Cells Tissues Organs最新文献

Introduction: Macrophages exist on a spectrum from pro-inflammatory (M1) to pro-healing (M2). Characterization of macrophage phenotype is important to understand tissue healing and response. The gold standard for assessing macrophage phenotypes is immunocytochemistry (ICC), which stains inducible nitric oxide synthase (iNOS) and arginase (Arg1), the proteins secreted before nitrite and urea production. The ICC method is an endpoint assay, time-consuming, and costly. Therefore, a more effective method to assess the phenotype of macrophages in vitro is needed. Based on the phenotype of the macrophage, the amino acid arginine gets metabolized differently. If arginine is metabolized by M1 macrophages, it produces nitrite, and if it is metabolized by M2 macrophages, it produces urea. A method that leverages arginine metabolism through secreted products (urea and nitrite) has the potential to determine macrophage phenotype in real time.

Methods: Rat bone marrow-derived macrophages were cultured to be naïve or polarized to M1-like or M2-like. The gold standard ICC method was used to determine the intensity of the iNOS and Arg1 staining. Nitrite and urea kits were utilized to measure the concentration of nitrite and urea in the media eluted from macrophages of various phenotypes. Nitrite and urea concentrations were compared to ICC results to validate the new method.

Results: ICC revealed the iNOS staining was significant and 2.5-folds higher in M1-like macrophages and the Arg1 staining was significant and 1.5-folds higher in the M2-like macrophages. The nitrite concentration was significant and 4-folds higher in the M1-like macrophage media, and the urea concentration was significant and 2.5-folds higher in the M2-like macrophage media. A correlation analysis showed that iNOS staining intensity and nitrite concentration levels had a linear correlation as well as Arg1 staining intensity and urea concentration levels.

Conclusion: Data confirm that the determination of nitrite and urea concentration can be utilized to assess macrophage phenotypes.

Introduction: The final step of the fertilization process involves gametes adhesion and fusion. JUNO is an essential folate receptor 4 protein present in the ooplasm of oocytes, which binds to IZUMO1, its receptor on the sperm surface. Both proteins are indispensable for the sperm-oocyte interaction, and their absence results in infertility. Despite the importance of JUNO in reproduction, there is still controversy about how different factors affect the functionality of JUNO. Therefore, the goal of this study was to provide a comprehensive overview of what we know so far about the presence and functionality of JUNO.

Methods: In order to accomplish this, a total of 198 articles were identified. Based on both inclusion and exclusion criteria, 40 articles were finally included in this study.

Results: The results showed that during oocyte maturation, the expression levels of JUNO undergo alterations and, in some instances, cross-species gamete fusion is possible. Additionally, it has been observed that exposure of oocytes to factors such as bisphenol A, 17α-ethynylestradiol, diazinon, benzo(a)pyrene, butylparaben, bis(2-ethylhexyl) phthalate, hydroxyurea, dichlorophenol, isoniazid, and para-phenylenediamine disrupt JUNO and decrease the fertilization process rates. Moreover, exposure to ionic radiation, vitrification, and synthetic materials as microplastics has the same effect. Nonetheless, other compounds such as melatonin, mogroside V, cholesterol-loaded methyl-β-cyclodextrin, methyl-β-cyclodextrin, protocatechuic acid, coenzyme Q10, resveratrol, and Shoutai pills have been shown to enhance female fertility in terms of JUNO functionality.

Conclusion: In summary, this update highlights the crucial role of JUNO during fertilization and reveals how different factors and experimental procedures affect its activity.

Introduction: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes.

Methods: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/TA+ fish do not mediate creb3l1-dependent transcription.

Results: We document that the creb3l1TA+/TA+ fish exhibit defects in the patterning of caudal fin lepidotrichia, with significantly distalized points of proximal bifurcation and decreased secondary bifurcations. Moreover, using the caudal fin amputation model, we show that creb3l1TA+/TA+ fish have decreased regeneration and that their regenerates replicate the distalization and bifurcation defects observed in intact fins of creb3l1TA+/TA+ animals. These defects correlate with altered expression of the shha and ptch2 components of the Sonic Hedgehog signaling pathway in creb3l1TA+/TA+ regenerates.

Conclusion: Together, our results uncover a previously unknown intersection between Creb3l1 and the Sonic Hedgehog pathway and document a novel role of Creb3l1 in tissue patterning.

Introduction: The formation of normal bone and bone healing requires the cAMP-responsive element binding protein 3-like-1 (Creb3l1) transmembrane transcription factor, as deletion of the murine CREB3L1 results in osteopenic animals with limited capacity to repair bone after a fracture. Creb3l1 undergoes regulated intramembrane proteolysis (RIP) to release the N-terminal transcription activating (TA) fragment that enters the nucleus and regulates the expression of target genes.

