Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity.

Tumor exosomes are emerging as antitumor immunity regulators; however, their effects on secondary exosome secretion by distal organs have not been explored. We have previously demonstrated that suppression of exosomes at the distal tumor site of pancreatic ductal adenocarcinoma (PDAC) ablated the development of salivary biomarker profile. Here, we explore the function of salivary exosomes from tumor-bearing mice in immune surveillance. We provide evidence that salivary exosomes from mice with PDAC exhibit a suppressive effect that results in reduced tumor-killing capacity by NK cells. Salivary exosomes from mice with PDAC where pancreatic tumors were engineered to suppress exosome biogenesis failed to suppress NK cell cytotoxic potential against tumor cells, as opposed to salivary exosomes from mice with PDAC with normal tumor exosome biogenesis. These results reveal an important and previously unknown mechanism of antitumor immune regulation and provide new insights into our understanding of the alterations of this biofluid during tumor development.-Katsiougiannis, S., Chia, D., Kim, Y., Singh, R. P., Wong, D. T. W. Saliva exosomes from pancreatic tumor-bearing mice modulate NK cell phenotype and antitumor cytotoxicity.

Cryopreservation effects on sperm function and fertility in two threatened crane species.

The capacity to cryopreserve semen from captive cranes facilitates production of offspring from behaviorally incompatible or geographically separated pairs, and allows for long-term preservation of valuable genetic materials. The present study sought to develop effective cryopreservation protocols for whooping (Grus americana) and white-naped (Grus vipio) cranes, through examining the influences of two permeating (DMA and Me2SO) and one non-permeating (sucrose) cryoprotectants, as well as vitamin E on post-thaw sperm survival. In Study 1, ejaculates (whooping: n = 10, white-naped: n = 8) were collected and cryopreserved in one of six cryo-diluents (crane extender with: DMA; DMA+0.1M sucrose; Me2SO; Me2SO+0.1M sucrose; 0.1M sucrose; 0.2M sucrose) using a two-step cooling method. Frozen samples were thawed and assessed for overall motility, motion characteristics, membrane integrity, morphology, and ability to bind to the inner perivitelline membrane (IPVM). In Study 2, whooping crane ejaculates (n = 17) were frozen in crane extender containing Me2SO alone or with vitamin E (5 µg/mL or 10 µg/mL). Frozen samples were thawed and assessed as in Study 1, except the binding assay. White-naped crane sperm were more tolerant to cryopreservation than whooping crane (15% vs 6% post-thawed motility). In both species, sperm cryopreserved in medium containing Me2SO alone displayed higher post thaw survival and ability to bind to IPVM than the other cryodiluent treatments. Vitamin E supplementation exerted no benefits to post thaw motility or membrane integrity. The findings demonstrated that there was species specificity in the susceptibility to cryopreservation. Nevertheless, Me2SO was a preferred cryoprotectant for sperm from both whooping and white-naped cranes.

Influence of cooling and thawing conditions and cryoprotectant concentration on frozen-thawed survival of white-naped crane (Antigone vipio) spermatozoa.

To assist in genetic resource management and recovery efforts of the white-naped crane (Antigone vipio), we conducted two experiments to evaluate the effect of cooling condition, thawing rate, and cryoprotectant concentration on sperm survival post-thaw. Semen was collected from four mature males during breeding season (March and April) and evaluated for volume, sperm concentration, motility, and membrane integrity. In Experiment 1, ejaculates (n = 8) were diluted with Beltsville Poultry Semen Extender (BPSE) containing 10% dimethylsulfoxide (Me2SO) and frozen using either one (average cooling rate = 2.5 °C/min) or two step (average cooling rate = 7 and 9 °C/min, respectively) cooling method. The frozen samples were thawed using one of two thawing rates: 37 °C 30 s vs. 4 °C 1 min. In Experiment 2, samples were diluted with crane semen extender containing either 6% or 10% Me2SO, frozen using two-step method and then thawed at 37 °C for 30 s. Both cooling condition (two-step > one-step) and thawing rate (37 °C 30 s > 4 °C 1 min) impacted sperm motility, progression and kinetic characteristics (P < 0.05), but did not (P > 0.05) affect plasma membrane or acrosomal integrity. Concentration of Me2SO did not impact frozen-thaw survival. We conclude that white-naped crane sperm cryopreserved using a combination of two-step cooling and thawing at 37 °C 30 s was superior to other cooling and thawing combinations regarding to sustaining sperm motility with good motility kinetics. Findings represent the first steps towards the development of effective cryopreservation protocols and establishment of a genome resource bank for this threatened species.

