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Background and purpose Neuromyelitis optica spectrum disorder (NMOSD) is mainly an anti-aquaporin 4 (anti-AQP4) autoantibodies-mediated idiopathic inflammatory demyelinating disease of the central nervous system. Systemic and local inflammatory responses play a key role in the pathophysiology of NMOSD. However, the role of the crucial immunomodulators CD4+CD25+ forkhead box P3+ (Foxp3) regulatory T cells (Tregs) has not been investigated in NMOSD. Methods Twenty-five patients with anti-AQP4-postive NMOSD undergoing an attack and 21 healthy controls (HCs) were enrolled. Frequencies of T cell subsets and Tregs in the peripheral blood were assessed by flow cytometry. Additionally, a model of NMOSD using purified immunoglobulin G from anti-AQP4-antibodies-positive patients with NMOSD and human complement injected into brain of female adult C57BL/6J mice was established. Infiltrated Tregs into NMOSD mouse brain lesions were analyzed by flow cytometry, histological sections, and real-time quantitative Polymerase Chain Reaction. Astrocyte loss, demyelination, and inflammatory response were also evaluated in our NMOSD mouse model. Finally, we examined the effects of both depletion and adoptive transfer of Tregs. Results The percentage of Tregs, especially naïve Tregs, among total T cells in peripheral blood was significantly decreased in NMOSD patients at acute stage when compared to HCs. Within our animal model, the number and proportion of Tregs among CD4+ T cells were increased in the lesion of mice with NMOSD. Depletion of Tregs profoundly enhanced astrocyte loss and demyelination in these mice, while adoptive transfer of Tregs attenuated brain damage. Mechanistically, the absence of Tregs induced more macrophage infiltration, microglial activation, and T cells invasion, and modulated macrophages/microglia toward a classical activation phenotype, releasing more chemokines and pro-inflammatory cytokines. In contrast, Tregs transfer ameliorated immune cell infiltration in NMOSD mice, including macrophages, neutrophils, and T cells, and skewed macrophages and microglia towards an alternative activation phenotype, thereby decreasing the level of chemokines and pro-inflammatory cytokines. Conclusion Tregs may be key immunomodulators ameliorating brain damage via dampening inflammatory response after NMOSD.

Often referred to as the silent killer, ovarian cancer is the most lethal gynecologic malignancy. This disease rarely shows any physical symptoms until late stages and no known biomarkers are available for early detection. Because ovarian cancer is rarely detected early, the physiology behind the initiation, progression, treatment, and prevention of this disease remains largely unclear. Over the past 2 decades, the laying hen has emerged as a model that naturally develops epithelial ovarian cancer that is both pathologically and histologically similar to that of the human form of the disease. Different molecular signatures found in human ovarian cancer have also been identified in chicken ovarian cancer including increased CA125 and elevated E-cadherin expression, among others. Chemoprevention studies conducted in this model have shown that decreased ovulation and inflammation are associated with decreased incidence of ovarian cancer development. The purpose of this article is to review the major studies performed in laying hen model of ovarian cancer and discuss how these studies shape our current understanding of the pathophysiology, prevention, and treatment of epithelial ovarian cancer.

Background Multiple sclerosis (MS) is an immune-mediated demyelinated disease of the central nervous system. Activation of microglia is involved in the pathogenesis of myelin loss. Objective This study is focused on the role of Hv1 in regulating demyelination and microglial activation through reactive oxygen species (ROS) production after lysophosphatidylcholine (LPC)-mediated demyelination. We also explored autophagy in this process. Methods A model of demyelination using two-point LPC injection into the corpus callosum was established. LFB staining, immunofluorescence, Western blot, and electron microscopy were used to study the severity of demyelination. Microglial phenotype and autophagy were detected by immunofluorescence and Western blot. Morris water maze was used to test spatial learning and memory ability. Results We have identified that LPC-mediated myelin damage was reduced by Hv1 deficiency. Furthermore, we found that ROS and autophagy of microglia increased in the demyelination region, which was also inhibited by Hv1 knockout. Conclusion These results suggested that microglial Hv1 deficiency ameliorates demyelination through inhibition of ROS-mediated autophagy and microglial phenotypic transformation.

MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. We evaluated the levels of family members relative to the internal control miR-103a in ovarian cancer and control blood specimens collected from American and Hong Kong Chinese institutions, as well as from a laying hen spontaneous ovarian cancer model. The levels of miR-200a, miR-200b and miR-200c were significantly elevated in all human cancer versus all control blood samples. Further analyses showed significantly higher miR-200 levels in Chinese control (except miR-429) and cancer (except miR-200a and miR141) samples than their respective American counterparts. Subtype-specific analysis showed that miR-200b had an overall elevated level in serous cancer compared with controls, whereas miR-429 was significantly elevated in clear cell and endometrioid cancer versus controls. MiR-429 was also significantly elevated in cancer versus control in laying hen plasma samples, consistent with the fact that endometrioid tumor is the prevalent type in this species. A neural network model consisting of miR-200a/200b/429/141 showed an area under the curve (AUC) value of 0.904 for American ovarian cancer prediction, whereas a model consisting of miR-200b/200c/429/141 showed an AUC value of 0.901 for Chinese women. Hence, miR-200 is informative as blood biomarkers for both human and laying hen ovarian cancer.

Background 2-methoxyestradiol (2MeOE2) is a natural metabolite of estradiol, which is generated by the action of CYP1A1 enzyme in the liver. We have previously shown that a flaxseed-supplemented diet decreases both the incidence and severity of ovarian cancer in laying hens, also induces CYP1A1 expression in liver. Recently, we have shown that as a biologically derived active component of flax diet, 2MeOE2 induces apoptosis in ovarian cancer cells which is partially dependent on p38 MAPK. The objective of this study was to elucidate the molecular mechanism of actions of 2MeOE2, a known microtubule disrupting agent, in inducing apoptosis in ovarian tumors. Results 2MeOE2 induces γH2Ax expression and apoptotic histone modifications in ovarian cancer cells, which are predicted downstream targets of protein kinase Cδ (PKCδ) during apoptosis. Overexpressing full length PKCδ alone does not induce apoptosis but potentiates 2MeOE2-mediated apoptosis. C3-domain mutated dominant-negative PKCδ (PKCδDN) significantly reduces 2MeOE2-induced caspase-3 cleavage and apoptotic histone modification. Silencing PKCδ diminishes 2MeOE2-mediated apoptosis. The catalytic fragment of PKCδ (PKCδCAT) evokes pro-apoptotic effects which are principally dependent on p38 MAPK phosphorylation. Conclusions The pro-apoptotic actions of 2MeOE2 are in part dependent on catalytic activation of PKCδ. Catalytic activation of PKCδ accelerates the 2MeOE2-induced apoptotic cascade. This study describes a novel molecular action of flaxseed diet in ovarian cancer.

Epidemiologic and histopathologic findings and the laying hen model support the long-standing incessant ovulation hypothesis and cortical inclusion cyst involvement in sporadic ovarian cancer development. MicroRNA-200 (miR-200) family is highly expressed in ovarian cancer. Herewith, we show that ovarian surface epithelial (OSE) cells with ectopic miR-200 expression formed stabilized cysts in three-dimensional (3D) organotypic culture with E-cadherin fragment expression and steroid hormone pathway activation, whereas ovarian cancer 3D cultures with miR-200 knockdown showed elevated TGF-β expression, mitotic spindle disorientation, increased lumenization, disruption of ROCK-mediated myosin II phosphorylation, and SRC signaling, which led to histotype-dependent loss of collective movement in tumor spread. Gene expression profiling revealed that epithelial-mesenchymal transition and hypoxia were the top enriched gene sets regulated by miR-200 in both OSE and ovarian cancer cells. The molecular changes uncovered by the in vitro studies were verified in both human and laying hen ovarian cysts and tumor specimens. As miR-200 is also essential for ovulation, our results of estrogen pathway activation in miR-200-expressing OSE cells add another intriguing link between incessant ovulation and ovarian carcinogenesis.

