Mihai Cenariu

Evaluation of Bovine Embryo Biopsy Techniques according to Their Ability to Preserve Embryo Viability

The purpose of this research was to evaluate three embryo biopsy techniques used for preimplantation genetic diagnosis (PGD) in cattle and to recommend the least invasive one for current use, especially when PGD is followed by embryo cryopreservation. Three hundred bovine embryos were biopsied by either one of the needle, aspiration or microblade method, and then checked for viability by freezing/thawing and transplantation to recipient cows. The number of pregnancies obtained after the transfer of biopsied frozen/thawed embryos was assessed 30 days later using ultrasounds. The results were significantly different between the three biopsy methods: the pregnancy rate was of 57% in cows that received embryos biopsied by needle, 43% in cows that received embryos biopsied by aspiration, and 31% in cows that received embryos biopsied by microblade. Choosing an adequate biopsy method is therefore of great importance in embryos that will undergo subsequent cryopreservation, as it significantly influences their viability after thawing.

In vivo generation of beta-cell-like cells from CD34(+) cells differentiated from human embryonic stem cells.

OBJECTIVE CD34(+) cells, present within the bone marrow, have previously been shown to possess pancreatic endocrine potential. Based on this observation, we explored the capacity of CD34(+) cells derived in culture from the differentiation of human embryonic stem cells (hESC), for their in vivo pancreatic endocrine capacity. MATERIALS AND METHODS Sheep were transplanted with hESC-derived CD34(+) cells, as well as nonsorted differentiated cultures. Transplantations were carried out with in utero intraperitoneal injections prior to development of the immune system in the fetus so that tolerance toward foreign antigens was acquired during gestation and persisted in the adult. RESULTS All cell populations that were tested demonstrated human cellular activity and long-term presence up to 5 years. However, the in vivo beta-cell-like activity achieved from the transplantation of the sorted CD34(+) cell population was not augmented by transplanting the entire cell population from which the CD34(+) cells were isolated. Human DNA and insulin messenger RNA were detected in sheep pancreases. An average of 1.51 ng/mL human C-peptide was detected in serum from eight animals transplanted with differentiated cell populations and assayed up to 55 months posttransplantation. Transplantation of as few as 23,500 cells resulted in long-term sustainable beta-cell-like activity. Teratomas were absent in the transplanted animals. CONCLUSION Our data suggest that hESC-derived CD34(+) cells have a potential for long-term in vivo endocrine cellular activity that could prove useful in regenerative medicine. Because the same cell population has previously been shown to contain hematopoietic potential, it could be used for the induction of immunological tolerance and bone marrow chimerism prior to cellular therapy for diabetes.

Immune Ontogeny and Engraftment Receptivity in the Sheep Fetus

Objective: The biologic explanation for fetal receptivity to donor engraftment and subsequent long-term tolerance following transplantation early in gestation is not known. We investigated the role fetal immune ontogeny might play in fetal transplantation tolerance in sheep. Methods: Engraftmentof allogeneic and xenogeneicHSC was determined 60 days following transplantation at different time points in sheep fetal gestation. Parallel analysis of surface differentiation antigen expression on cells from lymphoid organs of timed gestational age fetal sheep was determined by flow cytometry using available reagents. Results: An engraftment window was identified after day 52 gestation lasting until day 71 (term gestation: 145 days). This period was associated with the expression of the leukocyte common antigen CD45 on all cells in the thymus. Double-positive and single-positive CD4 and CD8 cells began appearing in the thymus just prior (day 45 gestation) to the beginning of the engraftment window, while single-positive CD4 or CD8 cells do not begin appearing in peripheral organs until late in the engraftment period, suggesting deletional mechanisms may be operative. In concert, surface IgM-positive cells express CD45 in the thymus at day 45, with a comparable delay in the appearance of IgM/CD45 cells in the periphery until late in the engraftment window. Conclusions: These findings support a central role for the thymus in multilineage immune cell maturation during the period of fetal transplantation receptivity. Further, they suggest that fetal engraftment receptivity is due to gestational age-dependent deletional tolerance.