Effects of bisphenol-A on male reproductive success in adult Kadaknath chicken.

Bisphenol-A (BPA) adversely affects human and animal reproductive success in many ways, but this information is scant on birds. In the present study, we investigated the reproductive toxicity of BPA in adult Kadaknath chicken using two BPA dosages orally (1 or 5 mg/kg body weight) for seven weeks. In order to assess BPA toxicity, sperm functions, fertilizing ability, serum testosterone concentration and testis histopathology were measured in treated and control chickens. The semen volume was highest in birds exposed to 1mg/kg body weight BPA compared to other groups. 5 mg/kg body weight BPA reduced sperm concentration significantly more than other treatment and controls. However, overall fertility and testis histology were unaffected. These results indicate that BPA adversely affects sperm characteristics in adult kadaknath chicken without affecting fertilization potential.

A Role for GABAA Receptor ß3 Subunits in Mediating Harmaline Tremor Suppression by Alcohol: Implications for Essential Tremor Therapy.

Background: Essential tremor patients may find that low alcohol amounts suppress tremor. A candidate mechanism is modulation of α6ß3δ extra-synaptic GABAA receptors, that in vitro respond to non-intoxicating alcohol levels. We previously found that low-dose alcohol reduces harmaline tremor in wild-type mice, but not in littermates lacking δ or α6 subunits. Here we addressed whether low-dose alcohol requires the ß3 subunit for tremor suppression. Methods: We tested whether low-dose alcohol suppresses tremor in cre-negative mice with intact ß3 exon 3 flanked by loxP, and in littermates in which this region was excised by cre expressed under the α6 subunit promotor. Tremor in the harmaline model was measured as a percentage of motion power in the tremor bandwidth divided by overall motion power. Results: Alcohol, 0.500 and 0.575 g/kg, reduced harmaline tremor compared to vehicle-treated controls in floxed ß3 cre- mice, but had no effect on tremor in floxed ß3 cre+ littermates that have ß3 knocked out. This was not due to potential interference of α6 expression by the insertion of the cre gene into the α6 gene since non-floxed ß3 cre+ and cre- littermates exhibited similar tremor suppression by alcohol. Discussion: As α6ß3δ GABAA receptors are sensitive to low-dose alcohol, and cerebellar granule cells express ß3 and are the predominant brain site for α6 and δ expression together, our overall findings suggest alcohol acts to suppress tremor by modulating α6ß3δ GABAA receptors on these cells. Novel drugs that target this receptor may potentially be effective and well-tolerated for essential tremor. Highlights: We previously found with the harmaline essential tremor model that GABAA receptors containing α6 and δ subunits mediate tremor suppression by alcohol. We now show that ß3 subunits in α6-expressing cells, likely cerebellar granule cells, are also required, indicating that alcohol suppresses tremor by modulating α6ß3δ extra-synaptic GABAA receptors.

Search for Novel Therapies for Essential Tremor Based on Positive Modulation of α6-Containing GABAA Receptors.

