Early transcriptomic changes in peripheral blood 7 days after embryo transfer in dairy cattle.

A common goal of the dairy industry is to shorten the calving interval to reap several benefits associated with improved fertility. Early pregnancy detection is crucial to shorten this interval, allowing for prompt re-insemination of cows that failed to conceive after the first service. Currently, the industry lacks a method to accurately predict pregnancy within the first 3 wk. The polypeptide cytokine IFN-tau (IFNT) is the primary signal for maternal recognition of pregnancy in ruminants. As IFNT is released from the early conceptus, it initiates a cascade of effects, including upregulation of IFN-stimulated genes (ISG). Expression of ISG can be detected in the peripheral blood. The present study aimed to characterize peripheral transcriptomic changes, including the ISG, as early as d 7 after embryo transfer. A total of 170 Holstein heifers received in vitro-produced embryos. Whole blood was collected from these heifers within 24 h of the embryo transfer (d 0), d 7, and d 14 after embryo transfer. The heifers were divided into 2 groups, pregnant and nonpregnant, based on pregnancy diagnosis on d 28 via ultrasound. Total RNA was extracted from the peripheral blood of pregnant and nonpregnant heifers, pooled and sequenced. Expression analysis on d 7 heifers resulted in 13 significantly differentially expressed genes mostly related to innate immunity. Differential expression analysis comparing pregnant heifers on d 0 to the same heifers on d 14 showed 51 significantly differentially expressed genes. Eight genes were further quantified through reverse-transcription quantitative real-time PCR for biological validation. On d 7 after embryo transfer, mRNA transcriptions of EDN1, CXCL3, CCL4, and IL1A were significantly upregulated in pregnant heifers (n = 14) compared with nonpregnant heifers (n = 14), with respective fold changes of 8.10, 18.12, 29.60, and 29.97. Although on d 14 after embryo transfer, mRNA transcriptions of ISG15, MX2, OASY1, and IFI6 were significantly upregulated in the blood of pregnant heifers (n = 14) compared with the same heifers on d 0, with respective fold changes of 5.09, 2.59, 3.89, and 3.08. These findings demonstrate that several immune-related genes and ISG are activated during the first 2 wk after embryo transfer, which may explain how the maternal immune system accommodates the allogenic conceptus. To further investigate the diagnostic potentials of these genes, future studies are warranted to analyze the specificity and sensitivity of these biomarkers to predict early pregnancy.

Mitochondrion-targeted antioxidant SkQ1 prevents rapid animal death caused by highly diverse shocks.

The response to stress involves the activation of pathways leading either to protection from the stress origin, eventually resulting in development of stress resistance, or activation of the rapid death of the organism. Here we hypothesize that mitochondrial reactive oxygen species (mtROS) play a key role in stress-induced programmed death of the organism, which we called "phenoptosis" in 1997. We demonstrate that the synthetic mitochondria-targeted antioxidant SkQ1 (which specifically abolishes mtROS) prevents rapid death of mice caused by four mechanistically very different shocks: (a) bacterial lipopolysaccharide (LPS) shock, (b) shock in response to intravenous mitochondrial injection, (c) cold shock, and (d) toxic shock caused by the penetrating cation C12TPP. Importantly, under all these stresses mortality was associated with a strong elevation of the levels of pro-inflammatory cytokines and administration of SkQ1 was able to switch off the cytokine storms. Since the main effect of SkQ1 is the neutralization of mtROS, this study provides evidence for the role of mtROS in the activation of innate immune responses mediating stress-induced death of the organism. We propose that SkQ1 may be used clinically to support patients in critical conditions, such as septic shock, extensive trauma, cooling, and severe infection by bacteria or viruses.

Local immune system in oviduct physiology and pathophysiology: attack or tolerance?

