Differential Secretion Research Articles

Establishing and characterizing lacrispheres from human lacrimal gland for potential clinical application.

Lacrimal gland (LG) dysfunction leading to dry eye syndrome (DES) is an important cause of ocular morbidity. One of the potential and promising long-term management therapies for restoration of LG function could be transplantation of autologous ex vivo expanded stem cells. The present study was aimed at exploring the 2D and 3D cultures of human LG, identifying inherent stem cells and evaluating their secretory potential. Fresh human lacrimal gland (HuLG) (n = 5) from patients undergoing therapeutic exenteration was harvested after ethical approval and informed consent. The gland was enzymatically digested and the isolated cells plated in Hepato-STIM media supplemented with l-glutamine, epidermal growth factor, fibroblast growth factor, and N-2 supplement. The native HuLG and the cultured spheres (DIV14-16) were evaluated for presence of stem cells (CD117 expression, quiescence, BrdU label retention, cell cycle, colony forming efficiency) and differentiation (secretion of tear proteins). Under the established culture conditions, suspension 3D cultures of human "lacrispheres" could be maintained and propagated for 3-4weeks. The spheres consist of both acinar as well as ductal cells with evidence of stem cells (0.8 ± 0.05% CD117+ cells), BrdU label retention (9.31 ± 0.41%), G0/G1 profile similar to native lacrimal cells at isolation (76.9 versus 79.9%) and colony forming units (3.1%). The lacrispheres also secreted quantifiable levels of tear proteins (lysozyme, lactoferrin, scIgA) into the conditioned media. The study provides promising, first-of-its-kind evidence for the generation of lacrispheres from fresh HuLG, with enriched population of stem cells and secretory competent differentiated cells. The dual properties of these spheres make them a highly suitable source of transplantable cells for restoring the structure and function of damaged lacrimal gland.

Condition-dependent generation of aquaporin-4 antibodies from circulating B cells in neuromyelitis optica

Autoantibodies to aquaporin-4 (AQP4) are pathogenic in neuromyelitis optica spectrum disorder (NMOSD). However, it is not known which B cells are the major contributors to circulating AQP4 antibodies nor which conditions promote their generation. Our experiments showed CD19+CD27++CD38++ circulating ex vivo antibody-secreting cells did not produce AQP4 antibodies under several culture conditions. To question whether other cells in circulation were capable of AQP4 antibody production, B cells were differentiated into antibody-secreting cells in vitro. Unfractionated peripheral blood mononuclear cells, isolated from 12 patients with NMOSD and a wide range of serum AQP4 antibody levels (91-26 610 units), were cultured with factors that mimicked established associations of NMOSD including T cell help, concurrent infections and cytokines reported to be elevated in NMOSD. Overall, the in vitro generation of CD19+CD27++CD38++ cells across several culture conditions correlated closely with the total IgG secreted (P < 0.0001, r = 0.71), but not the amount of AQP4 antibody. AQP4 antibody production was enhanced by CD40-ligand (P = 0.005), and by interleukin-2 plus toll-like receptor stimulation versus interleukin-21-predominant conditions (P < 0.0001), and did not require antigen. Across NMOSD patients, this in vitro generation of AQP4 antibodies correlated well with serum AQP4 antibody levels (P = 0.0023, r = 0.81). To understand how early within B cell lineages this AQP4 specificity was generated, purified B cell subsets were activated under these optimized conditions. Naïve pre-germinal centre B cells (CD19+CD27-IgD+) differentiated to secrete AQP4 antibodies as frequently as post-germinal centre cells (CD19+CD27+). Taken together, these human cell-culture experiments demonstrate that preformed B cells, rather than ex vivo circulating antibody-secreting cells, possess AQP4 reactivity. Their differentiation and AQP4 antibody secretion is preferentially driven by select cytokines and these cells may make the dominant contribution to serum AQP4 antibodies. Furthermore, as AQP4-specific B cells can derive from likely autoreactive naïve populations an early, pre-germinal centre loss of immunological tolerance appears present in some patients with NMOSD. This study has implications for understanding mechanisms of disease perpetuation and for rational choice of immunotherapies in NMOSD. Furthermore, the in vitro model presents an opportunity to apply condition-specific approaches to patients with NMOSD and may be a paradigm to study other antibody-mediated diseases.awy010media15732448284001.

