Mean Total Cell Number Research Articles

Inhibition of metastatic brain cancer in Sonic Hedgehog medulloblastoma using caged nitric oxide albumin nanoparticles.

Medulloblastoma is a tumor of the cerebellum that metastasizes to the leptomeninges of the central nervous system (CNS), including to forebrain and to spinal cord. The inhibitory effect of polynitroxylated albumin (PNA), a caged nitroxide nanoparticle, on leptomeningeal dissemination and metastatic tumor growth was studied in a Sonic Hedgehog transgenic mouse model. PNA treated mice showed an increased lifespan with a mean survival of 95 days (n = 6, P<0.05) compared with 71 days in controls. In primary tumors, proliferation was significantly reduced and differentiation was significantly increased (P<0.001) as shown by Ki-67+ and NeuN+ immunohistochemistry, while cells in spinal cord tumors appeared unaffected. Yet, histochemical analysis of metastatic tumor in spinal cord showed that the mean total number of cells in spinal cord was significantly reduced in mice treated with PNA compared to albumin vehicle (P<0.05). Examination of various levels of the spinal cord showed that PNA treated mice had significantly reduced metastatic cell density in the thoracic, lumbar and sacral spinal cord levels (P<0.05), while cell density in the cervical region was not significantly changed. The mechanism by which PNA may exert these effects on CNS tumors is discussed.

Oocyte in vitro maturation with eugenol improves the medium antioxidant capacity and total cell number per blastocyst

This study investigates the impact of eugenol (EU) supplementation on bovine oocyte in vitro maturation (IVM) and antioxidant capacity, as well as in vitro embryo production and quality after conventional in vitro fertilization (IVF). A total of 1077 cumulus oocyte complexes were cultured in TCM-199+ without EU supplementation (control treatment) or supplemented with EU at the concentrations of 10 μM (EU-10), 20 μM (EU-20), or 40 μM (EU-40). After IVM, the oocytes were subjected to IVF and embryo culture. The addition of EU at 40 μM to the IVM medium improved (P < 0.05) the antioxidant capacity and cleavage rate when compared to the control treatment. Moreover, a positive correlation (r = 0.61, P < 0.03) was observed between cleavage rate and EU concentration. The addition of EU at concentrations of 10 and 20 μM decreased (P < 0.05) the calreticulin (CALR) levels in expanded blastocysts when compared to the control treatment and EU-40 treatment. However, the EU-10 and EU-20 treatments had a greater (P < 0.05) mean total cell number (TCN) per expanded blastocyst when compared to the control treatment and EU-40 treatment. In conclusion, the addition of EU to the enriched culture medium during IVM of bovine oocytes improved the antioxidant capacity of the spent medium, as well as the cleavage rate and embryonic quality (i.e., TCN/expanded blastocyst), and reduced the endoplasmic reticulum stress (i.e., CALR levels) in the embryos. Thus, we recommend enriching the IVM medium with 10 μM EU for in vitro bovine embryo production.

Quantification and Comprehensive Analysis of Mesenchymal Stromal Cells in Bone Marrow Samples from Sickle Cell Disease Patients with Osteonecrosis.

The potential use of bone marrow mesenchymal stromal cells (BM-MSCs) for the treatment of osteonecrosis in sickle cell disease (SCD) patients is increasing. However, convenient BM-MSC quantification and functional property assays are critical factors for cell-based therapies yet to be optimized. This study was designed to quantify the MSC population in bone marrow (BM) samples from SCD patients with osteonecrosis (SCD group) and patients with osteoarticular complications not related to SCD (NS group), using flow cytometry for CD271+CD45-/low cell phenotype and CFU-F assay. We also compared expanded BM-MSC osteogenic differentiation, migration, and cytokine secretion potential between these groups. The mean total cell number, CFU-F count, and CD271+CD45-/low cells in BM mononuclear concentrate were significantly higher in SCD than in NS patients. A significant correlation between CD271+CD45-/low cell number and CFU-F counts was found in SCD (r = 0.7483; p = 0.0070) and NS (r = 0.7167; p = 0.0370) BM concentrates. An age-related quantitative reduction of CFU-F counts and CD271+CD45-/low cell number was noted. Furthermore, no significant differences in the morphology, replicative capacity, expression of surface markers, multidifferentiation potential, and secretion of cytokines were found in expanded BM-MSCs from SCD and NS groups after in vitro culturing. Collectively, this work provides important data for the suitable measurement and expansion of BM-MSC in support to advanced cell-based therapies for SCD patients with osteonecrosis.

Flow Cytometric Aldehyde Dehydrogenase Assay Enables a Fast and Accurate Human Umbilical Cord Blood Hematopoietic Stem Cell Assessment.

