated mice (p <0. 001). We have previously reported that treatment with PS depleted polycomb repressive complex proteins EZH2, SUZ12 and BMI1 but also reduced LSD1 expression in AML cells. Co-treatment with PS enhanced SP-2509-induced chromatin effects and differentiation of AML cells. Also, PS and SP-2509 synergistically induced apoptosis of the cultured AML OCI-AML3, MOLM13 and MV4-11cells (combination indices, CI <1.0). Additionally, co-treatment with SP-2509 sensitized AML cells to ATRA-induced differentiation. Notably, co-treatment with SP-2509 and PS also induced significantly greater loss of viability of primary AML BPCs but not of normal CD34+ cells. SP-2509 treatment (15 mg/kg b.i.w. IP) also dramatically improved survival of NSG mice with established human AML following tail-vein injection of primary AML blasts. Survival was further significantly improved upon co-treatment with SP-2509 and PS (5 mg/kg IP, MWF) (p < 0.001). Mice did not experience any toxicity or weight loss. Taken together, these findings demonstrate promising pre-clinical activity of combined therapy with SP-2509 and PS, warranting further in vivo development and testing of SP-2509 against human AML. Disclosures: Sharma: Salarius Pharmaceuticals: Equity Ownership.

Download Full-text

Evaluation of Patient-Derived Xenografts for Modeling Outcome of Pediatric B-Cell Precursor Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v126.23.3759.3759 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3759-3759

Author(s):

Abdulmohsen M Alruwetei ◽

Hernan Carol ◽

Rosemary Sutton ◽

Glenn M Marshall ◽

Richard B Lock

Keyword(s):

Gene Expression ◽

Drug Resistance ◽

Clinical Outcome ◽

Ex Vivo ◽

Lymphoblastic Leukemia ◽

Nsg Mice ◽

Relapse Prediction ◽

Pediatric All ◽

Selection Of

Abstract Introduction: Children with acute lymphoblastic leukemia (ALL) are stratified at diagnosis based on molecular/cytogenetic characteristics and their response to initial treatment to receive risk-adapted multi-agent chemotherapy. The majority of ALL patients are stratified as Intermediate Risk (IR) and present with moderate levels of minimal residual disease (MRD<5x104) after receiving induction therapy, although an unacceptably high proportion of these patients relapse. The lack of specific prognostic features makes it difficult to predict the response of IR patients to treatment. The early identification of patients who are destined to relapse would facilitate improvements in tailored treatments for IR ALL patients. Recent progress in the development of patient-derived xenografts (PDXs) in immune-deficient mice represents an opportunity to improve relapse prediction in ALL patients. The aims of this study were to: (1) optimize the engraftment conditions of IR pediatric ALL samples to predict patient response to treatment; and, (2) to assess the development and mechanisms of therapy-induced drug resistance. Methods: Two pairs of IR pediatric ALL patients were matched based on clinical and genetic features, except that one patient from each pair relapsed early while the other remains relapse-free (ALL-Rel and ALL-CR1, respectively). Three parameters were varied in establishing PDXs by inoculating one million bone marrow (BM) derived biopsy cells collected at diagnosis into groups of 4 mice: (1) mouse strain (NOD/SCID vs. NSG); (2) site of inoculation (intravenous vs. intra-femoral); and (3) early treatment of mice with a 2-week induction chemotherapy regimen of vincristine, dexamethasone, and L-asparaginase (VXL). Leukemia engraftment was monitored weekly based on the proportion of human versus mouse CD45+ cells in the murine PB, and the median times to engraftment were compared according to patient outcome. The median time to engraft was also compared between the VXL-treated and non-treated groups. PDXs harvested from mice were compared for ex vivo sensitivity to single agent vincristine, dexamethasone and L-asparaginase. PDX gene expression profiles were also compared to identify pathways associated with evasion of VXL treatment in vivo. Results: The efficiency of engraftment was greater for NSG mice (29/32 mice engrafted) versus NOD/SCID mice (20/32 mice), and primary ALL cells also engrafted significantly faster in NSG mice (median time to engraft 71.1 days) compared with NOD/SCID mice (83.5 days) (P < 0.01), with no apparent difference associated with clinical outcome. Intrafemoral inoculation did not improve the efficiency or speed of engraftment compared with intravenous inoculation, nor predicted clinical outcome. However, PDX responses to VXL induction chemotherapy reflected the clinical outcome of the patients from whom they were derived; those derived from the 2 ALL-Rel patients exhibited in vivo drug resistance (leukemia growth delay of 1 and 6.2 days) compared with those derived from the 2 ALL-CR1 patients (34.7 and >119.8 days). Further, ex vivo analysis showed that the PDXs derived from the ALL-Rel patients exhibited resistance to vincristine or L-asparaginase compared with those derived from the ALL-CR1 cases. Moreover, the in vivo VXL treatment of an ALL-CR1 PDX resulted in selection of cells that exhibited vincristine resistance. Gene expression profiling revealed significant up-regulation of microtubule associated proteins (MAPs) and tubulin isotypes (alpha and beta) in vincristine-resistant PDXs. Genes that were significantly upregulted in vincristine resistant PDXs with a false discovery rate (FDR) < 0.05 and P value < 0.02 include TUBB6, TUBA1A, TUBA1B, MAP1S, TUBA3D and TBCA. The increased expression of genes that affect microtubule functions suggest that changes in microtubule dynamics and/or stability led to decreased sensitivity to antimicrotubule agents. Conclusions: In vivo selection of PDXs with an induction-type regimen of chemotherapeutic drugs may lead to improved relapse prediction and identify novel mechanisms of drug resistance in IR pediatric ALL. Support: Steven Walter Foundation; NHMRC Australia, APP1057746 Disclosures No relevant conflicts of interest to declare.

