Cell Mitotic Activity Research Articles

172 Impact of maternal and postnatal 25-hydroxycholecalciferol supplementation on porcine and avian satellite cell mitotic activity and skeletal muscle growth characteristics

Abstract Vitamin D is an essential micronutrient most well-known for regulating calcium and phosphorus homeostasis and bone mineralization. However, over the last 20 yr, the extra-skeletal, pleotropic effects of vitamin D and its metabolites have gained greater attention. For livestock species reared indoors, vitamin D supplementation is especially important and commonly achieved by dietary addition of cholecalciferol, also known as vitamin D3 (D3). Cholecalciferol is hydroxylated in the liver to produce the circulating form, 25-hydroxycholecalciferol (25OHD3), also called calcidiol. Circulating 25OHD3 is subsequently converted to 1α,25-hydroxycholecalciferol (1,25OHD3) which binds a nuclear receptor found in many tissues, including skeletal muscle, to elicit its effects. This presentation will focus on the recent work regarding the effects of both maternal and postnatal 25OHD3 supplementation on porcine and avian satellite cell mitotic activity and skeletal muscle growth characteristics. Feeding broilers 25OHD3 posthatch results in a satellite cell-mediated skeletal muscle hypertrophic response in the fast-twitch pectoralis major but not the slow-twitch biceps femoris. Maternal dietary supplementation of gilts and hens with 25OHD3 in place of the majority of D3 increases circulating 25OHD3 concentrations in both dams and their offspring. The improvements in vitamin D status coincide with positive effects on skeletal muscle fiber number in fetal piglets as well as satellite cell mitotic activity and hypertrophic growth of fibers in both swine and broiler chickens. Though, interestingly, combining maternal and posthatch 25OHD3 supplementation in broilers does not appear to produce additive or synergistic effects on SC-mediated muscle growth. However, increasing circulating concentrations of 25OHD3 by supplementing either the maternal or postnatal diet with 25OHD3 has positive effects on meat yield in both porcine and avian models. Overall, these data shed light on the cellular mechanisms behind the efficacy of this vitamin D supplementation strategy using commercially available 25OHD3 sources in increasing meat yield and yet, leaves enough unanswered questions remaining to warrant further exploration.

Морфофункциональное состояние органов иммунной системы у поросят-гипотрофиков в ранний неонатальный период

Abstract. The purpose of the study was to study the state of the immune system organs in hypotrophic piglets in the early neonatal period in an industrial pig breeding complex. Methods. The experiment was conducted in 2023 in a large industrial pig farm in the Voronezh region on piglets of the wounded neonatal period received from sows 3-4 farrowing. The sows were kept at optimal microclimate parameters, taking into account their physiological state, and fed with SK-1 nutritionally balanced feed. At the initial stage of the experiment, the piglets obtained during farrowing underwent clinical examination and weighing. Animals under 800 g are counted as hypotrophic piglets (n = 30), animals over 800 g are normotrophic, respectively (n =30). After the groups were formed, animals were forced to be slaughtered before taking colostrum (n =5) and biological material was taken from each group (thymus, lymph nodes (inguinal), spleen for immunohistochemical studies. Results. The level of mitotic activity in the thymus of normotrophic piglets was 9.4 % higher (p < 0.05) than in piglets with body weight deficiency. In the spleen, the level of mitotic activity had no significant differences and was approximately the same in all animals participating in the experiment. In lymph nodes, the mitotic activity of cells in normotrophics was 12.7 % higher (p < 0.05). A study of positively expressed CD-3 cells in the spleen revealed significant differences, so, in normotrophic piglets, the number of cells positively stained with this marker was significantly higher than in piglets with a body weight deficiency by 10.2 % (p < 0.05). The number of CD-3 cells in the thymus differed by 5.6 %, and in the lymph nodes by 2.4 % between the groups, but there were no significant differences. The cellular expression of “immature” forms of B lymphocytes (PAX-5) in the lymph nodes of normotrophics was significantly higher by 12.9 % (p < 0.05) compared with hypotrophic piglets. The scientific novelty lies in the fact that for the first time a comprehensive immunohistochemical study of the organs of the immune system of piglets in the early neonatal period was carried out using monoclanal antibodies CD-3, Ki-67 and PAX-5. As a result of the experiment, it was revealed that piglets with body weight deficiency have a “depression” of the immune system, which manifests itself in hypoplasia of T-lymphocytes in the spleen, “maturing” B-lymphocytes in the lymph nodes and low mitotic potential in the thymus and lymph nodes.