Objective:Colony-forming units of granulocytes/macrophages (CFU-GM) analysis is the most widely used method to determine the hematopoietic stem cell (HSC) content of human umbilical cord blood (CB) for prediction of engraftment potential. The measurement of aldehyde dehydrogenase (ALDH) activity is a more recent method for HSC qualification. Our aim was to correlate phenotypic and functional assays to find the most predictive method.Materials and Methods:In this study, flow cytometric quantitation of CD34+ cells and ALDH positivity along with CFU-GM capacity were assessed in fresh and post-thaw CB units.Results:Among 30 post-processing samples, for each CB unit the mean total number of nucleated cells (TNCs) was (93.8±30.1)x107, CD34+ cells were (3.85±2.55)x106, ALDH+ cells were (3.14±2.55)x106, and CFU-GM count was (2.64±1.96)x105. Among an additional 19 post-thaw samples the cell counts were as follows: TNCs, (32.79±17.27)x107; CD34+, (2.18±3.17)x106; ALDH+, (2.01±2.81)x106; CFU-GM, (0.74±0.92)x105. Our findings showed that in fresh samples TNCs, CD34+ cells, and ALDH correlated highly with counts of CFU-GM, CFU-erythroids/granulocytes-macrophages/megakaryocytic cells (GEMM), and burst forming units of erythroids (BFU-E) as follows: TNCs, r=0.47, r=0.35, r=0.41; CD34+, r=0.44, r=0.54, r=0.41; and ALDH, r=0.63, r=0.45, r=0.6, respectively. In terms of post-thaw samples, the correlations were as follows: TNCs, r=0.59, r=0.46, r=0.56; CD34+, r=0.67, r=0.48, r=0.61; and ALDH, r=0.61, r=0.67, r=0.67, for CFU-GM, CFU-GEMM, and BFU-E, respectively. All correlations were statistically significant.Conclusion:In our experience, HSC assessment by ALDH activity yields the highest correlation with conventional analytical methods, particularly for post-thaw samples. Thus, this fast, inexpensive method has the potential to overcome the weaknesses of other techniques.

How many TCR clonotypes does a body maintain?

We consider the lifetime of a T cell clonotype, the set of T cells with the same T cell receptor, from its thymic origin to its extinction in a multiclonal repertoire. Using published estimates of total cell numbers and thymic production rates, we calculate the mean number of cells per TCR clonotype, and the total number of clonotypes, in mice and humans. When there is little peripheral division, as in a mouse, the number of cells per clonotype is small and governed by the number of cells with identical TCR that exit the thymus. In humans, peripheral division is important and a clonotype may survive for decades, during which it expands to comprise many cells. We therefore devise and analyse a computational model of homeostasis of a multiclonal population. Each T cell in the model competes for self pMHC stimuli, cells of any one clonotype only recognising a small fraction of the many subsets of stimuli. A constant mean total number of cells is maintained by a balance between cell division and death, and a stable number of clonotypes by a balance between thymic production of new clonotypes and extinction of existing ones. The number of distinct clonotypes in a human body may be smaller than the total number of naive T cells by only one order of magnitude.

Intrinsic and extrinsic innervation of the heart in zebrafish (Danio rerio).

In the vertebrate heart the intracardiac nervous system is the final common pathway for autonomic control of cardiac output, but the neuroanatomy of this system is not well understood. In this study we investigated the innervation of the heart in a model vertebrate, the zebrafish. We used antibodies against acetylated tubulin, human neuronal protein C/D, choline acetyltransferase, tyrosine hydroxylase, neuronal nitric oxide synthase, and vasoactive intestinal polypeptide to visualize neural elements and their neurotransmitter content. Most neurons were located at the venous pole in a plexus around the sinoatrial valve; mean total number of cells was 197 ± 23, and 92% were choline acetyltransferase positive, implying a cholinergic role. The plexus contained cholinergic, adrenergic, and nitrergic axons and vasoactive intestinal polypeptide-positive terminals, some innervating somata. Putative pacemaker cells near the plexus showed immunoreactivity for hyperpolarization-activated cyclic nucleotide-gated channel 4 (HCN4) and the transcription factor Islet-1 (Isl1). The neurotracer neurobiotin showed that extrinsic axons from the left and right vagosympathetic trunks innervated the sinoatrial plexus proximal to their entry into the heart; some extrinsic axons from each trunk also projected into the medial dorsal plexus region. Extrinsic axons also innervated the atrial and ventricular walls. An extracardiac nerve trunk innervated the bulbus arteriosus and entered the arterial pole of the heart to innervate the proximal ventricle. We have shown that the intracardiac nervous system in the zebrafish is anatomically and neurochemically complex, providing a substrate for autonomic control of cardiac effectors in all chambers.

High revivability of vitrified-warmed bovine mature oocytes after recovery culture with α-tocopherol.

The objective of this study was to investigate whether developmental competence of vitrified-warmed bovine oocytes can be improved by antioxidant treatment during recovery culture. In experiment 1, one of the two antioxidants (either l-ascorbic acid or α-tocopherol) was added as a supplement to the recovery culture medium to which postwarming oocytes were exposed for 2 h before IVF. The exposure to α-tocopherol had a positive effect on rescuing the oocytes as assessed by the blastocyst yield 8 days after the IVF (35.1-36.3% vs 19.2-25.8% in untreated postwarming oocytes). Quality of expanding blastocysts harvested on Day 8 was comparable between α-tocopherol-treated vitrification group and fresh control group in terms of total cell number and chromosomal ploidy. In experiment 2, level of reactive oxygen species, mitochondrial activity, and distribution of cortical granules in α-tocopherol-treated postwarming oocytes were assessed. No obvious differences from the control data were found in these parameters. However, the treatment with α-tocopherol increased the percentage of zygotes exhibiting normal single aster formation (90.3% vs 48.0% in untreated postwarming oocytes; 10 h post-IVF). It was concluded that α-tocopherol treatment of vitrified-warmed bovine mature oocytes during recovery culture can improve their revivability, as shown by the high blastocyst yield and the higher mean total cell number in the blastocysts.