Download Full-text

211 IN VITRO CULTURE CONDITIONS AFFECT GENE EXPRESSION PATTERN OF BOVINE BLASTOCYST IN A STAGE-SPECIFIC MANNER

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab211 ◽

2013 ◽

Vol 25 (1) ◽

pp. 254 ◽

Cited By ~ 1

Author(s):

A. Gad ◽

U. Besenfelder ◽

V. Havlicek ◽

M. Hölker ◽

M. U. Cinar ◽

...

Keyword(s):

Gene Expression ◽

Cell Death ◽

In Vitro Culture ◽

Blastocyst Stage ◽

Culture Conditions ◽

Control Group ◽

Transcriptome Profile ◽

Specific Manner

The aim of this study was to examine the effect of in vitro culture conditions at specific phases of early embryonic development on the transcriptome profile of bovine blastocysts. Simmental heifers were superovulated and artificially inseminated 2 times with the same frozen–thawed commercial bull semen. Using nonsurgical endoscopic oviductal flushing technology (Besenfelder et al. 2001 Theriogenology 55, 837–845), 6 different blastocyst groups were flushed out at different time points (2-, 4-, 8-, 16-, 32-cell and morula). After flushing, embryos cultured under in vitro conditions until the blastocyst stage. Blastocysts from each group were collected and pooled in groups of 10. Complete in vivo blastocysts were produced and used as control. A unique custom microarray (Agilent) containing 42 242 oligo probes (60-mers) was used over 6 replicates of each group v. the in vivo control group to examine the transcriptome profile of blastocysts. A clear difference in terms of the number of differentially expressed genes (DEG, fold change ≥2, false discovery rate ≤0.05) has been found between groups flushed out at 2-, 4-, and 8-cell (1714, 1918, 1292 DEG, respectively) and those flushed out at 16-, 32-cell and morula stages and cultured in vitro until blastocyst stage (311, 437, 773 DEG, respectively) compared with the complete vivo group. Ontological classification of DEG showed cell death to be the most significant function in all groups. However, the longer time embryos spent under in vitro conditions, the more the percentage of DEG involved in cell death and apoptosis processes are represented in those groups. In addition, genes related to post-translational modification and gene expression processes were significantly dysregulated in all groups. Pathway analysis revealed that protein ubiquitination pathway was the dominant pathway in the groups flushed out at 2-, 4-, and 8-cells but not in the other groups flushed at later stages compared with the in vivo control group. Moreover, retinoic acid receptor activation and apoptosis signalling pathways followed the same pattern. Embryos flushed out before the time of embryonic genome activation and subsequently cultured in vitro were highly affected by culture conditions. Overall, the results of the present study showed that despite the fact that embryos originated from the same source, in vitro culture condition affected embryo quality, measured in terms of gene expression, in a stage-specific manner.

