Factors defining developmental competence of bovine oocytes collected for in vitro embryo production†.

Despite the currently relatively low effectiveness of producing bovine embryos in vitro, there is a growing interest in applying this laboratory method in the field of reproduction. Many aspects of the procedure need to be improved. One of the main problems is the inferior developmental competence of in vitro matured oocytes that are collected using the ovum pick-up method. The mechanisms of oocyte capacitation and maturation, as well as the in vivo conditions in which they grow and mature, should be carefully analyzed. A deliberate application of the identified mechanisms and beneficial factors affecting the in vitro procedures seems to be essential for achieving higher developmental competence of the oocytes that are subjected to fertilization. The results may be improved by developing and employing a laboratory maturation protocol that corresponds with appropriate preparation of donors before the ovum pick-up, an optimized hormonal treatment program, the appropriate size of ovarian follicles at the time of aspiration, and a fine-tuned coasting period.

Schizophrenia patients perform as well as healthy controls on creative problem solving when fluid intelligence is accounted for.

INTRODUCTION: This study examined creative problem solving in schizophrenia. We aimed to verify three hypotheses: (H1) schizophrenia patients differ from healthy controls in the accuracy of creative problem solving; (H2) schizophrenia patients are less effective at evaluating and rejecting incorrect associations and (H3) have a more idiosyncratic way of searching for semantic associations compared to controls. METHODS: Six Remote Associates Test (RAT) items and three insight problems were applied to schizophrenia patients and healthy controls. We compared groups on the overall accuracy in the tasks to verify H1 and developed a novel method of comparing the patterns of errors in the RAT to verify H2 and H3. We controlled for fluid intelligence to eliminate this significant source of variation, as typically creativity and intelligence are significantly related. RESULTS: Bayesian factor analysis did not support the group differences in either insight problems and RAT accuracy or the patterns of RAT errors. CONCLUSIONS: The patients performed as well as the controls on both tasks. Analysis of RAT errors suggested that the process of searching for remote associations is comparable in both groups. It is highly improbable that individuals with schizophrenia benefit from their diagnosis during creative problem solving.

Signaling mechanisms and their regulation during in vivo or in vitro maturation of mammalian oocytes.

In vitro fertilization (IVF) is currently one of the most effective methods of infertility treatment. An alternative to commonly used ovarian hyperstimulation can become extracorporeal maturation of oocytes (in vitro maturation; IVM). Fertilization and normal development of the embryo depends on the cytoplasmic, nuclear and genomic maturity of the oocyte. The microenvironment of the ovarian follicle and maternal signals, which mediate bidirectional communication between granulosa, cumulus and oocyte cells, influence the growth, maturation and acquisition of oocyte development capability. During oogenesis in mammals, the meiosis is inhibited in the oocyte at the prophase I of the meiotic division due to the high cAMP level. This level is maintained by the activity of C-type natriuretic peptide (CNP, NPPC) produced by granulosa cells. The CNP binds to the NPR2 receptor in cumulus cells and is responsible for the production of cyclic guanosine monophosphate (cGMP). The cGMP penetrating into the oocyte through gap junctions inhibits phosphodiesterase 3A (PDE3A), preventing cAMP hydrolysis responsible for low MPF activity. The LH surge during the reproductive cycle reduces the activity of the CNP/NPR2 complex, which results in a decrease in cGMP levels in cumulus cells and consequently in the oocyte. Reduced cGMP concentration unblocks the hydrolytic activity of PDE3A, which decreases cAMP level inside the oocyte. This leads to the activation of MPF and resumption of meiosis. The latest IVM methods called SPOM, NFSOM or CAPA IVM consist of two steps: prematuration and maturation itself. Taking into account the role of cAMP in inhibiting and then unblocking the maturation of oocytes, they have led to a significant progress in terms of the percentage of mature oocytes in vitro and the proportion of properly developed embryos in both animals and humans.

Label-Free Mass Spectrometry-Based Quantitative Proteomics to Evaluate the Effects of the Calcium-Sensing Receptor Agonist Cinacalcet on Protein Expression in Rat Brains and Livers.