The detailed histopathological characteristics of ovarian fibroma compared with thecoma, granulosa cell tumor, and sclerosing stromal tumor

BackgroundOvarian fibromas are benign tumors that can present peculiar morphological features not studied sufficiently. Material and MethodsIn this retrospective study, 75 consecutive cases of ovarian fibroma were morphologically compared with 46 thecomas, 16 granulosa cell tumors, and 5 sclerosing stroma tumors for the following factors: the growth pattern as diffuse or nodular, the presence of hyaline plaques, necrosis, keloid-like sclerosis, calcifications, cystic degeneration, fibrous or edematous stroma, prominent vascularity, lutein cells, cellularity, scant or abundant cytoplasm, prominent cell membranes, nuclear grooves, atypia, and mitotic activity. ResultsThe tumors differed significantly in terms of hyaline plaques presence, nuclear grooves, growth pattern, stroma type, tumor cellularity, cytoplasm, prominence of cell membranes, atypia, mitotic activity, and prominent vascularity. ConclusionOvarian fibromas can present some maybe unexpected features rather frequently, such as cystic degeneration, hyaline plaques, prominent vascularity, increased cellularity, and some mitotic activity, thus their presence should not always prompt to an alternative diagnosis.

The effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on human cervical, colorectal, and mammary carcinoma cell lines

Targeting the inflammation-related molecules with nonsteroidal anti-inflammatory drugs (NSAIDs) represents a promising approach for cancer prevention/therapy. We evaluated the in vitro anticancer effects of meloxicam, lornoxicam, ketoprofen, and dexketoprofen on the proliferation, migration, and apoptosis of human cervical, colorectal, and mammary carcinoma cells. The antiproliferative activity and cytotoxicity of tested NSAIDs on HeLa, HT-29, and MCF-7 cell lines were assessed by the MTT test. The apoptosis-inducing potential was analyzed by fluorescent staining with acridine orange/ethidium bromide and DAPI. Migration activity was assessed by a wound-healing scratch assay. The tested NSAIDs reduced the viability of the used tumor cell lines. The cytomorphological analysis revealed reduced cell density and mitotic activity and the presence of cells with morphological features of early and late apoptosis. Significant inhibition of the migration capacity was established as well. In conclusion, NSAIDs could be candidates for the development of new pharmacological strategies for the treatment and prevention of cancer.

237 Evaluation of Thermal Variation During Early-Stage Incubation on Broiler Chicken Pectoralis Major Muscle Satellite Cell Mitotic Activity and Heterogeneity at 28 Days Post-Hatch

Abstract Muscle satellite cells (SC) have an essential role in post-hatch skeletal muscle growth. To evaluate the effects of thermal variation (TV) during early-stage incubation (ESI) on Pectoralis major (PM) muscle SC heterogeneity of broiler chickens reared to 28 d post-hatch a randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. Yield Plus × Ross 708 broiler breeder eggs (n = 2,160) ranging from 54 to 59 g were allotted equally by weight into 90-egg trays (n = 4 trays per incubator). All eggs were pre-warmed at 24 °C for 8 h and incubated at 37.5 °C from embryonic day (ED) 0 to 4. From ED 4 to 11, eggs were incubated at 1 of 3 temperatures (n = 2 incubators per TV treatment); 37.5 °C (CTL), 36.4 °C (COLD), or 38.6 °C (HOT). On ED 11, all incubators were returned to the CTL (37.5 °C) set point until ED 18. On ED 18, all eggs were transferred to baskets in hatchers set to 36.7 °C. After 514 h of incubation, all chicks were removed simultaneously, vent sexed, and weighed. Chicks were randomly allotted to floor pens (6 pens per treatment per sex) blocked by location and reared on a common diet. On d 28, birds (n = 10 per TV x sex treatment) were injected with bromodeoxyuridine (BrdU) before PM sample collection. Cryosections were immunofluorescence stained to facilitate taxonomy of SC populations expressing the myogenic regulatory factors Pax7 and MyoD as well as the proliferation marker BrdU by fluorescence microscopy. Data were analyzed as a 2-way ANOVA using SAS PROC GLIMMIX with TV treatment and broiler sex as main effects. Means were separated with the PDIFF option at P ≤ 0.05. Tendencies were declared when 0.0501 ≤ P ≤ 0.10. A TV × sex interaction was observed only for the MyoD+: Pax7+ SC density (P = 0.0232); however, none of the pairwise mean comparisons were significant at P < 0.05. Though CTL male and COLD female chicks had twice the density of MyoD+:Pax7+ SC as CTL females (P = 0.0535) and HOT males (P = 0.0645), respectively. There is no clear explanation for this observation. There were no interactive or main effects on density of MyoD+, Pax7+, MyoD+:BrdU+, Pax7+:BrdU+, MyoD+:Pax7+:BrdU+ SC (P ≥ 0.1258) or nuclear density (P = 0.1017) in 28-d-old broilers. These data suggest that if TV during ESI impacts SC myogenic regulatory factor heterogeneity and mitotic activity in broilers, the effects have concluded by d 28 post-hatch in commercial broilers. Further exploration of earlier time points will be necessary to determine whether the previously observed effects of TV during ESI on growth performance and meat yield are SC-mediated post-hatch.

