Search

Purpose To evaluate whether in the setting of negative diagnostic mammogram for breast pain additional ultrasound is necessary. Methods Retrospective IRB-approved review of our database identified 8085 women who underwent ultrasound evaluation for breast pain from 1/1/2013-12/31/2013. Of 8085 women, 559 women had mammogram evaluation preceding the ultrasound and these women comprise the basis of this study. The patient′s age, type of mammogram examination (screening or diagnostic), Breast Imaging-Reporting and Data System (BI-RADS) breast density (BD), type of breast pain (focal, diffuse, cyclical, unilateral, bilateral), additional breast symptoms (palpable concern, nipple discharge, skin changes, others), mammogram or ultrasound findings and final BI-RADS assessment, follow-up imaging, and follow-up biopsy results were reviewed and recorded. Results The median age of patients was 46 years old (range: 27-97). Patients recalled from negative screening mammogram were 29.8% (167/559). Patients with preceding negative diagnostic mammogram were 70.2% (392/559). The BI-RADS BD distribution was BD1: 5.5%, BD2: 39.9%, BD3: 46.0%, BD4: 8.6%. Final BI-RADS assessments were BI-RADS 1/2 (79%), BI-RADS 3 (12.9%), BI-RADS 4 (8.1%), BI-RADS 5 (0%). Majority (66.9%, 374/559) of the patient had breast pain alone. Additional breast symptoms were also noted as follows: palpable concern (24%), nipple discharge (3.9%), skin changes or other (5.2%). On follow-up evaluation, 26 findings were recommended for tissue sampling yielding 2 malignancies (0.4%, 2/559) in 2 patients. In the setting of negative mammogram and clinical symptom of breast pain alone yielded no malignances (NPV, 100%, 374/374) and was not impacted by BD. In patients with additional symptoms accompanying pain, malignancies were present despite negative mammogram in 2 patients; nipple discharge (4.5%, 1/22), and palpable concern (0.7%, 1/134). Conclusion In the setting of negative mammogram and breast pain alone, additional evaluation with ultrasound is likely low yield and may be unnecessary. However, with additional symptoms such as palpable concern or nipple discharge, ultrasound is likely an important adjunct modality for identifying mammographically occult tumors.

Pancreas ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival rate of 10%. Currently, chemotherapy remains the standard of care for systemic treatment. Immunotherapy with checkpoint inhibitors unfortunately has not been found to be effective in the treatment of PDAC to date, likely due to the highly desmoplastic and immunosuppressive tumor microenvironment (TME). Treatment targeting pathways against the immunosuppressive mechanisms of PDAC are of mounting interest to improve outcomes in PDAC. In this review, we discuss prior efforts and the current state of immunotherapy in PDAC. We will also review the emerging targets and treatments with significant clinical potential for the treatment of PDAC such as: CD40 pathway, the adenosine pathway, the CXCR4/CXCL12 axis, the CCR2/CCL2 axis, IDO pathway, and others.

Thymosin beta-4 (Tβ4) is an actin-sequestering peptide that plays important roles in regeneration and remodeling of injured tissues. However, its function in a naturally occurring pathogenic bacterial infection model has remained elusive. We adopted Tβ4-overexpressing transgenic (Tg) mice to investigate the role of Tβ4 in acute pulmonary infection and systemic sepsis caused by Legionella pneumophila. Upon infection, Tβ4-Tg mice demonstrated significantly lower bacterial loads in the lung, less hyaline membranes and necrotic abscess, with lower interstitial infiltration of neutrophils, CD4+, and CD8+ T cells. Bronchoalveolar lavage fluid of Tβ4-Tg mice possessed higher bactericidal activity against exogenously added L. pneumophila, suggesting that constitutive expression of Tβ4 could efficiently control L. pneumophila. Furthermore, qPCR analysis of lung homogenates demonstrated significant reduction of interleukin 1 beta (IL-1β) and tumor necrosis factor alpha (TNF-α), which primarily originate from lung macrophages, in Tβ4-Tg mice after pulmonary infection. Upon L. pneumophila challenge of bone marrow-derived macrophages (BMDM) in vitro, secretion of IL-1β and TNF-α proteins was also reduced in Tβ4-Tg macrophages, without affecting their survival. The anti-inflammatory effects of BMDM in Tβ4-Tg mice on each cytokine were affected when triggering with tlr2, tlr4, tlr5, or tlr9 ligands, suggesting that anti-inflammatory effects of Tβ4 are likely mediated by the reduced activation of Toll-like receptors (TLR). Finally, Tβ4-Tg mice in a systemic sepsis model were protected from L. pneumophila-induced lethality compared to wild-type controls. Therefore, Tβ4 confers effective resistance against L. pneumophila via two pathways, a bactericidal and an anti-inflammatory pathway, which can be harnessed to treat acute pneumonia and septic conditions caused by L. pneumophila in humans.