BACKGROUND Cinacalcet is a calcium-sensing receptor agonist that is clinically approved for the treatment of secondary hyperparathyroidism in chronic kidney disease and hypercalcemia in patients with parathyroid carcinoma. This study aimed to use quantitative mass spectrometry-based label-free proteomics to evaluate the effects of cinacalcet on protein expression in rat brains and livers. MATERIAL AND METHODS We randomly assigned 18 Wistar rats to 2 groups: an untreated control group (n=6) and a group treated with cinacalcet at a dose corresponding to the maximum dose used in humans (2 mg/kg/body weight, 5 days/week) divided into 7-day (n=6) and 21-day (n=6) treatment subgroups. A mass-spectrometry-based label-free quantitative proteomics approach using peptides peak area calculation was used to evaluate the changes in protein expression in examined tissues. Bioinformatics analysis of quantitative proteomics data was done using MaxQuant and Perseus environment. RESULTS No changes in protein expression were revealed in the 7-day treatment subgroup. We detected 10 upregulated and 3 downregulated proteins in the liver and 1 upregulated protein in the brain in the 21-day treatment subgroup compared to the control group. Based on Gene Ontology classification, all identified differentially expressed proteins were indicated as molecular functions involved in the enzyme regulator activity (36%), binding (31%), and catalytic activity (19%). CONCLUSIONS These findings indicate that long-term cinacalcet therapy can impair phase II of enzymatic detoxication and can cause disturbances in blood hemostasis, lipid metabolism, and inflammatory mediators or contribute to the acceleration of cognitive dysfunction; therefore, appropriate patient monitoring should be considered.

Gain of Function (GOF) Mutant p53 in Cancer-Current Therapeutic Approaches.

Continuous development of personalized treatments is undoubtedly beneficial for oncogenic patients' comfort and survival rate. Mutant TP53 is associated with a worse prognosis due to the occurrence of metastases, increased chemoresistance, and tumor growth. Currently, numerous compounds capable of p53 reactivation or the destabilization of mutant p53 are being investigated. Several of them, APR-246, COTI-2, SAHA, and PEITC, were approved for clinical trials. This review focuses on these novel therapeutic opportunities, their mechanisms of action, and their significance for potential medical application.

Fluorine-Containing Drug Administration in Rats Results in Fluorination of Selected Proteins in Liver and Brain Tissue.

In many pharmaceuticals, a hydrogen atom or hydroxyl group is replaced by a fluorine to increase bioavailability and biostability. The fate of fluorine released from fluorine-containing drugs is not well investigated. The aim of this study was to examine possible fluorination of proteins in rat liver and brain after administration of the fluorinated drug cinacalcet. We assigned 18 Wistar rats to a control group (n = 6) and a group treated with cinacalcet (2 mg kg-1/body weight, 5 days/week), divided into 7 day (n = 6) and 21 day (n = 6) treatment subgroups. Fluorinated proteins were identified using a free proteomics approach; chromatographic separation and analysis by high-resolution mass spectrometry; peptide/protein identification using the Mascot search algorithm; manual verification of an experimentally generated MS/MS spectrum with the theoretical MS/MS spectrum of identified fluorinated peptides. Three fluorinated proteins (spectrin beta chain; carbamoyl-phosphate synthase [ammonia], mitochondrial; 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 1) were identified in the liver and four (spectrin beta chain, dihydropyrimidinase-related protein 4, prominin-2, dihydropyrimidinase-related protein 4) in the brain tissue after 21 days of cinacalcet treatment, but not in the control group. Introduction of fluorine into an organism by administration of fluorinated drugs results in tissue-specific fluorination of proteins.

The Influence of Intravaginal Gestagens Treatment on the Morphological Features and Endometrial Steroid Hormone Receptors Content during Anestrus Type II in Dairy Cattle.