Effects of doxorubicin mediated by gold nanoparticles and resveratrol in two human cervical tumor cell lines

Green synthesis of gold nanoparticles capped with resveratrol (GNPs) and their physical and chemical characterization by UV-vis spectra, FTIR, DLS, XRD, TEM and AFM are reported. The GNPs are highly stable, with average diameter of about 20 nm. Then, supramolecular nanoassemblies of GNPs and doxorubicin (Dox), Dox-GNPs complexes, were prepared and morphologically characterized. The stability of these Dox nanocomplexes is high in phosphate buffer saline as estimated by UV-vis spectra, TEM and AFM analysis. Effects of resveratrol (Resv), Resv-Dox mixtures, GNPs and Dox-GNPs complexes on HeLa and CaSki cells, after 24h drug incubation, were assessed using MTT cell viability assay. Results showed strong anticancer activity for Resv-Dox mixtures and Dox-GNPs complexes in the two human cervical carcinoma cell lines. Clearly, both Resv and GNPs can mediate the anticancer activity of Dox at its very low concentration of 0.1 μg/mL, reaching the cytotoxicity of Dox alone, at its concentration up to 20 times higher. Cytotoxic effects of Resv-Dox mixtures and Dox-GNPs complexes have been found for the first time in HeLa and CaSki cells. Furthermore, the apoptosis induction in HeLa and CaSki cells was evidenced for Resv-Dox mixtures and Dox-GNPs complexes by flow cytometry using Annexin V-FITC/propidium iodide cellular staining. For CaSki cells, the apoptosis was also demonstrated, mainly for the treatment with Dox-GNPs complexes, by MTT formazan cellular staining visualized in phase contrast microscopy. Our results provide strong evidence that novel drug delivery vehicles developed on Dox-GNPs nanocomplexes and Resv could have wide applications in cancer diagnosis and treatment.

Raffinose and hypotaurine improve the post-thawed Merino ram sperm parameters ☆

The aim of this study was to determine the effects of raffinose and hypotaurine on sperm parameters after the freeze-thawing of Merino ram sperm. Totally 40 ejaculates of five Merino ram were used in the study. Semen samples, which were diluted with a Tris-based extender containing 10mM raffinose, 5mM hypotaurine, 5mM raffinose +2.5mM hypotaurine (H+R) and no antioxidant (control), were cooled to 5 °C and frozen in 0.25 ml French straws and stored in liquid nitrogen. Frozen straws were then thawed individually at 37 °C for 25s in a water bath for evaluation. The addition of raffinose led to higher percentages of subjective and CASA motilities (47.5 ± 12.2%, 46.3 ± 13.6%) compared to controls (38.8 ± 13.8%, 30.5 ± 11.7%, P<0.05). For the CASA progressive motility, 5mM raffinose (20.12 ± 8.82%) had increasing effect in comparison to control (10 ± 7.94%, P<0.05) following the freeze-thawing process. Raffinose and hypotaurine led to higher viability (40.8 ± 4.68%, 40.8 ± 4.7%), high sperm mitochondrial activity (29.5 ± 5.4%, 27.3 ± 4.9%) and acrosome integrity (50.8 ± 8.1, 50.7 ± 4.4) percentages, compared to control groups (31.5 ± 3.5%, 9.5 ± 8.2%, 42.8 ± 7.3%, P<0.05). H+R group only led to high sperm mitochondrial activity when compared to control group. In the comet test, raffinose and hypotaurine resulted in lower sperm with damaged DNA (6.2% and 3.9%) than that of control (9.1%), reducing the DNA damage. For TUNEL assay, The TUNEL-positive cell was distinguished by distinct nuclear staining. Raffinose and H+R groups resulted in lower sperm with TUNEL-positive cell (1.5 ± 1.2% and 2.1 ± 0.9%) than that of control (4.9 ± 2.5%) (P<0.05). In conclusion, findings of this study showed that raffinose and hypotaurine supplementation in semen extenders provided a better protection of sperm parameters against cryopreservation injury, in comparison to the control groups.

