Simple propagation method for resident macrophages by co-culture and subculture, and their isolation from various organs.

BACKGROUND: Resident macrophages (Mø) originating from yolk sac Mø and/or foetal monocytes colonise tissues/organs during embryonic development. They persist into adulthood by self-renewal at a steady state, independent of adult monocyte inputs, except for those in the intestines and dermis. Thus, many resident Mø can be propagated in vitro under optimal conditions; however, there are no specific in vitro culture methods available for the propagation of resident Mø from diverse tissues/organs. RESULTS: We provided a simple method for propagating resident Mø derived from the liver, spleen, lung, and brain of ICR male mice by co-culture and subculture along with the propagation of other stromal cells of the respective organs in standard culture media and successfully demonstrated the propagation of resident Mø colonising these organs. We also proposed a simple method for segregating Mø from stromal cells according to their adhesive property on bacteriological Petri dishes, which enabled the collection of more than 97.6% of the resident Mø from each organ. Expression analyses of conventional Mø markers by flow cytometry showed similar expression patterns among the Mø collected from the organs. CONCLUSION: This is the first study to clearly provide a practical Mø propagation method applicable to resident Mø of diverse tissues and organs. Thus, this novel practical Mø propagation method can offer broad applications for the use of resident Mø of diverse tissues and organs.

EphA receptors and ephrin-A ligands are upregulated by monocytic differentiation/maturation and promote cell adhesion and protrusion formation in HL60 monocytes.

BACKGROUND: Eph signaling is known to induce contrasting cell behaviors such as promoting and inhibiting cell adhesion/spreading by altering F-actin organization and influencing integrin activities. We have previously demonstrated that EphA2 stimulation by ephrin-A1 promotes cell adhesion through interaction with integrins and integrin ligands in two monocyte/macrophage cell lines. Although mature mononuclear leukocytes express several members of the EphA/ephrin-A subclass, their expression has not been examined in monocytes undergoing during differentiation and maturation. RESULTS: Using RT-PCR, we have shown that EphA2, ephrin-A1, and ephrin-A2 expression was upregulated in murine bone marrow mononuclear cells during monocyte maturation. Moreover, EphA2 and EphA4 expression was induced, and ephrin-A4 expression was upregulated, in a human promyelocytic leukemia cell line, HL60, along with monocyte differentiation toward the classical CD14++CD16- monocyte subset. Using RT-PCR and flow cytometry, we have also shown that expression levels of αL, αM, αX, and ß2 integrin subunits were upregulated in HL60 cells along with monocyte differentiation while those of α4, α5, α6, and ß1 subunits were unchanged. Using a cell attachment stripe assay, we have shown that stimulation by EphA as well as ephrin-A, likely promoted adhesion to an integrin ligand-coated surface in HL60 monocytes. Moreover, EphA and ephrin-A stimulation likely promoted the formation of protrusions in HL60 monocytes. CONCLUSIONS: Notably, this study is the first analysis of EphA/ephrin-A expression during monocytic differentiation/maturation and of ephrin-A stimulation affecting monocyte adhesion to an integrin ligand-coated surface. Thus, we propose that monocyte adhesion via integrin activation and the formation of protrusions is likely promoted by stimulation of EphA as well as of ephrin-A.

Truncated EphA2 likely potentiates cell adhesion via integrins as well as infiltration and/or lodgment of a monocyte/macrophage cell line in the red pulp and marginal zone of the mouse spleen, where ephrin-A1 is prominently expressed in the vasculature.

