Pathogenic Potential of Eomesodermin-Expressing T-Helper Cells in Neurodegenerative Diseases.

Eomesodermin-expressing (Eomes+) T-helper (Th) cells show cytotoxic characteristics in secondary progressive multiple sclerosis. We found that Eomes+ Th cell frequency was increased in the peripheral blood of amyotrophic lateral sclerosis and Alzheimer's disease patients. Furthermore, granzyme B production by Th cells from such patients was high compared with controls. A high frequency of Eomes+ Th cells was observed in the initial (acutely progressive) stage of amyotrophic lateral sclerosis, and a positive correlation between Eomes+ Th cell frequency and cognitive decline was observed in Alzheimer's disease patients. Therefore, Eomes+ Th cells may be involved in the pathology of amyotrophic lateral sclerosis and Alzheimer's disease. ANN NEUROL 2024;95:1093-1098.

Involvement of cytotoxic Eomes-expressing CD4+ T cells in secondary progressive multiple sclerosis.

Multiple sclerosis (MS), a putative autoimmune disease of the central nervous system (CNS), commonly presents as relapsing-remitting MS (RRMS), characterized by recurrent episodes of peripheral disabling symptoms resulting from inflammatory CNS damage. Many RRMS patients transition to a chronic disease course with progressive neurological dysfunctions (secondary progressive MS, SPMS), with the progression rate varying between patients and over time. SPMS pathogenesis is now linked to immune-cell-mediated processes, although the mechanisms driving SPMS transition and progression remain elusive, and SPMS lacks biomarkers and effective treatments. We report the crucial involvement of cytotoxic CD4+ T cells expressing Eomes (Eomes+ Th cells) in SPMS pathogenesis-a Th cell subset previously identified in a mouse model of late/chronic autoimmune CNS inflammation. Few Eomes+ Th cells circulate in RRMS patient peripheral blood (n = 44), primary progressive MS (PPMS) patients (n = 25), or healthy controls (n = 42), but Eomes+ Th cells were significantly increased in SPMS (n = 105, P < 0.0001). Strikingly, lymphocytes isolated from SPMS autopsy brain samples revealed CD4+ T cells infiltrating CNS that coexpressed Eomes and the cytotoxic molecule granzyme B. In particular, the Eomes+ Th cell levels were increased in SPMS patients in progressive disease phases versus SPMS patients without current disability increases (P < 0.0001). Moreover, Eomes level acted as a biomarker to predict SPMS patients at risk of disease worsening with over 80% accuracy (ROC-AUC = 0.8276). Overall, our results indicate that granzyme B-expressing Eomes+ T helper cells are involved in the pathogenesis of SPMS, with significant implications for SPMS biomarkers and therapeutic targets.

Harnessing autoimmunity with dominant self-peptide: Modulating the sustainability of tissue-preferential antigen-specific Tregs by governing the binding stability via peptide flanking residues.

Sensitization to self-peptides induces various immunological responses, from autoimmunity to tumor immunity, depending on the peptide sequence; however, the underlying mechanisms remain unclear, and thus, curative therapeutic options considering immunity balance are limited. Herein, two overlapping dominant peptides of myelin proteolipid protein, PLP136-150 and PLP139-151, which induce different forms of experimental autoimmune encephalomyelitis (EAE), monophasic and relapsing EAE, respectively, were investigated. Mice with monophasic EAE exhibited highly resistant to EAE re-induction with any encephalitogenic peptides, whereas mice with relapsing EAE were susceptible, and progressed, to EAE re-induction. This resistance to relapse and re-induction in monophasic EAE mice was associated with the maintenance of potent CD69+CD103+CD4+CD25high regulatory T-cells (Tregs) enriched with antigen specificity, which expanded preferentially in the central nervous system with sustained suppressive activity. This tissue-preferential sustainability of potent antigen-specific Tregs was correlated with the antigenicity of PLP136-150, depending on its flanking residues. That is, the flanking residues of PLP136-150 enable to form pivotally arranged strong hydrogen bonds that secured its binding stability to MHC-class II. These potent Tregs acting tissue-preferentially were induced only by sensitization of PLP136-150, not by its tolerance induction, independent of EAE development. These findings suggest that, for optimal therapy, "benign autoimmunity" can be critically achieved through inverse vaccination with self-peptides by manipulating their flanking residues.