67 Effect of Different Dietary Protein Sources on Skeletal Muscle Growth Characteristics and Satellite Cell Mitotic Activity of Young Piglets

Abstract The neonatal period in pigs is distinguished by rapid growth as a function of extensive protein synthesis. Muscle satellite cells also referred to as stem cells (SC) have an essential role in postnatal hypertrophic muscle growth by increasing myofibrillar protein synthesis potential through fusion with existing myofibers to increase the DNA template available for transcription of these important muscle proteins. Thus, the impact of protein sources and differences in their amino acid availability on SC-mediated hypertrophic growth over time in neonatal pigs is of interest. To evaluate how different dietary protein sources fed to young piglets affect muscle fiber cross-sectional area (CSA), fiber density, and SC proliferative activity, a randomized complete block design experiment was conducted using piglets (n = 123) from 5 different farrowing groups. At birth, piglets were assigned to 1 of 4 treatments consisting of a control group and 3 liquid dietary treatments. The commercial milk replacer-based diets contained: whey only (MLK), MLK + spray dried plasma (SDP), and MLK + soy protein concentrate (SPC). After birth, all piglets were allowed to nurse at least 36-h on the sow to allow colostrum intake, assigned to 1 of 3 dietary treatments, transferred to Rescue Decks, and reared until tissue collection on d 6, 14, 19, and 25. The control group was left in the farrowing crate and nursed by the sow until sampling (SOW). On sampling day, pigs were injected with 5’-bromo-2’-deoxyuridine (BrdU) to label mitotically active cells. One-hour post-injection, pigs were euthanized and longissimus dorsi (LM) and biceps femoris (BF) muscle samples were collected for immunofluorescence microscopy analysis. Cell populations were classified as either mitotically inactive SC (MyoD+:BrdU-; Pax7+:BrdU-; MyoD+:Pax7+:BrdU-) or mitotically active SC (MyoD+:BrdU+; Pax7+:BrdU+; MyoD+:Pax7:BrdU+). Data were analyzed as a 2-way ANOVA using SAS V9.4 PROC GLIMMIX and means were separated using PDIFF at P ≤ 0.05. Tendencies were declared when 0.0501 < P ≤ 0.10. At d 14 and 19, MLK-fed piglets had the least BWG (P ≤ 0.0810). On d 14 and 19, MLK-fed piglet LM fibers had the smallest CSA and greatest density (P ≤ 0.0459) though they were similar in fiber density to SOW piglets on d 14. The MLK-fed piglets exhibited the least density of Pax7+:BrdU+ SC in BF (P = 0.0185) and the greatest density of MyoD+ SC in LM (P = 0.0831) on d 6 as well as the greatest density of MyoD+ SC in LM (P = 0.0551) and proliferative MyoD+:BrdU+ SC in BF (P = 0.0799) on d 19, though these differences did not persist through d 25. Overall, these data indicate that supplementation of different dietary protein sources can impact satellite cell-mediated hypertrophic muscle growth of neonatal piglets though the mechanism will require further investigation.

Immunophenotypic endometrium profile in experimental hyperplasia

Aim. To study the topography and expression of cell cycle markers, as well as those of inflammation and immune response, by the method of immunohistochemistry using an experimental model of endometrial hyperplasia.Materials and methods. Endometrial hyperplasia was modeled in laboratory rats by ovariectomy and transdermal administration of estrogen to form hyperestrogenism. Morphological changes were verified by a histological method. The immunophenotypic profile was assessed by immunohistochemistry. The obtained digital values were processed by mathematical and statistical methods.Results. The modelled endometrial hyperplasia indicated an increase in the glandular component, a decrease in the expression of estrogen receptors in the uterine horns, an increase in the mitotic activity of epithelial cells in the uterine body, an activation of cell apoptosis in all its departments, as well as a decrease in the expression of plasmocyte markers (CD138) in the stroma of all parts of the uterus and T-lymphocytes (CD8) in the stroma the body of the uterus.Conclusion. Structural changes in endometrial hyperplasia in the setting of hyperestrogenism are caused by cell cycle dysregulation. At the same time, intracellular autoregulation systems are more effective in the uterine horns, with no changes being observed in the expression of estrogen receptors in the uterine body and the mitotic activity of cells being increased. Given the deficiency of immunocompetent T cells, the risk of tissue and cellular transformations increases in this area.