The local immune system in the oviduct has a unique ability to deal with pathogens, allogeneic spermatozoa, and the semi-allogeneic embryo. To achieve this, it seems likely that the oviduct possesses an efficient and strictly controlled immune system that maintains optimal conditions for fertilization and early embryo development. The presence of a proper sperm and/or embryo-oviduct interaction begs the question of whether the local immune system in the oviduct exerts beneficial or deleterious effects on sperm and early embryo; support or attack?. A series of studies has revealed that bovine oviduct epithelial cells (BOECs) are influenced by preovulatory levels of Estradiol-17ß, progesterone, and LH to maintain an immunologic homeostasis in bovine oviduct, via inhibition of proinflammatory responses that are detrimental to allogenic sperm. Under pathologic conditions, the mucosal immune system initiates the inflammatory response to the infection; the bacterial lipopolysaccharide (LPS) at low concentrations induces a proinflammatory response with increased expression of TLR-4, PTGS2, IL-1ß, NFκB1, and TNFα, resulting in tissue damage. At higher concentrations, however, LPS induces a set of anti-inflammatory genes (TLR-2, IL-4, IL-10, and PTGES) that may initiate a tissue repair. This response of BOECs is accompanied by the secretion of acute phase protein, suggesting that BOECs react to LPS with a typical acute proinflammatory response. Under physiological conditions, polymorphonuclear neutrophils (PMN) are existent in the oviductal fluid during preovulatory period in the bovine. Interestingly, the bovine oviduct downregulates sperm phagocytosis by PMN via prostaglandin E2 (PGE2) action. In addition, the angiotensin-endothelin-PGE2 system controlling oviduct contraction may fine-tune the PMN phagocytic behavior to sperm in the oviduct. Importantly, a physiological range of PGE2 supplies anti-inflammatory balance in BOEC. Our recent results show that the sperm binding to BOECs further shift the local immunity toward anti-inflammatory conditions with upregulation of IL-10, TGFß, and PGE2. In addition, this local environment leads PMN to express anti-inflammatory cytokines. In conclusion, the oviduct displays mucosal immunity that maintains an anti-inflammatory environment under physiological conditions that supports the sperm. Under pathologic condition, however, the oviduct supplies the innate immunity that may attack the sperm. Moreover, the oviduct-sperm interaction further suppresses the innate immune cells and strengthens the anti-inflammatory balance in the oviduct. Therefore, the oviduct immunity ensures sperm viability before fertilization.

Short communication: Urea induces T helper 2 (Th2) type environment at transcriptional level and prostaglandin E2 secretion in bovine oviduct epithelial cells in culture.

Excess dietary protein intake in early lactation dairy cows resulting in blood urea nitrogen of greater than 19 to 20mg/dL is associated with decreased fertility. Little is known about the local interference of urea in the normal immunological environment of the oviduct that provides optimal conditions for early reproductive events. A bovine oviduct epithelial cell (BOEC) culture was used to determine how urea influences immune environment. The BOEC monolayer was supplemented with low (20mg/dL) and high (40mg/dL) concentrations of urea together with ovarian steroids, estradiol (1ng/mL) and progesterone (1ng/mL), and LH (10ng/mL) at concentrations observed during the preovulatory period. The urea values used in this study were equivalent to 9.3 and 18.7mg/dL of blood urea nitrogen, which are typically common in lactating dairy cows with low or high protein intake, respectively. Stimulation of BOEC with 40mg/dL of urea induced gene expression of IL10 and IL4, epithelial-derived T helper type 2-driving (anti-inflammatory) cytokines as well as mPGES-1 expression and prostaglandin E2 (PGE2) secretion. However, urea concentrations of both 20 and 40mg/dL failed to alter the expression of IL1B and TNFA, Th1-driving cytokines, and the gene expression of TLR4. However, a concentration of 40mg/dL of urea stimulated α 1-acid glycoprotein expression, an acute phase protein. Data from this in vitro study suggest that urea, at least in part, contributes to influence the expression of some immune-related genes toward T helper type 2 type and prostaglandin E2 secretion, leading to disruption in local environment for fertilization and early embryonic development.