Characteristics of inflammatory reactions during development of liver abscess in hamsters inoculated with Entamoeba nuttalli.

BackgroundEntamoeba nuttalli is an intestinal protozoan with pathogenic potential that can cause amebic liver abscess. It is highly prevalent in wild and captive macaques. Recently, cysts were detected in a caretaker of nonhuman primates in a zoo, indicating that E. nuttalli may be a zoonotic pathogen. Therefore, it is important to evaluate the pathogenicity of E. nuttalli in detail and in comparison with that of E. histolytica.Methodology/Principal findingsTrophozoites of E. nuttalli GY4 and E. histolytica SAW755 strains were inoculated into liver of hamsters. Expression levels of proinflammatory factors of hamsters and virulence factors from E. histolytica and E. nuttalli were compared between the two parasites. Inoculations with trophozoites of E. nuttalli resulted in an average necrotic area of 24% in liver tissue in 7 days, whereas this area produced by E. histolytica was nearly 50%. Along with the mild liver tissue damage induced by E. nuttalli, expression levels of proinflammatory factors (TNF-α, IL-6 and IL-1β) and amebic virulence protein genes (lectins, cysteine proteases and amoeba pores) in local tissues were lower with E. nuttalli in comparison with E. histolytica. In addition, M2 type macrophages were increased in E. nuttalli-induced amebic liver abscesses in the late stage of disease progression and lysate of E. nuttalli trophozoites induced higher arginase expression than E. histolytica in vitro.Conclusions/SignificanceThe results show that differential secretion of amebic virulence proteins during E. nuttalli infection triggered lower levels of secretion of various cytokines and had an impact on polarization of macrophages towards a M1/M2 balance. However, regardless of the degree of macrophage polarization, there is unambiguous evidence of an intense acute inflammatory reaction in liver of hamsters after infection by both Entamoeba species.

Design of Magnetically Labeled Cells (Mag-Cells) for in Vivo Control of Stem Cell Migration and Differentiation.

Cell-based therapies are attractive for treating various degenerative disorders and cancer but delivering functional cells to the region of interest in vivo remains difficult. The problem is exacerbated in dense biological matrices such as solid tissues because these environments impose significant steric hindrances for cell movement. Here, we show that neural stem cells transfected with zinc-doped ferrite magnetic nanoparticles (ZnMNPs) can be pulled by an external magnet to migrate to the desired location in the brain. These magnetically labeled cells (Mag-Cells) can migrate because ZnMNPsgenerate sufficiently strong mechanical forces to overcome steric hindrances in the brain tissues. Once at the site of lesion, Mag-Cells show enhanced neuronal differentiation and greater secretion of neurotrophic factors than unlabeled control stem cells. Our study shows that ZnMNPs activate zinc-mediated Wnt signaling to facilitate neuronal differentiation. When implemented in a rodent brain stroke model, Mag-Cells led to significant recovery of locomotor performance in the impaired limbs of the animals. Our findings provide a simple magnetic method for controlling migration of stem cells with high therapeutic functions, offering a valuable tool for other cell-based therapies.

Reduced MBD2 expression enhances airway inflammation in bronchial epithelium in COPD.