Abstract 2580: Nuclear receptor coactivator 3 (NCOA3) fuels oncogenic pathways in uveal melanoma

Abstract Background: Uveal melanoma (UM) originates from melanocytes residing in the uveal tract of an eye. Despite effective local control, approximately half of patients develop metastatic disease resistant to systemic chemotherapy and presenting poor response to immunotherapy or targeted therapy. UM patients have a median overall survival of 6 to 12 months. The basis of the pathogenesis of UM are mutations in GNAQ and GNA11 genes resulting in constitutive activation of PKC/MAPK signaling. Here, we analyzed the role of NCOA3, a transcription factor coactivator that was previously shown to be modified by PKC in multiple cancer, as a factor potentiating oncogenic outcome of multiple oncogenic pathways activated in UM downstream of GNAQ or GNA11. Methods: We examined the impact of altered NCOA3 mRNA in UM, using publicly available TCGA dataset. Next, we used CRISPR/Cas9 technology to delete NCOA3 protein in MP41 UM cell line. We evaluated proliferation of MP41/NCOA3 KO cell subline and its parental counterpart in vitro by a colony formation assay. Next, we analyzed growth of xenografts of MP41/NCOA3 KO and parental cells in SCID/beige mice. To understand the molecular consequences of NCOA3 activity in UM, we performed a genome wide comparison of gene expression in MP41/NCOA3 KO and parental cells. Functional enrichment analysis was used to identify classes of genes associated with NCOA3 activity and determine similarities with datasets obtained by targeting proteins of GNAQ/GNA11 pathways and inhibiting activity of NCOA3 using a tool small molecule inhibitor SI-2 in UM cell lines. Results: We found that NCOA3 overexpression negatively correlated with UM patients’ progression free and overall survival. Knockout of NCOA3 gene in MP41 UM cells substantially reduced their proliferation in vitro and xenograft growth in mice. Global gene expression analysis revealed that NCOA3 activity is linked with transcriptional signatures associated with cell mitotic activity (cell cycle and DNA replication) and transcriptional machinery binding (especially affecting E2F and myc regulated programs). The MP41 NCOA3 KO normalized enrichment scores (NES) show high concordance with NES obtained after silencing GNAQ or GNA11 in UM cell lines and pharmacological targeting PKC or NCOA3. Conclusions: Inhibition of NCOA3 activity inhibits several oncogenic pathways in a concerted manner, limiting UM cell survival and tumor growth. Our results deliver proof of concept that NCOA3 is a master transcriptional co-regulator of oncogenic signaling in uveal melanoma and a potential therapeutic target. Citation Format: Aleksandra Rusin, Maria E. Ruiz Echartea, Darlene G. Skapura, Karen Berman deRuiz, Christel M. Davis, Eric A. Ehli, Sandra L. Grimm, Christian Coarfa, Salma Kaochar. Nuclear receptor coactivator 3 (NCOA3) fuels oncogenic pathways in uveal melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2580.

EVALUATION OF THE COMPLEX USE OF PLANT TEST-SYSTEMS TO DETERMINE THE NATURE AND INTENSITY OF THE MUTAGENIC BACKGROUND OF THE ENVIRONMENT.

Aim: A comprehensive evaluation of the effectiveness of the use of plant test-systems as biological objects for the identification of the specifitown of the effect of mutagens on the hereditary apparatus. Materials and methods: With the use of test-systems (determination of the gonadotoxitown of plant pollen grains, the Allium cepa-test and the use of celandine greater Chelidonium majus as an accumulative phytoindicator) was analyzed the ecological situation of the areas in Ivano-Frankivsk region. The samples of soil, water, generative and vegetative organs of plants were taken in the territories of the Carpathian region and tested in the following areas: ecologically safe, conditionally ecologically safe and ecologically dangerous areas. Results: Inhibition of mitotic activity in cells of the primary meristem of Allium cepa roots grown in water samples taken in chemically polluted Kalush and Burshtyn and stimulation of mitosis in water samples from Stetseva, was determined. The town Kosiv is considered conditionally ecologically clean, a control district. The indices of chromosomal aberrations frequency were high in plants from the chemically polluted Burshtyn (3.98±0.31) %, Kalush (2.95±0.25) % and village Stetseva (3.36±0.39) %, and dominated by controls 4.57-fold; 2.92-fold and 3.86-fold, respectively. Conclusions: With the help of a comprehensive study of two test-systems (determination of the fertility of plant reproductive structures and the Allium cepa test), we have proven an increased level of xenobiotics in the water, soil and air of the chemically polluted territoties of Precarpathian region. The use of Chelidonium majus as a test-object with metal-accumulating properties is a promising index for the detection of substances of a chemical nature (heavy metals) in ecological areas of the Ivano-Frankivsk region.