The Identification and Use of a Three Fungus-Antigen Combination to Generate T-Cell Products with Activity Against Pathogenic Filamentous Fungi and Yeasts for Clinical Cell Therapy

Invasive fungal diseases (IFDs) caused by the filamentous fungal pathogens of the genera Aspergillus, fusarium, zygomycetes, scedosporium and the yeast candida are the most important causes of fungus related deaths in immunosuppressed hematology patients1. Clinical efficacy of A. fumigatus specific T cells has been demonstrated in haploidentical hematopoietic stem cell transplant (HSCT) recipients2. We recently published a method to culture A. fumigatus specific T cells from normal allogeneic donors using procedures compliant with the code of Good Manufacturing Practice3. The safety and efficacy of cells generated using our method is currently being tested in a Phase 1 study in HSCT recipients. Here, we investigated the possibility of manufacturing a single T cell product with activity against a broad target range of fungal pathogens for clinical adoptive immunotherapy.We made water-soluble lysates from germinated spores of fungal pathogens, A. flavus, A. terreus, C. albicans, C. krusei, F. oxysporum, F. solani, R. oryzae and S. prolificans and used these to pulse monocyte derived dendritic cells (MoDC) from PBMC of 4 normal donors. Pulsed MoDC were washed, irradiated and used to stimulate autologous PBMC on Days 0 and 7. Cells were expanded with the addition of IL-2, IL-7 and IL-15 from Days 7 to 21. Cell numbers, phenotype and fungus-specificity were assessed on Day 21. Cross-reactivity of cultured T cells was tested against lysates of other fungi to identify a combination of antigens likely to induce broad anti-fungal T cell reactivity. Products were generated from PBMC using either no selection or TNFα capture on Day 7 of culture to select antigen-specific cells. Three products were generated from G-CSF mobilized peripheral blood stem cell products (PBSC) without selection. Antifungal activity was mapped to specific HLA molecules using blocking antibodies to HLA-DR, -DP and –DQ.In cultures from normal donor PBMC, expansion occurred with lysates from all fungi (range 2.3-109.6 fold) generating 85-97% T cells of which >80% were CD4+ T cells. The percentage of fungus-specific (TNFα+) CD4+ cells were A flavus 6.8±5.5%, A terreus 13.2±12.8%, C albicans 10.5±8.5%, C krusei 6.4±6.8%, F oxysporum 6.4±3.8%, F solani 5.2±7.8%, R oryzae 7.6±6.4 and S prolificans 4.4±3.8% (n=4). T cells also produced IFNγ and IL-2. T cells from cultures generated with Aspergillus, Fusarium and Scedosporium cross-reacted with one another and with lysates from most other fungi. We selected a combination of A. terreus, C. krusei and R. oryzae to generate multifungus T cell products from PBMC and PBSC. All but one multifungus culture generated >89% T cells. The majority of T cells had terminally differentiated effector and effector memory phenotypes. Regulatory T cells were <6%. Starting from a mean total cell number of 18.5±3.0, 14.0±4.9 and 12.0±4.0 x 106 cells, we obtained 12.5±4.9, 3.0±3.9 and 19.1±9.6 x 106 fungus-specific CD4+ T cells from PBMC (n=4), TNFα selected PBMC (n=4) and PBSC (n=3) cultures respectively. CD4+ T cells specific for most fungal antigens were present in all cultures. Cultured CD4+ T cells proliferated in response to all fungal lysates (n=3).Blocking HLA-DR, but not -DP or -DQ on antigen presenting cells abrogated fungus-specific cytokine production by cultured T cells (n=7). Antifungal activity was maintained when MoDC from partially HLA-DRB1 matched allogeneic donors were used to present fungal antigens to cultured T cells (n=7). In contrast, no antifungal activity was observed when MoDC from completely HLA-DRB1 mismatched donors were used to stimulate fungal T cells (n=4).We demonstrate that similarly to A. fumigatus specific T cells, T cells specific for other clinically relevant fungi can be expanded in vitro. We have developed a method to manufacture a T cell product with activity against multiple clinically relevant fungi, using blood or stem cells of healthy donors as starting material. The use of TNFα capture did not increase the number or purity of fungus-specific cells in cultures. The clinical utility of infusions of multifungus responsive T cell products in prophylaxis and treatment of invasive fungal disease needs to be tested clinically.References1Neofytos D, Horn D et al, Clinical Infectious Diseases 20092Perruccio K, Tosti A et al, Blood 20053Gaundar S, Clancy L et al, Cytotherapy 2012 DisclosuresNo relevant conflicts of interest to declare.