Background: Prior work using GABAA receptor subunit knockouts and the harmaline model has indicated that low-dose alcohol, gaboxadol, and ganaxolone suppress tremor via α6ßδ GABAA receptors. This suggests that drugs specifically enhancing the action of α6ßδ or α6ßγ2 GABAA receptors, both predominantly expressed on cerebellar granule cells, would be effective against tremor. We thus examined three drugs described by in vitro studies as selective α6ßδ (ketamine) or α6ßγ2 (Compound 6, flumazenil) receptor modulators. Methods: In the first step of evaluation, the maximal dose was sought at which 6/6 mice pass straight wire testing, a sensitive test for psychomotor impairment. Only non-impairing doses were used to evaluate for anti-tremor efficacy in the harmaline model, which was assessed in wildtype and α6 subunit knockout littermates. Results: Ketamine, in maximally tolerated doses of 2.0 and 3.5 mg/kg had minimal effect on harmaline tremor in both genotypes. Compound 6, at well-tolerated doses of 1-10 mg/kg, effectively suppressed tremor in both genotypes. Flumazenil suppressed tremor in wildtype mice at doses (0.015-0.05 mg/kg) far lower than those causing straight wire impairment, and did not suppress tremor in α6 knockout mice. Discussion: Modulators of α6ßδ and α6ßγ2 GABAA receptors warrant attention for novel therapies as they are anticipated to be effective and well-tolerated. Ketamine likely failed to attain α6ßδ-active levels. Compound 6 is an attractive candidate, but further study is needed to clarify its mechanism of action. The flumazenil results provide proof of principle that targeting α6ßγ2 receptors represents a worthy strategy for developing essential tremor therapies. Highlights: We tested for harmaline tremor suppression drugs previously described as in vitro α6ßδ or α6ßγ2 GABAA receptor-selective modulators. Well-tolerated flumazenil doses suppressed tremor in α6-wildtype but not α6-knockout mice. Compound 6 and ketamine failed to display this profile, likely from off-target effects. Selective α6 modulators hold promise as tremor therapy.

Alcohol and Ganaxolone Suppress Tremor via Extra-Synaptic GABAA Receptors in the Harmaline Model of Essential Tremor.

Background: A long-standing question is why essential tremor often responds to non-intoxicating amounts of alcohol. Blood flow imaging and high-density electroencephalography have indicated that alcohol acts on tremor within the cerebellum. As extra-synaptic δ-subunit-containing GABAA receptors are sensitive to low alcohol levels, we wondered whether these receptors mediate alcohol's anti-tremor effect and, moreover, whether the δ-associated GABAA receptor α6 subunit, found abundantly in the cerebellum, is required. Methods: We tested the hypotheses that low-dose alcohol will suppress harmaline-induced tremor in wild-type mice, but not in littermates lacking GABAA receptor δ subunits, nor in littermates lacking α6 subunits. As the neurosteroid ganaxolone also activates extra-synaptic GABAA receptors, we similarly assessed this compound. The harmaline mouse model of essential tremor was utilized to generate tremor, measured as a percentage of motion power in the tremor bandwidth (9-16 Hz) divided by background motion power at 0.25-32 Hz. Results: Ethanol, 0.500 and 0.575 g/kg, and ganaxolone, 7 and 10 mg/kg, doses that do not impair performance in a sensitive psychomotor task, reduced harmaline tremor compared to vehicle-treated controls in wild-type mice but failed to suppress tremor in littermates lacking the δ or the α6 GABAA receptor subunit. Discussion: As cerebellar granule cells are the predominant brain site intensely expressing GABAA receptors containing both α6 and δ subunits, these findings suggest that this is where alcohol acts to suppress tremor. It is anticipated that medications designed specifically to target α6ßδ-containing GABAA receptors may be effective and well-tolerated for treating essential tremor. Highlights: How does alcohol temporarily ameliorate essential tremor? This study with a mouse model found that two specific kinds of GABA receptor subunits were needed for alcohol to work. As receptors with both these subunits are found mainly in cerebellum, this work suggests this is where alcohol acts to suppress tremor.

Comparison of cycloplegia with atropine 1% versus cyclopentolate 1.