Venlafaxine increases aromatization, reduces apical V-ATPase in clear cells and induces increased number of mast cells and smooth muscle cells death in rat cauda epididymis

Depressive disorders (DD) have affected millions of people worldwide. Venlafaxine, antidepressant of the class of serotonin and norepinephrine reuptake inhibitors, has been prescribed for the treatment of DD. In rat testes, venlafaxine induces testosterone (T) aromatization and increases estrogen levels. Aromatase is a key enzyme for the formation of estrogen in the epididymis, an essential organ for male fertility. We investigated the impact of serotonergic/noradrenergic venlafaxine effect on the epididymal cauda region, focusing on aromatase, V-ATPase and EGF epithelial immunoexpression, smooth muscle (SM) integrity and mast cells number (MCN). Male rats were distributed into control (CG; n = 10) and venlafaxine (VFG, n = 10) groups. VFG received 30 mg/kg b.w. of venlafaxine for 35 days. The epididymal cauda was processed for light and transmission electron microscopy (TEM). The expression of connexin 43 (Cx43) and estrogen alpha (Esr1), adrenergic (Adra1a) and serotonergic (Htr1b) receptors were analyzed. Clear cells (CCs) area, SM thickness, viable spermatozoa (VS) and MCN were evaluated. Apoptosis was confirmed by TUNEL and TEM. The following immunoreactions were performed: T, aromatase, T/aromatase co-localization, V-ATPase, EGF, Cx43 and PCNA. The increased Adra1a and reduced Htr1b expressions confirmed the noradrenergic and serotonergic venlafaxine effects, respectively, corroborating the increased MCN, apoptosis and atrophy of SM. In VFG, the epithelial EGF increased, explaining Cx43 overexpression and basal cells mitotic activity. T aromatization and Esr1 downregulation indicate high estrogen levels, explaining CCs hypertrophy and changes in the V-ATPase localization, corroborating VS reduction. Thus, in addition to serotonergic/noradrenergic effects, T/estrogen imbalance, induced by venlafaxine, impairs epididymal structure and function.

ОСОБЕННОСТИ ЭКСПРЕССИИ ФАКТОРОВ ПРОЛИФЕРАЦИИТИРОЦИТОВ ПРИ ХРОНИЧЕСКОЙ ЭНДОГЕННОЙ ИНТОКСИКАЦИИ

Currently, thyroid diseases are one of the key problems of modern endocrinology, where the restoration of the thyroid parenchyma after exposure to negative factors and the direction of proliferative processes remain relevant. We conducted a study of the material obtained from deceased individuals as part of a standard pathoanatomical study, followed by animmunohistochemical staining of the expression of nonspecific markers of proliferative activity.It was found that the proliferative activity of thyrocytes increases from the upper to the lower part of the thyroid lobe. Also, in a comparative analysis of the central and peripheral zones, it was found that in normal conditions, the source of neoplasm of thyrocytes is located in the central zone, while when the tissue is involved in the pathological process, an increase in the expression of Ki-67 and PHH3 occursin the peripheral zone.The mitotic activity of cells can lead to the development of thyropathy and disruption of thyroid homeostasis,which serves as the basis for screening the thyroid gland in patients with chronic endogenous intoxication syndrome.

Effect of Xylene Oral Exposure on the Mouse Uterus – A Preliminary Study

Abstract Xylene is one of the environmental pollutants with a negative impact mainly on several organ systems. The aim of this study was to determine the effect of xylene on the uterus of mice. The study was performed on 12 adult female mice. Control mice (n = 6) were fed shredded pellets at a dose of 4 g per day. Xylene mice (n = 6) were fed the same diet at the same dose and orally administered xylene at 10 μl per day for 14 days. The mice were synchronized using the Whit-ten effect and introduced to males before the end of the procedure. Mice of both groups with no copulation plug were euthanized by cervical dislocation. The uteri were collected for routine histological and immunohistochemical analysis. The endometrial epithelium demonstrated vacuolar degeneration, mitotic cell activity, and the presence of leukocytes typical of metoestrus. Reductions of the endometrium, stroma, and myometrium were observed in the xylene mice. The xylene application did not have a significant effect on the superficial epithelium, or the size and number of uterine tubular glands. The immunohistochemical analysis of a proliferation marker PCNA revealed that the xylene increased its expression in the stroma, endometrial and myometrial cells, but did not significantly affect the superficial epithelial cells. The expression of an anti-apoptotic marker Bcl-xl in the xylene mice was stronger in the superficial epithelial, stromal, and endometrial cells. The Bcl-xl expression in the myometrial cells was similar to the controls. The results showed that the application of xylene stimulated the proliferation and exerted an anti-apoptotic effect on the uterine cells. However, the increased proliferation can lead to the malignant transformation of cells, resulting in their uncontrollable division.

Effect of combined maternal and post-hatch dietary 25-hydroxycholecalciferol supplementation on broiler chicken Pectoralis major muscle growth characteristics and satellite cell mitotic activity.