We report a fatal case of COVID-19 in a 51-year-old African American woman with multiple sclerosis on natalizumab. She had multiple risk factors for severe COVID-19 disease including race, obesity, hypertension, and elevated inflammatory markers, but the contribution of natalizumab to her poor outcome remains unknown. We consider whether altered dynamics of peripheral immune cells in the context of natalizumab treatment could worsen the cytokine storm syndrome associated with severe COVID-19. We discuss extended interval dosing as a risk-reduction strategy for multiple sclerosis patients on natalizumab, and the use of interleukin-6 inhibitors in such patients who contract COVID-19.

Introduction:Plasmablastic lymphoma (PBL) is a rare and aggressive lymphoma most commonly seen in the setting of chronic immunosuppression, such as HIV infection and organ transplantation, or in patients with pre-existing lymphoproliferative or autoimmune disorders. PBL commonly presents at extranodal sites and carries a poor prognosis with a global overall survival of 9-15 months after initial diagnosis. Despite poor prognosis for patients with PBL, therapeutic strategies to target this disease are limited, as CHOP-like regimens have failed to produce durable remission, and no standard of care has been established. The cell of origin for PBL is believed to be the plasmablast, as PBL cells possess immunoblastic morphology and contain an immunohistochemical profile positive for plasmablast markers, such as CD38, CD138, and MUM1/IRF4, and negative for B cell markers, such as CD20, CD19, and PAX5. The similarities between PBL cells and multiple myeloma (MM) cells, a plasma cell neoplasm, have led to investigations of the efficacy of MM therapeutics for the treatment of PBL. Daratumumab is a first-in-class monoclonal antibody directed against CD38 that has shown efficacy in treating relapsed/refractory and newly diagnosed MM. Here, we describe the treatment of four patients with advanced-stage PBL in the context of varying degrees of immunosuppression using combination treatment with daratumumab and EPOCH. Methods:Four consecutive patients were treated with daratumumab plus chemotherapy. Three of the four patients were treated in the frontline setting and received low-dose EPOCH (vincristine, doxorubicin, etoposide daily for 4 days; cyclophosphamide day 5) with 16mg/kg daratumumab dosed on days 1 and 8 for 6 cycles. Patient #4 showed partial CD20 positivity, prompting Rituximab co-administration (R-EPOCH). Patient #3 was treated in the relapsed setting with 16mg/kg daratumumab in combination with lenalidomide, dexamethasone, and doxorubicin. She was treated for 12 months. Responses were followed by PET/CT imaging. Results:The four consecutive patients (2 female, 2 male) ranged in age from 26-88 and all had advanced-stage PBL (Table 1). Three of the four patients had some degree of immunosuppression (Patient #1- post-transplantation lymphoproliferative disorder (PTLD), Patient #2- HIV/AIDS, Patient #3- Chron's disease, Patient #4- no history of immunosuppression). All patients had a Ki67 proliferation index over 70% and demonstrated extranodal involvement of disease (bone n=3, intestine n=2, liver n=2; kidney n=1; adrenal n=1). The three patients that received daratumumab in combination with EPOCH demonstrated a complete response at their first disease assessment by PET/CT scan after cycle 2 (Patient #1) or cycle 4 (Patients #2,4) (Fig1). Patient #3, who demonstrated a mixed response to previous therapy, achieved a complete response 5 months after starting treatment with daratumumab in combination with chemotherapy. As of July 2021, three of the four patients continued to have no evidence of disease for a median of 17 months (range 15-19 months). Patient #4 relapsed in July 2021, 3 months after demonstrating a complete response. Adverse events that required hospitalization were rarely noted following daratumumab treatment but included neutropenic fever (n=2, one event following treatment cycle 4 and the other event 2 months after completion of daratumumab administration), COVID-19 infection (n=1), and a PICC line-associated thrombus (n=1). Minor events were also noted and included self-limiting bradycardia (n=1), neuropathy (n=1), and rigor (n=1). Conclusions:We describe four patients with varying degrees of immunosuppression and HIV-status with aggressive-stage PBL that achieved complete response following treatment with daratumumab in combination with low-dose EPOCH or other chemotherapy. Three of four patients obtained durable responses. At the time of this writing, three additional HIV +patients have initiated treatment with daratumumab but have not yet reached their first disease assessment. Disease progression for these patients will be monitored and presented as part of this study. Our findings suggest a potential efficacy and warrant further investigation of using daratumumab for the treatment of HIV +and HIV - PBLs. Figure 1 Figure 1. Disclosures Amengual: Epizyme, Inc.: Speakers Bureau; Appia Pharmaceuticals: Research Funding; Daiichi Sankyo, Inc: Consultancy; Seagen: Consultancy. OffLabel Disclosure: our work uses daratumumab, a first-in-class monoclonal antibody against CD38 for the treatment of plasmablastic lymphoma

