Lnk/Sh2b3 controls the production and function of dendritic cells and regulates the induction of IFN-γ-producing T cells.

Dendritic cells (DCs) are proficient APCs that play crucial roles in the immune responses to various Ags and pathogens and polarize Th cell immune responses. Lnk/SH2B adaptor protein 3 (Sh2b3) is an intracellular adaptor protein that regulates B lymphopoiesis, megakaryopoiesis, and expansion of hematopoietic stem cells by constraining cytokine signals. Recent genome-wide association studies have revealed a link between polymorphism in this adaptor protein and autoimmune diseases, including type 1 diabetes and celiac disease. We found that Lnk/Sh2b3 was also expressed in DCs and investigated its role in the production and function of DC lineage cells. In Lnk(-/-) mice, DC numbers were increased in the spleen and lymph nodes, and growth responses of bone marrow-derived DCs to GM-CSF were augmented. Mature DCs from Lnk(-/-) mice were hypersensitive and showed enhanced responses to IL-15 and GM-CSF. Compared to normal DCs, Lnk(-/-) DCs had enhanced abilities to support the differentiation of IFN-γ-producing Th1 cells from naive CD4(+) T cells. This was due to their elevated expression of IL-12Rß1 and increased production of IFN-γ. Lnk(-/-) DCs supported the appearance of IFN-γ-producing T cells even under conditions in which normal DCs supported induction of regulatory T cells. These results indicated that Lnk/Sh2b3 plays a regulatory role in the expansion of DCs and might influence inflammatory immune responses in peripheral lymphoid tissues.

Lnk prevents inflammatory CD8⁺ T-cell proliferation and contributes to intestinal homeostasis.

The intracellular adaptor Lnk (also known as SH2B3) regulates cytokine signals that control lymphohematopoiesis, and Lnk(-/-) mice have expanded B-cell, megakaryocyte, and hematopoietic stem-cell populations. Moreover, mutations in the LNK gene are found in patients with myeloproliferative disease, whereas LNK polymorphisms have recently been associated with inflammatory and autoimmune diseases, including celiac disease. Here, we describe a previously unrecognized function of Lnk in the control of inflammatory CD8(+) T-cell proliferation and in intestinal homeostasis. Mature T cells from newly generated Lnk-Venus reporter mice had low but substantial expression of Lnk, whereas Lnk expression was downregulated during homeostatic T-cell proliferation under lymphopenic conditions. The numbers of CD44(hi) IFN-γ(+) CD8(+) effector or memory T cells were found to be increased in Lnk(-/-) mice, which also exhibited shortening of villi in the small intestine. Lnk(-/-) CD8(+) T cells survived longer in response to stimulation with IL-15 and proliferated even in nonlymphopenic hosts. Transfer of Lnk(-/-) CD8(+) T cells together with WT CD4(+) T cells into Rag2-deficient mice recapitulated a sign of villous abnormality. Our results reveal a link between Lnk and immune cell-mediated intestinal tissue destruction.

Zymosan Induces Immune Responses Comparable with Those of Adults in Monocytes, Dendritic Cells, and Monocyte-Derived Dendritic Cells from Cord Blood.

OBJECTIVE: To investigate the differences in toll-like receptor (TLR)-mediated immune responses between human neonates and adults, focusing on the cytokine profiles of monocytes, dendritic cells (DCs), and monocyte-derived DCs (MoDCs) in cord and adult blood. STUDY DESIGN: Purified monocytes, DCs, and MoDCs were stimulated with the following TLR ligands: lipopolysaccharide (TLR4), Pam3CSK4 (TLR1/2), flagellin (TLR5), zymosan (TLR2), polyinosinic:polycytidylic acid (TLR3), imiquimod (TLR7), and CpG (TLR9). Interleukin (IL)-8, IL-6, tumor necrosis factor, IL-1ß, and IL-10 concentrations were analyzed in culture supernatants. RESULTS: Compared with the effects in adult blood, lipopolysaccharide-, Pam3CSK4-, flagellin-, and polyinosinic:polycytidylic acid-stimulated inflammatory cytokine production in cord blood was generally weak in monocytes, comparable in DCs, and elevated in MoDCs. CpG- and imiquimod-stimulated cytokine production in DCs was comparable in cord blood and adult blood, but cytokine production was almost absent in monocytes and MoDCs in both cord blood and adult blood. In contrast, zymosan stimulation produced comparable inflammatory cytokine profiles in the monocytes, DCs, and MoDCs of cord blood and adult blood. CONCLUSION: The immaturity of TLR-mediated innate immunity in neonates depends on monocytes rather than on DCs. Our results indicate that zymosan-mediated TLR2 signaling may be useful for developing a neonatal vaccine adjuvant.