Skeletal muscle growth is largely dependent on the proliferation and differentiation of muscle-specific stem cells known as satellite cells (SC). Previous work has shown that dietary inclusion of the vitamin D3 metabolite, 25-hydroxycholecalciferol (25OHD3), also called calcidiol, can promote skeletal muscle growth in post-hatch broiler chickens. Improving vitamin D status of broiler breeder hens by feeding 25OHD3 in addition to vitamin D3 has also been shown to positively impact progeny. Yet, whether combined pre- and post-hatch supplementation with 25OHD3 produces an additive or synergistic SC-mediated, skeletal muscle growth response remains unanswered. To evaluate the effect of combined maternal and post-hatch dietary 25OHD3 supplementation on the growth and SC mitotic activity of the Pectoralis major (PM) muscles in broiler chickens, a randomized complete block design experiment with the main effects of maternal diet (MDIET) and post-hatch diet (PDIET) arranged in a 2 × 2 factorial treatment structure was conducted. From 25 to 36 wk of age, broiler breeder hens were fed 1 of 2 MDIET formulated to provide 5,000 IU D3 (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3). Their male broiler chick offspring (n = 400) hatched from eggs collected from 35 to 36 wk of age were reared in raised floor pens. Broilers were fed 1 of 2 PDIET formulated to provide 5,000 IU of D3 per kg of feed (PCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (P25OHD3). Muscle was collected at days 4, 8, 15, 22, and 29 and stored until immunofluorescence analysis. Data were analyzed as a 2-way ANOVA with SAS GLIMMIX. Dietary 25OHD3 was effectively transferred from hen plasma to egg yolks (P = 0.002) and to broiler progeny plasma (days 4 to 22; P ≤ 0.044). Including 25OHD3 in either MDIET or PDIET altered PM hypertrophic growth prior to day 29 (P ≥ 0.001) and tended to reduce Wooden Breast severity (P ≤ 0.089). Mitotic SC populations were increased in PM of MCTL:P25OHD3 and M25OHD:PCTL-fed broilers at d 4 (P = 0.037). At d 8, the PM mitotic SC populations were increased 33% by P25OHD3 (P = 0.054). The results of this study reveal that combined maternal and post-hatch 25OHD3 supplementation does not produce additive or synergistic effects on SC-mediated broiler muscle growth. However, vitamin D status improvement through dietary 25OHD3 inclusion in either the maternal or post-hatch diet stimulated broiler breast muscle growth by increasing proliferating SC populations.

MEDB-60. Medulloblastoma with extensive nodularity mimics cerebellar development and differentiates along the granular precursor lineage

BACKGROUND: Medulloblastoma with extensive nodularity (MBEN) represents a rare type of cerebellar tumors of infancy comprising two histologically distinct components that differ in cell differentiation and mitotic activity. Whereas some children suffering from MBEN experience disease recurrence, MBEN can also spontaneously differentiate and discontinue to grow. The underlying mechanisms of this variable biological behavior may offer insight into how embryonal tumors develop. METHODS: Fresh frozen and FFPE tumor tissue from nine MBEN-patients was subjected to multi-omics characterization including bulk sequencing, microdissection followed by RNA sequencing, single nucleus RNA-sequencing using the 10X Genomics- and SMART Seq. V2-protocols and spatial transcriptomics via RNAscope. RESULTS: All cases were molecularly classified as Sonic Hedgehog (SHH)-MB, and harbored somatic mutations within the SHH-pathway. After quality control, a total of ~30.000 cells were subjected to downstream analysis. Several non-malignant cell types, such as glial cells, were identified. In accordance with previous studies, we found only sparse immune infiltration. Unsupervised clustering identified cell clusters that differed in differentiation state and represented a continuum from embryonal-like cells with SHH-upregulation over intermediate cell states, to neuronal-like, postmitotic cells. Mapping to a single nucleus sequencing atlas of cerebellar development indicated that tumor cells reflected various stages of normally developing cerebellar granular precursors. Interestingly, one cluster of malignant cells with tumor-specific copy number alterations showed both transcriptomic features of astrocytes and embryonal cells. Using spatial transcriptomics, we were able to correlate different clusters of MBEN cells with distinct histologic MBEN compartments, with astrocyte-like tumor cells being located in the internodular compartment and in close proximity to mitotically active cancer cells. CONCLUSION: MBEN is formed by a continuum of malignant cell differentiation along the granular precursor lineage, with a subset of cells developing into cells that may represent tumor astrocytes. This differentiation process is reflected in the bicompartmental structure of MBEN.

Assessment of the functions of cultured lymphocytes when exposed to drugs used in cosmetology