Early postnatal exposure to general anesthetic agents causes a lasting impairment in learning and memory in animal models. One hypothesis to explain this finding is that exposure to anesthetic agents during critical points in neural development disrupts the formation of brain circuitry. Here, we explore the effects of sevoflurane on the neuronal growth cone, a specialization at the growing end of axons and dendrites that is responsible for the targeted growth that underlies connectivity between neurons.Dissociated neuronal cultures were prepared from embryonic mouse neocortex. Time-lapse images of live growth cones exposed to anesthetics were taken using differential interference contrast microscopy, and the rate of change of the area of the lamellipodia and the speed of the filopodial tip were quantified as measures of motility. The involvement of the p75 neurotropin receptor (p75NTR) was tested using inhibitors applied to the media and by a coimmunoprecipitation assay.The rate of lamellipodial area change and filopodial tip velocity in both axonal and dendritic growth cones was significantly reduced with sevoflurane exposure between 2% and 6%. Motility could be substantially restored by treatment with Y27632 and TAT-peptide 5, which are inhibitors of Rho Kinase and p75NTR, respectively. Sevoflurane results in reduced coimmunoprecipitation of Rho-Guanosine-5'-diphosphate dissociation inhibitor after pulldown with p75NTR.Sevoflurane interferes with growth cone motility, which is a critical process in brain circuitry formation. Our data suggest that this may occur through an action on the p75NTR, which promotes growth inhibitory signaling by the Rho pathway.

Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

// Jae-Wook Lee 1, * , Yun-Kyoung Ryu 1, * , Young-Hoon Ji 2 , Joo Hyun Kang 3 , Eun-Yi Moon 1 1 Department of Bioscience and Biotechnology, Sejong University, Seoul 143–747, Korea 2 Research Center for Radiotherapy, Korea Institute of Radiological and Medical Science, Seoul 139–709, Korea 3 Molecular Imaging Research Center, Korea Institute of Radiological and Medical Science, Seoul 139–709, Korea * These authors have contributed equally to this work Correspondence to: Eun-Yi Moon, e-mail: eunyimoon@sejong.ac.kr , eunyimoon@gmail.com Keywords: thymosin beta-4, Rac1-GTPase, Rap1-GTPase, cancer cell migration, tumor metastasis Received: September 28, 2014 Accepted: January 26, 2015 Published: March 23, 2015 ABSTRACT Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2′-O-methyladenosine-3′,5′-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo , and H/R-experienced cancer cell migration in vitro , more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

Vascular endothelial growth factor (VEGF) is an important regulator of neovascularization. Hypoxia inducible nitric oxide (NO) enhanced the expression of VEGF and thymosin beta-4 (Tβ4), actin sequestering protein. Here, we investigated whether NO-mediated VEGF expression could be regulated by Tβ4 expression in HeLa cervical cancer cells. Hypoxia inducible NO production and VEGF expression were reduced by small interference (si) RNA of Tβ4. Hypoxia response element (HRE)-luciferase activity and VEGF expression were increased by the treatment with N-(β-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, which was inhibited by the inhibition of Tβ4 expression with Tβ4-siRNA. In hypoxic condition, HRE-luciferase activity and VEGF expression were inhibited by the treatment with N(G)-monomethyl-L-arginine (L-NMMA), an inhibitor to nitric oxide synthase (NOS), which is accompanied with a decrease in Tβ4 expression. VEGF expression inhibited by L-NMMA treatment was restored by the transfection with pCMV-Tβ4 plasmids for Tβ4 overexpression. Taken together, these results suggest that Tβ4 could be a regulator for the expression of VEGF via the maintenance of NOS activity.