Effect of dienogest administration on angiogenesis and hemodynamics in a rat endometrial autograft model.

BACKGROUND: We aimed to establish an endometrial autograft model in rats that would allow for repetitive in vivo analyses of angiogenesis. Dienogest (DNG) is an orally active progestin used for the treatment of endometriosis. We investigated whether DNG would affect angiogenesis of the ectopic endometrium in our model. METHODS: Mechanically isolated endometrial fragments were transplanted into dorsal skinfold chambers in rats. We analyzed the effect of DNG on angiogenesis of the ectopic endometrium on Days 0, 2, 4, 7, 10 and 14 after transplantation using intravital fluorescence microscopy. RESULTS: The DNG-administered group showed significant suppression of angiogenesis of endometrial autografts, as indicated by the reduced size of the microvascular network and decreased microvessel density compared with those of control animals. The newly formed microvessels of the DNG-administered group showed consistently elevated diameters and centerline red blood cell velocity was decreased. Immunohistochemistry revealed a significant reduction in the level of perivascular α-smooth muscle actin within endometrial grafts of the DNG-administered group. CONCLUSIONS: DNG inhibited angiogenesis of the ectopic endometrium, with confirmed structural changes in the microvessels.

Time-interval changes of cardiac cycles in fetal growth restriction.

OBJECTIVE: The aims of this study were to investigate the time intervals of each component of cardiac flow velocity waveforms (FVWs) in fetuses with fetal growth restriction (FGR) and to compare these with those of normal fetuses using reference ranges. METHODS: The durations of atrioventricular (AV) valve opening (AVVO), AV valve closure (AVVC), total E- (total-E) and A- (total-A) waves, total ejection time (total-ET), acceleration time (acc-E for E-wave, acc-A for A-wave, and acc-ET for ejection time), and deceleration time (dec-E for E-wave, dec-A for A-wave, and dec-ET for ejection time) were measured in fetuses with FGR. All variables were analyzed using z-scores. RESULTS: Measurements of 17 growth-restricted fetuses were obtained. The time intervals between the last Doppler examination and delivery ranged from 0 to 6 days, with a median of 1 day. Significant increases were observed in AVVO, total-E, dec-E, and acc-A of the left heart. acc-E, acc-ET and AVVC of the left heart were significantly decreased. In the right heart, AVVO, total-E and dec-E were significantly increased. CONCLUSION: A prolonged time interval between early ventricular inflow and atrial contraction, as well as increased duration of AV valve opening, may reflect hemodynamic alterations in FGR fetuses.

Enhanced oral delivery of alendronate by sucrose fatty acids esters in rats and their absorption-enhancing mechanisms.

Oral delivery is the most fascinating route for interminable drug remedy. However, the intestinal absorption of alendronate (ALN), a bisphosphonate drug after oral administration is very poor. Absorption enhancers, which help to achieve the efficiency-safety balance, are considered one of the most promising agents for the improvement the intestinal absorption of drugs. In the current study, we focused on using sucrose fatty acid esters (SEs) as promising absorption enhancers to enhance the intestinal absorption of alendronate using an in situ closed-loop method in rats. The intestinal absorption of alendronate was significantly enhanced in the presence of SEs, especially L-1695. In addition, no considerable increase was observed in the activity of lactate dehydrogenase (LDH) or in protein release from the intestinal epithelium in the presence of sugar esters at concentrations equivalent to or lower than 1.0% (w/v), suggesting that these compounds are safe. Furthermore, mechanistic studies revealed increased membrane fluidity and loosening of the tight junctions (TJs) might be the underlying mechanism by which SEs improve the intestinal intake of alendronate, via transcellular and paracellular routes, respectively. These findings suggest that SEs are effective absorption enhancers for improving the intestinal absorption of alendronate, without causing serious damage to the enteric epithelium.