We previously established a J774.1 monocyte/macrophage subline expressing a truncated EphA2 construct lacking the kinase domain. We demonstrated that following ephrin-A1 stimulation, endogenous EphA2 promotes cell adhesion through interaction with integrins and integrin ligands such as ICAM1 and that truncated EphA2 potentiates the adhesion and becomes associated with the integrin/integrin ligand complex. Based on these findings, we hypothesized that the EphA/ephrin-A system, particularly EphA2/ephrin-A1, regulates transendothelial migration/tissue infiltration of monocytes/macrophages, because ephrin-A1 is widely recognized to be upregulated in inflammatory vasculatures. To evaluate whether this hypothesis is applicable in the spleen, we screened for EphA2/ephrin-A1 expression and reexamined the cellular properties of the J774.1 subline. We found that ephrin-A1 was expressed in the vasculature of the marginal zone and the red pulp and that its expression was upregulated in response to phagocyte depletion; further, CD115, F4/80, and CXCR4 were expressed in J774.1 cells, which serve as a usable substitute for monocytes/macrophages. Moreover, following ephrin-A1 stimulation, truncated EphA2 did not detectably interfere with the phosphorylation of endogenous EphA2, and it potentiated cell adhesion possibly through modulation of integrin avidity. Accordingly, by intravenously injecting mice with equal numbers of J774.1 and the subline cells labeled with distinct fluorochromes, we determined that truncated EphA2 markedly potentiated preferential cell infiltration into the red pulp and the marginal zone. Thus, modulation of EphA2 signaling might contribute to effective transplantation of tissue-specific resident macrophages and/or monocytes.

Complementary expression and repulsive signaling suggest that EphB2 and ephrin-B1 are possibly involved in epithelial boundary formation at the squamocolumnar junction in the rodent stomach.

Eph receptors and ephrin ligands are cell-cell communication molecules with well-defined roles in cell adhesion, migration, and tissue boundary formation. However, their expression levels in the squamocolumnar epithelial junction region at the distal esophagus are completely unknown. We examined EphB2 and ephrin-B1 localization in the squamocolumnar epithelial junction region between the proximal and distal stomach of the rodents. Immunostaining showed complimentary expression patterns along the proximal-to-distal axis of the gastric epithelia across the junction: EphB2 expression was maximal around the epithelial junction and sharply decreased in the stratified squamous epithelium at a short distance from the junction, whereas ephrin-B1 was strongly expressed in the stratified squamous epithelium at a distance from the junction and sharply decreased toward the junction. These expression patterns suggest that EphB2/ephrin-B1 signaling occurs preferentially in the epithelia across the junction, where the receptor and ligand expression highly overlap. We also show that (1) EphB2 preferentially binds ephrin-B1, and (2) cell repulsion/lateral migration was induced in primary cultured gastric keratinocytes on ephrin-B1-Fc- and EphB2-Fc-coated surfaces. On the basis of these findings, we propose that EphB2 and ephrin-B1 are possibly involved in epithelial boundary formation at the squamocolumnar junction.

Testosterone upregulates progesterone production in mouse testicular interstitial macrophages, whose niche likely provides properties of progesterone production to tissue-resident macrophages.

The niche of the macrophages (Mø) residence concept is now accepted; Mø colonize tissue/organ-specific microenvironments (niches) that shape Mø to perform tissue/organ-specific functions. Recently, we developed a simple propagation method for tissue-resident Mø by mixed culture with the respective tissue/organ-residing cells acting as the niche and demonstrated that testicular interstitial Mø propagated by mixed culture with testicular interstitial cells showing properties of Leydig cells in culture (we termed them "testicular Mø niche cells") produce progesterone (P4) de novo. Based on previous evidence of testosterone production downregulation in Leydig cells by P4 and androgen receptor expression in testicular Mø, we proposed a local feedback loop of testosterone production between Leydig cells and testicular interstitial Mø. To verify this hypothesis, we further examined P4 de novo production in propagated testicular interstitial Mø treated with testosterone using ELISA and found that exogenous testosterone upregulates P4 production in testicular interstitial Mø. Thus, testosterone production, which is controlled by the local feedback loop, likely becomes more reliable. Moreover, we examined whether tissue-resident Mø other than testicular interstitial Mø can be transformed into P4-producing cells by mixed culture with testicular Mø niche cells: using RT-PCR and ELISA we found that splenic Mø newly acquired P4 production properties by mixed-culturing with testicular Mø niche cells for 7 days. This likely indicates the substantiative in vitro evidence on the niche concept and possibly opens the door to using P4-secreting Mø as a transplantation tool for clinical application due to the migratory property of Mø into inflammatory sites.

Mixed-Culture Propagation of Uterine-Tissue-Resident Macrophages and Their Expression Properties of Steroidogenic Molecules.