Neonatal hypoxic-ischemic injury (HI) results in widespread cerebral encephalopathy and affects structures that are essential for neurocognitive function, such as the hippocampus. The dentate gyrus contains a reservoir of neural stem and progenitor cells (NSPCs) that are critical for postnatal development and normal adult function of the hippocampus, and may also facilitate the recovery of function after injury. Using a neonatal mouse model of mild-to-moderate HI and immunohistochemical analysis of NSPC development markers, we asked whether these cells are vulnerable to HI and how they respond to both injury and hypothermic therapy. We found that cleaved caspase-3 labeling in the subgranular zone, where NSPCs are located, is increased by more than 30-fold after HI. The population of cells positive for both proliferating cell nuclear antigen and nestin (PCNA+Nes+), which represent primarily actively proliferating NSPCs, are acutely decreased by 68% after HI. The NSPC population expressing NeuroD1, a marker for NSPCs transitioning to become fate-committed neural progenitors, was decreased by 47%. One week after HI, there was a decrease in neuroblasts and immature neurons in the dentate gyrus, as measured by doublecortin (DCX) immunolabeling, and at the same time PCNA+Nes+ cell density was increased by 71%. NSPCs expressing Tbr2, which identifies a highly proliferative intermediate neural progenitor population, increased by 107%. Hypothermia treatment after HI partially rescues both the acute decrease in PCNA+Nes+ cell density at 1 day after injury and the chronic loss of DCX immunoreactivity and reduction in NeuroD1 cell density measured at 1 week after injury. Thus, we conclude that HI causes an acute loss of dentate gyrus NSPCs, and that hypothermia partially protects NSPCs from HI.

Background: Wnt5a is important for the development of various organs and postnatal cellular function. Little is known, however, about the role of Wnt5a in kidney development, although WNT5A mutations were identified in patients with Robinow syndrome, a genetic disease which includes developmental defects in kidneys. Our goal in this study was to determine the role of Wnt5a in kidney development. Methods: Whole-mount in situ hybridization was used to establish the expression pattern of Wnt5a during kidney development. Zebrafish with wnt5a knockdown and Wnt5a global knockout mice were used to identify kidney phenotypes. Results: In zebrafish, wnt5a knockdown resulted in glomerular cyst formation and dilated renal tubules. In mice, Wnt5a global knockout resulted in pleiotropic, but severe, kidney phenotypes, including agenesis, fused kidney, hydronephrosis and duplex kidney/ureter. Conclusions: Our data demonstrated the important role of Wnt5a in kidney development. Disrupted Wnt5a resulted in kidney cysts in zebrafish and pleiotropic abnormal kidney development in mice.

There is growing concern that pediatric exposure to anesthetic agents may cause long-lasting deficits in learning by impairing brain development. Most studies to date on this topic have focused on the direct effects of anesthetics on developing neurons. Relatively little attention has been paid to possible effects of anesthetics on astrocytes, a glial cell type that plays an important supporting role in neuronal development.Astrocytes were exposed to isoflurane and then cocultured with unexposed neurons to test for astrocyte-specific toxic effects on neuronal growth. Axon length was measured in the cocultured neurons to assess neuronal growth.We found that neurons cocultured with astrocytes exposed to isoflurane exhibited a 30% reduction in axon outgrowth. Further experimentation showed that this effect is likely due to reduced levels of brain-derived neurotrophic factor in the coculture media.Isoflurane interferes with the ability of cultured astrocytes to support neuronal growth. This finding represents a potentially novel mechanism through which general anesthetics may interfere with brain development.

The results of several retrospective clinical studies suggest that exposure to anesthetic agents early in life is correlated with subsequent learning and behavioral disorders. Although ongoing prospective clinical trials may help to clarify this association, they remain confounded by numerous factors. Thus, some of the most compelling data supporting the hypothesis that a relatively short anesthetic exposure can lead to a long-lasting change in brain function are derived from animal models. The mechanism by which such changes could occur remains incompletely understood. Early studies identified anesthetic-induced neuronal apoptosis as a possible mechanism of injury, and more recent work suggests that anesthetics may interfere with several critical processes in brain development. The function of the mature brain requires the presence of circuits, established during development, which perform the computations underlying learning and cognition. In this review, we examine the mechanisms by which anesthetics could disrupt brain circuit formation, including effects on neuronal survival and neurogenesis, neurite growth and guidance, formation of synapses, and function of supporting cells. There is evidence that anesthetics can disrupt aspects of all of these processes, and further research is required to elucidate which are most relevant to pediatric anesthetic neurotoxicity.