259 DOES THE SENSITIVITY TO OXIDATIVE STRESS DEPEND ON THE SEX OF THE EMBRYO?

Male and female bovine embryos show several differences as early as the blastocyst stage. For example, differences are observed in metabolism, developmental kinetics, or gene expression that can lead to a shift in the sex ratio. Interestingly, the culture medium differentially affects male and female embryos. We previously showed that male Day 7 blastocysts present lower apoptotic rates than females (Ghys et al. 2013 Reprod. Fertil. Dev. 25, 194). The objective of the present study was to determine if such difference might be related to a differential sensitivity to oxidative stress, known to increase apoptosis in bovine blastocysts. In vitro-produced embryos were cultured in a SOF-based medium containing 0.4% BSA. At Day 5 post-insemination (pi) all the embryos were transferred in drops containing the same culture medium supplemented or not with 1 mM 2,2′-azobis(2-amidinopropane) dihydrochloride, an inducer of reactive oxygen species. Blastocysts were collected at Day 7, and apoptosis was evaluated by an immunofluorescent staining of cleaved caspase-3 (8 replicates, n = 175). Total and apoptotic cells were counted using an epifluorescence microscope. As expected, embryos cultured under stress conditions from Day 5 pi presented a lower blastocyst rate at Day 7 (10.9 ± 1.0% v. 23.1 ± 1.9% for the control group; standard least squares, P &lt; 0.0001). The stressed blastocysts also showed fewer cells (113 ± 3 v. 139 ± 4; P &lt; 0.0001) and higher apoptotic rates (15.3 ± 0.9% v. 9.4 ± 0.6%; P &lt; 0.0001). As previously observed, the mean total cell number of the blastocysts was higher for males than females, whatever the culture condition (stress: males: 119 ± 4, females: 108 ± 4; control: males: 144 ± 5, females: 131 ± 6; sex effect: P = 0.005; interaction of sex × condition: P = 0.9). Interestingly, the sex ratio of the blastocysts was significantly different between control and stress conditions (χ2, P = 0.02); whereas a deviation in favour of the male embryos was observed in the control group (males: n = 57, 61%, females: n = 37, 39%; P = 0.04), it disappeared when embryos were submitted to oxidative stress (males: n = 35, 43%, females: n = 46, 57%; P = 0.22). However, oxidative stress had a similar impact on male and female blastocysts regarding the apoptotic rates (stress: males: 15.3 ± 1.3%, females: 15.4 ± 1.3%; control: males: 8.9 ± 0.7%, females: 10.2 ± 1.1%; standard least squares, sex effect: P = 0.99; interaction of sex × condition: P = 0.3). In conclusion, female embryos seem more resistant to oxidative stress than male ones when the stress is induced from Day 5 pi. Oxidative stress has a similar impact on the apoptotic rates in male and female blastocysts. The higher rate of apoptosis previously observed in female blastocysts can thus not be explained by a higher sensitivity of female embryos to oxidative stress. This is in accordance with the higher level of expression of several X-linked genes related to antioxidant pathways in female blastocysts.

76 RELATIONSHIP BETWEEN BLASTOCYST CELL NUMBER AT EMBRYO TRANSFER AND CONCEPTUS ELONGATION ON DAY 14: CAN SUPPLEMENTARY PROGESTERONE RESCUE SMALL EMBRYOS?

Conceptus size on Day 14 following multiple embryo transfer of Day 7 in vitro-produced blastocysts varies greatly within animal. One explanation for this variation may be related to blastocyst cell number at the time of transfer. The aim of this study was to examine the effect of Day 7 blastocyst cell number on Day 14 conceptus size and to examine the effect of progesterone (P4) supplementation on embryo development following the transfer of Day 7 blastocysts containing a low total cell number. The oestrous cycles of crossbred beef heifers were synchronised using an 8-day CIDR (1.38 g of P4) treatment with administration of a prostaglandin F2α analogue (Estrumate™, Merck Animal Health, Summit, NJ, USA) on the day before CIDR removal. Only those heifers recorded in standing oestrus (Day 0) were used. Heifers were randomly assigned to 1 of 4 treatment groups: (1) Control: large blastocysts (high total cell number), (2) Control: small blastocysts (low total cell number), (3) small blastocysts plus a single intramuscular injection of 3000 IU of hCG (Chorulon®, Merck Animal Health) on Day 2 after oestrus, or (4) small blastocysts plus insertion of a vaginal progesterone insert (PRID, 1.55 g of P4) between Days 3 to 5 after oestrus. In vitro-produced blastocysts were transferred to each heifer on Day 7 (n = 10 blastocysts per heifer) and conceptuses were recovered at slaughter on Day 14. A sample of blastocysts was fixed and stained using Hoechst 33342 to determine the mean total cell number per group. Daily blood samples were collected from Day 0 to 14 to measure serum P4 concentrations. Data were analysed using the PROC MIXED procedure of SAS (SAS Institute Inc. Cary, NC, USA). Total cell number on Day 7 was significantly lower in small compared with large blastocysts (72.4 ± 3.93 v. 144.8 ± 3.90; P &lt; 0.05). Conceptus recovery rate was 53.8% overall (140/260) and was highest in the large blastocyst group (68.3%, 41/60) compared with the other groups (45.7–55.0%). Concentrations of serum P4 were similar in the 2 unmanipulated recipient groups but were significantly elevated (P &lt; 0.05) by Day 8 in hCG-treated heifers and on Days 4 and 5 in the PRID group (P &lt; 0.003). In the absence of supplemental P4, Day 14 conceptuses resulting from the transfer of small blastocysts (2.48 ± 0.54 mm) were smaller than those from large blastocysts (3.32 ± 0.52 mm). Administration of hCG on Day 2 approximately doubled conceptus length on Day 14 (4.94 ± 1.15 mm), whereas insertion of a PRID from Day 3 to 5 increased conceptus length ~5-fold (13.09 ± 2.11 mm; P &lt; 0.05) compared with controls. In conclusion, results indicate that supplemental P4 is capable of “rescuing” poor quality blastocysts, presumably via the now well-described actions on the endometrium and consequent effects on uterine lumen fluid composition. Supported by Science Foundation Ireland (07/SRC/B1156).