PURPOSE: Cycloplegic refraction is mandatory for children to know the eye's refractive status. In this study, we compared cycloplegia induced by cyclopentolate 1% to that induced by atropine 1% by means of retinoscopy. METHODS: In this parallel-designed interventional study, we included 67 children aged between 4 and 17 years. After the initial retinoscopy under cyclopentolate 1% (used twice in each eye), we repeated it a week later under atropine ointment 1% (used twice a day for 3 days); both were done by the same trained optometrist masked to the drug. Each eye's refraction was converted to spherical equivalents (SEs), and the values averaged between the two eyes of each child under each drug. We compared SE with paired t-test (JASP 16.4). In addition, we performed correlational analysis, and looked for agreement using the Bland-Altman plot. Significance was set at P < 0.05. Wherever possible, 95% confidence intervals (CIs) are quoted. RESULTS: The mean SE with atropine was +1.93 ± 2.0 D, compared to +1.75 ± 1.95 D under cyclopentolate. On average, atropine induced greater cycloplegia by a mere 0.18 D (95% CI: 0.07 to 0.29 D, P value 0.002). The two cycloplegic refractions correlated significantly (Pearson's r: 0.975, P < 0.001). The Bland-Altman plot revealed the limits of agreement as 1.06 and -0.71 D. CONCLUSION: Our study suggests that cyclopentolate works for the most part as well as atropine to attain cycloplegia. Atropine may be considered for children less than 15 years of age with greater than 5.0 D of hyperopia. Cycloplentolate, with its advantages of quick action and short duration, should form the first go-to topical cycloplegic in busy outpatient clinics.

Blockade of programmed death-1 in young (New Zealand black x New Zealand white)F1 mice promotes the activity of suppressive CD8+ T cells that protect from lupus-like disease.

The programmed death-1 (PD-1)/programmed death-1 ligand 1 (PD-L1) pathway regulates both stimulatory and inhibitory signals. In some conditions, PD-1/PD-L1 inhibits T and B cell activation, induces anergy, and reduces cytotoxicity in CD8(+) T cells. In other conditions, PD-l/PD-L1 has costimulatory effects on T cells. We recently showed that induction of suppressive CD8(+)Foxp3(+) T cells by immune tolerance of lupus-prone (New Zealand black × New Zealand white)F(1) (BWF(1)) mice with the anti-DNA Ig-based peptide pConsensus (pCons) is associated with significantly reduced PD-1 expression on those cells. In this study, we tested directly the role of PD-1 by administering in vivo neutralizing Ab to PD-1 to premorbid BWF(1) and healthy control mice. Anti-PD-1-treated mice were protected from the onset of lupus nephritis for 10 wk, with significantly improved survival. Although the numbers of T cells declined in aging control mice, they were maintained in anti-PD-1-treated mice, including CD8(+)Foxp3(+) T cells that suppressed syngeneic CD4(+)CD25(-) T cell proliferation and IFN-γ production, reduced production of IgG and anti-dsDNA IgG, induced apoptosis in syngeneic B cells, and increased IL-2 and TGF-ß production. The administration of anti-PD-1 Ab to BWF(1) mice after induction of tolerance with pCons abrogated tolerance; mice developed autoantibodies and nephritis at the same time as control mice, being unable to induce CD8(+)Foxp3(+) T suppressor cells. These data suggest that tightly regulated PD-1 expression is essential for the maintenance of immune tolerance mediated by those CD8(+)Foxp3(+) T cells that suppress both T(h) cells and pathogenic B cells. PD-1 regulation could represent a target to preserve tolerance and prevent autoimmunity.

Identification and Contribution of Inflammation-Induced Novel MicroRNA in the Pathogenesis of Systemic Lupus Erythematosus.