Inflammation is closely associated with tumor growth, which is mediated by the activation of bone marrow-derived CD11b(+) cells. Here, we investigated whether anti-inflammatory dexamethasone (Dex), a synthetic glucocorticoid (GC), could regulate tumor growth and CD11b(+) myeloid bone marrow cells (BMCs) in lymphocyte (R1), monocyte (R2) and granulocyte (R3) regions of FSC-SSC dot plot. The growth of B16F10 mouse melanoma tumor was inhibited in Dex-injected group. Lung metastasis was decreased and the lifespan was elongated in Dex-injected mice with tumor resection. Intravenous injection of B16F10 cells increased the percentage of CD11b(+) myeloid BMCs in R1 and R2 regions from 3h to 72h. In contrast, little changes in the percentage of CD11b(+) myeloid BMCs were detected in R3 region. Among CD11b(+) myeloid BMCs, the percentage of CD11b(+)Gr-1(+) cells was increased in R1, R2 and R3 regions. Absolute number of CD11b(+) and CD11b(+)Gr-1(+) cells was enhanced in R1 region from 3h to 72 h. B16F10 tumor growth was significantly increased by intravenous injection of CD11b(+) BMCs. Tumor-bearing mice showed an increase in the percentage of CD11b(+) myeloid BMCs in R2 region and CD11b(+)Gr-1(+) cells in R2 and R3 regions, which are reduced by intravenous injection with Dex. Absolute number of CD11b(+)Gr-1(+) cells was enhanced in R2 and R3 regions. Tumor growth was significantly inhibited by intravenous injection of BMCs collected from Dex-treated tumor-bearing mice. Taken together, data demonstrate that tumor regression by Dex was resulted from the alteration of CD11b(+) myeloid BMCs and their inhibitory function to tumor growth. It suggests that CD11b(+) myeloid BMCs could regulate antitumor efficacy of GCs such as Dex.

Macrophages play a role in innate immune responses to various foreign antigens. Many products from primary tumors influence the activation and transmigration of macrophages. Here, we investigated a migration of macrophages stimulated with cancer cell culture-conditioned medium (CM). Macrophage activation by treatment with CM of B16F10 cells were judged by the increase in protein levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase 2 (COX2). The location where macrophages were at 4 h-incubation with control medium or CM was different from where they were at 5 h-incubation in culture dish. Percentage of superimposed macrophages at every 1 h interval was gradually increased by CM treatment as compared to control. Total coverage of migrated track expressed in coordinates was smaller and total distance of migration was shorter in CM-treated macrophages than that in control. Rac1 activity in CM-treated macrophages was also decreased as compared to that in control. When macrophages were treated with CM in the presence of dexamethasone (Dex), an increase in COX2 protein levels, and a decrease in Rac1 activity and total coverage of migration were reversed. In the meanwhile, biphasic changes were detected by Dex treatment in section distance of migration at each time interval, which was more decreased at early time and then increased at later time. Taken together, data demonstrate that macrophage motility could be reduced in accordance with activation in response to cancer cell products. It suggests that macrophage motility could be a novel marker to monitor cancer-associated inflammatory diseases and the efficacy of anti-inflammatory agents.

Clinical and preclinical studies indicate that early postnatal exposure to anesthetics can lead to lasting deficits in learning and other cognitive processes. The mechanism underlying this phenomenon has not been clarified and there is no treatment currently available. Recent evidence suggests that anesthetics might cause persistent deficits in cognitive function by disrupting key events in brain development. The hippocampus, a brain region that is critical for learning and memory, contains a large number of neurons that develop in the early postnatal period, which are thus vulnerable to perturbation by anesthetic exposure. Using an in vivo mouse model we demonstrate abnormal development of dendrite arbors and dendritic spines in newly generated dentate gyrus granule cell neurons of the hippocampus after a clinically relevant isoflurane anesthesia exposure conducted at an early postnatal age. Furthermore, we find that isoflurane causes a sustained increase in activity in the mechanistic target of rapamycin pathway, and that inhibition of this pathway with rapamycin not only reverses the observed changes in neuronal development, but also substantially improves performance on behavioral tasks of spatial learning and memory that are impaired by isoflurane exposure. We conclude that isoflurane disrupts the development of hippocampal neurons generated in the early postnatal period by activating a well-defined neurodevelopmental disease pathway and that this phenotype can be reversed by pharmacologic inhibition.

The mechanism by which anesthetics might act on the developing brain in order to cause long term deficits remains incompletely understood. The hippocampus has been identified as a structure that is likely to be involved, as rodent models show numerous deficits in behavioral tasks of learning that are hippocampal-dependent. The hippocampus is an unusual structure in that it is the site of large amounts of neurogenesis postnatally, particularly in the first year of life in humans, and these newly generated neurons are critical to the function of this structure. Intriguingly, neurogenesis is a major developmental event that occurs during postulated windows of vulnerability to developmental anesthetic neurotoxicity across the different species in which it has been studied. In this review, we examine the evidence for anesthetic effects on neurogenesis in the early postnatal period and ask whether neurogenesis should be studied further as a putative mechanism of injury. Multiple anesthetics are considered, and both in vivo and in vitro work is presented. While there is abundant evidence that anesthetics act to suppress neurogenesis at several different phases, evidence of a causal link between these effects and any change in learning behavior remains elusive.