Tissue-resident macrophages (Mø) play tissue/organ-specific roles, and the physiological/pathological implications of uterine Mø in fertility and infertility are not yet fully understood. Herein, we report a simple propagation method for tissue-resident Mø by mixed culture with the respective tissue/organ-residing cells as the niche. We successfully propagated mouse uterine Mø by mixed culture with fibroblastic cells that exhibited properties of endometrial stromal cells. Propagated mouse uterine Mø were CD206- and arginase-1-positive; iNOS- and MHC-II-negative, indicating M2 polarization; and highly phagocytic, similar to endometrial Mø. Furthermore, uterine Mø were observed to express steroidogenic molecules including SRD5A1 and exhibited gap junction formation, likely with endometrial stromal cells. Accordingly, uterine Mø propagated by mixed culture may provide a new tool for studying immune-endocrine interactions related to fertility and infertility, particularly androgen's intracrine actions in preparing the uterine tissue environment to support implantation and pregnancy as well as in the etiology of endometriosis.

Eph/Ephrin Promotes the Adhesion of Liver Tissue-Resident Macrophages to a Mimicked Surface of Liver Sinusoidal Endothelial Cells.

Kupffer cells are maintained via self-renewal in specific microenvironmental niches, primarily the liver sinusoidal endothelial cells (LSECs). In this study, we propagated tissue-resident macrophages (Mø) from mouse liver using mixed culture with hepatic fibroblastic cells. Propagated liver Mø express Id3, Lxra and Spic transcription factors, which are required for Kupffer cell characterization. Thus, Kupffer cell properties are likely to be maintained in liver Mø propagated using mixed culture with fibroblastic cells. We revealed (i) gene expression of certain Eph receptors and ephrin ligands including EphA2, ephrin-A1, EphB4, and ephrin-B1 in propagated liver Mø and primary LSECs, (ii) immunohistochemical localization of these Eph/ephrin member molecules indicating common expression in Kupffer cells and LSECs, and (iii) surface expression of several integrin α and ß subunits, including α4ß1, αLß2, αMß2, and αXß2 integrin in propagated liver Mø and that of the corresponding ligands ICAM-1 and VCAM-1 in primary LSECs. Moreover, EphA/ephrin-A and EphB/ephrin-B interactions promoted liver Mø adhesion to the ICAM-1-adsorbed surface, which mimicked that of LSECs and may be implicated in the residence of Kupffer cells in the liver sinusoid. Further studies on regulating the residence and regeneration of Kupffer cells in related hepatic disorders are required to validate our findings.

Testicular Macrophages Produce Progesterone De Novo Promoted by cAMP and Inhibited by M1 Polarization Inducers.

Tissue-resident macrophages (Mø) originating from fetal precursors are maintained via self-renewal under tissue-/organ-specific microenvironments. Herein, we developed a propagation method of testicular tissue-resident Mø in mixed primary culture with interstitial cells composed of Leydig cells from the mouse testis. We examined Mø/monocyte marker expression in propagated testicular Mø using flow cytometry; gene expression involved in testosterone production as well as spermatogenesis in testicular Mø and interstitial cells propagated by mixed culture via RT-PCR; and progesterone (P4) de novo production in propagated testicular Mø treated with cyclic adenosine monophosphate, isoproterenol, and M1 polarization inducers using ELISA. Mø marker expression patterns in the propagated Mø were identical to those in testicular interstitial Mø with a CD206-positive/major histocompatibility complex (MHC) II-negative M2 phenotype. We identified the genes involved in P4 production, transcription factors essential for steroidogenesis, and androgen receptors, and showed that P4 production de novo was upregulated by cyclic adenosine monophosphate and ß2-adrenergic stimulation and was downregulated by M1 polarization stimulation in Mø. We also demonstrated the formation of gap junctions between Leydig cells and interstitial Mø. This is the first study to demonstrate de novo P4 production in tissue-resident Mø. Based on previous studies revealing inhibition of testosterone production by P4, we propose that local feedback machinery between Leydig cells and adjacent interstitial Mø regulates testosterone production. The results presented in this study can facilitate future studies on immune-endocrine interactions in gonads that are related to infertility and hormonal disorders.