Recently microRNAs (miRNAs) have been recognized as powerful regulators of many genes and pathways involved in the pathogenesis of inflammatory diseases including Systemic Lupus Erythematosus (SLE). SLE is an autoimmune disease characterized by production of various autoantibodies, inflammatory immune cells, and dysregulation of epigenetic changes. Several candidate miRNAs regulating inflammation and autoimmunity in SLE are described. In this study, we found significant increases in the expression of miR21, miR25, and miR186 in peripheral blood mononuclear cells (PBMCs) of SLE patients compared to healthy controls. However, miR146a was significantly decreased in SLE patients compared to healthy controls and was negatively correlated with plasma estradiol levels and with SLE disease activity scores (SLEDAI). We also found that protein levels of IL-12 and IL-21 were significantly increased in SLE patients as compared to healthy controls. Further, our data shows that protein levels of IL-12 were positively correlated with miR21 expression and protein levels of IL-21 positively correlated with miR25 and miR186 expression in SLE patients. In addition, we found that levels of miR21, miR25, and miR186 positively correlated with SLEDAI and miR146a was negatively correlated in SLE patients. Thus, our data shows a dynamic interplay between disease pathogenesis and miRNA expression. This study has translational potential and may identify novel therapeutic targets in patients with SLE.

A novel process driving Alzheimer's disease validated in a mouse model: Therapeutic potential.

Introduction: The neuronal mechanism driving Alzheimer's disease (AD) is incompletely understood. Methods: Immunohistochemistry, pharmacology, biochemistry, and behavioral testing are employed in two pathological contexts-AD and a transgenic mouse model-to investigate T14, a 14mer peptide, as a key signaling molecule in the neuropathology. Results: T14 increases in AD brains as the disease progresses and is conspicuous in 5XFAD mice, where its immunoreactivity corresponds to that seen in AD: neurons immunoreactive for T14 in proximity to T14-immunoreactive plaques. NBP14 is a cyclized version of T14, which dose-dependently displaces binding of its linear counterpart to alpha-7 nicotinic receptors in AD brains. In 5XFAD mice, intranasal NBP14 for 14 weeks decreases brain amyloid and restores novel object recognition to that in wild-types. Discussion: These findings indicate that the T14 system, for which the signaling pathway is described here, contributes to the neuropathological process and that NBP14 warrants consideration for its therapeutic potential.

Modulation of p38 MAPK activity in regulatory T cells after tolerance with anti-DNA Ig peptide in (NZB x NZW)F1 lupus mice.

Treatment of (NZB x NZW)F(1) (NZB/W) lupus-prone mice with the anti-DNA Ig-based peptide pConsensus prolongs the survival of treated animals and effectively delays the appearance of autoantibodies and glomerulonephritis. We have previously shown that part of these protective effects associated with the induction of CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) that suppressed autoantibody responses. Because the effects of pConsensus appeared secondary to qualitative rather than quantitative changes in Tregs, we investigated the molecular events induced by tolerance in Tregs and found that signaling pathways including ZAP70, p27, STAT1, STAT3, STAT6, SAPK, ERK, and JNK were not significantly affected. However, peptide tolerization affected in Tregs the activity of the MAPK p38, whose phosphorylation was reduced by tolerance. The pharmacologic inhibition of p38 with the pyridinyl imidazole inhibitor SB203580 in naive NZB/W mice reproduced in vivo the effects of peptide-induced tolerance and protected mice from lupus-like disease. Transfer experiments confirmed the role of p38 in Tregs on disease activity in the NZB/W mice. These data indicate that the modulation of p38 activity in lupus Tregs can significantly influence the disease activity.

Sex Hormones and Gender Influence the Expression of Markers of Regulatory T Cells in SLE Patients.