Requirement of carbon dioxide for initial growth of facultative methylotroph, Acidomonas methanolica MB58.

The facultative methylotrophic bacterium Acidomonas methanolica MB58 can utilize C1 compounds via the ribulose monophosphate pathway. A large gene cluster comprising three components related to C1 metabolism was found in the genome. From upstream, the first was an mxa cluster encoding proteins for oxidation of methanol to formaldehyde; the second was the rmp cluster encoding enzymes for formaldehyde fixation; and the third was the cbb gene cluster encoding proteins for carbon dioxide (CO2) fixation. Examination of CO2 requirements for growth of A. methanolica MB58 cells demonstrated that it did not grow on any carbon source under CO2-free conditions. Measurement of ribulose-1,5-bisphosphate carboxylase activity and RT-PCR analysis demonstrated enzymatic activity was detected in A. methanolica MB58 at growth phase, regardless of carbon sources. However, methanol dehydrogenase and 3-hexlose-6-phosphate synthase expression was regulated by methanol or formaldehyde; it were detected during growth and apparently differed from ribulose-1,5-bisphosphate carboxylase expression. These results suggested that A. methanolica MB58 may be initially dependent on autotrophic growth and that carbon assimilation was subsequently coupled with the ribulose monophosphate pathway at early- to mid-log phases during methylotrophic growth.

Serum Biopterin and Neopterin Levels as Predictors of Empty Follicles.

OBJECTIVE: This study measured serum and follicular fluid (FF) levels of biopterin, neopterin, vascular endothelial growth factor (VEGF), and macrophage colony-stimulating factor (M-CSF) in patients receiving mild ovarian stimulation for oocyte retrieval. PATIENTS AND METHODS: Infertile patients who underwent ovarian stimulation were divided into the following: Group 1, no oocyte retrieval (n = 12), and Group 2, retrieval of more than four oocytes (n = 13). Median total gonadotropin dose in both groups was 150 IU. Biopterin and neopterin levels were measured using high-performance liquid chromatography. VEGF and M-CSF levels were measured by enzyme-linked immunosorbent assay. RESULTS: Compared to Group 2, serum and FF levels of neopterin and VEGF and serum levels of M-CSF were significantly increased, and serum and FF levels of biopterin were significantly decreased in Group 1 (P < 0.05 each). CONCLUSION: Biopterin and neopterin levels showed similar differences in FF and serum of patients with empty follicles. Decreased biopterin and increased neopterin in serum could predict poor oocyte retrieval.

Sustained Decrease of Early-Phase Insulin Secretion in Japanese Women with Gestational Diabetes Mellitus Who Developed Impaired Glucose Tolerance and Impaired Fasting Glucose Postpartum.

OBJECTIVE: The aim of this study was to compare glucose intolerance in the antenatal and the postpartum periods using a 75-g oral glucose tolerance test (OGTT) in the Japanese women with gestational diabetes mellitus (GDM) using a retrospective design. PATIENTS AND METHODS: Data were obtained from 85 Japanese women with GDM who delivered from April 2011 through April 2015 and who underwent an OGTT 6-14 weeks postpartum. The women were divided into two groups based on the results of the postpartum OGTT: one group with normal glucose tolerance (NGT) and the other with impaired glucose tolerance (IGT) as well as impaired fasting glucose (IFG). We analyzed the associations between postpartum IGT-IFG and various factors. RESULTS: Antenatally, a significant difference was observed between the groups only in the 1-hour plasma glucose level of the 75-g OGTT. Postpartum results of plasma glucose level were significantly higher at 0.5, 1, and 2 hours in the IGT-IFG group than those in the NGT group. Moreover, a significant decrease in the levels of 0.5-hour immunoreactive insulin and insulinogenic index was observed in the IGT-IFG group compared to those in the NGT group. Homeostasis model assessment-insulin resistance and homeostasis model assessment ß-cell function of both groups were found to significantly decrease in the postpartum period; however, there was no significant change in the insulinogenic index of either group. CONCLUSIONS: Our study clearly showed that the postpartum IGT and IFG levels of Japanese women with GDM are affected by impaired early-phase insulin secretion; however, insulin resistance promptly improves.