We used an LC-MS/MS metabolomics approach to investigate one-carbon metabolism in the plasma of flaxseed-fed White Leghorn laying hens (aged 3.5 years). In our study, dietary flaxseed (via the activity of a vitamin B6 antagonist known as “1-amino d-proline”) induced at least 15-fold elevated plasma cystathionine. Surprisingly, plasma homocysteine (Hcy) was stable in flaxseed-fed hens despite such highly elevated cystathionine. To explain stable Hcy, our data suggest accelerated Hcy remethylation via BHMT and MS-B12. Also supporting accelerated Hcy remethylation, we observed elevated S-adenosylmethionine (SAM), an elevated SAM:SAH ratio, and elevated methylthioadenosine (MTA), in flaxseed-fed hens. These results suggest that flaxseed increases SAM biosynthesis and possibly increases polyamine biosynthesis. The following endpoint phenotypes were observed in hens consuming flaxseed: decreased physiological aging, increased empirical lifespan, 9–14% reduced body mass, and improved liver function. Overall, we suggest that flaxseed can protect women from ovarian tumor metastasis by decreasing omental adiposity. We also propose that flaxseed protects cancer patients from cancer-associated cachexia by enhancing liver function.

Seizures are common in humans with various etiologies ranging from congenital aberrations to acute injuries that alter the normal balance of brain excitation and inhibition. A notable consequence of seizures is the induction of aberrant neurogenesis and increased immature neuronal projections. However, regulatory mechanisms governing these features during epilepsy development are not fully understood. Recent studies show that microglia, the brain's resident immune cell, contribute to normal neurogenesis and regulate seizure phenotypes. However, the role of microglia in aberrant neurogenic seizure contexts has not been adequately investigated. To address this question, we coupled the intracerebroventricular kainic acid model with current pharmacogenetic approaches to eliminate microglia in male mice. We show that microglia promote seizure-induced neurogenesis and subsequent seizure-induced immature neuronal projections above and below the pyramidal neurons between the DG and the CA3 regions. Furthermore, we identify microglial P2Y12 receptors (P2Y12R) as a participant in this neurogenic process. Together, our results implicate microglial P2Y12R signaling in epileptogenesis and provide further evidence for targeting microglia in general and microglial P2Y12R in specific to ameliorate proepileptogenic processes.SIGNIFICANCE STATEMENTEpileptogenesis is a process by which the brain develops epilepsy. Several processes have been identified that confer the brain with such epileptic characteristics, including aberrant neurogenesis and increased immature neuronal projections. Understanding the mechanisms that promote such changes is critical in developing therapies to adequately restrain epileptogenesis. We investigated the role of purinergic P2Y12 receptors selectively expressed by microglia, the resident brain immune cells. We report, for the first time, that microglia in general and microglial P2Y12 receptors in specific promote both aberrant neurogenesis and increased immature neuronal projections. These results indicate that microglia enhance epileptogenesis by promoting these processes and suggest that targeting this immune axis could be a novel therapeutic strategy in the clinic.

In chronic-phase chronic myeloid leukemia (CP-CML) patients treated with frontline imatinib, failure to achieve early molecular response (EMR; EMR failure: BCR-ABL1 >10% on the international scale at 3 months) is predictive of inferior outcomes. Identifying patients at high-risk of EMR failure at diagnosis provides an opportunity to intensify frontline therapy and potentially avoid EMR failure. We studied blood samples from 96 CP-CML patients at diagnosis and identified 365 genes that were aberrantly expressed in 13 patients who subsequently failed to achieve EMR, with a gene signature significantly enriched for stem cell phenotype (eg, Myc, β-catenin, Hoxa9/Meis1), cell cycle, and reduced immune response pathways. We selected a 17-gene panel to predict EMR failure and validated this signature on an independent patient cohort. Patients classified as high risk with our gene expression signature (HR-GES) exhibited significantly higher rates of EMR failure compared with low-risk (LR-GES) patients (78% vs 5%; P < .0001), with an overall accuracy of 93%. Furthermore, HR-GES patients who received frontline nilotinib had a relatively low rate of EMR failure (10%). However, HR-GES patients still had inferior deep molecular response achievement rate by 24 months compared with LR-GES patients. This novel multigene signature may be useful for selecting patients at high risk of EMR failure on standard therapy who may benefit from trials of more potent kinase inhibitors or other experimental approaches.

Ischemic stroke is a leading cause of morbidity and mortality worldwide. Resident microglia are the principal immune cells of the brain, and the first to respond to the pathophysiological changes induced by ischemic stroke. Traditionally, it has been thought that microglial activation is deleterious in ischemic stroke, and therapies to suppress it have been intensively explored. However, increasing evidence suggests that microglial activation is also critical for neurogenesis, angiogenesis, and synaptic remodeling, thereby promoting functional recovery after cerebral ischemia. Here, we comprehensively review the dual role of microglia during the different phases of ischemic stroke, and the possible mechanisms controlling the post-ischemic activity of microglia. In addition, we discuss the dynamic interactions between microglia and other cells, such as neurons, astrocytes, oligodendrocytes, and endothelial cells within the brain parenchyma and the neurovascular unit.

Background We have previously shown that a whole flaxseed supplemented diet decreased the onset and severity of ovarian cancer in the laying hen, the only known animal model of spontaneous ovarian cancer. Flaxseed is rich in omega-3 fatty acids (OM3FA), mostly α-Linoleic acid (ALA), which gets converted to Docosahexaenoic acid (DHA) by the action of delta-6 desaturase enzyme. Ingestion of flaxseed also causes an increase in production of 2-methoxyestradiol (2MeOE2) via the induction of the CYP1A1 pathway of estrogen metabolism. We have previously reported that the flaxseed diet induces apoptosis via p38-MAPK pathway in chicken tumors. The objective of this study was to investigate the effect of the flaxseed diet on ovarian cancer in chickens, focusing on two hallmarks of cancer, apoptosis and angiogenesis. Results The anti-cancer effects of two active biologically derived compounds of flax diet, 2MeOE2 and DHA, were individually tested on human ovarian cancer cells and in vivo by the Chick Chorioallantoic Membrane (CAM) assay. Our results indicate that a flaxseed-supplemented diet promotes apoptosis and inhibits angiogenesis in chicken tumors but not in normal ovaries. 2MeOE2 promotes apoptosis in human ovarian cancer cells, inhibits angiogenesis on CAM and its actions are dependent on the p38-MAPK pathway. DHA does not have any pro-apoptotic effect on human ovarian cancer cells but has strong anti-angiogenic effects as seen on CAM, but not dependent on the p38-MAPK pathway. Conclusions Dietary flaxseed supplementation promotes a pro-apoptotic and anti-angiogenic effect in ovarian tumors, not in normal ovaries. The biologically derived active compounds from flaxseed diet act through different pathways to elicit their respective anti-cancer effects. A flaxseed-supplemented diet is a promising approach for prevention of ovarian cancer as well as having a significant potential as an adjuvant treatment to supplement chemotherapeutic agents for treatment of advanced stages of ovarian cancer. Electronic supplementary material The online version of this article (10.1186/s13048-019-0523-3) contains supplementary material, which is available to authorized users.