BackgroundChronic obstructive pulmonary disease (COPD) is a common inflammatory lung disease characterized by inflammatory cells activation and production of inflammatory mediators. Methyl-CpG-binding domain protein 2 (MBD2) plays an important role in diverse immunological disorders by regulating immune cell functions, such as differentiation and mediator secretion. However, the role of MBD2 in COPD remains unknown.MethodsMBD2 protein expression in lung tissues of patients with COPD and cigarette smoke (CS)-exposed mice were evaluated by Western blot and immunohistochemistry. The role of MBD2 in cigarette smoke extract (CSE)-induction of inflammatory mediator expression in the human bronchial epithelial (HBE) cell line was assessed by silencing MBD2 expression in vitro. The involvement of signaling pathways in mediation of inflammation was tested with signaling inhibitors.ResultsCompared with controls, MBD2 expression was distinctly reduced in the bronchial epithelium of both patients with COPD and CS-exposed mice. Moreover, MBD2 expression was decreased in HBE after CSE stimulation in vitro. Moreover, MBD2 knockdown enhanced interleukin (IL)-6 and IL-8 expression in HBE in the presence and absence of CSE treatment by the ERK signaling pathway.ConclusionMBD2 protein expression was reduced in the airway epithelium of COPD. In HBE, this reduced expression was associated with increased levels of IL-6 and IL-8 mediated by the ERK pathway. These results suggest that MBD2 could contribute to chronic airway inflammation in COPD.

B Cell-Intrinsic Role for IRF5 in TLR9/BCR-Induced Human B Cell Activation, Proliferation, and Plasmablast Differentiation.

Upon recognition of antigen, B cells undergo rapid proliferation followed by differentiation to specialized antibody secreting cells (ASCs). During this transition, B cells are reliant upon a multilayer transcription factor network to achieve a dramatic remodeling of the B cell transcriptional landscape. Increased levels of ASCs are often seen in autoimmune diseases and it is believed that altered expression of regulatory transcription factors play a role in this imbalance. The transcription factor interferon regulatory factor 5 (IRF5) is one such candidate as polymorphisms in IRF5 associate with risk of numerous autoimmune diseases and correlate with elevated IRF5 expression. IRF5 genetic risk has been widely replicated in systemic lupus erythematosus (SLE), and loss of Irf5 ameliorates disease in murine lupus models, in part, through the lack of pathogenic autoantibody secretion. It remains unclear, however, whether IRF5 is contributing to autoantibody production through a B cell-intrinsic function. To date, IRF5 function in healthy human B cells has not been characterized. Using human primary naive B cells, we define a critical intrinsic role for IRF5 in B cell activation, proliferation, and plasmablast differentiation. Targeted IRF5 knockdown resulted in significant immunoglobulin (Ig) D retention, reduced proliferation, plasmablast differentiation, and IgG secretion. The observed decreases were due to impaired B cell activation and clonal expansion. Distinct from murine studies, we identify and confirm new IRF5 target genes, IRF4, ERK1, and MYC, and pathways that mediate IRF5 B cell-intrinsic function. Together, these results identify IRF5 as an early regulator of human B cell activation and provide the first dataset in human primary B cells to map IRF5 dysfunction in SLE.

Ex Vivo Culture Assay to Measure Human Follicular Helper T (Tfh) Cell-Mediated Human B Cell Proliferation and Differentiation.

Upon priming by antigens, B cells undergo activation, proliferation, and differentiation into antibody-secreting cells. During thymus-dependent (TD) antibody responses, the proliferation and differentiation of antigen-primed B cells essentially rely on the helper function from CD4+ T cells. Follicular helper T (Tfh) cells constitute a specialized Th subset that localizes in close proximity to B cells and supports B cell proliferation and differentiation through co-stimulatory receptors and cytokines. Impaired Tfh-mediated B cell proliferation and differentiation were observed in patients with immunodeficiency, while overactivation of this process may lead to dysregulated immune responses seen in autoimmune disorders. Here, we describe an ex vivo coculture assay using circulating Tfh cells and B cells isolated from human blood. This method can be used to examine the function of patients' B cells for proliferation, differentiation, and antibody secretion, mediated by the physiological help from Tfh cells.