Download Full-text

129 POST-HATCHING DEVELOPMENT AND GENE EXPRESSION OF IN VITRO-PRODUCED BOVINE EMBRYOS IN RECIPIENT UTERUS AFTER MULTIPLE TRANSFER

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab129 ◽

2013 ◽

Vol 25 (1) ◽

pp. 212

Author(s):

G. Machado ◽

A. Ferreira ◽

I. Pivato ◽

A. Fidelis ◽

J. F. Srpicigo ◽

...

Keyword(s):

Gene Expression ◽

Expression Analysis ◽

Gene Expression Analysis ◽

T Test ◽

Molecular Profile ◽

Control Group ◽

Fluid Medium ◽

Uterine Flushing

This study aimed to compare post-hatching development of Day 7 in vitro and in vivo embryos cultured in recipient uterus until Day 14. For producing in vitro embryos (IVP), oocytes were matured, fertilized (Day 0) and cultured in vitro for 6 days (Day 7) in synthetic oviduct fluid medium supplemented with 5% of fetal bovine serum and incubated at 39°C in 5% CO2 in air. At Day 7, part of IVP blastocysts was transferred to recipient uterus and part was stored for gene expression analysis. As a control group, in vivo embryos were produced after ovarian stimulation, insemination and uterine flushing on Day 7 post insemination. Similarly to the IVP embryos, part of embryos was transferred to recipient uterus and part was stored for gene expression analysis. Day 7 in vivo (n = 53) and IVP (n = 64) expanded blastocysts were transferred to synchronized recipients (10/horn) and were collected by uterine flushing 7 days after transfer (Day 14). Recovered embryos were measured using Motic Image Plus software and evaluated for presence and size of embryonic disc (ED). A trophoblast biopsy was removed and stored for gene expression analysis. For the molecular profile evaluation of Day 7 and Day 14 in vivo and in vitro embryos, 8 genes related with placentation, implantation, oxidative stress, and glucose metabolism (PLAC8, CD9, GLUT-1, GLUT-3, KRT8, MnSOD, HSP70, and INFT, respectively) were quantified by RT-qPCR using ΔΔCT method and CYC-A gene as endogenous control. The recovery rate of Day 14 embryos, analyzed by chi-square test, was higher (P < 0.05) for in vitro than for in vivo embryos, being 50.0% (64/128) and 38.6% (53/137), respectively. No differences (P > 0.05; t-test) were observed in embryo length when comparing Day 14 in vitro (19.1 ± 2.4 mm) and in vivo embryos (24.2 ± 3.7 mm). ED was detected in 25% (16/64) of in vitro and in 26% (14/53) of in vivo embryos. No differences were found (P > 0.05; t-test) in diameter between the two types of embryos (0.3 ± 0.0 mm/in vitro and 0.3 ± 0.0 mm/in vivo). Regarding gene expression, Day 7 IVP embryos showed higher (P < 0.05, Mann–Whitney test) expression of HSP70 and SCL2A1 than in vivo embryos. However, at Day 14 no differences between embryos were observed in transcript levels for any of the studied genes. Therefore, the present study showed that although differences in Day 7 in vitro embryos were observed at the molecular level compared to in vivo counterpart, after transfer to the uterine environment, they showed similar morphology and gene expression profile. These results highlight the importance of evaluating embryos produced by assisted reproductive techniques in later stages of development to have a more precise evaluation of their quality. Financial support: Embrapa, CNPq, CAPES.