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in pain. However, there has not been direct evidence showing that selective microglial activation in vivo is sufficient to induce chronic pain. Here, we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity in both male and female mice. In addition, activation of microglial ReaChR up-regulated neuronal c-Fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased interleukin (IL)-1β production, which is likely mediated by inflammasome activation and calcium elevation. IL-1 receptor antagonist (IL-1ra) was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation. Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

Background Breast cancer is the most common cancer in women and the leading cause of female cancer deaths worldwide. Obesity causes chronic inflammation and is a risk factor for post-menopausal breast cancer and poor prognosis. Obesity is known to trigger increased infiltration of macrophages into adipose tissue, yet little research has focused on the effects of macrophages in the early stages of breast tumor development in obese patients. In this study, the effects of pro-inflammatory macrophages on breast cancer-adipocyte crosstalk were investigated. Methods An innovative human cell co-culture system was used to model the paracrine interactions among adipocytes, macrophages, and breast cancer cells, and how they can facilitate tumor progression. The effects on human breast cancer cells were examined using cell counts and migration assays. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) was used to measure the expression levels of several cytokines and proteases to analyze adipocyte cancer-association. Results Macrophage conditioned media intensified the effects of breast cancer-adipocyte crosstalk. More specifically, adipocytes became delipidated and increased production of pro-inflammatory cytokines, even in the absence of breast cancer cells, although the expression levels were highest with all three cell components. As a result, co-cultured breast cancer cells became more aggressive, with increased proliferation and migration potential when compared to adipocyte-breast cancer cell co-cultures treated with unconditioned media. Conclusions Macrophage conditioned media promotes adipocyte cancer-association and production of pro-inflammatory factors. These macrophage-adipocyte paracrine interactions promote human breast cancer cell proliferation and migration. Thus, macrophages may contribute to adipocyte inflammation and cancer-association and promote breast cancer progression.

Neuromyelitis optica (NMO) is an autoantibody-triggered neuro-inflammatory disease which preferentially attacks the spinal cord and optic nerve. Its defining autoantibody is specific for the water channel protein, aquaporin‐4 (AQP4), which primarily is localized at the end-feet of astrocytes. Histopathology studies of early NMO lesions demonstrated prominent activation of microglia, the resident immune sentinels of the central nervous system (CNS). Significant microglial reactivity is also observed in NMO animal models induced by introducing AQP4-IgG into the CNS. Here we review the potential roles for microglial activation in human NMO patients as well as different animal models of NMO. We will focus primarily on the molecular mechanisms underlying microglial function and microglia-astrocyte interaction in NMO pathogenesis. Understanding the role of microglia in NMO pathology may yield novel therapeutic approaches for this disease.

Triggering receptor expressed on myeloid cell 2 (TREM2) is a surface receptor that, in the central nervous system, is exclusively expressed on microglia. TREM2 variants have been linked to increased risk for neurodegenerative diseases, but the functional effects of microglial TREM2 remain largely unknown. To this end, we investigated TAR-DNA binding protein 43 kDa (TDP-43)-related neurodegenerative disease via viral-mediated expression of human TDP-43 protein (hTDP-43) in neonatal and adult mice or inducible expression of hTDP43 with defective nuclear localization signals in transgenic mice. We found that TREM2 deficiency impaired microglia phagocytic clearance of pathological TDP-43, and enhanced neuronal damage and motor function impairments. Mass cytometry analysis revealed that hTDP-43 induced a TREM2-dependent subpopulation of microglia with high CD11c expression and higher phagocytic ability. Using mass spectrometry and surface plasmon resonance analysis, we further demonstrated an interaction between TDP-43 and TREM2, in vitro and in vivo, in hTDP-43-expressing transgenic mouse brains. We computationally identified the region within hTDP-43 that interacts with TREM2 and observed the potential interaction in ALS patient tissues. Our data reveal the novel interaction between TREM2 and TDP-43, highlighting that TDP-43 is a possible ligand for microglial TREM2 and the interaction mediates neuroprotection of microglial TREM2 in TDP-43-related neurodegeneration.

Hemangioendothelioma (HE) is a type of angiomatous lesions that features endothelial cell proliferation. Understanding the mechanisms orchestrating HE angiogenesis can provide therapeutic insights. It has been shown that platelets can support normal and malignant endothelial cells during angiogenesis. Using the mouse endothelial-derived EOMA cell line as a model of HE, we explored the regulatory effect of platelets. We found that platelets stimulated EOMA proliferation but did not mitigate apoptosis. Furthermore, direct platelet-EOMA cell contact was required and the proliferation was mediated via integrin β3/Akt signaling in EOMA cells. SiRNA knockdown of integrin β3 and inhibition of Akt activity significantly abolished platelet-induced EOMA cell proliferation in vitro and tumor development in vivo. These results provide a new mechanism by which platelets support HE progression and suggest integrin β3 as a potential target to treat HE.

Gliosis is a histopathologic characteristic of epilepsy that comprises activated microglia and astrocytes. It is unclear whether or how crosstalk occurs between microglia and astrocytes in the evolution of epilepsy. Here, we report in a mouse model of status epilepticus, induced by intracerebroventricular injection of kainic acid (KA), sequential activation of microglia and astrocytes and their close spatial interaction in the hippocampal CA3 region. Microglial ablation reduced astrocyte activation and their upregulation of complement C3. When compared to wild-type mice, both C3(−/−) and C3aR(−/−) mice had significantly less microglia-astrocyte interaction in response to KA-induced status epilepticus. Additionally, KA-injected C3(−/−) mice had significantly less histochemical evidence of neurodegeneration. The results suggest that the C3-C3aR pathway contributes to KA-induced neurodegeneration by mediating microglia-astrocyte communication. The C3-C3aR pathway may prove to be a potential therapeutic target for epilepsy treatment.

Microglia play an important role in the central sensitization and chronic pain. However, a direct connection between microglial function and pain development in vivo remains incompletely understood. To address this issue, we applied chemogenetic approach by using CX(3)CR1(creER/+):R26(LSL-hM4Di/+) transgenic mice to enable expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (Gi DREADD) in microglia. We found that microglial Gi DREADD activation inhibited spinal nerve transection (SNT)-induced microglial reactivity as well as chronic pain in both male and female mice. Gi DREADD activation downregulated the transcription factor interferon regulatory factor 8 (IRF8) and its downstream target pro-inflammatory cytokine interleukin 1 beta (IL-1β). Using in vivo spinal cord recording, we found that activation of microglial Gi DREADD attenuated synaptic transmission following SNT. Our results demonstrate that microglial Gi DREADD reduces neuroinflammation, synaptic function and neuropathic pain after SNT. Thus, chemogenetic approaches provide a potential opportunity for interrogating microglial function and neuropathic pain treatment.

Spinal microglia are highly responsive to peripheral nerve injury and are known to be a key player in neuropathic pain. However, there has not been any direct evidence showing selective microglial activation in vivo is sufficient to induce chronic pain. Here we used optogenetic approaches in microglia to address this question employing CX3CR1creER/+: R26LSL-ReaChR/+ transgenic mice, in which red-activated channelrhodopsin (ReaChR) is inducibly and specifically expressed in microglia. We found that activation of ReaChR by red light in spinal microglia evoked reliable inward currents and membrane depolarization. In vivo optogenetic activation of microglial ReaChR in the spinal cord triggered chronic pain hypersensitivity lasting for 5-7 days. In addition, activation of microglial ReaChR upregulated neuronal c-fos expression and enhanced C-fiber responses. Mechanistically, ReaChR activation led to a reactive microglial phenotype with increased IL-1β production. IL-1 receptor antagonist was able to reverse the pain hypersensitivity and neuronal hyperactivity induced by microglial ReaChR activation.Therefore, our work demonstrates that optogenetic activation of spinal microglia is sufficient to trigger chronic pain phenotypes by increasing neuronal activity via IL-1 signaling.

Microglia are the resident immune cells of the center nervous system and participate in various neurological diseases. Here we determined the function of microglia in epileptogenesis using microglial ablation approaches. Three different microglia-specific genetic tools were used, CX3CR1(CreER/+):R26(iDTA/+), CX3CR1(CreER/+):R26(iDTR/+), and CX3CR1(CreER/+):Csf1r(Flox/Flox) mice. We found that microglial depletion led to worse kainic acid (KA)-induced status epilepticus, higher mortality rate, and increased neuronal degeneration in the hippocampus. In KA-induced chronic spontaneous recurrent seizures, microglial depletion increased seizure frequency, interictal spiking, and seizure duration. Therefore, microglia depletion aggravates the severity of KA-induced acute and chronic seizures. Interestingly, microglial repopulation reversed the effects of depletion upon KA-induced status epilepticus. Our results demonstrate a beneficial role of microglia in suppressing both acute and chronic seizures, suggesting that microglia are a potential therapeutic target for the management of epilepsy.