AB029. Physiological expression of HLA-G and pregnancy

: Human leukocyte antigen (HLA)-G is a nonclassical major histocompatibility complex (MHC) class I molecule with immune-modulatory properties. Such molecule predominantly possesses tolerogenic and anti-inflammatory functions. Until now, it has been verified the existence of seven isoforms of HLA-G, being that four are membrane-bound isoforms and the other three are soluble isoforms. During physiological pregnancy, it should be noted that HLA-G is expressed in high levels by extravillous cytotrophoblasts. It has been proposed that in the course of pregnancy period HLA-G induces fetus immune tolerance through direct binding to the inhibitory receptors immunoglobulin-like transcript (ILT)-2 present on lymphoid and myelomonocytic cells, and ILT-4 expressed by dendritic cells, macrophages and monocytes. In addition, in this context, the killer cell immunoglobulin-like receptor expressed by natural killer (NK) cells is also an HLA-G-specific receptor. Therefore, HLA-G induces the maintenance of maternal-fetal tolerance at different stages of the immune response, such as in differentiation, proliferation, cytolysis and cytokine secretion, given that it can directly interact with different immune cell subpopulations. Furthermore, by indirect mechanisms HLA-G can express the nonclassical HLA class I molecule HLA-E, which directly binds peptides derived from HLA-G. Consequently, HLA-E can interact with the inhibitory receptor CD94/NKG2A (present on NK cells and T lymphocytes) resulting in inhibition of cytolysis. However, despite this some controversies can be found in the literature about this subject. We expect that the original results of our ongoing research can provide new and relevant explanations concerning the role of HLA-G during pregnancy. We aim to publish them soon in a scientific journal.

Alterations of the Cytoskeleton in Breast Cancer Cells during Microgravity visualised by FLUMIAS Live-Cell Imaging

Alterations of the Cytoskeleton in Breast Cancer Cells during Microgravity visualised by FLUMIAS Live-Cell Imaging

Circulating microRNA (miRNA) Expression Profiling in Plasma of Patients with Gestational Diabetes Mellitus Reveals Upregulation of miRNA miR-330-3p.

Gestational diabetes mellitus (GDM) is characterized by insulin resistance accompanied by low/absent beta-cell compensatory adaptation to the increased insulin demand. Although the molecular mechanisms and factors acting on beta-cell compensatory response during pregnancy have been partially elucidated and reported, those inducing an impaired beta-cell compensation and function, thus evolving in GDM, have yet to be fully addressed. MicroRNAs (miRNAs) are a class of small endogenous non-coding RNAs, which negatively modulate gene expression through their sequence-specific binding to 3′UTR of mRNA target. They have been described as potent modulators of cell survival and proliferation and, furthermore, as orchestrating molecules of beta-cell compensatory response and function in diabetes. Moreover, it has been reported that miRNAs can be actively secreted by cells and found in many biological fluids (e.g., serum/plasma), thus representing both optimal candidate disease biomarkers and mediators of tissues crosstalk(s). Here, we analyzed the expression profiles of circulating miRNAs in plasma samples obtained from n = 21 GDM patients and from n = 10 non-diabetic control pregnant women (24–33 weeks of gestation) using TaqMan array microfluidics cards followed by RT-real-time PCR single assay validation. The results highlighted the upregulation of miR-330-3p in plasma of GDM vs non-diabetics. Furthermore, the analysis of miR-330-3p expression levels revealed a bimodally distributed GDM patients group characterized by high or low circulating miR-330 expression and identified as GDM-miR-330high and GDM-miR-330low. Interestingly, GDM-miR-330high subgroup retained lower levels of insulinemia, inversely correlated to miR-330-3p expression levels, and a significant higher rate of primary cesarean sections. Finally, miR-330-3p target genes analysis revealed major modulators of beta-cell proliferation and of insulin secretion, such as the experimentally validated genes E2F1 and CDC42 as well as AGT2R2, a gene involved in the differentiation of mature beta-cells. In conclusion, we demonstrated that plasma miR-330-3p could be of help in identifying GDM patients with potential worse gestational diabetes outcome; in GDM, miR-330-3p may directly be transferred from plasma to beta-cells thus modulating key target genes involved in proliferation, differentiation, and insulin secretion.

