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B cells and T cells collaborate in multiple sclerosis (MS) pathogenesis. IgH[MOG] mice possess a B cell repertoire skewed to recognize myelin oligodendrocyte glycoprotein (MOG). Here, we show that upon immunization with the T cell-obligate autoantigen, MOG[35-55], IgH[MOG] mice develop rapid and exacerbated experimental autoimmune encephalomyelitis (EAE) relative to wildtype (WT) counterparts, characterized by aggregation of T and B cells in the IgH[MOG] meninges and by CD4+ T helper 17 (Th17) cells in the CNS. Production of the Th17 maintenance factor IL-23 is observed from IgH[MOG] CNS-infiltrating and meningeal B cells, and in vivo blockade of IL-23p19 attenuates disease severity in IgH[MOG] mice. In the CNS parenchyma and dura mater of IgH[MOG] mice, we observe an increased frequency of CD4+PD-1+CXCR5- T cells that share numerous characteristics with the recently described T peripheral helper (Tph) cell subset. Further, CNS-infiltrating B and Tph cells from IgH[MOG] mice show increased reactive oxygen species (ROS) production. Meningeal inflammation, Tph-like cell accumulation in the CNS and B/Tph cell production of ROS were all reduced upon p19 blockade. Altogether, MOG-specific B cells promote autoimmune inflammation of the CNS parenchyma and meninges in an IL-23-dependent manner. B cells are crucial in multiple sclerosis (MS) pathology but the mechanisms are incompletely understood. In a mouse model of MS, the authors show that B cells make IL-23, and that IL-23 invokes meningeal inflammation and CNS presence of T peripheral helper cells. [ABSTRACT FROM AUTHOR]

Background: Cerebrospinal fluid (CSF) is an important sampling site for putative biomarkers and contains immune cells. CXCL10 is a multiple sclerosis (MS)-relevant chemokine that is present in the injured central nervous system and recruits CXCR3+ immune cells toward injured tissues. Objective: Perform a comprehensive evaluation to determine a potential relationship between CXCL10 and various immune cell subsets in the CNS of MS and control cases. Methods: In MS and control cases, CXCL10 was measured in the CSF and plasma by ELISA. Immune cells within both the CSF and peripheral blood were quantified by flow cytometry. Results: Compared to non-inflammatory neurological disease (NIND) cases, MS cases had significantly higher CXCL10 in CSF (p = 0.021); CXCL10 was also correlated with total cell numbers in CSF (p = 0.04) and T cell infiltrates (CD3+, p = 0.01; CD4+, p = 0.01; CD8+, p = 0.02); expression of CXCR3 on peripheral immune cell subsets was not associated with CSF CXCL10. Conclusions: Elevated levels of CXCL10 in the CSF of MS cases are associated with increased T cells but appear to be independent of peripheral CXCR3 expression. These results support the importance of elevated CXCL10 in MS and suggest the presence of an alternative mechanism of CXCL10 outside of solely influencing immune cell trafficking. [ABSTRACT FROM AUTHOR]

Innate immune signaling pathways are essential mediators of inflammation and repair following myelin injury. Inflammasome activation has recently been implicated as a driver of myelin injury in multiple sclerosis (MS) and its animal models, although the regulation and contributions of inflammasome activation in the demyelinated central nervous system (CNS) are not completely understood. Herein, we investigated the NLRP3 (NBD‐, LRR‐ and pyrin domain‐containing protein 3) inflammasome and its endogenous regulator microRNA‐223‐3p within the demyelinated CNS in both MS and an animal model of focal demyelination. We observed that NLRP3 inflammasome components and microRNA‐223‐3p were upregulated at sites of myelin injury within activated macrophages and microglia. Both microRNA‐223‐3p and a small‐molecule NLRP3 inhibitor, MCC950, suppressed inflammasome activation in macrophages and microglia in vitro; compared with microglia, macrophages were more prone to inflammasome activation in vitro. Finally, systemic delivery of MCC950 to mice following lysolecithin‐induced demyelination resulted in a significant reduction in axonal injury within demyelinated lesions. In conclusion, we demonstrate that NLRP3 inflammasome activity by macrophages and microglia is a critical component of the inflammatory microenvironment following demyelination and represents a potential therapeutic target for inflammatory‐mediated demyelinating diseases, including MS. Cover Image for this issue: https://doi.org/10.1111/jnc.15422 [ABSTRACT FROM AUTHOR]