Cell migration plays a role in many physiological and pathological processes. Reactive oxygen species (ROS) produced in mammalian cells influence intracellular signaling processes which in turn regulate various biological activities. Here, we investigated whether melanoma cell migration could be controlled by ROS production under normoxia condition. Cell migration was measured by wound healing assay after scratching confluent monolayer of B16F10 mouse melanoma cells. Cell migration was enhanced over 12 h after scratching cells. In addition, we found that ROS production was increased by scratching cells. ERK phosphorylation was also increased by scratching cells but it was decreased by the treatment with ROS scavengers, N-acetylcysteine (NAC). Tumor cell migration was inhibited by the treatment with PD98059, ERK inhibitor, NAC or DPI, well-known ROS scavengers. Tumor cell growth as judged by succinate dehydrogenase activity was inhibited by NAC treatment. When mice were intraperitoneally administered with NAC, the intracellular ROS production was reduced in peripheral blood mononuclear cells. In addition, B16F10 tumor growth was significantly inhibited by in vivo treatment with NAC. Collectively, these findings suggest that tumor cell migration and growth could be controlled by ROS production and its downstream signaling pathways, in vitro and in vivo.

Angiogenesis is induced by soluble factors such as vascular endothelial growth factor (VEGF) released from tumor cells in hypoxia. It enhances solid tumor growth and provides an ability to establish metastasis at peripheral sites by tumor cell migration. Thymosin beta-4 (TB4) is an actin-sequestering protein to control cytoskeletal reorganization. Here, we investigated whether angiogenesis and tumor metastasis are dependent on hypoxia conditioning-induced TB4 expression in B16F10 melanoma cells. TB4 expression in B16F10 cells was increased by hypoxia conditioning in a time-dependent manner. In addition, we found an increase of angiogenesis and HIF-1α expression in TB4-transgenic (Tg) mice as compared to wildtype mice. When wound healing assay was used to assess in vitro tumor cell migration, hypoxia conditioning for 1 h enhanced B16F10 cell migration. When TB4 expression in B16F10 cells was inhibited by the infection with small hairpin (sh) RNA of TB4 cloned in lentiviral vector, tumor cell migration was retarded. In addition, hypoxia conditioning-induced tumor cell migration was reduced by the infection of lentiviral shRNA of TB4. HIF-1α stabilization and the expression of VEGF isoform 165 and 121 in hypoxia were also reduced by the infection of lentiviral shRNA of TB4 in B16F10 cells. We also found an increase of tumor growth and lung metastasis count in TB4-Tg mice as compared to wildtype mice. Collectively, hypoxia conditioning induced tumor cell migration by TB4 expression-dependent HIF-1α stabilization. It suggests that TB4 could be a hypoxia responsive regulator to control tumor cell migration in angiogenesis and tumor metastasis.

Down syndrome is the most common chromosomal disorder affecting the nervous system in humans. To date, investigations of neural anomalies in Down syndrome have focused on the central nervous system, although dysfunction of the peripheral nervous system is a common manifestation. The molecular and cellular bases underlying peripheral abnormalities have remained undefined. Here, we report the developmental loss of sympathetic innervation in human Down syndrome organs and in a mouse model. We show that excess regulator of calcineurin 1 (RCAN1), an endogenous inhibitor of the calcineurin phosphatase that is triplicated in Down syndrome, impairs neurotrophic support of sympathetic neurons by inhibiting endocytosis of the nerve growth factor (NGF) receptor, TrkA. Genetically correcting RCAN1 levels in Down syndrome mice markedly improves NGF-dependent receptor trafficking, neuronal survival and innervation. These results uncover a critical link between calcineurin signalling, impaired neurotrophin trafficking and neurodevelopmental deficits in the peripheral nervous system in Down syndrome., Down syndrome is associated with both central and peripheral nervous system dysfunction. Here, the authors report reduced sympathetic neural innervation in human Down syndrome tissues and a mouse model, and propose a role for RCAN1 trisomy in disrupted NGF receptor trafficking and neurotrophic support.