The metabolic syndrome- associated small G protein ARL15 plays a role in adipocyte differentiation and adiponectin secretion

Common genetic variants at the ARL15 locus are associated with plasma adiponectin, insulin and HDL cholesterol concentrations, obesity, and coronary atherosclerosis. The ARL15 gene encodes a small GTP-binding protein whose function is currently unknown. In this study adipocyte-autonomous roles for ARL15 were investigated using conditional knockdown of Arl15 in murine 3T3-L1 (pre)adipocytes. Arl15 knockdown in differentiated adipocytes impaired adiponectin secretion but not adipsin secretion or insulin action, while in preadipocytes it impaired adipogenesis. In differentiated adipocytes GFP-tagged ARL15 localized predominantly to the Golgi with lower levels detected at the plasma membrane and intracellular vesicles, suggesting involvement in intracellular trafficking. Sequencing of ARL15 in 375 severely insulin resistant patients identified four rare heterozygous variants, including an early nonsense mutation in a proband with femorogluteal lipodystrophy and non classical congenital adrenal hyperplasia, and an essential splice site mutation in a proband with partial lipodystrophy and a history of childhood yolk sac tumour. No nonsense or essential splice site mutations were found in 2,479 controls, while five such variants were found in the ExAC database. These findings provide evidence that ARL15 plays a role in adipocyte differentiation and adiponectin secretion, and raise the possibility that human ARL15 haploinsufficiency predisposes to lipodystrophy.

Micromanaging Glucose Tolerance and Diabetes.

MicroRNAs (miRNAs) are endogenous non-coding RNAs that have significant roles in biological processes such as glucose homoeostasis. MiRNAs fine-tune target genes expression via sequence-specific binding of their seed sequence to the untranslated region of mRNAs and degrade target mRNAs. MicroRNAs in islet β-cells regulate β-cell differentiation, proliferation, insulin transcription and glucose-stimulated insulin secretion. Furthermore, miRNAs play key roles in the regulation of glucose and lipid metabolisms and modify insulin sensitivity by controlling metabolic functions in main target organs of insulin such as skeletal muscle, liver and adipose tissue. Moreover, since circulating miRNAs are detectable and stable in serum, levels of certain miRNAs seem to be novel biomarkers for prediction of diabetes mellitus. In this article, due to the prominent impact of miRNAs on diabetes, we overviewed the microRNAs regulatory functions in organs related to insulin resistance and diabetes and shed light on their potential as diagnostic and therapeutic markers for diabetes.

Abstract 26: Exosomal microRNA as salivary biomarkers of head and neck squamous cell carcinoma

Abstract Head and neck cancer is the 10th most common cancer overall and 5th most common among men in the United States, the vast majority of which are squamous cell carcinoma (HNSCC). Despite approximately two-thirds of cases being diagnosed at an advanced stage, the only routine screening approach currently in widespread use is visual inspection and palpation, which is provided by dentists and clinicians on an opportunistic basis, lacks sensitivity, and varies according to the skill of the clinician. This underscores the urgent and compelling need for establishment of novel and effective biomarkers to facilitate early detection. Exosomes may offer a new avenue for discovery and development of novel biomarkers of HNSCC. These nanosized (40-150 nm), membrane-encapsulated vesicles are released by both normal and malignant cells into the extracellular space and function as intercellular signaling vectors through the horizontal transfer of biologic molecules, including microRNA (miRNA), which can affect the phenotype of recipient cells. Importantly, exosomes are present in essentially all extracellular biofluids, including saliva, and thus can readily be collected by noninvasive means. Through previous cell culture work, we observed striking differences in exosomal miRNA secreted by HNSCC cells relative to healthy oral epithelial cells, with a high degree of overlap in exosomal miRNA profiles across HNSCC cell lines. Although salivary exosomes have been theorized to be a potentially powerful biomarker source for HNSCC, there has been an extreme paucity of work conducted in this arena. To begin to address this question, we conducted a small pilot study in which we collected 2-mL whole-saliva samples from 5 HNSCC cases (collected prior to initiation of treatment) and 5 cancer-free controls. Exosomes were isolated from each saliva sample via differential ultracentrifugation, and total RNA was extracted for miRNA-sequencing (miRNA-seq), which was performed by the University of Cincinnati Genomics, Epigenomics &amp; Sequencing Core (GESC). The number of reads for each sample during miRNA-seq ranged from 1.6 million to 27.4 million reads (median = 12.4 million). After aligning the reads to all human miRNA sequences catalogued in miRBase, there were a total of 1,334 mature miRNA transcripts detected across samples, with 307 transcripts (12%) detected solely in salivary exosomes from cases. Moreover, despite the small sample size of the pilot study, we observed significant differential exosomal secretion of miR-10b-5p (p = 0.006), with transcripts present at relatively high levels in salivary exosomes from 3 of the 5 cases but none detected in those from the 5 controls. We also preliminarily assessed the translational potential of 8 miRNA transcripts that were solely and universally expressed by our HNSCC cell lines (i.e. not secreted by healthy oral epithelial cells) in our previous cell culture work using the salivary exosome miRNA-seq data for the 5 cases and 5 controls. All 8 transcripts were detected in salivary exosomes obtained from the 5 HNSCC cases. In particular, miR-486-5p and miR-486-3p showed considerable promise, with 40% of cases expressing drastically higher levels of these transcripts relative to controls. When either miR-486-5p or miR-486-3p was combined with miR-10b-5p, the substantial separation of these markers could clearly distinguish 80% (4/5) of the HNSCC cases from controls. These findings demonstrate the potential of salivary miRNA as biomarkers of HNSCC and highlight the necessity of further workup and validation in full-scale studies involving human saliva. Citation Format: Scott M. Langevin, Damaris Kuhnell, Xiang Zhang, Trisha M. Wise-Draper, Keith A. Casper. Exosomal microRNA as salivary biomarkers of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Optimizing Survival and Quality of Life through Basic, Clinical, and Translational Research; April 23-25, 2017; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(23_Suppl):Abstract nr 26.