We utilized a vitamin D receptor (VDR) gene knockout model to study the effects of maternal and fetal absence of VDR on maternal fertility, fetal-placental calcium transfer, and fetal mineral homoeostasis. Vdr null mice were profoundly hypocalcemic, conceived infrequently, and had significantly fewer viable fetuses in utero that were also of lower body weight. Supplementation of a calcium-enriched diet increased the rate of conception in Vdr nulls but did not normalize the number or weight of viable fetuses. Among offspring of heterozygous (Vdr.sup.+/-] ) mothers (wild type, [Vdr.sup.+/-] , and Vdr null fetuses), there was no alteration in serum Ca, P, or Mg, parathyroid hormone, placental [sup.45]Ca transfer, Ca and Mg content of the fetal skeleton, and morphology and gene expression in the fetal growth plates. Vdr null fetuses did have threefold increased 1,25-dihydroxyvitamin D levels accompanied by increased 1[alpha]-hydroxylase mRNA in kidney hut not placenta; a small increase was also noted in placental expression of parathyroid hormone-related protein (PTHrP). Among offspring of Vdr null mothers, [Vdr.sup.+/-] and Vdr null fetuses had normal ionized calcium levels and a skeletal ash weight that was appropriate to the lower body weight. Thus our findings indicate that VDR is not required by fetal mice to regulate placental calcium transfer, circulating mineral levels, and skeletal mineralization. Absence of maternal VDR has global effects on fetal growth that were partly dependent on maternal calcium intake, but absence of maternal VDR did not specifically affect fetal mineral homeostasis. fetus; calcitriol; skeletal mineralization; parathyroid hormone-related protein; parathyroid hormone; calbindin-[D.sub.9k]; calcium-adenosinetriphosphatase; calcium transporter 1 and 2; 1[alpha]-hydroxylase; placenta

We used the calcitonin/calcitonin gene-related peptide (CGRP)-[alpha] gene knockout model (Ct/Cgrp null) to determine whether calcitonin and CGRP[alpha] are required for normal fetal mineral homeostasis and placental calcium transfer. Heterozygous (Ct/[Cgrp.sup.+/-]) and Ct/Cgrp null females were mated to Ct/[Cgrp.sup.+/-] males. One or two days before term, blood was collected from mothers and fetuses and analyzed for ionized Ca, Mg, P, parathyroid hormone (PTH), and calcitonin. Amniotic fluid was collected for Ca, Mg, and P. To quantity skeletal mineral content, fetuses were reduced to ash, dissolved in nitric acid, and analyzed by atomic absorption spectroscopy for total Ca and Mg. Placental transfer of [sup.45]Ca at 5 min was assessed. Ct/Cgrp null mothers had significantly fewer viable fetuses in utero compared with Ct/[Cgrp.sup.+/-] and wild-type mothers. Fetal serum Ca, P, and PTH did not differ by genotype, but serum Mg was significantly reduced in null fetuses. Placental transfer of [sup.45]Ca at 5 min was normal. The calcium content of the fetal skeleton was normal; however, total Mg content was reduced in Ct/Cgrp null skeletons obtained from Ct/Cgrp null mothers. In summary, maternal absence of calcitonin and CGRP[alpha] reduced the number of viable fetuses. Fetal absence of calcitonin and CGRP[alpha] selectively reduced serum and skeletal magnesium content but did not alter ionized calcium, placental calcium transfer, and skeletal calcium content. These findings indicate that calcitonin and CGRP[alpha] are not needed for normal fetal calcium metabolism but may regulate aspects of fetal Mg metabolism. fetus; placental calcium transfer; skeletal mineralization: mineral homeostasis

The expression of calcitropic genes and proteins was localized within murine placenta during late gestation (the time frame of active calcium transfer) with an analysis of several gene-deletion mouse models by immuno-histochemistry and in situ hybridization. Parathyroid hormone-related protein (PTHrP), the PTH/PTHrP receptor, calcium receptor, calbindin-[D.sub.9k], [Ca.sup.2+]-ATPase, and vitamin D receptor were all highly expressed in a localized structure of the murine placenta, the intraplacental yolk sac, compared with trophoblasts. In the PTHrP gene-deleted or Pthrp-null placenta in which placental calcium transfer is decreased, calbindin-[D.sub.9k] expression was downregulated in the intraplacental yolk sac but hot in the trophoblasts. These observations indicated that the intraplacental yolk sac contains calcium transfer and calcium-sensing capability and that it is a probable route of maternal-fetal calcium exchange in the mouse. parathyroid hormone receptors; fetus; fetal development; placental calcium transfer; calcium receptor; calcitonin; calcitriol; vitamin D receptor; calbindin; vitamin D-dependent calcium-binding protein; calcium-ATPase; trophoblasts; in situ hybridization; immunohistochemistry; gene knockout mice