BACKGROUND: Gestagens are the most widely used therapy in anestrus type II. The aim of this research is to evaluate the effectiveness of the vaginal progesterone inserts therapy in anestrus type II in cows. METHODS: The study was conducted on 33 cows. Progesterone (PR) and estrogen (ER) receptors expression in endometrium was assessed on a molecular level based on mRNA tissue expression. Additionally, blood 17ß-estradiol and progesterone levels were evaluated. RESULTS: A decrease in mRNA expression of A and B PR and ER α was noted in treated and untreated animals. In the treated group, an increase of ERß mRNA expression was observed, while a decreased was found in untreated animals. There was increased PR, ERα and ß expression in endometrial tissue in treated cows, and decreased expression of these factors in untreated cows. In the treated group, recurrence of ovarian cyclicity was noted in 52% of animals and pregnancy was obtained in 34.8% of them, while in the untreated group, recurrence did not occur. In the control group, spontaneous recurrence of ovarian cyclicity was not observed. An increase of PR expression was correlated with increased proliferation of endometrial cells. CONCLUSIONS: It seems likely that the endometrium is well developed and ready for placentation after removing the exogenous source of progesterone and preventing the recurrence of cyclicity of ovaries.

A novel approach to thermographic images analysis of equine thoracolumbar region: the effect of effort and rider's body weight on structural image complexity.

BACKGROUND: The horses' backs are particularly exposed to overload and injuries due to direct contact with the saddle and the influence of e.g. the rider's body weight. The maximal load for a horse's back during riding has been suggested not to exceed 20% of the horses' body weight. The common prevalence of back problems in riding horses prompted the popularization of thermography of the thoracolumbar region. However, the analysis methods of thermographic images used so far do not distinguish loaded horses with body weight varying between 10 and 20%. RESULTS: The superficial body temperature (SBT) of the thoracolumbar region of the horse's back was imaged using a non-contact thermographic camera before and after riding under riders with LBW (low body weight, 10%) and HBW (high body weight, 15%). Images were analyzed using six methods: five recent SBT analyses and the novel approach based on Gray Level Co-Occurrence Matrix (GLCM) and Gray Level Run Length Matrix (GLRLM). Temperatures of the horse's thoracolumbar region were higher (p < 0.0001) after then before the training, and did not differ depending on the rider's body weight (p > 0.05), regardless of used SBT analysis method. Effort-dependent differences (p < 0.05) were noted for six features of GLCM and GLRLM analysis. The values of selected GLCM and GLRLM features also differed (p < 0.05) between the LBW and HBW groups. CONCLUSION: The GLCM and GLRLM analyses allowed the differentiation of horses subjected to a load of 10 and 15% of their body weights while horseback riding in contrast to the previously used SBT analysis methods. Both types of analyzing methods allow to differentiation thermal images obtained before and after riding. The textural analysis, including selected features of GLCM or GLRLM, seems to be promising tools in considering the quantitative assessment of thermographic images of horses' thoracolumbar region.

Altrenogest affects the development and endocrine milieu of ovarian follicles in prepubertal and mature gilts†.

Altrenogest with gonadotropins is commonly used to synchronize the estrous cycle, but it can also lead to follicular cyst formation, especially in prepubertal gilts. Here, we aimed to investigate how maturity and altrenogest treatment affect the development, endocrine milieu, and molecular control of ovarian follicles. Crossbred prepubertal and mature gilts were challenged or not (control) with altrenogest, and ovaries were collected in the morning on the first day of behavioral estrus. In prepubertal gilts, altrenogest decreased the percentage of primordial and atretic small follicles, but increased large antral follicles when compared with controls. In mature gilts, altrenogest reduced the percentage of primary follicles and elevated the total number of antral follicles. Maturity affected the estradiol level in the follicular fluid of preovulatory follicles, luteinizing hormone (LH)-stimulated cyclic adenosine monophosphate (cAMP) generation, and LH receptor messenger RNA (mRNA) expression in granulosa. Moreover, cytochrome P45017A1 (CYP17A1) mRNA levels in the theca layer were affected and correlated with follicular androstendione and estradiol concentration. Altrenogest negatively affected follicular fluid progesterone concentration and decreased levels of prostaglandin (PG) E2 in prepubertal gilts and PGF2alpha metabolite in mature gilts. LH-stimulated cAMP release in granulosa cells of mature gilts as well as human chorionic gonadotropin- and forskolin-induced cAMP were also affected. In addition, altrenogest downregulated CYP17A1 mRNA in the prepubertal theca layer and PGF2alpha synthase expression in the granulosa and theca layer of mature gilts. To the best of our knowledge, this is the first study to report multiple effects of maturity and altrenogest on the endocrine milieu and molecular regulations governing ovarian follicle development in gilts.