Download Full-text

137 SPLITTING OF IVP BOVINE BLASTOCYST AFFECTS MORPHOLOGY AND GLOBAL GENE EXPRESSION OF RESULTING DEMI-EMBRYOS DURING ELONGATION

Reproduction Fertility and Development ◽

10.1071/rdv27n1ab137 ◽

2015 ◽

Vol 27 (1) ◽

pp. 161

Author(s):

A. E. Velásquez ◽

D. Veraguas ◽

J. F. Cox ◽

F. O. Castro ◽

L. l. Rodriguez

Keyword(s):

Gene Expression ◽

Embryo Development ◽

Average Length ◽

Global Gene Expression ◽

Differentially Expressed ◽

Control Group ◽

Molecular Characteristics ◽

Bovine Embryo ◽

Developmental Potential

Embryo splitting has been used since the early 1980s to produce identical twins and increase the pregnancy rate per available embryo. However, very little is known about the effect of splitting on embryo development and competence. Indeed splitting could provoke a negative effect on embryo survival and it can be presumed that each demi-embryo might respond differently to the injury. In this sense, even when embryos are genetically and morphologically identical at the moment of splitting, their developmental potential and molecular characteristics might change as a consequence of the intense manipulation or epigenetic differences due to the interaction with the environment. We have proposed an approach to evaluate the effect of blastocyst splitting on the morphological and gene expression in in vivo development up to the filamentous stage. For that, the effect of splitting on bovine embryo development was evaluated during the elongation period by transferring split and nonsplit IVF-derived blastocysts to cattle recipients and collecting them at Day 17 of development. The number of collected embryos, embryo size, and global gene expression was compared between both groups. Collected elongated embryos derived from split blastocyst were compared with time matched collected control embryos. From 14 transferred hemi-embryos, 5 (35.7%) were collected while 9 elongated from 17 controls were recovered (52.9%). Neither the recovery rate nor the average length of the elongated embryos was significantly different between the two treatments. However, when embryos were rated depending on their size, more than 50% of embryos from the control group had a length surpassing 100 mm, while only 33% of the split embryos reached that size. Global gene expression was performed using 2-colour microarray-based gene expression analysis. This was a whole-genome microarray study comparing 10 individual elongated embryos derived from split and nonsplit IVF blastocysts. Genes were considered differentially expressed if the fold change is greater than 2 (up or down-regulation) with P ≤ 0.05. A total of 29 585 transcripts were detected in all embryos. From those, 449 (1.5%) were differentially expressed between elongated embryos derived from split and nonsplit IVF blastocysts, among them, 248 (0.83%) genes were down-regulated and 201 (0.67%) genes were up-regulated in split embryos. Gene ontology analysis identified deregulated genes related with intrinsic component of membrane (ELOVL7, GJA1, LAPTM4B, LDLR, SLC18A2, SLC1A3, SLC38A5, TSPAN13), lipid transporter activity (RBP4, APOA1, MTTP), and organophosphate ester transport (GJA1, GJB1, ATP9B). In conclusion, we showed that splitting affect the in vivo developmental capability and gene expression profile during the elongation period of bovine embryos. However, further studies are needed to determine the long-term effect of this technique to produce viable offspring. This work was partially supported by Fondecyt No. 11100082 and Fondequip No. EQM12113 from the Ministry of Education of Chile.

Download Full-text

Leishmanicidal potentials of Gossypium hirsutum extract and its fractions on Leishmania major in a murine model: parasite burden, gene expression, and histopathological profile

Journal of Medical Microbiology ◽

10.1099/jmm.0.001333 ◽

2021 ◽

Vol 70 (6) ◽

Author(s):

Fatemeh Sharifi ◽

Fariba Sharififar ◽

Mostafa Pournamdari ◽

Mehdi Ansari ◽

Razieh Tavakoli Oliaee ◽

...