Methods: To expand our understanding of Creb3l1's role in skeletal development and skeletal patterning, we aimed to generate animals expressing only the TA fragment of Creb3l1 lacking the transmembrane domain and thereby not regulated through RIP. However, the CRISPR/Cas9-mediated genome editing in zebrafish Danio rerio caused a frameshift mutation that added 56 random amino acids at the C-terminus of the TA fragment (TA+), making it unable to enter the nucleus. Thus, TA+ does not regulate transcription, and the creb3l1TA+/

Introduction: The detection of chemical signals by the vomeronasal organ (VNO) is critical for mammals from an early age, influencing behaviors such as suckling and recognition of the mother. Located at the base of the nasal cavity, the VNO features a duct covered with a sensory epithelium. A critical aspect of VNO functionality is the efficient access of stimuli from the nasal and oral cavities to the receptors. In adult dogs, it has been demonstrated how the vomeronasal duct (VD) communicates to the environment through the incisive duct (ID). In newborn puppies, the existence of functional communication between the ID and the VD has not been confirmed to date, raising doubts about the potential physiological obliteration of the ID. Determining this aspect is necessary to evaluate the role played by chemocommunication in the survival and socialization of puppies.

Methods: This study employs serial histological staining to examine the presence and functionality of the ID in neonatal dogs. Additionally, a histochemical study was conducted using periodic acid-Schiff and Alcian Blue staining, along with labeling with six lectins to characterize the expression of glycoconjugates in the incisive papilla and in the area between the ID and the VD.

Results: The histological study has confirmed both the existence of functional communication between both ducts in perinatal puppies and the dual functional communication of the ID with the oral and nasal cavities. Lectin labeling has allowed for the characterization of the glycoconjugate expression profile in the papilla and ID, showing significant differences between lectins.

Conclusion: The ID is associated with a sophisticated cartilaginous complex that prevents its collapse, as well as erectile tissue that acts as a cushion, facilitating its action under pressure induced by sampling behaviors such as tonguing. This investigation demonstrates the communicative capabilities of the VNO during the perinatal stage in dogs.

Introduction: Androgenic alopecia (AGA), a hair loss condition caused by dihydrotestosterone binding to hair follicle receptors, negatively impacts quality of life for both men and women. Current treatments like minoxidil and finasteride have limitations, highlighting the need for alternative therapies, such as human umbilical cord blood-derived mesenchymal stem cells (HUCB-MSCs).

Methods: In this study, forty-eight adult male Wistar albino rats (3 months old) were used. The control group (Group I) received no treatment, while the other rats underwent AGA induction via daily subcutaneous testosterone injections (100 mg/kg). These rats developed alopecia and were divided into three groups: AGA (Group II), AGA plus daily minoxidil spray (Group III), and AGA plus a single intradermal injection of HUCB-MSCs (1 mL containing 1 × 105 cells, Group IV). After 4 weeks, the rats were sacrificed, and skin specimens were prepared for histological analysis using H&E, Masson's trichrome, and immunohistochemical staining for CK 19, vascular endothelial growth factor (VEGF), and TUNEL antibodies.

Results: It was shown that HUCB-MSC treatment reversed structural damage to hair and follicles, normalizing conditions within 1-week post-injection. The treatment enhanced the anagen phase, suppressed telogen and catagen phases, reduced apoptosis, and increased VEGF and CK 19 immune reactions. Observational follow-up for Groups III and IV revealed that while the minoxidil group experienced significant hair loss after 37 days, the stem cell group exhibited dense and long hair covering the treated area.

Conclusion: HUCB-MSC therapy demonstrated superior efficacy over minoxidil with no observed side effects, indicating its potential as a promising alternative for AGA treatment.

Background: The placenta is a temporary organ that develops throughout pregnancy, connecting a developing fetus to the maternal uterine wall. The placenta's structure is species-specific and complex, necessitating the advancement of in vitro models to study this dynamic organ. The main cell types composing the placenta, trophoblast cells, serve several roles and have been incorporated within biomaterials and devices to recapitulate the placental microenvironment.

Summary: This review highlights in vitro 2D, 3D, and on-a-chip models of two placental interfaces: the interface between the endometrium and extravillous trophoblast cells and the interface between the chorionic villi and intervillous space. First, an overview of placental cell types and in vitro model types used in the discussed studies is provided. Next, models of invasive trophoblasts cells at the endometrium where the placenta is anchored and the spiral arteries are remodeled are discussed. Next, the review highlights models of the chorionic villi and intervillous space, an interface of high gas and nutrient exchange where cytotrophoblast cells fuse into syncytiotrophoblasts. Finally, we discuss key takeaways and future directions in creating representative placental models.

Key messages: Combining biomaterial and engineering approaches has led to the development of physiologically relevant models, allowing placental trophoblast functions to be investigated with more clarity. Each cell type (e.g., trophoblast cell line vs. stem cells vs. primary placental cells) and biomaterial system (e.g., organoid vs. on-a-chip) that is selected for a given model has a unique combination of advantages and limitations, which are detailed within this review. Overall, the placental models discussed enable trophoblast cell behavior to be studied in vitro with the inclusion of extracellular matrix materials, growth factors, and other environmental cues. While one model alone does not fully recapitulate every function of the placenta, individual models are tailored to inform on specific placental trophoblast behaviors.