Gain-of-Function Mutations in p53 in Cancer Invasiveness and Metastasis.

Forty years of research has proven beyond any doubt that p53 is a key regulator of many aspects of cellular physiology. It is best known for its tumor suppressor function, but it is also a regulator of processes important for maintenance of homeostasis and stress response. Its activity is generally antiproliferative and when the cell is damaged beyond repair or intensely stressed the p53 protein contributes to apoptosis. Given its key role in preventing cancer it is no wonder that it is the most frequently mutated gene in human cancer. Surprisingly, a subset of missense mutations occurring in p53 (gain-of-function) cause it to lose its suppressor activity and acquire new functionalities that turn the tumor suppressor protein into an oncoprotein. A solid body of evidence exists demonstrating increased malignancy of cancers with mutated p53 in all aspects considered "hallmarks of cancer". In this review, we summarize current findings concerning the cellular processes altered by gain-of-function mutations in p53 and their influence on cancer invasiveness and metastasis. We also present the variety of molecular mechanisms regulating these processes, including microRNA, direct transcriptional regulation, protein-protein interactions, and more.

Computational multivariate modelling of electrical activity of the porcine uterus during spontaneous and hormone-induced oestrus.

NEW FINDINGS: What is the central question of this study? Does oestrous cycle synchronization influence myoelectrical activity of porcine myometrium? What is the main finding and its importance? Exogenous hormones used to synchronize oestrus in pigs altered myoelectrical activity, which was effectively modelled. Higher-order multivariate statistic modelling provided evidence of similar activity in both types of oestrus, but a larger order of EMG signals during induced oestrus. Higher-order statistical analysis of the probabilistic model suggests the beginning of the early follicular phase and the mid-luteal phase to be most important in evaluation of the natural patterns of myoelectrical activity. Higher-order multivariate cumulants are more informative than classical statistics in characterization of myoelectrical activity changes in porcine myometrium. ABSTRACT: In pig production units, control of the oestrous cycle and synchronization of ovulation have become routine herd management procedures. During the oestrous cycle, in both induced and spontaneous conditions, the ovaries and the uterus undergo hormone-dominated physiological changes, which are consistent with the hypothesis that there is a functional role of uterine contractions in promoting fertilization. We have used electromyography to determine whether the use of exogenous hormones, such as equine chorionic gonadotrophin and human chorionic gonadotrophin, which have the potential to control the timing of ovulation in female pigs, changes the multivariate relationships between parameters of electrical bursts and modulates the patterns of myoelectrical activity. We used the mathematical approach of higher-order multivariate cumulants in complex modelling of the myometrial electrical activity. The experiment was conducted on 12 mature Polish Landrace sows, and uterine activity was recorded during both spontaneous and induced oestrous cycles. The burst parameters were determined using six features in the time domain and, after Fast Fourier transformation, in the frequency domain. Evaluation of myoelectrical activity patterns was conducted based on classical univariate statistical methods and multivariate probabilistic modelling. The classical statistical approach indicated weaker myoelectrical activity after hormonal stimulation, whereas the higher-order multivariate statistical model showed evidence of similar status of activity and a larger order of signals during induced oestrus. Routine oestrous cycle synchronization affects the multivariate probabilistic model of myometrial electrical activity.

Transfer of orally administered ZnO:Eu nanoparticles through the blood-testis barrier: the effect on kinetic sperm parameters and apoptosis in mice testes.