Regulatory T cells have been implicated in the regulation and maintenance of immune homeostasis. Whether gender and sex hormones differentially influence the expression and function of regulatory T cell phenotype and their influence on FoxP3 expression remains obscure. We provide evidence in this study that the number and percent of human regulatory T cells (Tregs) expressing CD4+ and CD8+ are significantly reduced in healthy females compared to healthy males. In addition, both CD4+CD25+hi and CD8+CD25+hi subsets in healthy males have a 2-3 fold increase in FoxP3 mRNA expression compared to healthy females. Female SLE patients, compared to healthy women, have elevated plasma levels of estradiol and decreased levels of testosterone. Higher levels of testosterone correlate with higher expression of FoxP3 in CD4+CD25hiCD127low putative Tregs in women with SLE. Incubation of CD4+ regulatory T cells with 17ß-estradiol at physiological levels generally decreased FoxP3 expression in females with SLE. These data suggest that females may be more susceptible than males to SLE and other autoimmune diseases in part because they have fewer Tregs and reduced FoxP3 expression within those cells due to normal E2 levels which suppress FoxP3 expression. In addition, low levels of plasma testosterone in women may further reduce the ability of the Tregs to express FoxP3. These data suggest that gender and sex hormones can influence susceptibility to SLE via effects on regulatory T cells and FoxP3 expression.

Effects of Peptide-Induced Immune Tolerance on Murine Lupus.

The regulation of autoimmunity and the molecular mechanisms by which different immune cells, including T cells, polymorphonuclear leukocytes (PMN-granulocytes), and B cells suppress autoimmune diseases is complex. We have shown previously that BWF1 lupus mice are protected from autoimmunity after i.v. injection or oral administration of tolerogenic doses of pCons, an artificial synthetic peptide based on sequences containing MHC class I and MHC class II determinants in the VH region of a J558-encoded BWF1 anti-DNA Ab. Several T cell subsets can transfer this tolerance. In this study, we determined the potential roles of granulocytes, B cells and regulatory T cells altered by pCons treatment in the BWF1 (NZB/NZW) mouse model of lupus. Immunophenotyping studies indicated that pCons treatment of BWF1 mice significantly increased CD4+FoxP3+ T cells, reduced the percent of B cells expressing CD19+CD5+ but increased the percent of CD19+CD1d+ regulatory B cells and increased the ability of the whole B cell population to suppress IgG anti-DNA production in vitro. pCons treatment significantly decreased the expression of CTLA-4 (cytotoxic T-lymphocyte-associated protein-4) in CD8+ T cells. In addition, peptide administration modified granulocytes so they became suppressive. We co-cultured sorted naïve B cells from mice making anti-DNA Ab (supported by addition of sorted naive CD4+ and CD8+ T cells from young auto-antibody-negative BWF1 mice) with sorted B cells or granulocytes from tolerized mice. Both tolerized granulocytes and tolerized B cells significantly suppressed the production of anti-DNA in vitro. In granulocytes from tolerized mice compared to saline-treated littermate controls, real-time PCR analysis indicated that expression of interferon-induced TNFAIP2 increased more than 2-fold while Ptdss2 and GATA1 mRNA were up-regulated more than 10-fold. In contrast, expression of these genes was significantly down-regulated in tolerized B cells. Further, another IFN-induced protein, Bcl2, was reduced in tolerized B cells as determined by Western blot analyses. In contrast, expression of FoxP3 was significantly increased in tolerized B cells. Together, these data suggest that B cells and granulocytes are altered toward suppressive functions by in vivo tolerization of BWF1 mice with pCons and it is possible these cell types participate in the clinical benefits seen in vivo.

CD8+ T regulatory cells in lupus.

T regulatory cells (Tregs) have a key role in the maintenance of immune homeostasis and the regulation of immune tolerance by preventing the inflammation and suppressing the autoimmune responses. Numerical and functional deficits of these cells have been reported in systemic lupus erythematosus (SLE) patients and mouse models of SLE, where their imbalance and dysregulated activities have been reported to significantly influence the disease pathogenesis, progression and outcomes. Most studies in SLE have focused on CD4+ Tregs and it has become clear that a critical role in the control of immune tolerance after the breakdown of self-tolerance is provided by CD8+ Tregs. Here we review the role, cellular and molecular phenotypes, and mechanisms of action of CD8+ Tregs in SLE, including ways to induce these cells for immunotherapeutic modulation in SLE.

Cellular and Molecular Phenotypes of pConsensus Peptide (pCons) Induced CD8+ and CD4+ Regulatory T Cells in Lupus.