Microglia play an important role in the central sensitization and chronic pain. However, a direct connection between microglial function and the development of neuropathic pain in vivo remains incompletely understood. To address this issue, we applied chemogenetic approach by using CX3CR1creER/+:R26LSL-hM4Di/+ transgenic mice to enable expression of inhibitory Designer Receptors Exclusively Activated by Designer Drugs (Gi DREADD) exclusively in microglia. We found that microglial Gi DREADD activation inhibited spinal nerve transection (SNT)-induced microglial reactivity as well as chronic pain initiation and maintenance. Gi DREADD activation downregulated the transcription factor interferon regulatory factor 8 (IRF8) and its downstream target pro-inflammatory cytokine interleukin 1 beta (IL-1β). Using in vivo spinal cord recording, we found that activation of microglial Gi DREADD attenuated synaptic transmission following SNT. Our results demonstrate that microglial Gi DREADD reduces neuroinflammation, synaptic function and neuropathic pain after peripheral nerve injury. Thus, chemogenetic approaches provide a potential opportunity for interrogating microglial function and neuropathic pain treatment.

Epilepsy is a major neurological condition that affects millions of people globally. While a number of interventions have been developed to mitigate this condition, a significant number of patients are refractory to these treatments. Consequently, other avenues of research are needed. One such avenue is modulation of the immune system response to this condition, which has mostly focused on microglia, the resident immune cells of the central nervous system (CNS). However, other immune cells can impact neurological conditions, principally blood-borne monocytes that can infiltrate into brain parenchyma after seizures. As such, this review will first discuss how monocytes can be recruited to the CNS and how they can be distinguished from there immunological cousins, microglia. Then, we will explore what is known about the role monocytes have within seizure pathogenesis and epilepsy. Considering how little is known about monocyte function in seizure- and epilepsy-related pathologies, further studies are warranted that investigate infiltrated blood-borne monocytes as a potential therapeutic target for epilepsy treatment.

Microglia have been shown to be of critical importance to the progression of temporal lobe epilepsy. However, the broad transcriptional changes that these cells undergo following seizure induction is not well understood. As such, we utilized RNAseq analysis upon microglia isolated from the hippocampus to determine expression pattern alterations following kainic acid induced seizure. We determined that microglia undergo dramatic changes to their expression patterns, particularly with regard to mitochondrial activity and metabolism. We also observed that microglia initiate immunological activity, specifically increasing interferon beta responsiveness. Our results provide novel insights into microglia transcriptional regulation following acute seizures and suggest potential therapeutic targets specifically in microglia for the treatment of seizures and epilepsy. Electronic supplementary material The online version of this article (10.1186/s13041-018-0376-5) contains supplementary material, which is available to authorized users.

Duplication of chromosome 22q11.2 (LCR A-D) has been reported at higher frequencies in clinical samples than the general population, but phenotypes vary widely. Triplication (4 copies) is rare, but studying the associated phenotype may provide insight into dosage-sensitivity of the genes in this chromosomal interval. We describe a proband with a triplication, specifically a “double duplication” (two copies per chromosome) of the 22q11.2 region, while his parents and two siblings each have a single duplication (3 copies). The proband had a heart malformation, dysmorphic features, and learning and socialization deficits, whereas the other family members did not. This family illustrates that while duplication of the 22q11.2 may not be sufficient to cause clinically significant neurodevelopmental or health-related phenotypes, triplication of the same region may result in a phenotype characterized by a mild neurodevelopmental disorder, facial dysmorphism, and possibly cardiac anomalies.

The study reported here demonstrates that a flaxseed-supplemented diet causes ovarian tumors in the laying hen to undergo apoptosis, resulting in a reduction of tumor burden, reducing the frequency and severity of ovarian cancer. We have previously shown in normal ovaries that flaxseed and its components down-regulate ERalpha and alter the expression of enzymes that metabolize estrogen. In this study, we analyzed the effects of the two main components of whole flaxseed, ligan and omega 3 fatty acids on estrogen metabolism and the estrogen receptor in ovarian tumors. ER alpha expression was up-regulated in the ovarian tumors and was not affected by diet. Liver CYP1A1 expression was significantly increased by the whole flaxseed diet with a corresponding increase in 2-methoxyestradiol plasma levels. We also observed increased p38 and ERK 1/2 MAPK activation in the ovary as well as an increase in apoptosis in the tumor epithelium. SMAD 7, a factor involved in the 2-methoxyestradiol-mediated apoptosis pathway was also up-regulated in tumors from the whole flaxseed diet group. 2-methoxyestradiol-induced antitumor effects were further validated by in human ovarian cancer cells. This study details the effect of flaxseed diet on estrogen metabolism and demonstrates the antiovarian cancer effects of 2-methoxyestradiol.

Neuromyelitis optica (NMO) is a severe inflammatory autoimmune CNS disorder triggered by binding of an IgG autoantibody to the aquaporin 4 (AQP4) water channel on astrocytes. Activation of cytolytic complement has been implicated as the major effector of tissue destruction that secondarily involves myelin. We investigated early precytolytic events in the evolving pathophysiology of NMO in mice by continuously infusing IgG (NMO patient serum-derived or AQP4-specific mouse monoclonal), without exogenous complement, into the spinal subarachnoid space. Motor impairment and sublytic NMO-compatible immunopathology were IgG dose dependent, AQP4 dependent, and, unexpectedly, microglia dependent. In vivo spinal cord imaging revealed a striking physical interaction between microglia and astrocytes that required signaling from astrocytes by the C3a fragment of their upregulated complement C3 protein. Astrocytes remained viable but lost AQP4. Previously unappreciated crosstalk between astrocytes and microglia involving early-activated CNS-intrinsic complement components and microglial C3a receptor signaling appears to be a critical driver of the precytolytic phase in the evolving NMO lesion, including initial motor impairment. Our results indicate that microglia merit consideration as a potential target for NMO therapeutic intervention.

OBJECTIVES: In 2012, ovarian cancer accounted for 239,000 new cancer cases and 152,000 cancer-related deaths worldwide. Epidemiological studies around the world suggest that high dietary consumption of polyunsaturated fatty acids (PUFAs) reduces a woman's odds of developing ovarian cancer. We hypothesize that PUFA-enriched diets will decrease ovarian cancer risk in White Leghorn laying hens. METHODS: The specific aim of our research was to investigate ovarian cancer risk and molecular events in ovarian tumors of White Leghorn laying hens consuming PUFA-enriched diets. We exposed laying hens to long-term (46-week) dietary PUFA treatment, using Whole Flaxseed (WFX), Flaxseed Oil (FXO), Fish Oil (FHO), Corn Oil (CNO), and a low-PUFA diet, Defatted Flaxseed (DFX). At the end of the dietary treatment we collected 88 ovarian tumors and conducted qPCR analysis for gene expression. RESULTS: In comparison to a control diet lacking a PUFA supplement, the odds ratios of hens developing ovarian cancer decreased with PUFA enrichment (Odd Ratios: DFX = 0.74, WFX = 0.69, FXO = 0.61, FHO = 0.57, CNO = 0.57), corroborating human epidemiological observations of ovarian cancer risk. Gene expression analysis from hen ovarian tumors suggests that PUFA-rich diets (ie WFX, FXO, FHO, and CNO) improve mitochondrial electron transport chain function by upregulating Cytochrome B (Complex 3) and Cyctochrome C Oxidases 1, 2, and 3 (Complex 4), and likely reduce superoxide emission from Complex 1 as evidenced by the downregulation of NADH Dehydrogenase 2. PUFA enrichment also reduced the expression of enzymes associated with reactive oxygen species (ROS), reactive nitrogen species (RNS), and lipid peroxides, suggesting overall major improvement in the redox state of tumors from PUFA-enriched diets. The FXO diet was uniquely distinguished by increasing the expression of ATP5B1 (catalytic subunit of ATP Synthase) and the ADP/ATP Translocase (ANT1), suggesting increased mitochondrial cristae space and improved oxidative phosphorylation capacity in FXO-enriched ovarian tumors. CONCLUSIONS: Dietary PUFA enrichment (omega-3 or omega-6) reduces end point risk for ovarian cancer in laying hens. The underlying etiology appears to center on decreasing oxidative stress and increasing mitochondrial function in ovarian tumors. FUNDING SOURCES: National Institutes of Health.