Introduction: Members of the carbonic anhydrase gene family, responsible for the reversible hydration of carbon dioxide, participate in several important biological processes including processes involved in fertilization. CAIV has been shown to play a role in sperm cell capacitation and regulation of sperm motility and is present in mature murine placentae. The present study specifically analyzes the distribution of CAIV in female reproductive organs and during early placenta development.

Methods: Immunostaining for CAIV was performed on female reproductive organs (ovary, fallopian tube, uterus, vagina) of nonpregnant mice and on implantation sites of early pregnancy between 4.5 and 9.5 days post-coitum (dpc). Sex typing of embryos was performed by PCR using three separated gene combinations for X and Y chromosomes, respectively. Additionally, reproductive outcome of CAIV-deficient mice was determined.

Results: CAIV is largely absent in the female reproductive organs of nonpregnant mice. Immunostaining for CAIV was present in the blastocyst and in consecutive stages of the developing embryo. In the endometrial epithelium distant from the implantation chamber, CAIV is induced from 8.5 dpc onward. Moreover, the yolk sac epithelium, the trophoblast giant cells, and the labyrinthine compartment of the developing hemochorial placenta show a strong immunostaining for CAIV. In heterozygous mating, the number of CAIV knockout pups is significantly reduced than was to be expected according to the mendelian rules, while homozygous mating of CAIV knockout mice results in a significant reduction of litter size, which is mainly due to a reduced number of female mice born. Since at 9.5 dpc the number of female embryos is rather higher than that of males, the observed reduction of female offspring appears to be due to a defect in placentation after 9.5 dpc.

Conclusion: Thus, CAIV seems to be involved in the signaling network of embryo development, implantation, and placentation.

Introduction: Hemolymph nodes, characterized by erythrocyte rosettes, are found in humans and animals, including rats. The cytoarchitectural features that these nodes exhibit compared with those of ordinary lymph nodes and spleen are unknown. Herein, we describe the cytoarchitecture of rat hemolymph nodes.

Methods: We performed immunohistochemical analyses with antibodies against CD68, Iba-1, CD3, CD20, and S-100. Hematoxylin and eosin staining was used to compare findings with sections from ordinary lymph nodes and spleen.

Results: Hemolymph nodes exhibited erythrocyte rosettes with macrophages immunopositive for CD68, Iba-1, and CD3, which were rare in the physiologically normal spleen and lymph nodes. Additionally, sinusoidal macrophages often showed close apposition to erythrocytes and mast cells. Accumulation of cells immunoreactive to CD20, a B-lymphocyte marker, was seen only in the germinal centers of ordinary lymph nodes, not in the hemolymph nodes or spleen. Ordinary lymph nodes and spleen showed well-developed reticular configurations of cells with immunoreactivity for S-100, a marker for dendritic cells, unlike hemolymph nodes, suggesting less-developed antigen-presenting ability in the latter. Despite similarities to ordinary lymph nodes and spleen, the direct contact with erythrocytes and mast cells in the hemolymph nodes suggests a facilitation of direct cell-to-cell communication for macrophages, erythrocytes, and mast cells.

Conclusion: Our findings imply that the hemolymph nodes are a unique immune organ, differing from ordinary lymph nodes and spleen.

Introduction: This study aimed to examine the transforming growth factor (TGF)-β signaling pathway during secondary palate fusion by transfecting single and double small interfering RNA (siRNAs) for TGF-β2 and -β3. This investigation also focused on understanding the phenotype of palatal development.

Methods: siRNAs targeting TGF-β2 and -β3 were used in an organ culture model of fusion of the secondary palate of 13-day embryonic ICR mice cultured for up to 72 h. The palatal shelves were collected at different times following the initiation of organ culture and were examined for TGF-β2 and -β3 gene expression. Downstream signaling was characterized using Western blotting and PCR.

Results: In the double siRNA-treated palatal shelves, approximately 90% (91% anterior, 89% posterior with phenotype A) showed fusion failure in hematoxylin and eosin staining. Phosphorylation of Smad-dependent and -independent signaling showed a significant reduction in phosphorylation in double knockdown palate organ cultures when compared to single knockdown cultures. Although, the expression of matrix metalloproteinase 13 and TIMP2 were small influenced by siTGF-β2, the extracellular matrix and transcription factor expressions showed to be significantly reduced in double knockdown palate compared to single knockdown palates.

Conclusions: This study demonstrates that double siRNAs targeting TGF-β2 and -β3 results in phenotypes during secondary palatal fusion and that they could be affected phosphorylation of Smad-dependent and -independent signaling synergistically compared to single knockdown of TGF-β2 and -β3. The results of this study demonstrate important functions during secondary palatal fusion and will contribute to our understanding of the etiology of cleft palate.