Continuous acceleration of neural activity of the GnRH pulse generator during chronic peripheral infusion of neurokinin 3 receptor agonist in goats.

Secretion of pulsatile gonadotropin-releasing hormone (GnRH) is essential for reproduction. Kisspeptin neurons in the arcuate nucleus (ARC), which coexpress neurokinin B (NKB) and its receptor (NK3R), are believed to be components of the GnRH pulse generator that regulates pulsatile GnRH secretion. We examined the effects of peripheral infusion of senktide, an NK3R selective agonist, on GnRH pulse generator activity by monitoring multiple unit activity (MUA) in the goat ARC. Previous studies have shown that characteristic increases in MUA (MUA volleys) reflect GnRH pulse generator activity. Senktide was infused intravenously or intravaginally for 2 h while recording MUA. Both infusions significantly increased the MUA volley frequency compared with the control. These results demonstrate that peripherally administered senktide acts centrally to sustainably accelerate the neural activity of the GnRH pulse generator throughout the infusion period. This suggests the possibility of practical applications of NK3R agonists for improving reproductive activity in farm animals.

Alterations of the gut ecological and functional microenvironment in different stages of multiple sclerosis.

Multiple sclerosis (MS), an autoimmune disease of the central nervous system, generally starts as the relapsing remitting form (RRMS), but often shifts into secondary progressive MS (SPMS). SPMS represents a more advanced stage of MS, characterized by accumulating disabilities and refractoriness to medications. The aim of this study was to clarify the microbial and functional differences in gut microbiomes of the different stages of MS. Here, we compared gut microbiomes of patients with RRMS, SPMS, and two closely related disorders with healthy controls (HCs) by 16S rRNA gene and whole metagenomic sequencing data from fecal samples and by fecal metabolites. Each patient group had a number of species having significant changes in abundance in comparison with HCs, including short-chain fatty acid (SCFA)-producing bacteria reduced in MS. Changes in some species had close association with clinical severity of the patients. A marked reduction in butyrate and propionate biosynthesis and corresponding metabolic changes were confirmed in RRMS compared with HCs. Although bacterial composition analysis showed limited differences between the patient groups, metagenomic functional data disclosed an increase in microbial genes involved in DNA mismatch repair in SPMS as compared to RRMS. Together with an increased ratio of cysteine persulfide to cysteine in SPMS revealed by sulfur metabolomics, we postulate that excessive DNA oxidation could take place in the gut of SPMS. Thus, gut ecological and functional microenvironments were significantly altered in the different stages of MS. In particular, reduced SCFA biosynthesis in RRMS and elevated oxidative level in SPMS were characteristic.

Trial of Satralizumab in Neuromyelitis Optica Spectrum Disorder.