Extracellular vesicles (EVs) are secreted from cells under physiological and pathological conditions, and are found in biological fluids while displaying specific surface markers that are indicative of their cell of origin. EVs have emerged as important signaling entities that may serve as putative biomarkers for various neurological conditions, including multiple sclerosis (MS). The objective of this study was to measure and compare immune cell-derived EVs within human plasma between untreated RRMS patients and healthy controls. Using blood plasma and peripheral blood mononuclear cells (PBMCs) collected from RRMS patients and controls, PBMCs and EVs were stained and quantified by flow cytometry using antibodies against CD9, CD61, CD45, CD3, CD4, CD8, CD14, and CD19. While several immune cell-derived EVs, including CD3+, CD4+, CD8+, CD14+, and CD19+ were significantly increased in RRMS vs. controls, no differences in immune cell subsets were observed with the exception of increased circulating CD19+ cells in RRMS patients. Our study demonstrated that plasma-derived EVs secreted from T cells, B cells, and monocytes were elevated in untreated RRMS cases with low disability, despite very limited changes in circulating immune cells, and suggest the utility of circulating EVs as biomarkers in MS. [ABSTRACT FROM AUTHOR]

Antiretroviral therapy (ART) effectively extends the life expectancy of human immunodeficiency virus (HIV)-infected individuals; however, agerelated morbidities have emerged as major clinical concerns. In this context, coinfection with cytomegalovirus (CMV) accelerates immune senescence and elevates risk for other agerelated morbidities, possibly through increased inflammation. We investigated potential relationships between CMV memory inflation, immune senescence, and inflammation by measuring markers of inflammation and telomere lengths of different lymphocyte subsets in HIVinfected individuals seropositive for antiCMV antibodies. Our study cohort consists mainly of middle aged men who have sex with men (MSM) and heterosexuals who are stable under longterm ART. Median levels of IL6, TNFα, and CRP were significantly higher in those coinfected with CMV. Lymphocyte telomere length in general correlated with age, but for 32/32 subjects tested, there was a consistent hierarchy of telomere lengths with CD8+ T cells' shorter than the general lymphocyte population, CD57+CD8+ T cells' shorter than CD8+ T cells' and CMVspecific CD57+CD8+ T cells' the shortest of all. Telomeres of HIVspecific CD8+ T cells were longer than those of CMVspecific CD8+ T cells in all cases tested and over 10 years, CMV-specific CD8+ T cell telomeres of two HIVinfected individuals eroded faster than those of HIVspecific CD8+ T cells. These data indicate that CMVspecific CD8+ T cells of HIVinfected individuals are the lymphocytes closest to telomereimposed replicative senescence. Exhaustive proliferation of CMVspecific CD8+ T cells in HIVinfected individuals is a potential source of senescent lymphocytes affecting systemic immune function and inflammation. [ABSTRACT FROM AUTHOR]

Natural killer (NK) cells are cytotoxic lymphocytes that selectively respond against abnormal cells. Human cytomegalovirus (HCMV) infection causes expansion of NKG2C+CD57+ NK cells in vivo and NKG2C+ NK cells proliferate when cultured with HCMV-infected cells. This raises the possibility of an NK-cell subset selectively responding against a specific pathogen and accruing memory. To test this possibility, we compared proliferation, natural cytotoxicity and interferon-γ (IFN-γ) production of NK cells from HCMV-seropositive and HCMV-seronegative individuals co-cultured with HCMV-infected or uninfected MRC-5 cells. There was no significant difference in proliferation of NK cells from HCMV-seropositive or seronegative individuals against uninfected MRC-5 cells, but significantly more NK cells from the HCMV-seropositive group proliferated in response to HCMV-infected MRC-5 cells. Natural cytotoxicity of NK cells against K562 cells increased following co-culture with HCMV-infected versus uninfected MRC-5 only for the HCMV-seropositive group. After co-culture with HCMV-infected MRC-5 cells, proliferating NK cells from HCMV-seropositive donors selectively produced IFN-γ when re-exposed to HCMV-infected MRC-5 cells. Both NKG2C+ and NKG2C− NK cells proliferated in co-culture with HCMV-infected MRC-5 cells, with the fraction of proliferating NKG2C+ NK cells directly correlating with the circulating NKG2C+ fraction. These data illustrate an at least partly NKG2C-independent human NK-cell memory-type response against HCMV. [ABSTRACT FROM AUTHOR]