Microglial activation has been recognized as a major contributor to inflammation of the epileptic brain. Seizures are commonly accompanied by remarkable microgliosis and loss of neurons. In this study we utilize the CX3CR1(GFP/+) CCR2(RFP/+) genetic mouse model, in which CX3CR1(+) resident microglia and CCR2(+) monocytes are labeled with GFP and RFP, respectively. Using a combination of time-lapse two-photon imaging and whole-cell patch clamp recording, we determined the distinct morphological, dynamic, and electrophysiological characteristics of infiltrated monocytes and resident microglia, and the evolution of their behavior at different time points following kainic acid-induced seizures. Seizure activated microglia presented enlarged somas with less ramified processes, whereas, infiltrated monocytes were smaller, highly motile cells that lacked processes. Moreover, resident microglia, but not infiltrated monocytes, proliferate locally in the hippocampus after seizure. Microglial proliferation was dependent on the colony stimulating factor 1 receptor (CSF-1R) pathway. Pharmacological inhibition of CSF-1R reduced seizure-induced microglial proliferation, which correlated with attenuation of neuronal death without altering acute seizure behaviors. Taken together, we demonstrated that proliferation of activated resident microglia contributes to neuronal death in the hippocampus via CSF-1R after status epilepticus, providing potential therapeutic targets for neuroprotection in epilepsy.

This study examined the ability of flax-based ingredients to attenuate nonalcoholic fatty liver disease ( NAFLD: ) in aged laying hens-a novel and more physiologically relevant model of human disease. Our results showed only hens supplemented with whole flaxseed ( WFX: ) reduced steatosis and hepatocellular ballooning. Serum AST was also reduced in hens provided WFX and defatted flaxseed meal ( DFM: ). Hepatic ω-3 PUFA enrichment was improved with supplementation of WFX, DFM, and flaxseed oil ( FXO: ). However, this effect was more evident in the WFX group. In contrast, transcript abundance of genes linked to NAFLD were predominantly modified with FXO supplementation. Taken together, our data indicate a potential synergistic relationship between the fatty acid and lignan content in flaxseed which attenuated the progression of NAFLD in aged laying hens. Although more research is necessary, these findings demonstrate the potential use of whole flaxseed for the treatment and prevention of NAFLD in humans.

Early molecular response (EMR, BCR-ABL1 (IS)≤10% at 3 months) is a strong predictor of outcome in imatinib-treated chronic phase chronic myeloid leukemia (CP-CML) patients, but for patients who transform early, 3 months may be too late for effective therapeutic intervention. Here, we employed multiplex cytokine profiling of plasma samples to test newly diagnosed CP-CML patients who subsequently received imatinib treatment. A wide range of pro-inflammatory and angiogenesis-promoting cytokines, chemokines and growth factors were elevated in the plasma of CML patients compared with that of healthy donors. Most of these normalized after tyrosine kinase inhibitor treatment while others remained high in remission samples. Importantly, we identified TGF-α and IL-6 as novel biomarkers with high diagnostic plasma levels strongly predictive of subsequent failure to achieve EMR and deep molecular response, as well as transformation to blast crisis and event-free survival. Interestingly, high TGF-α alone can also delineate a poor response group raising the possibility of a pathogenic role. This suggests that the incorporation of these simple measurements to the diagnostic work-up of CP-CML patients may enable therapy intensity to be individualized early according to the cytokine-risk profile of the patient. Refereed/Peer-reviewed

The objective of this study is to determine if docosahexaenoic acid (DHA), a 22 carbon long-chain omega-3 fatty acid found in flaxseed, causes a reduction in cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) by blocking the activation of Nuclear Factor Kappa light chain enhancer of activated B cells (NF-κB). NF-κB is a transcription factor that regulates expression of many stress response genes. Many cancers, including ovarian cancer, have a dysregulated NF-κB pathway. The NF-κB transcriptional complex is sequestered in an inactivate state in the cytoplasm. I Kappa Beta Kinase (IKK) phosphorylation of I Kappa Beta (IKB) permits the translocation of the NF-κB complex into the nucleus and activation of transcription. COX-2, an enzyme that catalyzes the conversion of arachidonic acid to prostaglandins, such as PGE2, is regulated by NF-κB. PGE2 helps regulate and promote inflammation associated with increased incidence and severity of ovarian cancer. Our laboratory studies the laying hen because it is the only known animal model to spontaneously develop ovarian cancer. Flaxseed, the richest vegetable source of omega-3 fatty acids, may be effective in the prevention of ovarian cancer. Our previous studies have shown that in hens, a long-term flaxseed supplemented diet decreases the incidence and severity of ovarian cancer and decreases COX-2 and PGE2 levels. It is hypothesized that DHA will decrease inflammation by suppressing the activation of COX-2 and the production of PGE2 through inhibition of the NF-κB pathway. For this study, an NF-κB reporter plasmid was transfected into HEK293T cells using polyethylenimine (PEI). The reporter plasmid (“met-luc” with metridia luciferase driven by the NF-κB promoter) produces a secreted luciferase allowing sequential analysis of media from treated cells to assess changes in NF-κB activation. Tumor necrosis factor alpha (TNF-α)-induced activation of NF-κB is a positive control. NF-κB activation is assessed by immunocytochemistry (ICC) and Western blot by measuring its translocation into the nucleus. Data show that DHA reduces TNFα-induced NF-κB reporter activity. HEK293T cells treated with TNFα alone showed an increase in nuclear NF-κB p65 subunits, suggesting pathway activation. ICC suggests DHA treatment causes cytoplasmic sequestration of the NF-κB p105 and p65 subunits, indicating inhibition of TNFα-induced NF-κB activation. Western blot data indicate an increase in cytoplasmic NF-κB p65 subunit after TNFα and DHA treatment compared to cells treated with only TNFα. These data suggest that DHA could prevent the release of the NF-κB subunits for nuclear translocation, therefore decreasing COX-2 and PGE2 levels associated with inflammation seen in ovarian cancer. This could be a possible mechanism of action for the chemosuppressive role of long-term flaxseed consumption in ovarian cancer in laying hens. (Funding: NIH RO1AT005295). Citation Format: Kara Starkweather, Karen Hales, Dale Hales. Anti-inflammatory actions of DHA via inhibition of the NF-κB pathway [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr B45.

Bone marrow-derived macrophages (BMDMs) are mature leukocytes that serve a critical physiological role as professional phagocytes capable of clearing a variety of particles. Normally, BMDMs are restricted from the central nervous system (CNS), but following an injury, they can readily infiltrate. Once within the injured CNS tissue, BMDMs are the primary cell type responsible for the clearance of injury-derived cellular debris, including large quantities of lipid rich myelin debris. The neuropathological ramifications of BMDM infiltration and myelin debris phagocytosis within the CNS are complex and not well understood. The protocols described here, allow for the direct in vitro study of BMDMs in the context of CNS injury. We cover murine BMDM isolation and culture, myelin debris preparation, and assays to assess BMDM myelin debris phagocytosis. These techniques produce robust quantifiable results without the need for significant specialized equipment or materials, yet can be easily customized to meet the needs of researchers.

Multidrug resistance (MDR) represents a major hindrance to the efficacy of cancer chemotherapeutics. While surgical resection, radiation, and chemotherapy can be used to reduce tumor size, the subsequent appearance of drug resistant cells is a frequent problem. One of the main contributors to the development of MDR is increased expression of multi-drug resistant protein 1 (MDR1), also known as P-glycoprotein (P-gp). P-gp is a membrane-associated efflux pump that can efficiently remove internalized taxane-base chemotherapeutics thus preventing drug accumulation and maintaining cellular viability. Consequently, investigation into the molecular mechanisms responsible for regulation of P-gp expression is necessary to facilitate treatment of MDR tumors. Using molecular and biochemical approaches, we identified that the micro-RNA, miRNA149, contributes to the development of MDR within malignant mesothelioma cells by regulating the expression of MDR1.