Reactive Oxygen Species Link Gonadotropin-Releasing Hormone Receptor Signaling Cascades in the Gonadotrope.

Biological rhythms lie at the center of regulatory schemes that control many aspects of living systems. At the cellular level, meaningful responses to external stimuli depend on propagation and quenching of a signal to maintain vigilance for subsequent stimulation or changes that serve to shape and modulate the response. The hypothalamus–pituitary–gonad endocrine axis that controls reproductive development and function relies on control through rhythmic stimulation. Central to this axis is the pulsatile stimulation of the gonadotropes by hypothalamic neurons through episodic release of the neuropeptide gonadotropin-releasing hormone. Alterations in pulsatile stimulation of the gonadotropes result in differential synthesis and secretion of the gonadotropins LH and FSH and changes in the expression of their respective hormone subunit genes. The requirement to amplify signals arising from activation of the gonadotropin-releasing hormone (GnRH) receptor and to rapidly quench the resultant signal to preserve an adaptive response suggests the need for rapid activation and feedback control operating at the level of intracellular signaling. Emerging data suggest that reactive oxygen species (ROS) can fulfill this role in the GnRH receptor signaling through activation of MAP kinase signaling cascades, control of negative feedback, and participation in the secretory process. Results obtained in gonadotrope cell lines or other cell models indicate that ROS can participate in each of these regulatory cascades. We discuss the potential advantage of reactive oxygen signaling for modulating the gonadotrope response to GnRH stimulation and the potential mechanisms for this action. These observations suggest further targets of study for regulation in the gonadotrope.

Cytocompatibility and antibacterial activity of nanostructured H2Ti5O11\xb7H2O outlayered Zn-doped TiO2 coatings on Ti for percutaneous implants

To improve skin-integration and antibacterial activity of percutaneous implants, the coatings comprising an outer layer of H2Ti5O11·H2O (HTO) nanoarrays and an inner layer of microporous Zn-doped TiO2 were fabricated on Ti by micro-arc oxidation (MAO) followed with hydrothermal treatment (HT). During HT process, a large proportion of Zn2+ migrated out from TiO2 layer. TiO2 reacted with OH− and H2O, resulting in the nucleation of HTO. The nuclei grew to nanoplates, nanorods and nanofibres with HT process prolonged. Simultaneously, the orientation of nanoarrays changed from quasi-vertical to parallel to substrate. Compared to Ti, adhesion and proliferation of fibroblasts were enhanced on as-MAOed TiO2 and HTed coatings. The phenotype, differentiation and extracellular collagen secretion were obviously accelerated on vertical nanorods with proper interspace (e.g. 63 nm). HTed coatings showed enhanced antibacterial activity, which should be ascribed to the nano-topography of HTO.