BACKGROUND: Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disease of the central nervous system and is associated with autoantibodies to anti-aquaporin-4 (AQP4-IgG) in approximately two thirds of patients. Interleukin-6 is involved in the pathogenesis of the disorder. Satralizumab is a humanized monoclonal antibody targeting the interleukin-6 receptor. The efficacy of satralizumab added to immunosuppressant treatment in patients with NMOSD is unclear. METHODS: In a phase 3, randomized, double-blind, placebo-controlled trial, we randomly assigned, in a 1:1 ratio, patients with NMOSD who were seropositive or seronegative for AQP4-IgG to receive either satralizumab, at a dose of 120 mg, or placebo, administered subcutaneously at weeks 0, 2, and 4 and every 4 weeks thereafter, added to stable immunosuppressant treatment. The primary end point was the first protocol-defined relapse in a time-to-event analysis. Key secondary end points were the change from baseline to week 24 in the visual-analogue scale (VAS) pain score (range, 0 to 100, with higher scores indicating more pain) and the Functional Assessment of Chronic Illness Therapy-Fatigue (FACIT-F) score (range, 0 to 52, with lower scores indicating more fatigue). Safety was also assessed. RESULTS: A total of 83 patients were enrolled, with 41 assigned to the satralizumab group and 42 to the placebo group. The median treatment duration with satralizumab in the double-blind period was 107.4 weeks. Relapse occurred in 8 patients (20%) receiving satralizumab and in 18 (43%) receiving placebo (hazard ratio, 0.38; 95% confidence interval [CI], 0.16 to 0.88). Multiple imputation for censored data resulted in hazard ratios ranging from 0.34 to 0.44 (with corresponding P values of 0.01 to 0.04). Among 55 AQP4-IgG-seropositive patients, relapse occurred in 11% of those in the satralizumab group and in 43% of those in the placebo group (hazard ratio, 0.21; 95% CI, 0.06 to 0.75); among 28 AQP4-IgG-seronegative patients, relapse occurred in 36% and 43%, respectively (hazard ratio, 0.66; 95% CI, 0.20 to 2.24). The between-group difference in the change in the mean VAS pain score was 4.08 (95% CI, -8.44 to 16.61); the between-group difference in the change in the mean FACIT-F score was -3.10 (95% CI, -8.38 to 2.18). The rates of serious adverse events and infections did not differ between groups. CONCLUSIONS: Among patients with NMOSD, satralizumab added to immunosuppressant treatment led to a lower risk of relapse than placebo but did not differ from placebo in its effect on pain or fatigue. (Funded by Chugai Pharmaceutical; ClinicalTrials.gov number, NCT02028884.).

Th1 - CD11c+ B Cell Axis Associated with Response to Plasmapheresis in Multiple Sclerosis.

OBJECTIVE: Although plasmapheresis is a treatment option for patients with autoimmune neurological diseases, treatment response varies greatly among patients. The main objective of this study was to find out if biological/immune traits correlate with a beneficial response. METHODS: We thoroughly analyzed immune phenotypes in paired blood samples from a cohort of 31 patients with multiple sclerosis before and after plasmapheresis, in parallel with clinical evaluation of treatment response. RESULTS: The frequency of IFN-γ+ Th1 cells was persistently higher in those who obtained benefit from plasmapheresis (responders) than nonresponders. The Th1 cell frequency before plasmapheresis provided a high predictive value for beneficial response, achieving area under the curve (AUC) of 0.902. Plasmapheresis treatment decreased inflammation-related gene expressions in Th1 cells. Meanwhile, IFNG expression in Th1 cells positively correlated with the frequency of CD11c+ B cells, of which a pathogenic role has been suggested in several autoimmune diseases. In line with this, in vitro experiments showed that CD11c+ B cells would increase in response to exogenous IFN-γ compared to IL-4, and secrete high amounts of IgG. B cell receptor analysis indicated that clonal expansion of CD11c+ B cells takes place in patients with multiple sclerosis. Interestingly, CD11c+ B cells, which showed unique gene expression profile, decreased after plasmapheresis treatment along with all the immunoglobulin subsets in the circulation. INTERPRETATION: Taken together, we postulate that Th1 cell - CD11c+ B cell axis is involved in treatment response to plasmapheresis, giving us clues to better understanding of complicated pathogenesis of autoimmune diseases, and getting closer to a personalized therapy. ANN NEUROL 2021;90:595-611.

Extrapituitary prolactin promotes generation of Eomes-positive helper T cells mediating neuroinflammation.