41 EFFICIENT STRATEGY FOR INTERSPECIFIC CLONING IN FELIDS

Most of the 36 species of wild felids are at a level of threat, and interspecific SCNT (iSCNT) comes as a strategy to contribute to these species conservation. The aim of this study was to evaluate the effect of embryo aggregation in cheetah (Ch, Acinonyx jubatus), bengal (Ben, a hybrid between Felis silvestris and Prionailurus bengalensis), and domestic cat (DC, Felis silvestris) embryos generated by cloning. DC oocytes were in vitro matured and zona-free SCNT (with DC fibroblasts) or iSCNT (with Ch or Ben fibroblasts) was performed. The reconstructed embryos were activated with 5 μM ionomycin and 1.9 mM 6-DMAP, and cultured in SOF using microwells. Cloned embryos were cultured individually or as 2-embryo aggregates. The experimental groups were Ch1X, Ch2X, Ben1X, Ben2X, and the control groups were DC1X and DC2X. Embryo development was compared by Fisher's exact test (P ≤ 0.05). Embryo aggregation improved cleavage (Day 2) and blastocyst (Day 7) rates per well in all the groups (87.2% v. 96.7%, 83.8% v. 93.3% and 87.6% v. 98.2% for cleavage; and 13.7% v. 28.6%, 33.3% v. 43.8% and 27.4% v. 47.7% for blastocyst, for Ch1X (n = 102), Ch2X (n = 91), Ben1X (n = 154), Ben2X (n = 105), DC1X (n = 113), and DC2X (n = 109), respectively. Moreover, the Ch2X blastocyst rate was statistically similar as the control group DC1X. The mean total cell numbers of the blastocysts obtained were 264 ± 211 and 400.8 ± 97 for Ch1X and Ch2X, 278 ± 62 and 517 ± 104 for Ben1X and Ben2X, 385 ± 127 and 625 ± 183 for DC1X and DC2X, respectively. Although no statistical differences were obtained between the 1X and 2X groups, the 2X groups nearly doubled the average number of cells compared with the 1X groups. Blastocysts were also classified as grade 1 (expanded blastocysts with a well-defined ICM), grade 2 (expanded blastocysts without a well-defined ICM), and grade 3 (not expanded blastocysts). This classification showed an increase in grade 1 DC2X blastocyst compared with DC1X blastocysts (36.7% v. 16.1%), but no differences were observed in the other species. Expression of OCT-4 was assessed by inmunocytochemistry. The cheetah blastocysts markedly over-expressed this protein: the percentage of cells that expressed OCT-4 in Ch1X, Ch2X, Ben1X, Ben2X, DC1X, and DC2X was 88.2, 80.2, 46.3, 45.4, 51, and 47.4%, respectively, with statistical differences among all the groups except Ben1X and Ben2X. The proportion of OCT-4 expressing cells over total cell numbers was analysed by the difference of proportions test (P ≤ 0.05). In conclusion, iSCNT resulted in high rates of blastocyst formation, especially when embryo aggregation was applied. This strategy has not been previously evaluated in felids or iSCNT procedures, and has been demonstrated to improve blastocyst formation, the number of cells in the 3 groups, and the blastocyst quality in the DC. Other pluripotent genes besides OCT-4 should be studied to determine whether the overexpression of this gene in cheetah embryos is the consequence of an inefficient nuclear reprogramming that prevents a correct regulation. Finally, the iSCNT and embryo aggregation could contribute to species conservation in felids.

Development and partial validation of a mouse model for predicting drug hypersensitivity reactions

Animal models that can be used to predict the allergenic potential of drug candidates have not been adequately optimized, validated, or characterized. While initial validation data from an inter-laboratory study of the mouse lymph node proliferation assay (LNPA) appeared promising, no additional investigations in this model have been reported. The objectives of this study were to use positive and negative control drugs to further optimize and validate the LNPA utilizing a non-radioactive endpoint and determine the sensitivity, specificity, and predictivity of the model. Drugs associated with hypersensitivity reactions in the literature were chosen to test in the model in addition to drugs with few or no reports of hypersensitivity. Mice received a subcutaneous injection of drug or vehicle into the scruff of the neck once daily for a period of 3 days. On Day 6, draining lymph nodes were harvested, single cell suspensions prepared, and total cell numbers determined for each animal by flow cytometry. A stimulation index was calculated by dividing the mean total cell number for the drug-treated group by the mean total cell number for the vehicle-treated animals. Based on statistical analysis of the data, animals with a total cell number ≥2.5× the mean of the vehicle group were classified as ‘responders’. Based on data generated to date with 12 positive control and six negative control drugs, the model had a sensitivity of 75%, a specificity of 74%, and a relatively good predictive value (measured by the Receiver Operating Characteristic AUC of 0.80). The data here suggest that this model may be a useful tool for identifying drug candidates with the potential to produce allergic responses in the clinic. Future studies will investigate the mechanism(s) for the lymph node responses in order to develop additional endpoints that may increase the sensitivity and specificity of the model.