Integrative Temporo-Spatial, Mineralogic, Spectroscopic, and Proteomic Analysis of Postnatal Enamel Development in Teeth with Limited Growth.

Tooth amelogenesis is a complex process beginning with enamel organ cell differentiation and enamel matrix secretion, transitioning through changes in ameloblast polarity, cytoskeletal, and matrix organization, that affects crucial biomineralization events such as mineral nucleation, enamel crystal growth, and enamel prism organization. Here we have harvested the enamel organ including the pliable enamel matrix of postnatal first mandibular mouse molars during the first 8 days of tooth enamel development to conduct a step-wise cross-sectional analysis of the changes in the mineral and protein phase. Mineral phase diffraction pattern analysis using single-crystal, powder sample X-ray diffraction analysis indicated conversion of calcium phosphate precursors to partially fluoride substituted hydroxyapatite from postnatal day 4 (4 dpn) onwards. Attenuated total reflectance spectra (ATR) revealed a substantial elevation in phosphate and carbonate incorporation as well as structural reconfiguration between postnatal days 6 and 8. Nanoscale liquid chromatography coupled with tandem mass spectrometry (nanoLC-MS/MS) demonstrated highest protein counts for ECM/cell surface proteins, stress/heat shock proteins, and alkaline phosphatase on postnatal day 2, high counts for ameloblast cytoskeletal proteins such as tubulin β5, tropomyosin, β-actin, and vimentin on postnatal day 4, and elevated levels of cofilin-1, calmodulin, and peptidyl-prolyl cis-trans isomerase on day 6. Western blot analysis of hydrophobic enamel proteins illustrated continuously increasing amelogenin levels from 1 dpn until 8 dpn, while enamelin peaked on days 1 and 2 dpn, and ameloblastin on days 1–5 dpn. In summary, these data document the substantial changes in the enamel matrix protein and mineral phase that take place during postnatal mouse molar amelogenesis from a systems biological perspective, including (i) relatively high levels of matrix protein expression during the early secretory stage on postnatal day 2, (ii) conversion of calcium phosphates to apatite, peak protein folding and stress protein counts, and increased cytoskeletal protein levels such as actin and tubulin on day 4, as well as (iii) secondary structure changes, isomerase activity, highest amelogenin levels, and peak phosphate/carbonate incorporation between postnatal days 6 and 8. Together, this study provides a baseline for a comprehensive understanding of the mineralogic and proteomic events that contribute to the complexity of mammalian tooth enamel development.

Aiolos Overexpression in Systemic Lupus Erythematosus B Cell Subtypes and BAFF-Induced Memory B Cell Differentiation Are Reduced by CC-220 Modulation of Cereblon Activity.

BAFF is a B cell survival and maturation factor implicated in the pathogenesis of systemic lupus erythematosus (SLE). In this in vitro study, we describe that soluble BAFF in combination with IL-2 and IL-21 is a T cell contact-independent inducer of human B cell proliferation, plasmablast differentiation, and IgG secretion from circulating CD27+ memory and memory-like CD27-IgD- double-negative (DN) B cells, but not CD27-IgD+ naive B cells. In contrast, soluble CD40L in combination with IL-2 and IL-21 induces these activities in both memory and naive B cells. Blood from healthy donors and SLE patients have similar circulating levels of IL-2, whereas SLE patients exhibit elevated BAFF and DN B cells and reduced IL-21. B cell differentiation transcription factors in memory, DN, and naive B cells in SLE show elevated levels of Aiolos, whereas Ikaros levels are unchanged. Treatment with CC-220, a modulator of the cullin ring ligase 4-cereblon E3 ubiquitin ligase complex, reduces Aiolos and Ikaros protein levels and BAFF- and CD40L-induced proliferation, plasmablast differentiation, and IgG secretion. The observation that the soluble factors BAFF, IL-2, and IL-21 induce memory and DN B cell activation and differentiation has implications for extrafollicular plasmablast development within inflamed tissue. Inhibition of B cell plasmablast differentiation by reduction of Aiolos and Ikaros may have utility in the treatment of SLE, where elevated levels of BAFF and Aiolos may prime CD27+ memory and DN memory-like B cells to become Ab-producing plasmablasts in the presence of BAFF and proinflammatory cytokines.