Induction of eomesodermin-positive CD4+ T cells (Eomes+ T helper [Th] cells) has recently been correlated with the transition from an acute stage to a later stage of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis. Moreover, these cells' pathogenic role has been experimentally proven in EAE. While exploring how the pathogenic Eomes+ Th cells are generated during the course of EAE, we unexpectedly found that B cells and MHC class II+ myeloid cells isolated from the late EAE lesions strikingly up-regulated the expression of prolactin (PRL). We demonstrate that such PRL-producing cells have a unique potential to induce Eomes+ Th cells from naïve T cells ex vivo, and that anti-MHC class II antibody could block this process. Furthermore, PRL levels in the cerebrospinal fluid were significantly increased in the late phase of EAE, and blocking the production of PRL by bromocriptine or Zbtb20-specific siRNA significantly reduced the numbers of Eomes+ Th cells in the central nervous system (CNS) and ameliorated clinical signs in the later phase of EAE. The PRL dependency of Eomes+ Th cells was confirmed in a series of in vitro and ex vivo experiments. Collectively, these results indicate that extrapituitary PRL plays a crucial role in the CNS inflammation mediated by pathogenic Eomes+ Th cells. Cellular interactions involving PRL-producing immune cells could be considered as a therapeutic target for the prevention of chronic neuroinflammation.

Skewing of the B cell receptor repertoire in myalgic encephalomyelitis/chronic fatigue syndrome.

Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is a debilitating condition characterized by fatigue and post-exertional malaise, accompanied by various signs of neurological and autonomic dysfunction. ME/CFS is often triggered by an infectious episode and associated with an aberrant immune system. Here we report that ME/CFS is a disorder characterized by skewed B cell receptor gene usage. By applying a next-generation sequencing to determine the clone-based IGHV/IGHD/IGHJ repertoires, we revealed a biased usage of several IGHV genes in peripheral blood B cells from ME/CFS patients. Results of receiver operating characteristic (ROC) analysis further indicated a possibility of distinguishing patients from healthy controls, based on the skewed B cell repertoire. Meanwhile, B cell clones using IGHV3-30 and IGHV3-30-3 genes were more frequent in patients with an obvious infection-related episode at onset, and correlated to expression levels of interferon response genes in plasmablasts. Collectively, these results imply that B cell responses in ME/CFS are directed against an infectious agents or priming antigens induced before disease onset.

Local administration of Neurokinin B in the arcuate nucleus accelerates the neural activity of the GnRH pulse generator in goats.

Kisspeptin neurons in the arcuate nucleus (ARC), which co-express neurokinin B (NKB) and dynorphin A, are termed KNDy neurons. These neurons are candidates for the intrinsic gonadotropin-releasing hormone (GnRH) pulse generator. The central and peripheral administration of NKB or its receptor (NK3R) agonist evokes GnRH pulse generator activity and the subsequent pulsatile GnRH/luteinizing hormone (LH) secretion. However, the mechanism responsible for neural activation of the GnRH pulse generator in goats is unclear. We conducted electrophysiological and histochemical experiments to test the hypothesis that KNDy neurons receive NKB and that the signal is transmitted bilaterally to a population of KNDy neurons. Bilateral electrodes aimed at a cluster of KNDy neurons were inserted into the ovariectomized goat ARC. We observed the GnRH pulse generator activity, represented by characteristic increases in multiple-unit activity (MUA volleys). The unilateral administration of NKB or vehicle in the close vicinity of KNDy neurons under simultaneous MUA recording from both sides revealed that only NKB evoked MUA volley(s) immediately after administration. The timing of the MUA volley(s) evoked on the ipsilateral side was synchronized to that on the contralateral side. The double-labeled ISH for KISS1 and TACR3, which encode kisspeptin and NK3R, respectively, revealed that most KNDy neurons co-expressed TACR3. Therefore, NKB could directly stimulate KNDy neurons, following which the stimulatory signal is immediately transmitted to the entire population of KNDy neurons via connection with their fibers. This mechanism helps synchronize burst activity among KNDy neurons, thereby generating neural signals that govern pulsatile GnRH secretion.

Silkworm recombinant bovine zona pellucida protein 4 (bZP4) as a potential female immunocontraceptive antigen; impaired sperm-oocyte interaction and ovarian dysfunction.