38 GENERATION OF INTERSPECIFIC CLONED BLASTOCYSTS BY ZONA PELLUCIDA-FREE NUCLEAR TRANSFER IN WILD FELIDS

Somatic cell nuclear transfer is an assisted reproductive technique that could help to preserve endangered species. Because it is difficult to obtain wild felid oocytes, interspecific cloning using domestic cat (DC) oocytes is an alternative to produce cloned embryos in these species. The aim of this study was to evaluate different cloning strategies in the DC (Felis silvestris catus) and to use the most efficient strategy to generate wild felid embryos by interspecific cloning. First, we evaluated 3 different cloning strategies: (1) enucleation of DC oocytes with zona pellucida (ZP) followed by fusion of a DC fibroblast that was injected into the perivitelline space (ZP-enclosed group), (2) the same enucleation procedure followed by intracytoplasmic injection of a DC fibroblast (ZPi group), and (3) enucleation of ZP-free oocytes followed by adhesion and fusion of a DC fibroblast (ZP-free group). After 2 h of nuclear reprogramming, the reconstructed embryos were activated with 5 µM ionomycin and 1.9 mM DMAP, and cultured in SOF. The ZP-free embryos were cultured in wells of the well system. Embryo development among treatment groups was compared by the Fisher exact test (P ≤ 0.05). The blastocyst rates were similar among the 3 groups: 11.1% (2/18), 11.1% (5/45), and 12.7% (9/71), for ZP-enclosed, ZPi, and ZP-free, respectively. However, the quantity of reconstructed embryos after the procedure was higher in the ZP-free clones because of a higher fusion rate (82.7 vs. 25.4%) and the use of a less-invasive technique than the injection. Moreover, the percentage of expanded blastocysts was also higher (0, 16.2, and 77.8% for ZP-enclosed, ZPi, and ZP-free, respectively). Parthenogenetic controls, with and without ZP, did not differ in blastocyst rates: 47.7% (42/88) and 49.4% (38/77), respectively. After this, the ZP-free strategy was chosen for the successive experiment. In experiment 2, the wild felid species selected for interspecific cloning were Bengal (a hybrid between Felis silvestris and Prionailurus bengalensis; FP group), cheetah (Acinonyx jubatus; AJ group), and tiger (Panthera tigris; PT group). The morula and blastocyst rates were higher in the FP group: 34.3% (36/105), 16.2% (16/99), and 17.5% (11/63) for morulae, and 33.3% (35/105), 1% (1/99), and 3.2% (2/63) for blastocysts of FP, AJ, and PT, respectively. Additionally, total cell number and the expression pattern of octamer-binding transcription factor 4 (Oct-4) were examined in the blastocysts by immunocytochemistry. The mean total cell number in DC, FP, AJ, and PT blastocysts was 177.9 ± 53, 229 ± 40, 53, and 41, respectively. All blastocysts expressed Oct-4 but in different proportions. The percentage of cells expressing Oct-4 in DC, FP, AJ, and PT blastocysts was 48, 66, 100, and 98%, respectively. In summary, ZP-free cloning was found to be an efficient technique in DC, with potential to be used in wild felid species. We also demonstrated that DC oocytes were able to reprogram cells of other genera. This is the first report of felid ZP-free cloning and also the first time that tiger and cheetah embryos were produced by interspecific cloning.

Lacking expression of paternally-expressed gene confirms the failure of syngamy after intracytoplasmic sperm injection in swamp buffalo (Bubalus bubalis)

The objectives were to: (1) examine the efficiency of intracytoplasmic sperm injection (ICSI) technique, with or without chemical activation of in vitro matured buffalo oocytes, on sperm head decondensation; and (2) compare the subsequent development of embryos after activation of ICSI (ICSI (+) activation group) and sham injection (Sham (+) activation group) oocytes (embryos obtained by in vitro fertilization of IVM oocytes served as a control group). Pronuclear formation rates in ICSI (+) activation and Sham (+) activation groups were higher than that of ICSI without activation (P < 0.05). However, because 90.9% of presumptive zygotes in ICSI (+) activation group demonstrated pronuclear formation with an intact sperm head, we inferred that most were parthenotes. Neither developmental competence (morula and blastocyst formation rates) nor mean total cell number of blastocysts was significantly different among ICSI (+) activation, Sham (+) activation, and IVF groups. To clarify whether blastocysts were derived from syngamy or parthenogenesis, expression of Nnat, a paternally expressed gene in blastocysts derived from IVF, ICSI and oocyte activation without sperm or sham injection was additionally examined using reverse transcription polymerase chain reaction (RT-PCR). Expression of Nnat mRNA was not detected in ICSI (+) activation blastocysts, indicating failure of male genome activation. Although blastocyst development after ICSI combined with chemical activation was similar to IVF oocytes, these blastocysts were generated by parthenogenesis, due to failure of male pronucleus formation.

UNBIASED STEREOLOGICAL ESTIMATION OF DIFFERENT CELL TYPES IN RAT CEREBRAL CORTEX

The total numbers of neurons, glial cells, and endothelial cells in rat cerebral cortex were estimated using unbiased stereological counting techniques and systematic sampling. The reference volume chosen was the entire neocortex, most of the allocortex and parts of claustrum using the rhinal fissure as the macroscopical anatomical landmark. These regions are referred to collectively as syncortex. A method has been devised for reducing problems associated with the uncertainties that arise when distinguishing between various types of cells. At the light microscopic level, using the detailed criteria described in this article, the total numbers of neurons, glial cells, and endothelial cells, respectively, were estimated for the entire syncortex as the product of the estimate of the volume of the syncortex, made with point counting techniques, and the estimates of the numerical density for each group of cells, made with optical disectors. In a sample of three brains, the mean total number of cells (neurons, glial and endothelial) in the syncortex of the rat brain is 128 x 106. This number is made up of 47% neurons, 24% glial cells, 17% endothelial cells, and 11% uncertain cell types (probably mostly glial cells).