In spite of ovarian cancer mortality rates have tended to level off and decrease during the last years in several countries, the disease is still the gynecological cancer with the highest death rate. The current therapies for ovarian cancer include surgery and chemotherapy based on platinum and a taxane. Nevertheless, these therapies are only efficient at the first stages of the disease and the treatments are lack of specificity, further contributing to the high mortality rates of ovarian cancer. The adult laying hen is a very interesting model for studying the development of the epithelial ovarian cancer since hen develops intense ovulation, spontaneous ovarian cancers and metabolic progression similar to women. It is known that normal chicken cells and human cells have no metabolic pathway for NeuGc biosynthesis due to a partial deletion in the gene that encodes CMP-Neu5Ac hydroxylase. Gangliosides are interesting type of molecules, different from proteins that have been identified as tumor antigens. Mammalian cells have these types of glycosphingolipids on their plasmatic membranes. Due to gangliosides are over-expressed in many tumors compared with the corresponding normal tissues, they are considered as very attractive compounds for immunotherapy, attributing to them a very important role in the growth and metastatic tumor processes. The aim of this study is to elucidate by immunohistochemistry the existence of NeuGc-GM3 in hen ovarian cancers. Slides with normal and cancer ovarian tissues from 3.5 years old chickens were incubated with the very specific anti- NeuGcGM3 ganglioside 14F7 Mab for immunohistochemical studies. These studies showed that the 14F7 Mab immunorecognition was mainly evidenced in epithelial ovarian tumors and not in normal tissues. The presence of NeuGc-containing gangliosides in chicken and human epithelial ovarian cancers makes the chicken an interesting and relevant animal model for further experimental and translational investigations on ganglioside based immunotherapy.

Prevention of ovarian cancer is the best approach for reducing the impact of this deadly disease. The laying hen is a robust model of spontaneous ovarian cancer that recapitulates the human disease. Dietary intervention with flaxseed, the richest vegetable source of omega-3 fatty acids (OM-3FAs) and phytoestrogen lignans, demonstrate the potential for effective prevention and amelioration of ovarian cancer by targeting inflammatory prostaglandin pathways. Prostaglandin E2 (PGE2) is the most pro-inflammatory ecoisanoid and one of the downstream products of two isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Our objective was to investigate the effect of flaxseed supplementation for one year on ovarian cancer and correlate its effects to expression of COX enzymes and concentrations of prostaglandins. White Leghorn hens were fed 10% flaxseed-enriched or standard diet for one year. The severity of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 localization and protein and mRNA expression and PGE2 and PGE3 concentrations in ovaries were measured by IHC, western blot, quantitative real-time PCR and LC–MS–MS, respectively. The results demonstrated a significant reduction in late stage ovarian tumors in the flaxseed-fed hens compared with the control diet-fed hens. In correlation with decreased ovarian cancer severity, concentrations of PGE2 and expression of COX-2 were diminished in ovaries of flaxseed-fed hens. PGE3 concentrations were below the level of detection. The results demonstrated that in normal ovaries, COX-1 was localized to the granulosa cell layer surrounding the follicles and ovarian surface epithelium (OSE) whereas COX-2 protein was localized to the granulosa cell layer in the follicle. Extensive COX-1 and COX-2 protein expression was found throughout the ovarian carcinoma. Our findings suggest that the flaxseed-mediated reduction in the severity of ovarian cancer in hens is correlated to the reduction in PGE2 in the ovaries of flaxseed-fed hens. These findings may provide the basis for clinical trials of dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Epithelial ovarian cancer (OvCa) is associated with high mortality and, as the majority (>75%) of women with OvCa have metastatic disease at the time of diagnosis, rates of survival have not changed appreciably over 30 years. A mechanistic understanding of OvCa initiation and progression is hindered by the complexity of genetic and/or environmental initiating events and lack of clarity regarding the cell(s) or tissue(s) of origin. Metastasis of OvCa involves direct extension or exfoliation of cells and cellular aggregates into the peritoneal cavity, survival of matrix-detached cells in a complex ascites fluid phase, and subsequent adhesion to the mesothelium lining covering abdominal organs to establish secondary lesions containing host stromal and inflammatory components. Development of experimental models to recapitulate this unique mechanism of metastasis presents a remarkable scientific challenge and many approaches used to study other solid tumors (lung, colon, and breast, for example) are not transferable to OvCa research given the distinct metastasis pattern and unique tumor microenvironment. This review will discuss recent progress in the development and refinement of experimental models to study OvCa. Novel cellular, three-dimensional organotypic, and ex vivo models are considered and the current in vivo models summarized. The review critically evaluates currently available genetic mouse models of OvCa, the emergence of xenopatients, and the utility of the hen model to study OvCa prevention, tumorigenesis, metastasis, and chemoresistance. As these new approaches more accurately recapitulate the complex tumor microenvironment, it is predicted that new opportunities for enhanced understanding of disease progression, metastasis and therapeutic response will emerge.

The inflammatory biomarker high-sensitivity C-reactive protein (hs-CRP) has emerged as a predictor of future cardiovascular events. Screening for coronary artery calcium (CAC) is an alternative method for stratifying subjects by their cardiovascular risk. It is unclear, however, how hs-CRP compares with CAC scoring for the detection of obstructive coronary artery stenoses. We, thus, evaluated the association, if any, between hs-CRP and CAC scores for the detection of obstructive stenoses in a low-risk population with well-controlled traditional cardiovascular risk factors. In the present study of 1,079 stable subjects, 38 (3.5%) severely obstructive stenoses were found initially by coronary computed tomographic angiography and confirmed subsequently using invasive coronary angiography. The univariate predictors of severely obstructive coronary artery disease included the use of antihypertensive agents (p = 0.03), angina (p0.001), and an elevated CAC score (p0.001). The biomarker hs-CRP was not significantly associated with the presence of a severely obstructive stenosis. As the CAC scores increased, the frequency of obstructive stenosis also increased (p for trend0.001). In contrast, the frequency of obstructive stenoses was low when CAC was not detected. This relation remained significant after adjustment for antihypertensive medication use and angina. In conclusion, hs-CRP was not useful for the prediction of obstructive stenoses in stable subjects. CAC was found to be a better predictor of obstructive heart disease than hs-CRP.

Spinal cord injury (SCI) causes functional impairment as a result of the initial injury followed by secondary injury mechanism. SCI provokes an inflammatory response that causes secondary tissue damage and neurodegeneration. While the use of neural stem cell (NSC) engraftment to mitigate secondary injury has been of interest to many researchers, it still faces several limitations. As such, we investigated if NSC-conditioned medium (NSC-M) possesses therapeutic potential for the treatment of SCI. It has been proposed that many of the beneficial effects attributed to stem cell therapies are due to secreted factors. Utilizing primary cell culture and murine models of SCI, we determined that systemic treatment with NSC-M was able to significantly improve motor function and lesion healing. In addition, NSC-M demonstrated significant anti-inflammatory potential in vitro and in vivo, reducing inflammatory cytokine expression in both activated macrophages and injured spinal cord tissues. NSC-M was also able to reduce the expression of inducible nitric oxide synthase (iNOS) within the spleen of injured animals, indicating an ability to reduce systemic inflammation. Thus, we believe that NSC-M offers a possible alternative to direct stem cell engraftment for the treatment of SCI.