Zinc-based nanoparticles are promising materials for various applications, including in biomedicine. The aim of our study was to determine the effect of fluorescent europium-doped zinc oxide nanoparticles (ZnO:Eu NPs) on sperm parameters, cell apoptosis and integrity of the blood-testis barrier (BTB) in mice. Nanostructures were orally administered to adult mice (n = 34). Animals were sacrificed after 3 h, 24 h, 7 d and 14 d following oral administration. Sperm was collected and analysed for viability and kinetic parameters. Collected testes were quantitatively analysed for accumulation of ZnO:Eu NPs. Microscopic evaluation based on immunofluorescence and histopathological studies were also conducted. Results showed that ZnO:Eu NPs were able to overcome the BTB with their subsequent accumulation in the testis. No toxic or pro-apoptotic effects of nanoparticles on the male reproductive system were observed. The results suggested that ZnO:Eu NPs were able to accumulate in the testis with no negative impact on sperm parameters, tissue architecture or the integrity of the BTB.

Limited accuracy of transurethral and periurethral intrasphincteric injections of cellular suspension.

AIMS: The efficacy of cell therapy in patients with stress urinary incontinence (SUI) is lower than expected. The aim of this study was to determine the injection accuracy rate both with transurethral and periurethral route. METHODS: Autologous intraurethral cell transplantation was performed in female goats. The cells were injected either periurethrally (PERI group, two depots/animal, n = 8) or transurethrally (TRANS group, eight depots/animal, n = 11). Transurethral injections were performed under endoscopic guidance. The number and distribution of cell depots in urethras were analyzed in the three-step protocol: 1) screening of whole explants by in vivo imaging system; 2) systematic microscopic analysis of raw 10 µm cross-sections; 3) immunohistochemistry. As control, four urethras collected 1 day after transurethral transplantation were used. Episodes of cell suspension leakages after needle withdrawal were noted. RESULTS: In all experimental animals depots were identified in the urethral wall 28 days after transplantation. The mean percentage of depots located in the urethral wall in relation to all performed injections amounted to 68.7% and 67.0% for PERI and TRANS groups, respectively. The mean proportions of depots which were identified in external urethral sphincter (EUS) amounted 18.8% and 17.1%, respectively. Suspension leakage was observed in 19% of transurethral injections. CONCLUSIONS: Although majority of cell depots were administrated accurately into the urethral wall, the precise delivery of cells into EUS is limited regardless of injection method. The insufficient accuracy of cell delivery into EUS and cell suspension leakage can contribute to the low efficacy of cell therapy in human patients with SUI.

Uterine EMG activity in the non-pregnant sow during estrous cycle.

BACKGROUND: Uterine myoactivity is crucial for successful reproductive performance of the sow. Spontaneous contractions of the uterus are strictly controlled and coordinated. Uterine electromyographic (EMG) activity undergoes hormonal regulation with rapid and long-term effects. What is more, interstitial Cajal-like Cells (ICLC) appear essential for smooth muscle contractility in the reproductive tract where they are suspected to be playing a major role in generating, coordinating, modulating and synchronizing slow triggering waves. The aim of this study was to investigate the myoelectrical activity of sow's uterus during estrus cycle. RESULTS: Study was conducted on 10 Polish Landrace sows. Propagation mechanisms and their connection with the uterine EMG activity were considered in correlation with expression of c-kit, progesterone and oxytocin receptors of the non-pregnant sow. ICLC were labeled with antibody directed against c-kit receptor and visualized by confocal microscopy and scanning cytometer for positive cells percentage assessment. EMG signal was recorded directly from the myometrium with telemetry transmitters and electrodes located in different topographic regions of reproductive tracts. The stages of estrus cycle were determined by monitoring levels of luteinizing hormone, progesterone and estrogen with radioimmunoassays. Significant differences of the EMG signal parameters between diestrus and estrus and the correlations with density of labelled receptors were demonstrated. Moreover, the electrophysiological studies indicated that ICLC in the myometrium in the tip of uterine horn may participate in the regulation of slow waves duration and frequency. CONCLUSIONS: The pattern of EMG signal propagation in the wall of the non-pregnant porcine uterus occurs in an orderly, bidirectional fashion and at distinctive speed, with no differences between diestrus and estrus.