EphB signaling inhibits gap junctional intercellular communication and synchronized contraction in cultured cardiomyocytes.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with important roles not only in development but also in the physiology of many adult organs. However, their cellular localization and functions in the myocardium are virtually unknown and therefore, we have investigated the expression of EphB receptors and ephrin-B ligands in the rodent heart ventricles and their functions in the rodent cardiomyocytes of primary culture. Examinations by RT-PCR, immunohistochemistry and in situ hybridization revealed that the EphB receptors are preferentially expressed in cardiomyocytes and ephrin-B ligands in the vasculature in adult mouse heart ventricles. Interestingly, we found that inducing high levels of EphB receptor activation in primary cultures of rodent cardiomyocytes by stimulation with ephrin-B1-Fc desynchronized the contraction of adjacent clusters of cardiomyocytes that had contracted synchronously before the treatment. Co-immunoprecipitation experiments revealed that EphB4 physically associates with connexin43, a major component of gap junctions in the myocardium, and that EphB activation inhibits gap junctional intracellular communication between cardiomyocytes. The present findings suggest that ephrin-B-EphB signaling can modulate the electrical coupling of cardiomyocytes through effects on gap junctions.

Complementary expression of EphB receptors and ephrin-B ligand in the pyloric and duodenal epithelium of adult mice.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules that regulate the spatial organisation of cells in various tissues by repulsive or adhesive signals arising from contact between EphB- and ephrin-bearing cells. However, the expression and functions of Eph receptors in the gastric epithelium and Brunner's glands are virtually unknown. We detected several EphB receptors and ephrin-B ligands in the pyloric and duodenal mucosa of the adult mouse by RT-PCR amplification. Immunostaining showed complementary expression patterns, with ephrin-B1 being preferentially expressed in the superficial part and EphB receptors in the deeper part of both epithelia. In the gastric pylorus, ephrin-B1 was expressed in pit cells and proliferating cells of the isthmus. In contrast, EphB2, EphB3, and EphB4 were expressed in pyloric glandular cells and proliferating cells of the isthmus. In the duodenum, ephrin-B1 was expressed in cells lining the ducts of Brunner's glands as well as those covering villi and the upper portion of the crypts of Lieberkühn. In contrast, EphB2 and EphB3 were expressed in the gland segment of Brunner's glands and the lower portion of the crypts and EphB4, in the crypts. In both mucosae, EphB2, EphB3, and EphB4 were found to be tyrosine phosphorylated, suggesting that EphB/ephrin-B signalling might occur preferentially in the isthmus, crypts, and duct-gland transition of Brunner's glands, where the receptor and ligand expression overlaps. Based on these findings, we propose that EphB/ephrin-B signalling may regulate cell positioning within the pyloric and duodenal epithelium.

Complementary expression and repulsive signaling suggest that EphB receptors and ephrin-B ligands control cell positioning in the gastric epithelium.

Eph receptors and ephrin ligands are membrane-bound cell-cell communication molecules with well-defined roles in development. However, their expression and functions in the gastric epithelium are virtually unknown. We detected several EphB receptors and ephrin-Bs in the gastric corpus mucosa of the adult rodent stomach by RT-PCR amplification. Immunostaining showed complementary expression patterns, with EphB receptors preferentially expressed in the deeper regions and ephrin-Bs in the superficial regions of the gastric units. EphB1, EphB2 and EphB3 are expressed in mucous neck, chief and parietal cells, respectively. In contrast, ephrin-B1 is in pit cells and proliferating cells of the isthmus. In a mouse ulcer model, EphB2 expression was upregulated in the regenerating epithelium and expanded into the isthmus. Thus, EphB/ephrin-B signaling likely occurs preferentially in the isthmus, where receptor-ligand overlap is highest. We show that EphB signaling in primary gastric epithelial cells promotes cell retraction and repulsion at least in part through RhoA activation. Based on these findings, we propose that the EphB-positive progeny of gastric stem cells migrates from the isthmus toward the bottom of the gastric glands due to repulsive signals arising from contact with ephrin-Bs, which are preferentially expressed in the more superficial regions of the isthmus and gastric pits.