Objective. Measuring NKG2C(+)CD57(+) natural killer (NK) cell expansion to investigate NK responses against human cytomegalovirus (HCMV) and assessing relationships with adaptive immunity against HCMV. Methods. Expansion of NKG2C(+)CD57(+) NK was measured in peripheral blood mononuclear cells (PBMC) from groups distinguished by HCMV and human immunodeficiency virus (HIV) infection status. Anti-HCMV antibody levels against HCMV-infected MRC-5 cell lysate were assessed by ELISA and HCMV-specific CD8(+) T cell responses characterized by intracellular flow cytometry following PBMC stimulation with immunodominant HCMV peptides. Results. Median NK, antibody, and CD8(+) T cell responses against HCMV were significantly greater in the HCMV/HIV coinfected group than the group infected with CMV alone. The fraction of CMV-specific CD8(+) T cells expressing CD28 correlated inversely with NKG2C(+)CD57(+) NK expansion in HIV infection. Conclusion. Our data reveal no significant direct relationships between NK and adaptive immunity against HCMV. However, stronger NK and adaptive immune responses against HCMV and an inverse correlation between NKG2C(+)CD57(+) NK expansion and proliferative reserve of HCMV-specific CD8(+) T cells, as signified by CD28 expression, indicate parallel evolution of NK and T cell responses against HCMV in HIV infection. Similar aspects of chronic HCMV infection may drive both NK and CD8(+) T cell memory inflation. [ABSTRACT FROM AUTHOR]

Background: In our previous investigations of the role of the extracellular matrix (ECM) in promoting neurite growth we have observed that a permissive laminin (LN) substrate stimulates differential growth responses in subpopulations of mature dorsal root ganglion (DRG) neurons. DRG neurons expressing Trk and p75 receptors grow neurites on a LN substrate in the absence of neurotrophins, while isolectin B4-binding neurons (IB4+) do not display significant growth under the same conditions. We set out to determine whether there was an expression signature of the LN-induced neurite growth phenotype. Using a lectin binding protocol IB4+ neurons were isolated from dissociated DRG neurons, creating two groups - IB4+ and IB4-. A small-scale microarray approach was employed to screen the expression of a panel of ECM-associated genes following dissociation (t=0) and after 24 hr culture on LN (t=24LN). This was followed by qRT-PCR and immunocytochemistry of selected genes. Results: The microarray screen showed that 36 of the 144 genes on the arrays were consistently expressed by the neurons. The array analyses showed that six genes had lower expression in the IB4+ neurons compared to the IB4- cells at t=0 (CTSH, Icam1, Itgß1, Lamb1, Plat, Spp1), and one gene was expressed at higher levels in the IB4+ cells (Plaur). qRT-PCR was carried out as an independent assessment of the array results. There were discrepancies between the two methods, with qRT-PCR confirming the differences in Lamb1, Plat and Plaur, and showing decreased expression of AdamTs1, FN, and Icam in the IB4+ cells at t=0. After 24 hr culture on LN, there were no significant differences detected by qRT-PCR between the IB4+ and IB4- cells. However, both groups showed upregulation of Itgß1 and Plaur after 24 hr on LN, the IB4+ group also had increased Plat, and the IB4- cells showed decreased Lamb1, Icam1 and AdamTs1. Further, the array screen also detected a number of genes (not subjected to qRT-PCR) expressed similarly by both populations in relatively high levels but not detectably influenced by time in culture (Bsg, Cst3, Ctsb, Ctsd, Ctsl, Mmp14, Mmp19, Sparc. We carried out immunohistochemistry to confirm expression of proteins encoded by a number of these genes. Conclusions: Our results show that 1B4+ and IB4- neurons differ in the expression of several genes that are associated with responsiveness to the ECM prior to culturing (AdamTs1, FN, Icam1, Lamb1, Plat, Plaur). The data suggest that the genes expressed at higher levels in the IB4- neurons could contribute to the initial growth response of these cells in a permissive environment and could also represent a common injury response that subsequently promotes axon regeneration. The differential expression of several extracellular matrix molecules (FN, Lamb1, Icam) may suggest that the IB4- neurons are capable of maintaining /secreting their local extracellular environment which could aid in the regenerative process. Overall, these data provide new information on potential targets that could be manipulated to enhance axonal regeneration in the mature nervous system. [ABSTRACT FROM AUTHOR]