The aim of the study was to determine the number of lymphocytes, intracellular cytokines produced by lymphocytes, and the cell cycle of lymphocytes isolated from the blood of patients when exposed to various drugs, as well as to assess the functions of cultured lymphocytes when exposed to drugs in vivo and in vitro.Materials and methods. The study involved lymphocytes isolated from the blood of healthy women under various conditions. At the first stage of the study, T-lymphocytes were isolated from the blood of patients before exposure to the drug. The absolute and relative lymphocyte count, the number of intracellular cytokines, and the cell cycle were determined.At the second stage, the drugs were added to the nutrient medium, where lymphocytes isolated from the blood of patients who did not receive systemic drugs were cultured. The placental extract preparation was added to the lymphocytes isolated from the first group of patients, while the hyaluronic acid preparation was added to the lymphocytes isolated from the second group of patients.At the third stage, the lymphocytes isolated from the blood of patients after systemic exposure to the placental extract preparation or hyaluronic acid preparation were isolated and cultured, after which the same lymphocyte parameters were determined.Results. The number of T-lymphocytes increased with the systemic use of the placental extract and hyaluronic acid preparations and practically did not change compared with the baseline data, when these drugs were added to the nutrient medium. Placental extract and hyaluronic acid had a positive effect on the mitotic activity of cells; it is worth noting that the effect of placental extract was greater than that of hyaluronic acid. Both drugs did not have a negative effect on apoptosis of T-lymphocytes. Under the effect of placental extract, lymphocytes secreted more interleukins, which contributed to proliferation of keratinocytes.Conclusion. The placental extract and hyaluronic acid preparations have a stimulating effect on keratinocytes. The placental extract preparation has a stimulating effect on T-lymphocytes after systemic exposure of the body to it.

Dietary Protein Source and Litter Condition Alter Broiler Chicken Intestinal Macrophage and Mitotically Active Cell Populations.

As antibiotic-free (ABF) broiler production continues to increase, understanding the development and local immune response in the intestines of ABF broilers is essential. Mitotically active cells, the majority of which will become enterocytes, help maintain the intestinal epithelial barrier. Macrophages prevent pathogen invasion by their phagocytic activity, functioning as immune response amplifying cells to aid in the recruitment of additional immune cells, and stimulating cytokine production in other adjacent cells. The objective of this experiment was to evaluate commonly used practical production practices on intestinal cell mitotic activity and local intestinal immunological responses. A randomized complete block design experiment with a 3 × 2 factorial treatment structure was conducted. The 3 dietary protein sources were: soybean meal (SBM), a mix of 50% poultry by-product meal and 50% feather meal (PFM), and porcine meat and bone meal (MBM) and broilers were reared on either new litter (NL) or used litter (UL). On d 3, 8, 11, 15, and 21, 6 birds per treatment from 6 blocks (total n = 36 per d) were randomly selected for sampling. Broilers were injected intraperitoneally with 5'-bromo-2'-deoxyuridine (BrdU) 1 h prior to sampling to label mitotically active cells. Samples were analyzed using cryohistology and immunofluorescence to determine the density of mitotically active cells and macrophages. Mitotically active cell and macrophage densities changed in both the duodenum and ileum over time. Neither dietary protein source nor litter condition affected mitotically active cell or macrophage densities in the duodenum on d 11 and 21 or in the ileum on d 3, 8, 11, and 15. However, on d 3 and 15 in the duodenum (P ≤ 0.0126) and d 21 in the ileum (P ≤ 0.0009), broilers reared on UL had greater mitotically active cell densities than those reared on NL. On d 8 in the duodenum, broilers fed MBM had increased macrophage density compared with those fed PFM and SBM (P ≤ 0.0401). These results indicate dietary protein source and litter condition may impact the physiology of the broiler small intestine, though additional work with this model is necessary to understand the underlying mechanisms.

Combined Maternal and Post-Hatch Dietary Supplementation of 25-Hydroxycholecalciferol Alters Early Post-Hatch Broiler Chicken Duodenal Macrophage and Crypt Cell Populations and Their Mitotic Activity.

The previous work has demonstrated that maternal supplementation of the circulating metabolite of vitamin D3 (D3), 25-hydroxycholecalciferol (25OHD3), enhances the immunocompetence of broiler chick offspring. In post-hatch broiler diets, 25OHD3 has been shown to affect intestinal morphology and improve the immune status of broilers. An experiment with a 2 × 2 factorial treatment arrangement was conducted to assess the effects of combining maternal (MDIET) and post-hatch (PDIET) dietary 25OHD3 inclusion on duodenal crypt and macrophage cell populations and mitotic activity in young broiler chickens. All diets were formulated to provide 5,000 IU of vitamin D. Broiler breeder hens were offered 1 of 2 MDIET: 5,000 IU D3 per kg of feed (MCTL) or 2,240 IU of D3 + 2,760 IU of 25OHD3 per kg of feed (M25OHD3) from week 25 to 41. Male broiler offspring (n = 480) hatched from eggs collected during week 41 of breeding age were allotted in raised floor pens (4 birds per pen from day 0 to 7 and individually allotted from day 8 to 21). Chicks were fed 1 of 2 PDIET (starter day 0 to 21): 5,000 IU D3 per kg of feed (PCTL) or 2,240 IU D3 + 2,760 IU 25OHD3 (P25OHD3). DUO samples (n = 12 birds per treatment per day) were collected on days 3, 6, 9, 12, 15, 18, and 21 for cryohistological and immunofluorescence analysis to facilitate the enumeration of the total macrophages, CD80+ macrophages (pro-inflammatory macrophages), and mitotically active cells (BrdU+) to calculate the proportion of proliferating cells (PPC) per duodenal crypt. Bird age impacted crypt PPC with the greatest PPC per duodenal crypt observed on days 3 and 9, and the lowest PPC per crypt was observed on day 21 (P < 0.0001). Broilers from the M25OHD3:PCTL treatment had a greater PPC (P =.002) than birds from the MCTL:PCTL treatment at day 3. An interaction among MDIET and PDIET was observed for proliferating macrophages at day 21 (P = 0.029) where M25OHD3:P25OHD3 birds had more proliferating macrophages than M25OHD3:PCTL-fed birds. These results indicate that combined MDIET and PDIET 25OHD3 supplementation may alter early post-hatch duodenal development and innate immunity.