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease with widespread inflammation, immune dysregulation, and is associated with the generation of destructive anti-DNA autoantibodies. We have shown previously the immune modulatory properties of pCons peptide in the induction of both CD4+ and CD8+ regulatory T cells which can in turn suppress development of the autoimmune disease in (NZB/NZW) F1 (BWF1) mice, an established model of lupus. In the present study, we add novel protein information and further demonstrate the molecular and cellular phenotypes of pCons-induced CD4+ and CD8+ Treg subsets. Flow cytometry analyses revealed that pCons induced CD8+ Treg cells with the following cell surface molecules: CD25highCD28high and low subsets (shown earlier), CD62Lhigh, CD122low, PD1low, CTLA4low, CCR7low and 41BBhigh. Quantitative real-time PCR (qRT-PCR) gene expression analyses revealed that pCons-induced CD8+ Treg cells downregulated the following several genes: Regulator of G protein signaling (RGS2), RGS16, RGS17, BAX, GPT2, PDE3b, GADD45ß and programmed cell death 1 (PD1). Further, we confirmed the down regulation of these genes by Western blot analyses at the protein level. To our translational significance, we showed herein that pCons significantly increased the percentage of CD8+FoxP3+ T cells and further increased the mean fluorescence intensity (MFI) of FoxP3 when healthy peripheral blood mononuclear cells (PBMCs) are treated with pCons (10 µg/ml, for 24-48 hours). In addition, we found that pCons reduced apoptosis in CD4+ and CD8+ T cells and B220+ B cells of BWF1 lupus mice. These data suggest that pCons stimulates cellular, immunological, and molecular changes in regulatory T cells which in turn protect against SLE autoimmunity.

Interferon Genes Are Influenced by 17ß-Estradiol in SLE.

Recent evidence suggests the existence of a nexus between inflammatory pathways and the female sex hormone 17ß-estradiol, resulting in increased interferon-stimulated genes (ISGs), autoantibodies, and dysregulation of immune cells in SLE. However, the molecular mechanisms and the effect of estradiol on candidate target genes and their pathways remains poorly understood. Our previous work suggests that female SLE patients have increased estradiol levels compared to healthy controls. In the present study, we explored the effects of 17ß-estradiol treatment on expression of IFN (interferons)-stimulated genes and pro-inflammatory cytokines/chemokines. We found significantly increased (5-10-fold) expression of IFN-regulated genes in healthy females. Furthermore, we found significantly increased plasma levels of IL-6, IL-12, IL-17, IL-18, stem cell factor (SCF), and IL-21/IL-23 in SLE patients compared to healthy controls, and those levels positively correlated with the plasma levels of 17ß-estradiol. In addition, levels of IL-21 positively correlated with the SLE disease activity index (SLEDAI) score of SLE patients. In vitro treatment of PBMCs from either SLE patients or healthy controls with 17ß-estradiol at physiological concentration (~50 pg/ml) also significantly increased secretion of many pro-inflammatory cytokines and chemokines (IL-6, IL-12, IL-17, IL-8, IFN-γ; MIP1α, and MIP1ß) in both groups. Further our data revealed that 17ß-estradiol significantly increased the percentage of CD3+CD69+ and CD3+IFNγ+ T cells; whereas, simultaneous addition of 17ß-estradiol and an ERα inhibitor prevented this effect. Collectively, our findings indicate that 17ß-estradiol participates in the induction of pro-inflammatory cytokines and chemokines and further influences interferon genes and pathways.

Immune Checkpoint Blockade Enhances Immune Activity of Therapeutic Lung Cancer Vaccine.