[Stem Cells in the Brain of Mammals and Human: Fundamental and Applied Aspects].

Brain stem cells represent an extremely intriguing phenomenon. The aim of our review is to present an integrity vision of their role in the brain of mammals and humans, and their clinical perspectives. Over last two decades, investigations of biology of the neural stem cells produced significant changes in general knowledge about the processes of development and functioning of the brain. Researches on the cellular and molecular mechanisms of NSC differentiation and behavior led to new understanding of their involvement in learning and memory. In the regenerative medicine, original therapeutic approaches to neurodegenerative brain diseases have been elaborated due to fundamental achievements in this field. They are based on specific regenerative potential of neural stem cells and progenitor cells, which possess the ability to replace dead cells and express crucially significant biologically active factors that are missing in the pathological brain. For the needs of cell substitution therapy in the neural diseases, adequate methods of maintaining stem cells in culture and their differentiation into different types of neurons and glial cells, have been developed currently. The success of modern cellular technologies has significantly expanded the range of cells used for cell therapy. The near future may bring new perspective and distinct progress in brain cell therapy due to optimizing the cells types most promising for medical needs.

Evidence for a novel, local acute-phase response in the bovine oviduct: progesterone and lipopolysaccharide up-regulate alpha 1-acid-glycoprotein expression in epithelial cells in vitro.

Little is known about the local production and function of alpha 1-acid glycoprotein (AGP), an acute-phase protein, in the female reproductive tract. This study aimed to investigate the regulation and immune function of AGP in cultured bovine oviduct epithelial cells. Analysis by Western blotting and immunohistochemistry revealed that bovine oviduct tissue expresses AGP protein in epithelial cells and the smooth muscle layer. Stimulation of bovine oviduct epithelial cells in culture with either progesterone (1 ng/ml) or lipopolysaccharide (LPS, 10 ng/ml) induced both mRNA expression and secretion of AGP. Estradiol (1 ng/ml), progesterone (1 ng/ml), and luteinizing hormone (10 ng/ml), which are observed during the peri-ovulatory period in oviduct tissues (steroids) or in circulation (luteinizing hormone), suppressed LPS-induced expression and secretion of AGP, which in turn induced the expression of Toll-like receptor-4 (TLR-4) and interleukin-1ß (IL-1B), but suppressed TLR-2 and tumor necrosis factor-α (TNFA) expression. AGP also inhibited LPS-induced TLR-2 and TNFA expression, but had no effect on LPS-induced TLR-4 and IL-1B expression. These findings suggest that oviductal epithelial cells can participate in antimicrobial processes through the secretion of AGP, which is partly regulated by ovarian steroids. Moreover, oviductal AGP may regulate the response of epithelial cells, thereby reducing the expression of the acute pro-inflammatory cytokine TNFA, which could contribute to the local homeostasis during the acute response to endotoxin release in the oviduct's anti-infection process.

Effect of cell therapy on recovery of cognitive functions in rats during the delayed period after brain injury.

We studied the effect of systemic transplantation of human stem cells from various tissues on cognitive functions of the brain in rats during the delayed period after experimental brain injury. Stem cells were shown to increase the efficacy of medical treatment with metabolic and symptomatic drugs for recovery of cognitive functions. They accelerated the formation of the conditioned defense response. Fetal neural stem cells had a stronger effect on some parameters of cognitive function 2 months after brain injury. The efficacy of bone marrow mesenchymal stem cells from adult humans or fetuses was higher 3 months after brain injury.

Cell-to-cell cross-talk between mesenchymal stem cells and cardiomyocytes in co-culture.