Immune checkpoint blockade: the role of PD-1-PD-L axis in lymphoid malignancies

The co-inhibitory receptor programmed cell death (PD)-1, expressed by immune effector cells, is credited with a protective role for normal tissue during immune responses, by limiting the extent of effector activation. Its presently known ligands, programmed death ligands (PD-Ls) 1 and 2, are expressed by a variety of cells including cancer cells, suggesting a role for these molecules as an immune evasion mechanism. Blocking of the PD-1-PD-L signaling axis has recently been shown to be effective and was clinically approved in relapsed/refractory tumors such as malignant melanoma and lung cancer, but also classical Hodgkin’s lymphoma. A plethora of trials exploring PD-1 blockade in cancer are ongoing. Here, we review the role of PD-1 signaling in lymphoid malignancies, and the latest results of trials investigating PD-1 or PD-L1 blocking agents in this group of diseases. Early phase studies proved very promising, leading to the clinical approval of a PD-1 blocking agent in Hodgkin’s lymphoma, and Phase III clinical studies are either planned or ongoing in most lymphoid malignancies.

Efficiency of photodynamic therapy on WM35 melanoma with synthetic porphyrins: Role of chemical structure, intracellular targeting and antioxidant defense.

Photodynamic therapy (PDT) could be an adjuvant therapy in melanoma, an aggressive cancer that arises from melanocytes. Several reports showed encouraging results of the efficacy of PDT in melanoma on experimental models and in clinical trials. Therefore, we studied the efficacy of two derivatives of tetraphenylporphyrin (TPP): meso-5,10,15,20-tetrakis (4-hydroxyphenyl) porphyrin (THOPP) and meso-5-(4-hydroxyphenyl)-10,15,20-tris (4-methoxyphenyl) porphyrin (THOMPP) as photosensitizers for PDT, compared to FDA approved delta aminolevulinic acid (ALA) against a lightly pigmented, melanoma cell line, WM35, in vitro. Both porphyrins were more efficient as photosensitizers, compared to ALA, without dark toxicity. The efficiency depended on the intracellular localization and the molecule structure. THOPP, the most efficient porphyrin localized mainly in mitochondria, while THOMPP accumulated in lysosomes; both showed melanosomal localization. The symmetric THOPP molecule was able to generate increased oxidative stress damage and apoptosis. THOPP also induced a low effect on the defense mechanisms like antioxidant enzyme SOD (superoxide dismutase), NF-kB (nuclear transcription factor kB) activation and MITF (microphthalmia transcription factor). The lower efficiency of the asymmetric molecule, THOMPP was probably due to a diminished photoactivation, which led to a lower ROS induced damage, combined with higher activation of the defense mechanisms.

In vitro studies on the mechanisms involved in chemoprevention using Calluna vulgaris on vascular endothelial cells exposed to UVB.

The study aims to investigate the mechanisms involved in the in vitro effect of UVB on endothelial vascular cells (HUVECs) pretreated with a photochemopreventive agent, the Calluna vulgaris (Cv) extract. Two concentrations of Cv, below the limit of cytotoxicity IC50 (2.5 and 7.5 μg GAE/ml) and two doses of UVB (50 and 100 mJ/cm(2)) were used. Oxidative stress parameters were quantified at 1 h and 24 h after irradiation and apoptosis, DNA damage and the induction/activation of NF-κB were evaluated at 24 h. UVB exposure led to the formation of lipid peroxides in a dose dependent manner (p<0.001), induced apoptosis, increased the γ-H2AX levels and the activation of NF-κB. Pretreatment with 2.5 μg GAE/ml Cv improved the antioxidant defense, protected against DNA lesions and was able to decrease cellular death at low dose of irradiation. 7.5 μg GAE/ml Cv was prooxidant, favored the formation of DNA lesions, amplified the NF-κB activation UVB-induced (p<0.01) and led to high levels of cellular death. Both doses of Cv inhibited caspase-3 activation. The modulatory effect of Cv extract on endothelial cells exposed to UVB depend on the concentration of Cv used. This study provides insides into the mechanisms triggered by UVB and antioxidants on skin endothelial cells.