Porcine zona pellucida proteins (ZPs) have been utilized as female immunocontraceptive antigens. The purpose of this study was to explore the potential use of silkworm recombinant bovine ZP4 as an alternative. When the protein was injected with monophosphoryl lipid A (MPL) - an immuno-stimulative agent - into two female goats, marked elevation of the anti-ZP4 titer was detected. Application of the purified specific IgG to a porcine in vitro fertilization system reduced the sperm penetration rate. In one goat, the cyclic profile of serum progesterone disappeared as the anti-ZP4 titer increased. Histological examination of the ovaries revealed degeneration of antral follicles with sparse infiltration of inflammatory cells in the theca, indicating that autoimmune oophoritis had been induced. Together, the present results suggest that recombinant ZP4 disturbs fertilization and exerts a pathogenic effect on follicle development in goats, thus indicating its potential as a female immunocontraceptive antigen.

Gut microbiota-dependent CCR9+CD4+ T cells are altered in secondary progressive multiple sclerosis.

The mechanism underlying the progression of relapsing-remitting multiple sclerosis to secondary progressive multiple sclerosis (SPMS), characterized by accumulating fixed disability, is yet to be fully understood. Although alterations in the gut microbiota have recently been highlighted in multiple sclerosis pathogenesis, the mechanism linking the altered gut environment with the remote CNS pathology remains unclear. Here, we analyse human CD4+ memory T cells expressing the gut-homing chemokine receptor CCR9 and found a reduced frequency of CCR9+ memory T cells in the peripheral blood of patients with SPMS relative to healthy controls. The reduction in the proportion of CCR9+ cells among CD4+ memory T cells (%CCR9) in SPMS did not correlate with age, disease duration or expanded disability status scale score, although %CCR9 decreased linearly with age in healthy controls. During the clinical relapse of both, relapsing-remitting multiple sclerosis and neuromyelitis optica, a high proportion of cells expressing the lymphocyte activating 3 gene (LAG3) was detected among CCR9+ memory T cells isolated from the CSF, similar to that observed for mouse regulatory intraepithelial lymphocytes. In healthy individuals, CCR9+ memory T cells expressed higher levels of CCR6, a CNS-homing chemokine receptor, and exhibited a regulatory profile characterized by both the expression of C-MAF and the production of IL-4 and IL-10. However, in CCR9+ memory T cells, the expression of RORγt was specifically upregulated, and the production of IL-17A and IFNγ was high in patients with SPMS, indicating a loss of regulatory function. The evaluation of other cytokines supported the finding that CCR9+ memory T cells acquire a more inflammatory profile in SPMS, reporting similar aspects to CCR9+ memory T cells of the elderly healthy controls. CCR9+ memory T cell frequency decreased in germ-free mice, whereas antibiotic treatment increased their number in specific pathogen-free conditions. Here, we also demonstrate that CCR9+ memory T cells preferentially infiltrate into the inflamed CNS resulting from the initial phase and that they express LAG3 in the late phase in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. Antibiotic treatment reduced experimental autoimmune encephalomyelitis symptoms and was accompanied by an increase in CCR9+ memory T cells in the peripheral blood. Antibodies against mucosal vascular addressin cell adhesion molecule 1 (MADCAM1), which is capable of blocking cell migration to the gut, also ameliorated experimental autoimmune encephalomyelitis. Overall, we postulate that the alterations in CCR9+ memory T cells observed, caused by either the gut microbiota changes or ageing, may lead to the development of SPMS.

Intravaginal administration of progesterone using a new technique for sustained drug release in goats.

The objective of the present study was to develop and evaluate a sustained release vaginal progesterone (P4) capsule containing a mixture of mucoadhesive polymer and silicone fluid. Goats were administered a gelatin capsule containing 0.4 g of P4 mixed in silicone fluid and either a hydroxypropylmethylcellulose (HM) or polyaclil starch (PA) base. The mean plasma P4 concentrations at 2 and 12 h after administration were significantly higher in goats treated with PA capsules than in those with HM capsules. The plasma P4 concentrations in goats treated with HM capsules increased and remained above 1.0 ng/ml for 96 h after administration, whereas the plasma P4 concentrations in goats treated with PA capsules remained above 1.0 ng/ml for only 24 h after administration. In the next experiment, an HM capsule was attached to a silicone device and inserted in the vagina for 10 days. The plasma P4 concentration remained similar to that of the natural luteal phase for 9 days. These results suggest that a mixture of mucoadhesive polymer and silicone fluid has the potential to be applied clinically as a sustained release base for estrus synchronization or hormonal therapy.