Ischemic preconditioning attenuates of ischemia-induced degradation of spectrin and tau: implications for ischemic tolerance.

Global ischemia selectively induces CA1 neuronal death in the hippocampus. Pretreatment with non-lethal ischemia (i.e. ischemic preconditioning) prevents CA1 neuronal death induced by lethal ischemia. While ischemic tolerance is a well-known phenomenon, the underlying molecular mechanisms are not fully understood. Cytoskeletal proteins including α-spectrin, tau, and microtubule-associated protein 2 (MAP-2) are indispensable for the maintenance of neuronal homeostasis. Here, we report the effects of ischemic preconditioning on the ischemia-induced degradation of cytoskeletal proteins α-spectrin, tau, and MAP-2 in the rat CA1 region. We found that most neurons of the CA1 region had died after 5 min of ischemia. However, exposing the brain to 3 min of ischemic preconditioning 3 days earlier significantly reduced the number of neuronal death. A significant degradation of α-spectrin and tau, but not of MAP-2, was found in the CA1 region after 5 min of ischemia. Ischemic preconditioning attenuated the ischemia-induced massive degradation of α-spectrin and tau. Our results suggest that the attenuation of ischemia-induced degradation of α-spectrin and tau by ischemic preconditioning may be associated with the neuroprotective mechanism of the ischemic tolerance.

Expression and localisation of ephrin-B1 and EphB4 in steroidogenic cells in the naturally cycling mouse ovary.

Ephrin receptors and ligands are membrane-bound molecules that modulate diverse cellular functions such as cell adhesion, epithelial-mesenchymal transition, motility, differentiation and proliferation. We recently reported the co-expression of ephrin-B1 and EphB4 in adult and foetal Leydig cells of the mouse testis, and thus speculated that their co-expression is a common property in gonadal steroidogenic cells. Therefore, in this study we examined the expression and localisation of ephrin-B1 and EphB4 in the naturally cycling mouse ovary, as their expression patterns in the ovary are virtually unknown. We found that ephrin-B1 and EphB4 were co-expressed in steroidogenic cells of all kinds, i.e. granulosa cells and CYP17A1-positive steroidogenic theca cells as well as in 3ß-HSD-positive luteal cells and the interstitial glands; their co-expression potentially serves as a good marker to identify sex steroid-producing cells even in extra-gonadal organs/tissues. We also found that ephrin-B1 and EphB4 expression in granulosa cells was faint and strong, respectively; ephrin-B1 expression in luteal cells was weak in developing and temporally mature corpora lutea (those of the current cycle) and likely strong in regressing corpora lutea (those of the previous cycle) and EphB4 expression in luteal cells was weak in corpora lutea of the current cycle and likely faint/negative in the corpora lutea of the previous cycle. These findings suggest that a luteinising hormone surge triggers the upregulation of ephrin-B1 and downregulation of EphB4, as this expression fluctuation occurs after the surge. Overall, ephrin-B1 and EphB4 expression patterns may represent benchmarks for steroidogenic cells in the ovary.

Cell Properties of Lung Tissue-Resident Macrophages Propagated by Co-Culture with Lung Fibroblastic Cells from C57BL/6 and BALB/c Mice.