Comparative Assessment of Oral Mesenchymal Stem Cells Isolated from Healthy and Diseased Tissues

The aim of the present study was to isolate human mesenchymal stem cells (MSCs) from palatal connective and periodontal granulation tissues and to comparatively evaluate their properties. MSCs were isolated using the explant culture method. Adherence to plastic, specific antigen makeup, multipotent differentiation potential, functionality, and ultrastructural characteristics were investigated. The frequency of colony-forming unit fibroblasts for palatal-derived mesenchymal stem cells (pMSCs) was significantly higher than that of granulation tissue-derived mesenchymal stem cells (gtMSCs). A significantly higher population doubling time and lower migration potential were recorded for gtMSCs than for pMSCs. Both cell lines were positive for CD105, CD73, CD90, CD44, and CD49f, and negative for CD34, CD45, and HLA-DR, but the level of expression was different. MSCs from both sources were relatively uniform in their ultrastructure. Generally, both cell lines possessed a large, irregular-shaped euchromatic nucleus, and cytoplasm rich in mitochondria, lysosomes, and endoplasmic reticulum. The periphery of the plasma membrane displayed many small filopodia. MSCs from both cell lines were successfully differentiated into osteogenic, adiopogenic, and chondrogenic lineages. Both healthy and diseased tissues may be considered as valuable sources of MSCs for regenerative medicine owing to the high acceptance and fewer complications during harvesting.

Combined regimen of photodynamic therapy mediated by Gallium phthalocyanine chloride and Metformin enhances anti-melanoma efficacy

Background Melanoma therapy is challenging, especially in advanced cases, due to multiple developed tumor defense mechanisms. Photodynamic therapy (PDT) might represent an adjuvant treatment, because of its bimodal action: tumor destruction and immune system awakening. In this study, a combination of PDT mediated by a metal substituted phthalocyanine—Gallium phthalocyanine chloride (GaPc) and Metformin was used against melanoma. The study aimed to: (1) find the anti-melanoma efficacy of GaPc-PDT, (2) assess possible beneficial effects of Metformin addition to PDT, (3) uncover some of the mechanisms underlining cell killing and anti-angiogenic effects. Methods Two human lightly pigmented melanoma cell lines: WM35 and M1/15 subjected to previous Metformin exposure were treated by GaPc-PDT. Cell viability, death mechanism, cytoskeleton alterations, oxidative damage, were assessed by means of colorimetry, flowcytometry, confocal microscopy, spectrophotometry, ELISA, Western Blotting. Results GaPc proved an efficient photosensitizer. Metformin addition enhanced cell killing by mechanisms dependent on the cell line, namely apoptosis in the metastatic M1/15 and necrosis in the radial growth phase, WM35. Cell death mechanism relied on the inhibition of nuclear transcription factor (NF)-κB activation and tumor necrosis factor (TNF)—related apoptosis-inducing ligand (TRAIL) sensitization, leading to TRAIL and TNF-α induced apoptosis. Metformin diminished the anti-angiogenic effect of PDT. Conclusions Metformin addition to GaPc-PDT increased tumor cell killing through enhanced oxidative damage and induction of proapoptotic mechanisms, but altered PDT anti-angiogenic effects. General significance Combination of Metformin and PDT might represent a solution to enhance the efficacy, leading to a potential adjuvant role of PDT in melanoma therapy.