Brain abnormalities in myalgic encephalomyelitis/chronic fatigue syndrome: Evaluation by diffusional kurtosis imaging and neurite orientation dispersion and density imaging.

BACKGROUND: Diffusional kurtosis imaging (DKI) and neurite orientation dispersion and density imaging (NODDI) metrics provide more specific information regarding pathological changes than diffusion tensor imaging (DTI). PURPOSE: To detect microstructural abnormalities in myalgic encephalomyelitis (ME) / chronic fatigue syndrome (CFS) patients by using DKI and NODDI metrics. STUDY TYPE: Prospective. POPULATION: Twenty ME/CFS patients and 23 healthy controls were recruited. FIELD STRENGTH/SEQUENCE: Three-b value DWI (b-values = 0, 1000, and 2000 sec/mm2 ) and 3D T1 -weighted images were at 3.0T. ASSESSMENT: Mean kurtosis (MK), neurite density index (NDI), orientation dispersion index (ODI), fractional anisotropy (FA), and mean diffusivity (MD) were calculated. STATISTICAL TESTING: The two-sample t-test analysis in SPM12 software was used to compare the differences between ME/CFS and control groups. RESULTS: In the ME/CFS patients, we observed significant FA decreases in the genu of the corpus callosum and the anterior limb of the right internal capsule (P < 0.05), but no significant difference in MD (P = 0.164); there were also significant MK decreases in the right frontal area, anterior cingulate gyrus, superior longitudinal fasciculus (SLF), and left parietal area (P < 0.05). Significant NDI decreases were observed in the right posterior cingulate gyrus, SLF, and left frontal area of the ME/CFS patients (P < 0.05). Significant ODI decreases were seen in the bilateral occipital areas, right superior temporal gyrus, the anterior limb of internal capsule, and the posterior cingulate gyrus (P < 0.05), and significant ODI increases were revealed in the bilateral occipital and right temporal areas (P < 0.05). DATA CONCLUSION: Right SLF abnormalities may be a diagnostic marker for ME/CFS. LEVEL OF EVIDENCE: 1 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2019;49:818-824.

A randomized placebo-controlled trial of delayed-release dimethyl fumarate in patients with relapsing-remitting multiple sclerosis from East Asia and other countries.

BACKGROUND: Delayed-release dimethyl fumarate (DMF) has demonstrated efficacy and a favorable benefit-risk profile in phase 2 and 3 studies that enrolled predominantly white patients with relapsing-remitting multiple sclerosis (RRMS). In this study (APEX, Part I), we evaluated the efficacy/safety outcomes of DMF in a predominantly East Asian population of patients with RRMS. METHODS: In this 24-week, randomized, double-blind, placebo-controlled phase 3 study, 225 patients, 142 of which were East Asian (63.4%), were enrolled: Japan (n = 114), South Korea (n = 20), Taiwan (n = 8), the Czech Republic (n = 42), and Poland (n = 40). Key exclusion criteria included diagnosis of neuromyelitis optica spectrum disorder. Stratified by country, patients were randomized 1:1 to receive DMF 240 mg twice daily or placebo. Clinical assessments, including neurological examination and EDSS scoring, were conducted at baseline and at weeks 12 and 24. RESULTS: A total of 213 patients (95.1%) completed the study. From weeks 12 - 24, the total number of new gadolinium-enhancing (Gd+) lesions was reduced by 84% (p < 0.0001) in DMF compared with placebo. For the secondary endpoint, from baseline to week 24, the total number of new Gd+ lesions was reduced by 75% and the mean number of new/newly enlarging T2 hyperintense lesions was reduced by 63% (both p < 0.0001). Flushing and flushing-related symptoms, and gastrointestinal events were adverse events related to DMF treatment. Efficacy and safety results in the Japanese subgroup and the East Asian subgroup (which included patients from Japan, Taiwan, and South Korea) were consistent with the overall study population. CONCLUSION: The strong efficacy and favorable benefit-risk profile of DMF extends to Japanese, and more broadly, East Asian patients with RRMS. TRIAL REGISTRATION: This trial is registered on ClinicalTrials.gov (identifier: NCT01838668 ), April 20, 2013 (retrospectively registered). The registration can be found at the following URL: https://clinicaltrials.gov/ct2/show/NCT01838668.