Functional identification of abductor and adductor branches for laryngeal transplantation.

PURPOSE: This is a feasibility study of functional identification of the abductor and adductor recurrent laryngeal nerve branches, which could be used in the donor's larynx for functional laryngeal transplantation. METHODS: The study was performed on swine (n = 3) and human (n = 4) models of a donor larynx. The recurrent laryngeal nerve and its branches were found. Using stimulator, abductor and adductor branches were identified, and glottis closing and opening were captured with an endoscope. RESULTS: The result was positive if two ENT specialists noticed at least one adduction and one abduction movement in the same subject. It was obtained in three out of three swine and three out of four humans. CONCLUSIONS: This study shows a way of reinnervation of a transplanted larynx which might result in a functional organ. It describes the first step of the procedure: functional identification of the abductor and adductor branches of the recurrent laryngeal nerve in the donor before the larynx is excised for transplantation.

Interferon-tau promotes luteal endothelial cell survival and inhibits specific luteolytic genes in bovine corpus luteum.

Interferon-tau (IFNT), a maternal recognition of pregnancy (MRP) signals in domestic ruminants, suppresses the release of luteolytic pulses of uterine prostaglandin F2a (PGF2a), thus extending the corpus luteum (CL) life span. We hypothesized that IFNT also exerts anti-luteolytic actions in bovine CL. To examine the direct effects of IFNT on bovine CL, luteal slices and enriched luteal endothelial cells (LECs) were utilized. We found that recombinant ovine IFNT (roIFNT) markedly elevates interferon-associated genes (STAT1, STAT2 and IRF9) and interferon-stimulated genes (ISGs: MX2, ISG15 and OAS1Y) in both models. Furthermore, IFNT time-dependently induced STAT1 phosphorylation in LECs without affecting total STAT1. roIFNT-stimulated viable LECs numbers and the knockdown of protein inhibitor of activated STAT1 (PIAS1) abolished this effect, suggesting that PIAS1 may mediate the proliferative effect of IFNT. IFNT significantly downregulated luteolytic genes such as TGFB1, thrombospondin-1 (THBS1), endothelin-1 (EDN1) and serpin family E member-1 (SERPINE1) in LECs. However, less robust effects were observed in luteal slices. Moreover, PGF2a alone induced THBS1, SERPINE1 and EDN1 mRNA in CL slices whereas in the presence of IFNT, THBS1 and SERPINE1 stimulation was abolished. Collectively, these results indicate that IFNT acts via STAT1- IRF9-dependent and independent pathways and affects diverse luteal functions. Most interestingly, this study suggests the existence of an anti-luteolytic effect of IFNT in bovine CL, namely, inhibiting key PGF2a-induced luteolytic genes. The proliferative effect of IFNT may constitute an additional mechanism that promotes luteal cell survival, thus, extending the luteal life span during early pregnancy in cows.

Characteristics of bioelectrical activity of oviducts and uterus during early pregnancy in sows recorded by telemetry method.

NEW FINDINGS: What is the central question of this study? The aim of present study was to record and analyse the myoelectrical activity in the female pig reproductive tract (uterus and oviduct) during early pregnancy. What is the main finding and its importance? Understanding the contractile activity of the uterus and oviducts is indispensable for understanding the physiological mechanisms as well as all irregularities associated with the period of conception and early pregnancy. The aim of the present study was to record the myoelectrical activity of the reproductive tract in sows during the oestrous phase and early pregnancy via a telemetry recording system. In a total of eight non-pregnant pigs, the bioelectrical activity was recorded through three silicone electrodes sutured on the oviduct (isthmus and ampulla) and the uterine horn. Blood samples were collected to monitor the concentrations of progesterone (P4) and luteinizing hormone (LH). The oestrous cycle was synchronized with equine chorionic gonadotrophin (eCG) and human chorionic gonadotrophin (hCG), and the animals were subjected to artificial insemination. Analysis of the EMG activity of the oviduct and uterus in the oestrous phase and in early stages of pregnancy suggests explicitly that telemetry could enable in vivo assessment of myoelectrical activity of parts of the reproductive system in sows. Off-line analysis of the duration of EMG activity bursts in the uterus, isthmus and ampulla were significantly higher during early pregnancy (phases II and III) than in the oestrous phase. The EMG signals demonstrated low mean amplitudes of activity in the oviduct and uterus during early pregnancy (phases I-III). Significant differences between the root mean square signals were observed in the isthmus and ampulla both during oestrus and in early pregnancy (phase I; P < 0.01). During the oestrous phase, the P4 concentration was estimated at <1 ng ml-1 , whereas the LH concentration was >4 ng ml-1 . In contrast, during early pregnancy, the P4 and LH concentrations were estimated at >4 and <1 ng ml-1 , respectively.