BACKGROUND: Immune checkpoint blockade that downregulates T cell evasion for effective immunity has provided a renewed interest in therapeutic cancer vaccines. METHODS: Utilizing murine lung cancer models, we determined: tumor burden, TIL cytolysis, immunohistochemistry, flow cytometry, RNA Sequencing, CD4 T cells, CD8 T cells, CXCL9 chemokine, and CXCL10 chemokine neutralization to evaluate the efficacy of Programmed cell death protein 1 (PD-1) blockade combined with chemokine (C-C motif) ligand 21-dendritic cell tumor antigen (CCL21-DC tumor Ag) vaccine. RESULTS: Anti-PD1 combined with CCL21-DC tumor Ag vaccine eradicated 75% of 12-day established tumors (150 mm3) that was enhanced to 90% by administering CCL21-DC tumor Ag vaccine prior to combined therapy. The effect of combined therapy was blocked by CD4, CD8, CXCL9, and CXCL10 neutralizing antibodies. CONCLUSION: PD-1 blockade therapy plus CCL21-DC tumor Ag vaccine could be beneficial to lung cancer patients.

Influence of Metabolic Status and Diet on Early Pregnant Equine Histotroph Proteome: Preliminary Findings.

Insulin resistance (IR) is characterized by an increase in biomarkers of systemic inflammation and susceptibility to laminitis in horses. Impacts on reproduction include a lengthened interovulatory period in horses. Dietary omega-3 (docosahexaenoic acid [DHA]) promotes anti-inflammatory processes, has been implicated in health benefits, and can reduce cytokine secretion. This preliminary study investigated the impact of IR as well as the influence of dietary supplementation (DHA) on the uterine fluid proteome in early pregnant horses. Mares were artificially inseminated; uterine fluid and embryos were collected on d 12.5 after ovulation. Uterine fluid was pooled for metabolic and diet categories (n = 8; n = 2 per metabolic and dietary status) and concentrated, and the proteome was analyzed using tandem mass spectrometry (iTRAQ). Five proteins met differential abundance criteria (±1.5-fold change, P < .05) in all comparisons (Control C, IS vs. C, IR; C, IS vs. DHA, IS; C, IR vs. DHA, IR). Serum amyloid A, afamin, and serotransferrin were upregulated in C, IR mares but downregulated in DHA, IR mares when compared to C, IS and C, IR, respectively. Quantitative PCR supported mass spectrometry results. The presence of serum amyloid A and serotransferrin in histotroph of IR mares potentially indicates an inflammatory response not seen in IS counterparts. These preliminary findings provide novel evidence on the potential impact of insulin resistance and DHA supplementation on the secreted equine uterine proteome during early pregnancy.

Reproductive seasonality and sperm cryopreservation in the male tufted deer (Elaphodus cephalophus).

The tufted deer is a small deer, listed as near threatened on the International Union for Conservation of Nature Red List, and there is no information on the fundamental reproductive biology of this species. In this study, we report for the first time, characterization of male reproductive traits and cryopreservation of semen in this species. Males were subjected to electroejaculation during each season (autumn, winter, spring, and summer), and ejaculates were assessed for motility and quality traits. Fecal samples were collected 3 to 5 times weekly for 2 years and analyzed for androgen metabolites using enzyme immunoassay. Ejaculates with greater than 70% motility were cryopreserved using Beltsville extender (BF5F) or Triladyl. Straws were thawed and assessed subjectively as well as swim-up processed to isolate motile spermatozoa for computer-assisted sperm analysis and acrosome integrity at hourly interval. Tufted deer male reproductive and semen traits peaked in autumn. Mean fecal androgen concentrations were highest in the summer compared with baseline values during rest of the year. Sperm motility and acrosome integrity were lower immediately after thawing in both cryodiluents compared with raw ejaculates. Motility characteristics after swim-up were higher in BF5F compared with Triladyl. Four hours after thawing, both percent sperm motility and progression decreased further and were similar between BF5F and Triladyl. However, the proportion of spermatozoa with intact acrosomal membranes was higher in BF5F than Triladyl. Results indicate that tufted deer exhibit seasonal variations in reproductive traits and that BF5F better preserves sperm motility and acrosomal integrity after cryopreservation compared with Triladyl.