MicroRNA-101a regulates microglial morphology and inflammation.

BACKGROUND: Microglia, as well as other tissue-resident macrophages, arise from yolk sac progenitors. Thus, it is likely that the central nervous system environment is critical for the acquisition of a distinct microglial phenotype. Several microRNAs that are enriched in the brain play crucial roles in brain development and may also play a role in the differentiation of microglia. METHODS: To track the differentiation of hematopoietic cells into microglia, lineage-negative bone marrow cells were co-cultured with astrocytes in the absence or presence of microRNAs or their inhibitors. Microglia-like cells were identified as small, round cells that were immunopositive for CD11b, Iba1, CX3CR1, and triggering receptor expressed on myeloid cells (TREM)-2. RESULTS: Five microRNAs (miR-101a, miR-139-3p, miR-214*, miR-218, and miR-1186) were identified as modifiers of the differentiation of bone marrow-derived microglia-like cells. Among them, miR-101a facilitated the differentiation of bone marrow cells into microglia-like cells most potently. Small, round cells expressing CD11b, Iba1, CX3CR1, and TREM-2 were predominant in cells treated by miR-101a. miR-101a was abundantly expressed in non-microglial brain cells. Transfection of miR-101a into microglia significantly increased the production of IL-6 in response to LPS. Finally, miR-101a downregulated the expression of MAPK phosphatase-1. CONCLUSIONS: miR-101a, which is enriched in the brain, promotes the differentiation of bone marrow cells into microglia-like cells.

A neurokinin 3 receptor-selective agonist accelerates pulsatile luteinizing hormone secretion in lactating cattle.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion, which is indispensable for follicular development, is suppressed in lactating dairy and beef cattle. Neurokinin B (NKB) neurons in the arcuate nucleus of the hypothalamus are considered to play an essential role in generating the pulsatile mode of GnRH/luteinizing hormone (LH) secretion. The present study aimed to clarify the role of NKB-neurokinin 3 receptor (NK3R) signaling in the pulsatile pattern of GnRH/gonadotropin secretion in postpartum lactating cattle. We examined the effects of the administration of an NK3R-selective agonist, senktide, on gonadotropin secretion in lactating cattle. The lactating cattle, at approximately 7 days postpartum, were intravenously infused with senktide (30 or 300 nmol/min) or vehicle for 24 h. The administration of 30 or 300 nmol/min senktide significantly increased LH pulse frequency compared to in the control group during 0-4 or 20-24 h after infusion, respectively. Moreover, LH and follicle-stimulating hormone levels were gradually increased by 300 nmol/min administration of senktide during the 0-4-h sampling period. Ultrasonography of the ovaries was performed to identify the first postpartum ovulation in senktide-administered lactating cattle. The interval from calving to first postpartum ovulation was significantly shorter in the 300 nmol/min senktide-administered group than in the control group. Taken together, these findings suggest that senktide infusion elicits an increase in LH pulse frequency that may stimulate follicular development and, in turn, induce the first postpartum ovulation in lactating cattle.

Mapping of KNDy neurons and immunohistochemical analysis of the interaction between KNDy and substance P neural systems in goat.