132 EFFECTS OF OXYGEN TENSION, MEDIUM, AND WELL OF THE WELL ON IN VITRO DEVELOPMENT OF THE MOUSE EMBRYO

The objectives of the present research were to investigate whether embryo culture media have preferences for oxygen tension, to explore the feasibility of using physical lung air to support the in vitro development of mouse embryos, and to evaluate the effect of well of the well (WOW) culture on in vitro preimplantational development of mouse embryos. The results are as follows. First, cleavage rate and blastocyst rate were not significantly different between medium CZB and mKSOM regardless of using 3 gas phases: 4% CO2 + 16% O2 + 78% N2 + 2% H2O (lung air), 5% CO2 + 5% O2 + 90% N2 (5% O2, low oxygen), and 5% CO2 + 95% air (20% O2, high oxygen; P &gt; 0.05), but mean total cell numbers per blastocyst cultured in CZB medium were higher than those in mKSOM when the lung air was used (P &lt; 0.05). Second, when mKSOM was used as the basic medium, the blastocyst rate (22.6%) in the 5% O2 gas phase was notably higher than that in other 2 gas phases (P &lt; 0.05). Third, for the CZB medium, the blastocyst rate was not different significantly among 3 gas environments (P &gt; 0.05). Fourth, both the blastocyst rate (74.6 ± 5.1%) and the mean total cell numbers per blastocyst (76 ± 2) cultured in the WOW system were obviously higher than those in the group culture system (38.2 ± 6.6% and 58 ± 4). Taken together, our results indicate that mKSOM and CZB have preferences for oxygen tension during in vitro culture of mouse embryos, the lung air was reaffirmed to be able to effectively support in vitro preimplantation development of mouse embryos, and the WOW culture system can apparently enhance in vitro developmental competence and blastocyst quality of mouse embryos. L. YS, C. ZB: equal contribution; supported by NSFC (30700574), 863 (2008AA101003).

Stimulatory effect of Rho-associated coiled-coil kinase (ROCK) inhibitor on revivability of in vitro-produced bovine blastocysts after vitrification

Inhibition of Rho-associated coiled-coil kinase (ROCK) activity promoted recovery and growth of frozen-thawed human embryonic stem cells. The primary objective was to determine if a ROCK inhibitor (Y-27632) in post-thaw culture medium improved revivability of vitrified IVP bovine blastocysts. Expanding or expanded blastocysts (7 d after IVF) were vitrified (minimum volume cooling procedure, using a Cryotop) in 15% ethylene glycol, 15% DMSO and 0.5 M sucrose. When post-warm blastocysts were cultured in mSOF medium, survival rate (re-expansion of blastocoel at 24 h of culture) was improved ( P < 0.05) by the addition of 10 μM Y-27632 (94.9 ± 2.4%, mean ± SEM) compared to a control (78.0 ± 6.0%). Conversely, after 48 h of culture, there were no significant differences in hatching rate (62.8 ± 11.1 vs. 59.6 ± 9.4%) and mean total cell number (135.2 ± 13.1 vs. 146.7 ± 13.3). In non-vitrified IVP bovine blastocysts, the hatching rate on Day 9 was improved by Y-27632 (91.7 ± 3.8 vs. 54.7 ± 8.9%, P < 0.05), with no difference in mean total cell number of blastocysts (230.0 ± 23.0 vs. 191.2 ± 22.2, P = 0.23). In an additional experiment, Y-27632 was added to culture medium on either Day 0, Day 2, or Day 4 (and remained present until Day 8), resulting in no improvement in blastocyst yield compared to a control group (7.5 ± 2.1, 31.4 ± 2.3, 36.2 ± 3.2, and 28.6 ± 6.9%, respectively). In conclusion, adding a ROCK inhibitor to post-thaw culture medium improved revivability of IVP bovine blastocysts after vitrification and warming.

Comparison of the Cellsearch Profile Kit (CPK) with the Standard Cellsearch Epithelial Kit (CEK) Demonstrates the CPK Produces Higher Circulating Tumor Cell (CTC) Yields and Is Better Suited for Use in Obtaining CTC's for Molecular Characterization.

Abstract Background: CTC offer a potential non-invasive approach to obtain and characterize metastatic tumor cells from solid cancers. However, conventional methods for isolating CTC, which use Epcam specific immunomagnetic beads, have been limited by low CTC yields and contaminating leukocytes. We sought to optimize and characterize CTC isolation using the CPK(Veridex LLC) and compare it to conventional methods using the CEK and automated CellTracks counting system, the FDA approved standard. We hypothesized that the CPK, which avoids the potential CTC loss and destruction associated with the multiple washes and labeling steps included in the CEK, would offer improved cell recovery.Methods: We collected two peripheral blood samples from patients (Pts) with metastatic breast cancer (n = 75) and processed them in parallel. Following automated enumeration of the samples with the CEK method, the cells were washed, fixed, and processed as cytospin slides followed by H&amp;E and FISH. Uncounted samples processed with the CPK method were similarly washed, fixed, and processed as cytospin slides, followed by either H&amp;E, IHC (AE1/AE3), IF (HER2, pHER2, EGFR, pEGFR) or FISH using probes against CEP7, EGFR, HER2, and MET.Results: The median cell count from pts with breast cancer using the CPK method was 120 per 7.5 mL of blood (range 4-4967) compared to 4 (range 0-57) using the CEK method (p &amp;lt; 0.0001; t-test). The purity of these cells was high: 59.7 % of cells isolated by CPK (range: 20.9-95.0) were identified as tumor cells by cytokeratin staining while 6.4% (range: 0-21.8) were identified as WBCs by CD45 staining. The remaining 32.3% (range: 4.8-68.0) of cells stained with DAPI only and could not be classified as either tumor cells or lymphocytes, and appeared to have lost their membrane during the isolation. We confirmed the purity of the CTC by HER2 FISH. Among pts whose primary cancer was HER2+ (n=70), 72.4% (range: 44-100%) of CTC had an HER2 gene copy number &amp;gt; 4. From samples isolated from healthy controls (n = 40) and processed using the CPK method the mean total number of cells was 5 per 7.5 mL of blood (range 0-20). These cells were positive for CD45 with only 2 copies of HER2 by FISH. The CPK had low intrapatient variability (CV 9.6%) and samples were stable for at least 72hr. Recovered CTC were well suited for evaluation by IF for phospho antibodies (e.g. pHER2, pAkt), TUNEL (apoptosis), and Ki67 (proliferation). Studies using BC cell lines demonstrated that viable cells could be recovered and propagated with an input as few as 50 cells consistently yielding viable cultures.Conclusion: The CPK technique's relatively high yield, purity, and sample stability make it well suited for use in obtaining CTC's for molecular characterization, including FISH and IF. Assays for mutation detection are currently being evaluated. This technology potentially has a large number of applications in investigating the biology of metastatic cancer as well as in drug development where it can be used to identify predictive biomarkers, mechanisms of resistance, and facilitate pharmacodynamic studies. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 3003.