Delayed Activation Kinetics of Th2- and Th17 Cells Compared to Th1 Cells.

During immune responses, different classes of T cells arise: Th1, Th2, and Th17. Mobilizing the right class plays a critical role in successful host defense and therefore defining the ratios of Th1/Th2/Th17 cells within the antigen-specific T cell repertoire is critical for immune monitoring purposes. Antigen-specific Th1, Th2, and Th17 cells can be detected by challenging peripheral blood mononuclear cells (PBMC) with antigen, and establishing the numbers of T cells producing the respective lead cytokine, IFN-γ and IL-2 for Th1 cells, IL-4 and IL-5 for Th2, and IL-17 for Th-17 cells, respectively. Traditionally, these cytokines are measured within 6 h in flow cytometry. We show here that 6 h of stimulation is sufficient to detect peptide-induced production of IFN-γ, but 24 h are required to reveal the full frequency of protein antigen-specific Th1 cells. Also the detection of IL-2 producing Th1 cells requires 24 h stimulation cultures. Measurements of IL-4 producing Th2 cells requires 48-h cultures and 96 h are required for frequency measurements of IL-5 and IL-17 secreting T cells. Therefore, accounting for the differential secretion kinetics of these cytokines is critical for the accurate determination of the frequencies and ratios of antigen-specific Th1, Th2, and Th17 cells.

Cartilage oligomeric matrix protein improves in vivo cartilage regeneration and compression modulus by enhancing matrix assembly and synthesis

Cartilage oligomeric matrix protein (COMP), an abundant cartilage extracellular matrix protein, plays an important role in mesenchymal chondrogenesis. To test our hypothesis that COMP could promote tissue engineering cartilage regeneration as well as improve cartilaginous mechanical properties, COMP gene transfected rabbit bone marrow mesenchymal stem cells (BMSCs) were used for chondrogenic differentiation in vitro and were implanted with biphasic scaffolds into osteochondral defects of New Zealand white rabbit trochlear grooves for cartilage regeneration in vivo. In vitro, over expressed COMP could enhance the chondrogenic differentiation and ECM secretion of BMSCs. After euthanasia at 12 weeks post implantation, macroscopic observation, histological staining, mechanical tests and micro-CT were performed for the assessment of repaired cartilage. Overexpression of COMP leads to more newly formed hyaline cartilage and significantly improved mechanical property. These results demonstrated the significant role of COMP in the cartilage regeneration in vivo and offered inspiring advantage of COMP in the application of tissue engineering.

Bone marrow IRF4 level in multiple myeloma: an indicator of peripheral blood Th17 and disease.

Interferon regulator factor 4 (IRF4) is characterized to be a member of interferon regulatory family, which is predominantly expressed in bone marrow plasma cells of patients with multiple myeloma (MM). Recent studies indicated IRF4 is critical for T-help cells (Th17) differentiation and interleukin-17 (IL-17) secretion. Here, a total of 58 MM patients were enrolled in this study, the proportions of Th17 cells and T regulatory (Treg) cells in peripheral blood mononuclear cells (PBMCs) were determined by flow cytometric analysis. Immunohistochemistry was employed to detect the IRF4 expression in bone marrow. Herein, we observed a significant increase of IRF4 in bone marrow accompanied with a notable up-regulation of Th17 cells in PBMC within MM patients compared with healthy donors. Furthermore, the proportions of Th17 cells and serum IL-17 levels were higher in patients with stage III than stage I & II MM patients, and those parameters were positively correlated with the expression of IRF4 in these cases. These results for the first time indicate that a crosstalk between IRF4 and Th17 cells is associated with MM prognosis, and IRF4 may be served an important target for MM immunotherapy.