The goals of the study were: (1) to explore the communication between human mesenchymal stem cells (MSC) and rat cardiac myocytes resulting in differentiation of the stem cells and, (2) to evaluate the role of mitochondria in it. Light and fluorescence microscopy as well as scanning electron microscopy revealed that after co-cultivation, cells formed intercellular contacts and transient exchange with cytosolic elements could be observed. The transport of cytosolic entity had no specific direction. Noticeably, mitochondria also could be transferred to the recipient cells in a unidirectional fashion (towards cardiomyocytes only). Transmission electron microscopy revealed significant variability in both the diameter of intercellular contacting tubes and their shape. Inside of these nanotubes mitochondria-resembling structures were identified. Moreover, after co-cultivation with cardiomyocytes, expression of human-specific myosin was revealed in MSC. Thus, we speculate that: (1) transport of intracellular elements to MSC possibly can determine the direction of their differentiation and, (2) mitochondria may be involved in the mechanism of the stem cell differentiation. It looks plausible that mitochondrial transfer to recipient cardiomyocytes may be involved in the mechanism of failed myocardium repair after stem cells transplantation.

Human neural stem cells normalize rat behavior after hypoxia.

Transplants of cultured neural stem cells from human brain survived, retained multipotent activity, and produced a neuroprotective effect on degenerating neurons in the brain of adult rats subjected to hypoxic hypoxia. They normalized animal behavior and improved conditioning in two-way avoidance response paradigm in a shuttle box.

Immunohistochemical study of fetal stem/progenitor cells from human brain transplanted into traumatized spinal cord of adult rats.

Neural stem/progenitor cells from human fetal brain were grown in a tissue culture and transplanted into traumatized spinal cord of adult rats. The behavior and differentiation of transplanted cells were studied morphologically by means of histological and immunohistochemical methods and confocal microscopy. Human neural stem/progenitor cells were viable for not less than 3 months. They migrated and differentiated into neurons and glia in the traumatized spinal cord of adult rats.

Transplantation of cultured human neural stem cells in rabbits with experimental laser-induced damage to the retina.

Cultured neural stem/progenitor cells from human fetal brain were transplanted into the retrobulbar and suprachoroid space in rabbits with laser-induced damage to the retina. Transplanted cells survived, retained multipotent activity, migrated into the zone of injury, and stimulated reparation and regeneration in the traumatized retina.

Xenotransplantation of stem/progenitor cells from human fetal brain to adult rats with spinal trauma.

In vitro grown neural stem cells from human fetal brain were transplanted to adult rats with spinal trauma. The spinal cord was examined morphologically using histological and immunohistochemical methods on days 5, 15, 30, and 110. Human neural stem/progenitor cells were viable, migrated, and differentiated into neurons and glia in the traumatized spinal cord in adult rats.

Evaluation of progenitor cell cultures from human embryos for neurotransplantation.

Human neural stem cells (HNSCs) are used in studies of neural development and differentiation, and are regarded as an alternative source of tissue for neural transplantation in degenerative diseases. Selection and standardization of HNSC samples is an important task in research and clinical approaches. We evaluated embryonal brain matter obtained from human 8-12-week-old fetuses by means of flow cytometry on a panel including: nestin; vimentin; NeuN; GFAP; beta-tubulin III; CD56; N-Cad; OB-Cad; HLA-ABC; HLA-DR; CD34, and annexin. Samples from embryos of even the same gestation differ dramatically regarding neural cell development, their phenotype and viability. The samples containing the highest proportion of stem cells and multipotent progenitors of neural types, and the least of definitive cells and antigens of histocompatibility, were selected for further expansion in serum-free medium. Secondary phenotyping 14 days later revealed again a marked heterogeneity of the cultures. For the final culturing for 24 h in a serum-containing medium we selected only samples having following phenotype: nestin+, and vimentin+ no less than 25%; HLA-DR+ and CD34+ no more than 5%; GFAP+ no more than 10%; beta-tubulin+ no more than 20%; CD56+, N-Cad+, OB-Cad+, HLA-A,B,C+, and annexin+ no more than 15%; cell viability no less than 60%. Immunocytochemical study of selected samples proved that numerous neural stem cells, and neuro- and glioblasts necessary for transplantation were present. Our results demonstrate that the flow cytometry phenotyping allows the screening and standardization of HNSC samples for further expansion and transplantation.