Biodegradation of the ZnO:Eu nanoparticles in the tissues of adult mouse after alimentary application.

Biodegradable zinc oxide nanoparticles (ZnO NPs) are considered promising materials for future biomedical applications. To fulfil this potential, biodistribution and elimination patterns of ZnO NPs in the living organism need to be resolved. In order to investigate gastrointestinal absorption of ZnO NPs and their intra-organism distribution, water suspension of ZnO or fluorescent ZnO:Eu (Europium-doped zinc oxide) NPs (10mg/ml; 0.3ml/mouse) was alimentary-administered (IG: intra-gastric) to adult mice. Internal organs collected at key time-points after IG were evaluated by AAS for Zn concentration and analysed by cytometric techniques. We found that Zn-based NPs were readily absorbed and distributed (3 h post IG) in the nanoparticle form throughout the organism. Results suggest, that liver and kidneys were key organs responsible for NPs elimination, while accumulation was observed in the spleen and adipose tissues. We also showed that ZnO/ZnO:Eu NPs were able to cross majority of biological barriers in the organism (including blood-brain-barrier).

Dynamics of acute local inflammatory response after autologous transplantation of muscle-derived cells into the skeletal muscle.

The vast majority of myoblasts transplanted into the skeletal muscle die within the first week after injection. Inflammatory response to the intramuscular cell transfer was studied in allogeneic but not in autologous model. The aim of this study was to evaluate immune reaction to autotransplantation of myogenic cells and to assess its dynamics within the first week after injection. Muscle-derived cells or medium alone was injected into the intact skeletal muscles in autologous model. Tissue samples were collected 1, 3, and 7 days after the procedure. Our analysis revealed the peak increase of the gene expression of all evaluated cytokines (Il-1α, Il-1 ß, Il-6, Tgf-ß, and Tnf-α) at day 1. The mRNA level of analyzed cytokines normalized in subsequent time points. The increase of Il- ß gene expression was further confirmed at the protein level. Analysis of the tissue sections revealed rapid infiltration of injected cell clusters with neutrophils and macrophages. The inflammatory infiltration was almost completely resolved at day 7. The survived cells were able to participate in the muscle regeneration process. Presented results demonstrate that autotransplanted muscle-derived cells induce classical early immune reaction in the site of injection which may contribute to cellular graft elimination.

Application of flow cytometry in diagnosing lymphomas in dogs and cats.

Classification of types of lymphomas is done by interpreting cell morphology results obtained in cytological and/or histological examinations. In recent years, additional methods like immunocytochemistry (ICC), immunohistochemistry (IHC), immunophenotyping by flow cytometry and polymerase chain reaction (PCR) for antigen receptor rearrangements (PARR), have been used to diagnose and classify lymphomas. Unfortunately, none of these methods is completely specific and sensitive. Thus, a combination of several diagnostic methods or use of all available techniques allows for evaluation of morphological properties of cells like their maturity and diversification. Owing to the use of sets of antibodies it is possible to identify the phenotype of hyperplastic cells as well as their origin. Combination of results obtained through phenotypical analysis with flow cytometry examination with morphological, histological and genetic testing enables a detailed analysis of, in this case, lymphoproliferative diseases including reaction changes, primary and secondary immunological deficits as well as autoimmune diseases.