Chronic inflammation has been linked to cancer. Prostaglandin E₂ (PGE₂) is the most pro-inflammatory lipid and one of the downstream products of 2 isoforms of cyclooxygenase (COX) enzymes: COX-1 and COX-2. Ovarian cancer is the most lethal gynecological malignancy and mainly occurs in older women. The factors that contribute to the correlation of age and ovarian cancer are unknown. The purpose of this study was to examine the expression of COX enzymes and PGE₂ levels in ovaries and correlate them to ovarian cancer and aging. White Leghorn hens aged 1, 1.5, 2, 2.5, 3 and 3.5 years were used. The incidence of ovarian cancer was determined by gross pathology and histology. COX-1 and COX-2 protein and mRNA expression and PGE₂ concentrations in ovaries were measured using Western blot, quantitative real-time PCR and ELISA, respectively. Our results indicated an increase in ovarian cancer incidence and expression of both COX enzymes in ovaries of older hens. In correlation with ovarian cancer incidence and COX enzymes expression, PGE₂ concentrations were elevated with age. Ovaries with tumor had elevated COX-1 expression and PGE₂ concentration compared to normal ovaries. Our findings suggest that the up-regulation of COX enzymes with age is the main contributing factor in the age associated increase in PGE₂. Furthermore, elevated PGE₂ in ovaries of hens concomitant with age suggests its important role in early stages of ovarian carcinogenesis. These finding may provide the basis for clinical trials utilizing COX specific inhibitors or dietary intervention targeting prostaglandin biosynthesis for the prevention and treatment of ovarian cancer.

Focal dystonia can result in a variety of technical problems in the performing musician, most often affecting control of finger movement, and embouchure. Less common is the effect of focal dystonia on the vibrato of string players. The professional cellist in our study presented with difficulty controlling her vibrato, which fluctuated both in speed and amplitude, causing an inconsistency of sound. This study investigated whether instrumental retraining could alleviate her condition. We report the novel finding that instrumental retraining can significantly improve the symptoms of a dystonic vibrato in a cellist.

There are three currently identified secondary resistance mechanisms observed in patients with chronic myeloid leukemia (CML) receiving tyrosine kinase inhibitors (TKIs). These are BCR-ABL kinase domain (KD) mutations, increased BCR-ABL expression, and overexpression of drug-efflux proteins (ABCB1 and ABCG2). To investigate the interplay between these three modes of resistance, three CML blast crisis cell lines (K562, its ABCB1-overexpressing variant K562 Dox, and KU812) were cultured in gradually increasing concentrations of imatinib to 2 mu M, or dasatinib to 200 nM. Eight imatinib-and two dasatinib-resistant cell lines were established. Two imatinib-resistant K562 lines both had increased BCR-ABL expression as the apparent mode of resistance. However, when a dasatinib-resistant K562 culture was generated we observed gradually increasing BCR-ABL expression which peaked prior to identification of the T315I mutation. BCR-ABL overexpression followed by mutation development was observed in a further 4/10 cell lines, each with different KD mutations. In contrast, three imatinib-resistant K562 Dox lines exhibited only a further increase in ABCB1 expression. All TKI-resistant cell lines generated had increased IC(50) (dose of drug required to reduce phosphorylation of the adaptor protein p-Crkl by 50%) to imatinib, dasatinib, and nilotinib, regardless of which TKI was used to induce resistance. This suggests that currently available TKIs share the same susceptibilities to drug resistance. Refereed/Peer-reviewed

Focal dystonia (FD) is a devastating neurological condition which causes involuntary muscle contractions and often results in the loss of a musician’s playing ability. Our study investigated whether retraining via video conferencing could be helpful in the treatment of a professional pianist with a 5-year history of FD. Although full recovery was not seen, improvement was observed at slow tempi, and his hand was visibly less cramped as training sessions progressed. We conclude that video conferencing could be an acceptable medium to assist pianism retraining in pianists with FD when location prevents on-site retraining. However, in this study it did not seem as effective as previously reported, similar, one-on-one retraining in the same location.

Objective With the exception of the laying hen, no other animal model of spontaneous ovarian surface epithelial cancer replicates the human disease. Flaxseed is the richest vegetable source of omega-3 fatty acids, which are chemopreventive in breast cancer and may be important in other cancers. The objective of this study was to determine if a flaxseed-enriched diet had a chemopreventive effect on ovarian cancer in the laying hen. Methods White Leghorn hens were fed with 10% flaxseed-enriched or standard diet for 1 year. The incidence and severity of ovarian cancer were determined by gross pathology and histology in the two groups. General health markers were also measured. Eggs were collected and analyzed by gas chromatography to determine omega-3 fatty acid levels. Results A significant reduction in late stage ovarian tumors was detected in the flaxseed-fed hens. Incidence rates of ovarian cancer were not significantly different between the two groups. The results indicate that a flaxseed diet increases overall survival in the laying hen. Flaxseed-fed hens' eggs incorporated significantly more omega-3 fatty acids compared to control hens. Conclusions These findings show that 10% flaxseed supplementation for 1 year in the laying hen results in a significant reduction in the severity of ovarian cancer, but no change in the incidence of the disease. Hens fed flaxseed had overall better health and reduced mortality. These findings may provide the basis for a clinical trial that evaluates the efficacy of flaxseed as a chemosuppressant of ovarian cancer in women.

Immunotherapy targeting of amyloid beta (Abeta) peptide in transgenic mouse models of Alzheimer disease (AD) has been widely demonstrated to resolve amyloid deposition as well as associated neuronal, glial, and inflammatory pathologies. These successes have provided the basis for ongoing clinical trials of immunotherapy for treatment of AD in humans. Acute as well as chronic Abeta-targeted immunotherapy has also been demonstrated to reverse Abeta-related behavioral deficits assessing memory in AD transgenic mouse models. We observe that three antibodies targeting the same linear epitope of Abeta, Abeta(3-7), differ in their ability to reverse contextual fear deficits in Tg2576 mice in an acute testing paradigm. Reversal of contextual fear deficit by the antibodies does not correlate with in vitro recognition of Abeta in a consistent or correlative manner. To better define differences in antigen recognition at the atomic level, we determined crystal structures of Fab fragments in complex with Abeta. The conformation of the Abeta peptide recognized by all three antibodies was highly related and is also remarkably similar to that observed in independently reported Abeta:antibody crystal structures. Sequence and structural differences between the antibodies, particularly in CDR3 of the heavy chain variable region, are proposed to account for differing in vivo properties of the antibodies under study. These findings provide a structural basis for immunotherapeutic strategies targeting Abeta species postulated to underlie cognitive deficits in AD.

Epithelial ovarian carcinoma (EOC) is a leading cause of cancer deaths in women. Until recently, a significant lack of an appropriate animal model has hindered the discovery of early detection markers for ovarian cancer. The aging hen serves as an animal model because it spontaneously develops ovarian adenocarcinomas similar in histological appearance to the human disease. E-cadherin is an adherens protein that is down-regulated in many cancers, but has been shown to be up-regulated in primary human ovarian cancer. Our objective was to evaluate E-cadherin expression in the hen ovary and compare its expression to human ovarian cancer.White Leghorn hens aged 185 weeks (cancerous and normal) were used for sample collection. A human ovarian tumor tissue array was used for comparison to the human disease. E-cadherin mRNA and protein expression were analyzed in cancerous and normal hen ovaries by immunohistochemistry (IHC), Western blot, and quantitative real-time PCR (qRT-PCR). Tissue fixed in neutral buffered formalin was used for IHC. Protein from tissue frozen in liquid nitrogen was analyzed by Western blot. RNA was extracted from tissue preserved in RNAlater and analyzed by qRT-PCR. The human ovarian tumor tissue array was used for IHC.E-cadherin mRNA and protein expression were significantly increased in cancerous hen ovaries as compared to ovaries of normal hens by qRT-PCR and Western blot. Similar expression of E-cadherin was observed by IHC in both human and hen ovarian cancer tissues. Similar E-cadherin expression was also observed in primary ovarian tumor and peritoneal metastatic tissue from cancerous hens.Our findings suggest that the up-regulation of E-cadherin is an early defining event in ovarian cancer and may play a significant role in the initial development of the primary ovarian tumor. E-cadherin also appears to be important in the development of secondary tumors within the peritoneal cavity. Our data suggest that E-cadherin may prove to be an important target in the preventative treatment of metastatic ovarian cancer and further confirm that the laying hen is a good model for the study of human epithelial ovarian carcinoma.