Tissue-resident macrophages (Mø) originating from foetal precursors are maintained by self-renewal under tissue/organ-specific microenvironments (niches). We recently developed a simple propagation method applicable to tissue-resident Mø by co-culturing. Here, we examined the properties of lung tissue-resident Mø propagated by co-culturing with lung interstitial cells. The intracardially and intratracheally perfused lung from BALB/c and C57BL/6 mice could minimise the contamination of alveolar Mø and lung monocytes. Lung tissue-resident Mø could be largely propagated under standard culture media along with the propagation of lung interstitial cells demonstrating a fibroblastic morphology. Propagated lung Mø showed characteristic expression properties for Mø/monocyte markers: high expressions of CD11b, CD64 and CD206; substantial expressions of Mertk; and negative expressions of Ly6C, MHC II and Siglec-F. These properties fit with those of lung interstitial Mø of a certain population that can undergo self-renewal. Propagated fibroblastic cells by co-culturing with lung Mø possessed niche properties such as Csf1 and Tgfb1 expression. Propagated lung Mø from both the mouse types were polarised to an M2 phenotype highly expressing arginase 1 without M2 inducer treatment, whereas the M1 inducers significantly increased the iNOS-positive cell percentages in C57BL/6 mice relative to those in BALB/c mice. This is the first study to demonstrate fundamental properties of lung tissue-resident Mø propagated by co-culturing. Propagated lung Mø showing features of lung interstitial Mø can serve as an indispensable tool for investigating SARS-CoV-2 diseases, although lung interstitial Mø have gained little attention in terms of their involvement in SARS-CoV-2 disease pathology, in contrast to alveolar and recruited Mø.

Effective and steady differentiation of a clonal derivative of P19CL6 embryonal carcinoma cell line into beating cardiomyocytes.

The P19CL6 cell line is a useful model to study cardiac differentiation in vitro. However, large variations were noticed in the differentiation rates among previous reports as well as our individual experiments. To overcome the unstable differentiation, we established P19CL6-A1, a new clonal derivative of P19CL6 that could differentiate into cardiomyocytes more efficiently and stably than the parent using the double stimulation with 5-Aza and DMSO based on the previous report. We also introduced a new software, Visorhythm, that can analyze the temporal variations in the beating rhythms and can chart correlograms displaying the oscillated rhythms. Using P19CL6-A1-derived cardiomyocytes and the software, we demonstrated that the correlograms could clearly display the enhancement of beating rates by cardiotonic reagents. These indicate that a combination of P19CL6-A1 and Visorhythm is a useful tool that can provide invaluable assistance in inotropic drug discovery, drug screening, and toxicity testing.

Expression and localisation of ephrin-B1, EphB2, and EphB4 in the mouse testis during postnatal development.

The erythropoietin-producing hepatocellular receptor B (EphB) class and ephrin-B ligand have been implicated in boundary formation in various epithelia. We recently found that ephrin-B1 and EphB2/EphB4 exhibit complementary expression in the epithelia along the excurrent duct system in the adult mouse testis. Moreover, the organisation and integrity of the duct system is indispensable for the transport of spermatozoa. Here, we examined ephrin-B1, EphB2 and EphB4 expression in the mouse testis during postnatal development. RT-PCR analysis revealed that the relative expression levels of these molecules decreased with age in early postnatal development, and were similar to those of adults by four weeks of age. Furthermore, immunostaining revealed that the excurrent duct system compartments exhibiting complementary expression of ephrin-B1 and EphB2/EphB4 were formed by two weeks of age. Meanwhile, ephrin-B1 and EphB4 were effective markers for spermatogonia in the neonatal testis due to their negative expression in gonocytes. Alternatively, EphB2 was a suitable marker for assessing completion of the first wave of spermatogenesis in puberty, due to its strong expression in the elongated spermatids of seminiferous tubules. Lastly, ephrin-B1 and EphB4 proved to be markers of both foetal and adult Leydig cells during postnatal development, as they were expressed in CYP17A1-positive cells. This study is the first to investigate the expression of ephrin-B1, EphB2, and EphB4 in normal mouse testes during postnatal development. The expression patterns of ephrin-B and EphBs may represent suitable tools for examining organisation of the excurrent duct system and monitoring reproductive toxicity during postnatal development.

Inhibition of male chick phenotypes and spermatogenesis by Bisphenol-A.