A population of neurons in the arcuate nucleus (ARC) coexpresses kisspeptin, neurokinin B (NKB), and dynorphin, and therefore they are referred to as KNDy neurons. It has been suggested that KNDy neurons participate in several brain functions, including the control of reproduction. The present study aimed to advance our understanding of the anatomy of the KNDy neural system. We first produced an antiserum against goat kisspeptin. After confirming its specificity, the antiserum was used to histochemically detect kisspeptin-positive signals. Using the colocalization of kisspeptin and NKB immunoreactivity as a marker for KNDy neurons, we mapped distributions of their cell somata and fibers in the whole brain (except the cerebellum) of ovariectomized (OVX) goats. KNDy neuronal somata were distributed throughout the ARC, and were particularly abundant in its caudal aspect. KNDy neuronal fibers projected into several areas within the septo-preoptic-hypothalamic continuum, such as the ARC, median eminence, medial preoptic nucleus, and bed nucleus of the stria terminalis. Kisspeptin immunoreactivity was not found outside of the continuum. We then addressed to the hypothesis that substance P (SP) is also involved in the KNDy neural system. Double-labeling immunohistochemistry for kisspeptin and SP revealed that KNDy neurons did not coexpress SP, but nearly all of the KNDy neuronal somata were surrounded by fibers containing SP in the OVX goats. The present results demonstrate anatomical evidence for a robust association between the KNDy and SP neural systems.

Effects of intravenous administration of neurokinin receptor subtype-selective agonists on gonadotropin-releasing hormone pulse generator activity and luteinizing hormone secretion in goats.

Recent evidence suggests that neurokinin B (NKB), a member of the neurokinin (tachykinin) peptide family, plays a pivotal role in gonadotropin-releasing hormone (GnRH) pulse generation. Three types of neurokinin receptors (NKRs), NK1R, NK2R and NK3R, are found in the brain. Although NKB preferentially binds to NK3R, other NKRs are possibly also involved in NKB action. The present study examined the effects of intravenous administration of the NKR subtype-selective agonists GR73632 (NK1R), GR64349 (NK2R), and senktide (NK3R) on GnRH pulse generator activity and luteinizing hormone (LH) secretion. Multiple-unit activity (MUA) was monitored in ovariectomized goats (n = 5) implanted with recording electrodes. Characteristic increases in MUA (MUA volleys) were considered GnRH pulse generator activity. Although three NKR agonists dose-dependently induced an MUA volley and an accompanying increase in LH secretion, the efficacy in inducing the volley markedly differed. As little as 10 nmol of senktide induced an MUA volley in all goats, whereas a dose of 1000 nmol was only effective for the NK1R and NK2R agonists in two and four goats, respectively. When the treatment failed to evoke an MUA volley, no apparent change was observed in the MUA or LH secretion. Similar effects of the NK2R and NK3R agonists were observed in the presence of estradiol. The results demonstrated that NK3R plays a predominant role in GnRH pulse generation and suggested that the contributions of NK1R and NK2R to this mechanism may be few, if any, in goats.

Development of a culture system to induce microglia-like cells from haematopoietic cells.

AIMS: Microglia are the resident immune cells in the central nervous system, originating from haematopoietic-derived myeloid cells. A microglial cell is a double-edged sword, which has both pro-inflammatory and anti-inflammatory functions. Although understanding the role of microglia in pathological conditions has become increasingly important, histopathology has been the only way to investigate microglia in human diseases. METHODS: To enable the study of microglial cells in vitro, we here establish a culture system to induce microglia-like cells from haematopoietic cells by coculture with astrocytes. The characteristics of microglia-like cells were analysed by flow cytometry and functional assay. RESULTS: We show that triggering receptor expressing on myeloid cells-2-expressing microglia-like cells could be induced from lineage negative cells or monocytes by coculture with astrocytes. Microglia-like cells exhibited lower expression of CD45 and MHC class II than macrophages, a characteristic similar to brain microglia. When introduced into brain slice cultures, these microglia-like cells changed their morphology to a ramified shape on the first day of the culture. Moreover, we demonstrated that microglia-like cells could be induced from human monocytes by coculture with astrocytes. Finally, we showed that interleukin 34 was an important factor in the induction of microglia-like cells from haematopoietic cells in addition to cell-cell contact with astrocytes. Purified microglia-like cells were suitable for further culture and functional analyses. CONCLUSION: Development of in vitro induction system for microglia will further promote the study of human microglial cells under pathological conditions as well as aid in the screening of drugs to target microglial cells.