Nitric oxide production and blood corpuscle dynamics in response to the endocrine status of female rats.

INTRODUCTION: Menopause is associated with marked changes in the endocrine profile, and increases the risk of vascular disease. However, the effect of hormones on the vascular system is still unclear. Therefore, the aim of this study was to examine the effects of endocrine status in female rats on nitric oxide (NO) production, inflammatory reactions and thrombus organization potency in the mesenteric microcirculation. MATERIALS AND METHODS: Female Wistar rats were divided into four groups: proestrus, metestrus, ovariectomized (OVX) and OVX plus estradiol treatment (OVX+E2). NO was imaged using an NO-sensitive dye. The leukocyte and platelet velocities relative to the erythrocyte velocity (VW/VRC and VP/VRE, respectively) and thrombi sizes created by laser radiation were measured as thrombogenesis indices. RESULTS: Changes in endocrine status did not affect vascular function in the arterioles. However, in venules, NO production, VW/VRC and VP/VRE were decreased in the OVX group compared with the proestrus and metestrus states. Thrombus size was significantly greater in the OVX group than in the proestrus and metestrus states. Administration of E2 for 2 weeks restored NO production, VW/VRC and VP/VRE to control levels. CONCLUSIONS: Changes in endocrine status did not affect arterioles. In contrast, in venules, reduced estrogen levels led to a decrease in NO production, thereby increasing thrombogenesis. Estrogen replacement restored NO production and leukocyte and platelet velocities, reducing thrombus formation relative to OVX. Although it is unclear how E2 reduces thrombus formation, our results indicate that leukocyte and platelet adhesion to the endothelium is a target for E2 via NO.

Gene expression analysis of atopic dermatitis-like skin lesions induced in NC/Nga mice by mite antigen stimulation under specific pathogen-free conditions.

BACKGROUND: Atopic dermatitis (AD) is a chronic relapsing inflammation characterized by pruritic and eczematous skin lesions usually observed in patients with a familial history of atopic diseases, but its exact etiology is unclear. An animal model is indispensable for the analysis of the pathogenesis and the development of new drugs to treat this disease. Here, we compare changes in gene expression profiles in the AD-like skin lesions of NC/Nga or BALB/c mice stimulated intradermally by mite antigen under specific pathogen-free (SPF) conditions. METHODS: Mite Extract-Dp was injected intradermally into the right and left pinnae and into the skin of the back of NC/Nga or BALB/c mice in 2 places once per 3 days, and the clinical symptoms and the ear thickness were measured. On day 14 or day 28 after starting mite extract injection, we collected plasma and pinnae from NC/Nga or BALB/c mice. The amount of total immunoglobulin E (IgE) in plasma was assayed. We analyzed mRNA transcripts in pinnae using real-time quantitative PCR for the murine counterparts of several known allergy-related genes. Moreover, genome-wide gene expression in pinnae from NC/Nga mice was analyzed using high-density oligonucleotide arrays (GeneChip, Affymetrix). RESULTS: From 2 weeks after stimulation, marked skin inflammation was induced in the pinnae of NC/Nga but not BALB/c mice. However, IgE levels in sera rose equally in both strains. Quantitative PCR analysis and comprehensive GeneChip analysis of the AD-like pinna skin lesions revealed that their development was accompanied by changes in expression of more than 1,000 genes. These included cytokines, cytokine receptors, proteases, and adhesion molecules. Furthermore, genes thus far not reported in association with AD were also affected. CONCLUSION: From these results, the NC/Nga mouse model using mite sensitization under SPF conditions could be useful for elucidating the mechanisms of AD pathogenesis and developing more effective therapy for AD.