154 THE EFFECT OF OXYGEN TENSION ON IN VITRO DEVELOPMENT OF BOVINE EMBRYOS IN POLYDIMETHYLSILOXANE-BASED WELL OF THE WELL DISHES PREPARED UNDER ATMOSPHERIC OR REDUCED AIR PRESSURE

Polydimethylsiloxane (PDMS) is a non-toxic silicon compound. Its excellent optical characteristics and easy preparation make it a good candidate material for the molding of custom-shaped dishes for embryo culture. We investigated the feasibility of PDMS-based well of the well (WOW) dishes for in vitro culture of bovine embryos under different oxygen tensions. The WOW dishes with 25 micro-wells (each of 175 μm depth and 250 μm width in diameter arranged in 5 columns and 5 rows) were molded from PDMS prepared either under atmospheric (Experiment 1) or reduced (0.1 MPa) (Experiment 2) air pressure to remove air bubbles. Presumptive zygotes obtained by the in vitro maturation and fertilization of follicular oocytes were placed and cultured for 7 days in traditional micro-drops of culture medium (Control) or in the micro-wells of PDMS-based WOW dishes (PDMS-WOW), both covered by paraffin oil. The culture medium was CR1aa supplemented with 5% calf serum. The culture drop size was 125 μL (5 μL/oocyte) in both groups. Embryo development and blastocyst cell numbers between Control and PDMS-WOW groups were compared either under 20% or 5% O2 tensions. There was no statistical difference in cleavage and blastocyst rates (ranging between 82.3-86.4% and 34.0-45.8%, respectively) between Control and PDMS-WOW embryos irrespective of oxygen tension and dish production method. In Experiment 1, the mean total cell numbers in blastocysts were lower in the PDMS-WOW group than that in Control under 20% O2 (105.0 ± 5.5 and 130.4 ± 9.9, respectively) (P &lt; 0.05, ANOVA); however, the application of 5% O2 significantly improved the cell numbers and eliminated the difference between the PDMS-WOW and Control groups (135.4 ± 6.2 and 148.0 ± 9.0, respectively). In Experiment 2, there was no significant difference in mean total cell numbers in blastocysts between the PDMS-WOW and Control either under 20% O2 (97.2 ± 5.7 and 103.9 ± 8.9, respectively) or 5% O2 (147.5 ± 12.1 and 157.3 ± 3.9, respectively). The numbers and rates of inner cell mass and trophectoderm cells did not differ between the Control and PDMS-WOW groups, irrespective of O2 tension and production method. Our results demonstrate that bovine embryos can develop to the blastocyst stage in PDMS-based WOW dishes; however, it may express detrimental effects on embryonic cell numbers, which can be neutralized by the application of low O2 tension during culture or reduced air pressure during the PDMS preparation. This work was supported by the Research and Development Program for New Bio-Industry Initiatives.

Production of good-quality porcine blastocysts by in vitro fertilization of follicular oocytes vitrified at the germinal vesicle stage

We investigated survival, meiotic competence, cytoplasmic maturation, in vitro fertilization, and development of immature porcine (Sus scrofa) oocytes cryopreserved by a modified solid surface vitrification protocol. Cumulus-oocyte complexes (COCs) collected from follicles 3 to 6 mm in diameter in abattoir-derived ovaries of prepubertal gilts were either vitrified (Vitrified group), subjected to cryoprotectant treatment (CPA group), or used without any treatment (Control group). Oocyte viability was assayed by staining with fluorescein diacetate. Live oocytes were matured in vitro and their meiotic progression investigated by nuclear staining. In a series of experiments, the glutathione (GSH) content of in vitro–matured oocytes and viability of cumulus cells were assayed simultaneously. The in vitro–matured oocytes were also fertilized and cultured in vitro to assess their ability to be fertilized and to develop to the blastocyst stage, respectively. The proportion of viable oocytes in the Vitrified group was significantly lower than that in the CPA and Control groups (27.7%, 90.4%, and 100%, respectively). Among the three groups, there were no differences in meiotic competence, cumulus viability, and GSH levels at the end of in vitro maturation. Fertilization parameters (i.e., rates of male pronucleus formation, monospermy, and second polar body extrusion) were also similar among groups. However, comparison of the developmental abilities of oocytes in the Vitrified, CPA, and Control groups revealed that the Vitrified group had a significantly reduced ability to undergo first cleavage (34.4%, 63.3%, and 69.0%) and to develop to the blastocyst stage (5.1%, 25.5%, and 34.6%). The mean total cell numbers in blastocysts after 6 d of culture were not significantly different among the Vitrified, CPA, and Control groups (40.3, 42.8, and 43.4). In conclusion, despite low survival rates and impaired development in the Vitrified group, meiotic competence, cytoplasmic maturation, and subsequent fertilization characteristics of surviving germinal vesicle oocytes were unaffected by vitrification, and high-quality blastocysts were produced from vitrified immature oocytes.