Keyword(s):

Gene Expression ◽

Gossypium Hirsutum ◽

Murine Model ◽

Complex Disease ◽

Leishmania Major ◽

Parasite Burden ◽

Control Group ◽

Standard Drug ◽

Soxhlet Apparatus

Introduction. Leishmaniasis is a neglected tropical and subtropical disease caused by over 20 protozoan species. Hypothesis. Treatment of this complex disease with traditional synthetic drugs is a major challenge worldwide. Natural constituents are unique candidates for future therapeutic development. Aim. This study aimed to assess the in vivo anti-leishmanial effect of the Gossypium hirsutum extract, and its fractions compared to the standard drug (Glucantime, MA) in a murine model and explore the mechanism of action. Methodology. Footpads of BALB/c mice were infected with stationary phase promastigotes and treated topically and intraperitoneally with G. hirsutum extract, its fractions, or Glucantime, 4 weeks post-infection. The extract and fractions were prepared using the Soxhlet apparatus with chloroform followed by the column procedure. Results. The crude extract significantly decreased the footpad parasite load and lesion size compared to the untreated control group (P<0.05), as revealed by dilution assay, quantitative real-time PCR, and histopathological analyses. The primary mode of action involved an immunomodulatory role towards the Th1 response in the up-regulation of IFN-γ and IL-12 and the suppression of IL-10 gene expression profiling against cutaneous leishmaniasis caused by Leishmania major. Conclusion. This finding suggests that the extract possesses multiple combinatory effects of diverse bioactive phytochemical compositions that exert its mechanisms of action through agonistic-synergistic interactions. The topical extract formulation could be a suitable and unique candidate for future investigation and pharmacological development. Further studies are crucial to evaluate the therapeutic potentials of the extract alone and in combination with conventional drugs using clinical settings.

Download Full-text

Abstract P141: Cardiac TRH Partly Mediates Angiotensin II-induced Fibrotic and Hypertrophy Effects in "in vivo" and "in vitro" Models

Hypertension ◽

10.1161/hyp.66.suppl_1.p141 ◽

2015 ◽

Vol 66 (suppl_1) ◽

Author(s):

Silvia I García ◽

Ludmila S Peres Diaz ◽

Maia Aisicovich ◽

Mariano L Schuman ◽

María S Landa

Keyword(s):

Gene Expression ◽

Body Weight ◽

Cardiac Hypertrophy ◽

Structural Changes ◽

In Vitro Models ◽

Heart Weight ◽

Saline Control ◽

Control Group

Cardiac TRH (cTRH) is overexpressed in the hypertrophied ventricle (LV) of the SHR. Additionally in vivo siRNA-TRH treatment induced downregulation of LV-TRH preventing cardiac hypertrophy and fibrosis demonstrating that TRH is involved in hypertrophic and fibrotic processes. Moreover, in a normal heart, the increase of LV TRH expression alone could induce structural changes where fibrosis and hypertrophy could be involved, independently of any other system alterations. Is well-known the cardiac hypertrophy/ fibrotic effects induced by AII, raising the question of whether specific LV cTRH inhibition might attenuates AII induced cardiac hypertrophy and fibrosis in mice. We challenged C57 mice with AII (osmotic pumps,14 days; 2 mg/kg) to induce cardiac hypertrophy vs saline. Groups were divided and , simultaneously to pump surgery, injected intracardiac with siRNA-TRH and siRNA-Con as its control. Body weight, water consume and SABP were measured daily. As expected, AII significantly increased SABP (p<0.05) in both groups treated , although cardiac hypertrophy (heart weight/body weight) was only evident in the group with the cardiac TRH system undamaged, suggesting that the cardiac TRH system function as a necessary mediator of the AII-induced hypertrophic effect. As hypothesized, we found an AII-induced increase of TRH (p<0.05) gene expression (real-t PCR) confirmed by immunofluorescence that was not observed in the group AII+siRNA-TRH demonstrating the specific siRNA treatment efficiency. Furthermore, AII significantly increase (p<0.05) BNP (hypertrophic marker), III collagen and TGFB (fibrosis markers) expressions only in the group with AII with the cardiac TRH system intact. On the contrary, the group with AII and the cTRH system inhibited, shows genes expressions similar to the saline control group. We confirmed these results by immunofluorescence. Similar fibrotic results were observed with NIH3T3 cell culture where we demonstrated that AII induced TRH gene expression (p<0.05) and its inhibition impedes AII-induced increase of TGFB and III/I collagens expressions telling us about the role of the cTRH in the AII fibrosis effects. Our results point out that the cardiac TRH is involved in the AII-induced hypertrophic and fibrotic effects.

Download Full-text