We used the calcitonin/calcitonin gene-related peptide (CGRP)-α gene knockout model (Ct/Cgrp null) to determine whether calcitonin and CGRPα are required for normal fetal mineral homeostasis and placental calcium transfer. Heterozygous (Ct/Cgrp+/-) and Ct/Cgrp null females were mated to Ct/Cgrp+/- males. One or two days before term, blood was collected from mothers and fetuses and analyzed for ionized Ca, Mg, P, parathyroid hormone (PTH), and calcitonin. Amniotic fluid was collected for Ca, Mg, and P. To quantify skeletal mineral content, fetuses were reduced to ash, dissolved in nitric acid, and analyzed by atomic absorption spectroscopy for total Ca and Mg. Placental transfer of 45Ca at 5 min was assessed. Ct/Cgrp null mothers had significantly fewer viable fetuses in utero compared with Ct/Cgrp+/- and wild type mothers. Fetal serum Ca, P, and PTH did not differ by genotype, but serum Mg was significantly reduced in null fetuses. Placental transfer of 45Ca at 5 min was normal. The calcium content of the fetal skeleton was normal; however, total Mg content was reduced in Ct/Cgrp null skeletons obtained from Ct/Cgrp null mothers. In summary, maternal absence of calcitonin and CGRPα reduced the number of viable fetuses. Fetal absence of calcitonin and CGRPα selectively reduced serum and skeletal magnesium content but did not alter ionized calcium, placental calcium transfer, and skeletal calcium content. These findings indicate that calcitonin and CGRPα are not needed for normal fetal calcium metabolism but may regulate aspects of fetal Mg metabolism. [ABSTRACT FROM AUTHOR]

Background: The calcium sensing receptor (CaSR) regulates serum calcium by suppressing secretion of parathyroid hormone; it also regulates renal tubular calcium excretion. Inactivating mutations of CaSR raise serum calcium and reduce urine calcium excretion. Thyroid C-cells (which make calcitonin) express CaSR and may, therefore, be regulated by it. Since calcium stimulates release of calcitonin, the higher blood calcium caused by inactivation of CaSR should increase serum calcitonin, unless CaSR mutations alter the responsiveness of calcitonin to calcium. To demonstrate regulatory effects of CaSR on calcitonin release, we studied calcitonin responsiveness to calcium in normal and CaSR heterozygous-ablated (Casr+/-) mice. Casr+/- mice have hypercalcemia and hypocalciuria, and live normal life spans. Each mouse received either 500 μl of normal saline or one of two doses of elemental calcium (500 μmol/kg or 5 mmol/kg) by intraperitoneal injection. Ionized calcium was measured at baseline and 10 minutes, and serum calcitonin was measured on the 10 minute sample. Results: At baseline, Casr+/- mice had a higher blood calcium, and in response to the two doses of elemental calcium, had greater increments and peak levels of ionized calcium than their wild type littermates. Despite significantly higher ionized calcium levels, the calcitonin levels of Casr+/- mice were consistently lower than wild type at any ionized calcium level, indicating that the dose response curve of calcitonin to increases in ionized calcium had been significantly blunted or shifted to the right in Casr+/- mice. Conclusions: These results confirm that the CaSR is a physiological regulator of calcitonin; therefore, in response to increases in ionized calcium, the CaSR inhibits parathyroid hormone secretion and stimulates calcitonin secretion. [ABSTRACT FROM AUTHOR]

Expansion of natural killer (NK) cells expressing NKG2C occurs following human cytomegalovirus (HCMV) infection and is amplified by human immunodeficiency virus (HIV) co-infection. These NKG2C-expressing NK cells demonstrate enhanced CD16-dependent cytokine production and downregulate FcεRIγ and promyelocytic leukemia zinc finger protein (PLZF). Lacking NKG2C diminishes resistance to HIV infection, but whether this affects NK cell acquisition of superior antibody-dependent function is unclear. Therefore, our objective was to investigate whether HCMV-driven NK cell differentiation is impaired in NKG2Cnull HIV-infected individuals. Phenotypic (CD2, CD16, CD57, NKG2A, FcεRIγ, and PLZF expression) and functional (cytokine induction and cytotoxicity) properties were compared between HIV–infected NKG2Cnull and NKG2C-expressing groups. Cytokine production was compared following stimulation through natural cytotoxicity receptors or through CD16. Cytotoxicity was measured by anti-CD16-redirected lysis and by classical antibody-dependent cell-mediated cytotoxicity (ADCC) against anti-class I human leukocyte antigen (HLA) antibody-coated cells. Our data indicate highly similar HCMV-driven NK cell differentiation in HIV infection with or without NKG2C. While the fraction of mature (CD57pos) NK cells expressing CD2 (p = 0.009) or co-expressing CD2 and CD16 (p = 0.03) was significantly higher in NKG2Cnull HIV-infected individuals, there were no significant differences in NKG2A, FcεRIγ, or PLZF expression. The general phenotypic and functional equivalency observed suggests NKG2C-independent routes of HCMV-driven NK cell differentiation, which may involve increased CD2 expression. [ABSTRACT FROM AUTHOR]