RT is commonly used to treat malignant tumors. However, tumor regrowth is a major limitation to RT as an antitumor treatment. In the present study, we investigated the tumor-promoting effects of high-dose (or ablative) RT treatments on tumor-bearing mice. We focused on the role of macrophages that interact with IR-CCs in the TME, which cause tumor regrowth. We observed that CT26(H-2d) tumor growth was enhanced by i.v. injection of IR-CT26 cells compared with NR control CT26 cells. The levels of iNOS gene expression and NO production from RAW264.7 macrophages (H-2d) in response to the interaction with IR-CT26 cells were higher than with NR-CT26 cells. When CT26 tumor-bearing mice were treated i.v. with L-NMMA, a NOS inhibitor, the reduction in in vivo tumor growth was higher in the IR-CT26-injected group compared with the NR-CT26-injected control group. In vivo CT26 tumor growth was decreased after transplanting PEM extracted from L-NMMA-treated, tumor-bearing mice. Although iNOS activity was reduced by inhibiting TLR1 expression with TLR1-siRNA, it was enhanced by TLR1 overexpression. Transcriptional activation and protein expression levels of iNOS were also decreased in the presence of TLR1-siRNA but increased as a result of TLR1 overexpression. These results demonstrate that postradiotherapeutic tumor regrowth may be caused by interaction of IR-CCs with macrophages that induce TLR1-mediated iNOS expression and NO production. Our data suggest that iNOS in macrophages could be a useful target to regulate postradiotherapeutic responses in hosts and subsequently limit tumor regrowth.

With the advent of modern techniques, drugs, and monitoring, general anesthesia has come to be considered an unlikely cause of harm, particularly for healthy patients. While this is largely true, newly emerging clinical and laboratory studies have suggested that exposure to anesthetic agents during early childhood may have long-lasting adverse effects on cognitive function. This concern has been the focus of intense study in the field of anesthesia research. A recent high-profile review by Rappaport et al. (2015) concluded that while many questions remain unanswered, there is strong evidence from laboratory studies that commonly used anesthetics interfere with brain development and that clinical studies suggest a correlation between early childhood exposure to these agents and subsequent effects on learning and cognition. The issue is of sufficient public health importance that a public-private partnership known as SmarTots (Strategies for Mitigating Anesthesia-Related Neurotoxicity in Tots) was developed by the FDA to study pediatric anesthetic neurotoxicity. The mechanism of injury underlying this phenomenon has yet to be fully elucidated, and there is evidence to suggest that anesthetics may have direct cytotoxic effects on neurons leading to cell death or suppressed neurogenesis (Stratmann et al., 2010) and that they may interfere with key processes in neuronal growth and development that underlie brain circuit development (Wagner et al., 2014).

During nervous system development, axon branching at nerve terminals is an essential step in the formation of functional connections between neurons and target cells. It is known that target tissues exert control of terminal arborization through secretion of trophic factors. However, whether the in-growing axons themselves produce diffusible cues to instruct target innervation remains unclear. Here, we use conditional mutant mice to show that Wnt5a derived from sympathetic neurons is required for their target innervation in vivo. Conditional deletion of Wnt5a resulted in specific deficits in the extension and arborization of sympathetic fibers in their final target fields, while no defects were observed in the overall tissue patterning, proliferation, migration or differentiation of neuronal progenitors. Using compartmentalized neuronal cultures, we further demonstrate that the Ror receptor tyrosine kinases are required locally in sympathetic axons to mediate Wnt5a-dependent branching. Thus, our study suggests an autocrine Wnt5a-Ror signaling pathway that directs sympathetic axon branching during target innervation.

Wnts make up a large family of extracellular signaling molecules that play crucial roles in development and disease. A subset of noncanonical Wnts signal independently of the transcription factor β-catenin by a mechanism that regulates key morphogenetic movements during embryogenesis. The best characterized noncanonical Wnt, Wnt5a, has been suggested to signal via a variety of different receptors, including the Ror family of receptor tyrosine kinases, the Ryk receptor tyrosine kinase, and the Frizzled seven-transmembrane receptors. Whether one or several of these receptors mediates the effects of Wnt5a in vivo is not known. Through loss-of-function experiments in mice, we provide conclusive evidence that Ror receptors mediate Wnt5a-dependent processes in vivo and identify Dishevelled phosphorylation as a physiological target of Wnt5a–Ror signaling. The absence of Ror signaling leads to defects that mirror phenotypes observed in Wnt5a null mutant mice, including decreased branching of sympathetic neuron axons and major defects in aspects of embryonic development that are dependent upon morphogenetic movements, such as severe truncation of the caudal axis, the limbs, and facial structures. These findings suggest that Wnt5a–Ror–Dishevelled signaling constitutes a core noncanonical Wnt pathway that is conserved through evolution and is crucial during embryonic development.