Oleic acid is an endogenous ligand of TLX/NR2E1 that triggers hippocampal neurogenesis

Neural stem cells, the source of newborn neurons in the adult hippocampus, are intimately involved in learning and memory, mood, and stress response. Despite considerable progress in understanding the biology of neural stem cells and neurogenesis, regulating the neural stem cell population precisely has remained elusive because we have lacked the specific targets to stimulate their proliferation and neurogenesis. The orphan nuclear receptor TLX/NR2E1 governs neural stem and progenitor cell self-renewal and proliferation, but the precise mechanism by which it accomplishes this is not well understood because its endogenous ligand is not known. Here, we identify oleic acid (18:1ω9 monounsaturated fatty acid) as such a ligand. We first show that oleic acid is critical for neural stem cell survival. Next, we demonstrate that it binds to TLX to convert it from a transcriptional repressor to a transcriptional activator of cell-cycle and neurogenesis genes, which in turn increases neural stem cell mitotic activity and drives hippocampal neurogenesis in mice. Interestingly, oleic acid-activated TLX strongly up-regulates cell cycle genes while only modestly up-regulating neurogenic genes. We propose a model in which sufficient quantities of this endogenous ligand must bind to TLX to trigger the switch to proliferation and drive the progeny toward neuronal lineage. Oleic acid thus serves as a metabolic regulator of TLX activity that can be used to selectively target neural stem cells, paving the way for future therapeutic manipulations to counteract pathogenic impairments of neurogenesis.

The Wnt Pathway: From Signaling Mechanisms to Synthetic Modulators.

The Wnt pathway is central to a host of developmental and disease-related processes. The remarkable conservation of this intercellular signaling cascade throughout metazoan lineages indicates that it coevolved with multicellularity to regulate the generation and spatial arrangement of distinct cell types. By regulating cell fate specification, mitotic activity, and cell polarity, Wnt signaling orchestrates development and tissue homeostasis, and its dysregulation is implicated in developmental defects, cancer, and degenerative disorders. We review advances in our understanding of this key pathway, from Wnt protein production and secretion to relay of the signal in the cytoplasm of the receiving cell. We discuss the evolutionary history of this pathway as well as endogenous and synthetic modulators of its activity. Finally, we highlight remaining gaps in our knowledge of Wnt signal transduction and avenues for future research.

Fine Needle Aspiration Cytology of Giant Cell Tumour of Tendon Sheath

Introduction: Giant Cell Tumour of Tendon Sheath (GCTTS), also known as localised nodular tenosynovitis, is a slow growing benign soft tissue tumour arising from synovium of tendon sheath, bursa or joint. Clinically, the lesions occur as skin-coloured nodules typically on the extremities. These tumours occur more frequently on the upper limbs especially hands (77%) where they form the second most common tumour following simple ganglion cyst. Aim: To describe the cytomorphologic findings in GCTTS and their histopathological features. Materials and Methods: This retrospective study was conducted at Department of Pathology, ESI Hospital, Delhi, India, in 12 diagnosed GCTTS cases for their cytological features from January 2015 to December 2017. Fine Needle Aspiration Cytology (FNAC) was performed with a 22-gauge needle attached to a 10 mL syringe. Smears were air-dried and stained with Giemsa stain. Cytomorphology of all the selected cases were analysed and descriptive statistics were used to evaluate the cases. Results: A total of 12 cases of GCTTS were retrieved. Diagnosis of GCTTS was made by FNAC in all the cases and confirmed by histopathological examination in half of the cases. The mean age of presentation was 39 years. Of these, 8 (66.67%) were women and 4 (33.33%) were men. The lesions were found most commonly over the index finger (n=5) followed by the ring finger (n=3), thumb (n=2), middle finger (n=1) and little finger (n=1). The most frequent clinical presentation was a painless, nodular, slow growing firm swelling over the finger. FNAC revealed cellular smears with few clusters and numerous scattered stromal cells along with interspersed multinucleated giant cells. Conclusion: A definitive preoperative diagnosis of GCTTS obtained through FNAC helps in formulating appropriate treatment plan. Histopathologic examination will confirm the cytological diagnosis and can help to predict recurrence by providing information on the resection margin, any satellite nodules, variable cell types and mitotic activity.