The high mortality rate due to ovarian cancer is attributed to the lack of an effective early detection method. Due to the non-specificity of symptoms at early stage, most of the ovarian cancer cases are detected at late stages. This makes the access to women with early stage disease problematic and presents a barrier to development and validation of tests for detection of early stage of ovarian cancer in humans. Animal models are used to elucidate disease etiologies and pathogenesis that are difficult to study in humans. Laying hen is the only available animal that develops ovarian cancer spontaneously; however, detail information on ovarian tumor histology is not available. The goal of this study was to determine the histological features of malignant ovarian tumors in laying hens. A total of 155 young and old (1-5 years of age) laying hens (Gallus domesticus) were selected randomly and evaluated gross and microscopically for the presence of ovarian tumors. Histological classification of tumors with their stages and grades were performed with reference to those for humans. Similar to humans, all four types including serous, endometrioid, mucinous and clear cell or mixed carcinomas were observed in hen ovarian tumors. Some early neoplastic as well as putative ovarian lesions were also observed. Similarities in histology, metastasis and stages of hen ovarian cancer to those of humans demonstrate the feasibility of the hen model for additional delineation of the mechanism underlying ovarian carcinogenesis, preclinical testing of new agents for the prevention and therapy of this disease.

Ovarian carcinoma is the most lethal gynecological malignancy. The genetic and molecular mechanisms that cause it still remain largely unknown. CYP1B1 is a cytochrome P450 enzyme that catalyzes the conversion of estrogens to genotoxic catechol estrogens which may cause DNA mutations and initiate ovarian epithelial cancer. Our objectives were to evaluate CYP1B1 expression, distribution and localization in the hen ovary and to determine if there is an increased CYP1B1 expression associated with, and possibly involved in the initiation of ovarian cancer.Two groups of hens were used: 1. young (50 weeks of age; devoid of cancer) and 2. old (165 weeks of age; divided into two groups: age-matched normal and ovarian cancer). CYP1B1 mRNA and protein expression were analyzed in cancerous ovaries, ovaries of age-matched normal and/or young hens by quantitative real-time PCR (qRT-PCR), in situ hybridization (ISH) and immunohistochemistry (IHC). RNA was extracted from tissue preserved in RNAlater for qRT-PCR. Tissue frozen in liquid nitrogen was used for ISH. Tissue fixed in neutral buffered formalin was subjected to IHC.Higher expression of CYP1B1 mRNA was observed in cancerous ovaries as compared to ovaries of young and age-matched normal hens by qRT-PCR. ISH and IHC confirmed that the expression of CYP1B1 was much higher in ovarian tumors compared to ovaries of age-matched normal hens. CYP1B1 mRNA and protein were distributed extensively throughout the carcinoma, while primarily localized to the granulosa layer surrounding the follicle in age-matched normal hens. IHC also showed nuclear localization of CYP1B1. Highly expressed CYP1B1 was found in POF-3 from young and age-matched normal hens as compared to POF-1 and POF-2 by qRT-PCR. No significant difference was found in the expression of CYP1B1 between the distal (site of rupture) and the proximal (site of attachment to the ovary) of POF-1 from young and age-matched normal hens.High expression of CYP1B1 in the hen ovary is associated with ovarian cancer and our data suggest that CYP1B1 may play an important role in the initiation of ovarian cancer and may prove to be a target for intervention. Moreover, the results of this study further confirm that the laying hen is a good model to study human ovarian cancer.

Objective To investigate the diagnostic accuracy of commonly used signs of dehydration in marathon runners. Design The diagnostic accuracy of 5 clinical signs/symptoms thought to indicate dehydration (altered skin turgor, dry oral mucous membranes, sunken eyes, an inability to spit and the sensation of thirst) was assessed by comparing the presence of these markers with the criterion standard of body weight change over a marathon footrace. Setting 2006 Auckland Marathon Participants 606 competitors in the full marathon Assessment: Body weight was measured before and immediately after the marathon. The 5 clinical signs/symptoms were assessed immediately after the marathon. Main outcome measures Diagnostic accuracy of clinical signs/symptoms to detect dehydration greater than 3% of body weight. Results 606 complete data sets were obtained. 3 clinical signs were associated with greater percentage weight loss: sunken eyes (mean percentage weight loss with symptom 2.6% (standard deviation 1.5), without 2.3% (1.5)); decreased skin turgor (with 3.0% (1.4), without 2.3% (1.5)) and the sensation of thirst (with 2.5% (1.5), without 2.3% (1.5)). The ability to spit and dry oral mucous membranes were unrelated to percentage weight loss. No signs/symptoms showed acceptably high validity for detecting a weight loss equal to or greater than 3% of body weight. Conclusions The 5 parameters (decreased skin turgor, sensation of thirst, sunken eyes, inability to spit and dry mucous membranes) tested in this study did not precisely identify runners with total weight loss.3% at the end of a marathon.

The relationship between lower urinary tract symptoms and depressive symptoms was assessed using data from the Male Attitudes Regarding Sexual Health study.Lower urinary tract symptoms, depressive symptoms and erectile dysfunction were assessed using International Prostate Symptom Score, Center for Epidemiologic Studies Depression Scale and a validated question from the Massachusetts Male Aging Study. Sociodemographic, clinical and other data were also collected. Odds ratios and 95% CIs were determined using weighted multivariate logistic regression stratified by race/ethnicity and age.Of 3,291 randomly selected men 2,173 completed the interview. Overall odds of lower urinary tract symptoms were increased in men who reported depressive symptoms (OR 2.68, 95% CI 1.60-4.47, p0.01), erectile dysfunction (OR 1.73, 95% CI 1.11-2.71, p0.05) and unhappiness/dissatisfaction on the International Prostate Symptom Score quality of life item (OR 10.72, 95% CI 5.56-20.69, p0.01), and those 60 to 69 years old (OR 1.99, 95% CI 1.14-3.46, p0.05) and 70 years or older (OR 1.91, 95% CI 1.06-3.43, p0.05). Increased odds of lower urinary tract symptoms were associated with depressive symptoms for white (OR 2.60, 95% CI 1.39-4.85, p0.01) and Hispanic men (OR 4.14, 95% CI 1.15-14.95, p0.05). Odds of depressive symptoms were increased in men reporting lower urinary tract symptoms (OR 2.64, 95% CI 1.57-4.43, p0.001), especially Hispanic men 50 to 59 years old (OR 133.17, 95% CI 18.40-963.87, p0.01) and black men older than 60 years (OR 21.61, 95% CI 3.04-153.55, p0.01), as well as men 40 to 49 years old expressing unhappiness/dissatisfaction on the International Prostate Symptom Score quality of life item (OR 6.80, 95% CI 1.77-26.16, p0.01), and Hispanic (OR 10.76, 95% CI 3.88-29.80, p0.01) and black men reporting erectile dysfunction (OR 4.77, 95% CI 1.15-19.78, p0.05), but not white men reporting erectile dysfunction (OR 1.05, 95% CI 0.48-2.28, p0.91).Lower urinary tract symptoms and depressive symptoms are strongly associated, and exhibit reciprocal relationships. Erectile dysfunction increases the odds of both disorders.

Summary Aims: Relationships were evaluated between treatment-related improvements in overactive bladder (OAB) symptoms as recorded in bladder diaries and patient-reported symptom bother, bladder-related problems and health-related quality of life (HRQL). Methods: A post hoc analysis was performed on data from patients with OAB (n = 863) enrolled in a 12-week open-label trial of tolterodine extended release (ER) in a primary care setting. At baseline and week 12, patients recorded every micturition, urgency episode and urgency urinary incontinence episode in 3-day bladder diaries. Patients also completed the Overactive Bladder Questionnaire (OAB-q) and Patient Perception of Bladder Condition (PPBC). Relationships between week 12 changes in bladder diary variables and OAB-q and PPBC scores were evaluated using Spearman correlations. Results: By week 12, tolterodine ER-related improvements in all bladder diary variables were significantly correlated with improvements on the PPBC (r = 0.26–0.36; p