Thymosin beta-4 (Tβ4), actin-sequestering protein, plays important roles in many cellular functions including cancer cell migrations. Glycogen synthase kinase (GSK) in Wnt signaling pathway is a key molecule to control intercellular interaction. Here, we investigated whether GSK-3 activity is regulated by Tβ4 and it is associated with Tβ4-mediated migration in gastric cancer cells. Various expression level of Tβ4 was observed in human gastric tumor tissues. Migration in gastric cancer cells, SNU638 and SNU668, was dependent on a relative expression level of Tβ4. Cell migration was higher in SNU668 with a higher expression level of Tβ4 than that in SNU638 with a lower Tβ4. Although the level of phosphorylated(p)-GSK-3α (inactive), β-catenin, E-cadherin and E-cadherin:β-catenin complex was relatively higher, p-GSK-3β (inactive) was lower in SNU638 compared to those in SNU668 cells. LiCl, GSK-3α/β inhibitor, reduced lung metastasis of B16F10 mouse melanoma cells and SNU668 cell migration. Small interference (si)RNA of GSK-3α increased SNU638 cell migration in accordance with the reduction of E-cadherin:β-catenin complex formation through a decrease in β-catenin and E-cadherin. Expression level of GSK-3α/β, β-catenin and E-cadherin in SNU668 and SNU638 was reversed by Tβ4-siRNA and by the treatment with acetylated-serine-aspartic acid-lysine-proline (SDKP) tetrapeptide of Tβ4, respectively. E-cadherin expression in SNU638 cells was decreased by β-catenin-siRNA. PD98059, MEK inhibitor, or U0126, ERK inhibitor, reduced SNU668 cell migration accompanying an increase in p-GSK-3α, β-catenin and E-cadherin. Taken together, data indicated that the expression of GSK-3α, β-catenin and E-cadherin could be negatively regulated by Tβ4-induced ERK phosphorylation. It suggests that Tβ4 could be a novel regulator to control Wnt signaling pathways.

The sympathetic nervous system has served as an amenable model system to investigate molecular mechanisms underlying developmental processes in the nervous system. While much attention has been focused on neurotrophic factors controlling survival and connectivity of postmitotic sympathetic neurons, relatively little is known about signaling mechanisms regulating development of sympathetic neuroblasts. Here, we report that Frizzled3 (Fz3), a member of the Wnt receptor family, is essential for maintenance of dividing sympathetic neuroblasts. InFz3−/−mice, sympathetic neuroblasts exhibit decreased proliferation and premature cell cycle exit.Fz3−/−sympathetic neuroblasts also undergo enhanced apoptosis, which could not be rescued by eliminating the proapoptotic factor,Bax. These deficits result in reduced generation of sympathetic neurons and pronounced decreases in the size of sympathetic chain ganglia. Furthermore, the axons of sympathetic neurons that persist inFz3−/−ganglia are able to extend out of sympathetic ganglia toward distal targets, but fail to fully innervate final peripheral targets. The cell cycle exit, but not target innervation, defects inFz3−/−mice are phenocopied in mice with conditional ablation of β-catenin, a component of canonical Wnt signaling, in sympathetic precursors. Sympathetic ganglia and innervation of target tissues appeared normal in mice lacking a core planar cell polarity (PCP) component, Vangl2. Together, our results suggest distinct roles for Fz3 during sympathetic neuron development; Fz3 acts at early developmental stages to maintain a pool of dividing sympathetic precursors, likely via activation of β-catenin, and Fz3 functions at later stages to promote innervation of final peripheral targets by postmitotic sympathetic neurons.

The actin-sequestering protein thymosin beta-4 (T beta 4) is involved in various cellular and physiological processes such as proliferation, motility, growth and metastasis. Nitric oxide (NO) promotes tumor invasiveness and metastasis by activating various enzymes. Herein, we investigated whether hypoxia-inducible NO regulates T beta 4 expression and cancer cell migration using HeLa cervical cancer cells. NO production and T beta 4 expression were increased in a hypoxic condition. The treatment with N-(beta-D-Glucopyranosyl)-N2-acetyl-S-nitroso-D, L-penicillaminamide (SNAP-1), to generate NO, enhanced the transcription of T beta 4 and cancer cell migration. SNAP-1-induced cell migration was decreased by the inhibition of T beta 4 with small interference (si) RNA. In a hypoxic condition, treatment with N-G-monomethyl-L-arginine (L-NMMA), nitric oxide synthase (NOS) inhibitor, reduced T beta 4 transcriptional activity, and hypoxia-inducible factor (HIF)-1 alpha. Hypoxia-induced cancer cell migration was also decreased by L-NMMA treatment. In a normoxic condition, T beta 4 transcriptional activity was decreased in the cells incubated in the presence of L-NMMA after co-transfection with T beta 4 promoter and GST-conjugated HIF-1 alpha. Collectively, these results suggest that NO could regulate the expression of T beta 4 by direct or indirect effect of HIF-1 alpha on T beta 4 promoter.