Bisphenol-A (BPA) has been reported to bind to the estrogen receptor (ER) and also to act as a xenoestrogen on the reproductive system of many species. In our previous study, a high dose of BPA disturbed the growth of the comb and testes of male chickens. In this study, the exposure of relatively low doses of BPA on the growth of the male chicken phenotypes was investigated. White Leghorn male chicks were orally administered various doses of BPA (2 microg to 200 mg/kg) from 2 weeks of age, and thereafter the comb, wattle and testes were examined at 5, 10, 15, 20 and 25 weeks of age. Although the body weight showed no significant difference among the birds of all ages, the growth of above organs was significantly affected in the chicks even with a minimal dose of 2-microg BPA. These inhibitory effects appeared in a dose-dependent manner. Histologically, the growth of the testes was negatively affected by exposure to over 20-microg/kg BPA: namely, the development of seminiferous tubuli and spermatogenesis were severely inhibited. The mRNA expressions of ERalpha and the aromatase gene (p450arom) increased in the testes in a dose-dependent manner after BPA administration. Accordingly, even low doses of BPA delayed the growth of the male chicken phenotype either by a direct effect or by an indirect response resulting in an increase in both of the endogenous estrogen levels and hyper-sensitivity to estrogen.

Age-related changes in growth hormone-immunoreactive cells in the anterior pituitary gland of Jcl: Wistar-TgN (ARGHGEN) 1Nts rats (Mini rats).

Rats of the Jcl: Wistar-TgN (ARGHGEN) 1Nts strain (Mini rats) are transgenic animals carrying an antisense RNA transgene for rat growth hormone (GH); they show poor somatic growth and a low blood GH level compared to age-matched wild-type Wistar (non-Mini) rats. The purpose of the present study was to investigate age-related changes in growth hormone-immunoreactive (GH-IR) cells in the anterior pituitary gland (AP) of Mini rats at four, six, and eight weeks of age. The body weight and size of the GH-IR cells of Mini rats was significantly lower than that of non-Mini rats at six and eight weeks of age; however, this difference was not observed at four weeks of age. The AP volume and the number of GH-IR cells in Mini rats were significantly smaller than those of the age-matched non-Mini rats at the three ages. These results suggest that the abnormal development of GH-IR cells in the AP induced by the GH antisense RNA transgene is responsible for the poor somatic growth and the low blood GH levels in Mini rats.

Neurite extension of DRG neurons by gicerin expression is enhanced by nerve growth factor.

Gicerin, a cell adhesion molecule, is expressed in dorsal root ganglion (DRG) and sciatic nerves during chick development. This molecule re-appears in these tissues after an injury to the sciatic nerve. In the present study, we investigated the expression of nerve growth factor (NGF) in the regenerating sciatic nerve of chicks and the effects of NGF on the expression and neurite activities of gicerin in DRG. In the sciatic nerve after a crush injury, the expression of NGF and gicerin increased in the Schwann cells and in the nerve fibers, respectively. NGF promoted the neurite projections from in vitro DRG on the gicerin ligands, which were inhibited by anti-NGF antibody. The gicerin mRNA expression increased in the DRG with NGF, which was inhibited by the co-incubation with anti-NGF antibody. These results indicate that NGF might therefore enhance the expression of gicerin in DRG, thereby promoting the gicerin-dependent neurite extension during sciatic nerve regeneration.

Aberrant EphB/ephrin-B expression in experimental gastric lesions and tumor cells.

AIM: To determine whether the expression profiles of EphB receptor and ephrin-B ligand can be used as markers for dysplastic/oncogenic transformation in gastric mucosa. METHODS: The protein expression and localization of EphB and ephrin-B in normal, ulcerated regenerating, and dysplastic gastric mucosa were examined in a rat experimental model by immunolabeling, and mRNA expression was assessed in four human gastric carcinoma cell lines by reverse transcription-polymerase chain reaction. RESULTS: Ephrin-B- and EphB-expressing regions were divided along the pit-gland axis in normal gastric units. EphB2 was transiently upregulated in the experimental ulcer, and its expression domain extended to gastric pits and/or the luminal surface where ephrin-B-expressing pit cells reside. EphB2, B3, and B4 and ephrin-B1 were coexpressed in the experimental gastric dysplasia, and more than one ligand-receptor pair was highly expressed in each of the gastric carcinoma cell lines. CONCLUSION: Robust and stable coexpression of EphB and ephrin-B is a feature common to experimentally induced gastric dysplasia and human gastric carcinoma cell lines as compared to normal gastric and ulcerated regenerating epithelia. Thus, EphB/ephrin-B may be a useful marker combination for dysplastic/oncogenic transformation in gastric cancer.