Development of a time-resolved mirrorless scintillation detector.

PURPOSE: We developed a compact and lightweight time-resolved mirrorless scintillation detector (TRMLSD) employing image processing techniques and a convolutional neural network (CNN) for high-resolution two-dimensional (2D) dosimetry. METHODS: The TRMLSD comprises a camera and an inorganic scintillator plate without a mirror. The camera was installed at a certain angle from the horizontal plane to collect scintillation from the scintillator plate. The geometric distortion due to the absence of a mirror and camera lens was corrected using a projective transform. Variations in brightness due to the distance between the image sensor and each point on the scintillator plate and the inhomogeneity of the material constituting the scintillator were corrected using a 20.0 × 20.0 cm2 radiation field. Hot pixels were removed using a frame-based noise-reduction technique. Finally, a CNN-based 2D dose distribution deconvolution model was applied to compensate for the dose error in the penumbra region and a lack of backscatter. The linearity, reproducibility, dose rate dependency, and dose profile were tested for a 6 MV X-ray beam to verify dosimeter characteristics. Gamma analysis was performed for two simple and 10 clinical intensity-modulated radiation therapy (IMRT) plans. RESULTS: The dose linearity with brightness ranging from 0.0 cGy to 200.0 cGy was 0.9998 (R-squared value), and the root-mean-square error value was 1.010. For five consecutive measurements, the reproducibility was within 3% error, and the dose rate dependency was within 1%. The depth dose distribution and lateral dose profile coincided with the ionization chamber data with a 1% mean error. In 2D dosimetry for IMRT plans, the mean gamma passing rates with a 3%/3 mm gamma criterion for the two simple and ten clinical IMRT plans were 96.77% and 95.75%, respectively. CONCLUSION: The verified accuracy and time-resolved characteristics of the dosimeter may be useful for the quality assurance of machines and patient-specific quality assurance for clinical step-and-shoot IMRT plans.

Organotypic primary blood vessel models of clear cell renal cell carcinoma for single-patient clinical trials.

Clear cell renal cell carcinoma (ccRCC) is a common genitourinary cancer associated with the development of abnormal tumor angiogenesis. Although multiple anti-angiogenic therapies have been developed, responses to individual treatment are highly variable between patients. Thus, the use of one-patient clinical trials has been suggested as an alternative to standard trials. We used a microfluidic device to generate organotypic primary patient-specific blood vessel models using normal (NEnC) and tumor-associated primary CD31+ selected cells (TEnC). Our model was able to recapitulate differences in angiogenic sprouting and vessel permeability that characterize normal and tumor-associated vessels. We analyzed the expression profile of vessel models to define vascular normalization in a patient-specific manner. Using this data, we identified actionable targets to normalize TEnC vessel function to a more NEnC-like phenotype. Finally, we tested two of these drugs in our patient-specific models to determine the efficiency in restoring vessel function showing the potential of the model for single-patient clinical trials.

Phenomenological examinations of delirium in advanced cancer patients: exploratory structural equation modelling and latent profile analysis.

BACKGROUND: This study examined phenomenological manifestations of delirium in advanced cancer patients by examining the factor structure of the Delirium Rating Scale-Revised-98 (DRS-R-98) and profiles of delirium symptoms. METHODS: Ninety-three patients with advanced cancer admitted to inpatient palliative care units in South Korea were examined by psychiatrists using the DRS-R-98 and the Confusion Assessment Method (CAM). The factor structure of the DRS-R-98 was examined by exploratory structural equation modelling analysis (ESEM) and profiles of delirium were examined by latent profile analysis (LPA). RESULTS: CAM-defined delirium was present in 66.6% (n = 62) of patients. Results from the ESEM analysis confirmed applicability of the core and noncore symptom factors of the DRS-R-98 to advanced cancer patients. LPA identified three distinct profiles of delirium characterizing the overall severity of delirium and its core and noncore symptoms. Class 1 (n = 55, 59.1%) showed low levels of all delirium symptoms. Class 2 (n = 17, 18.3%) showed high levels of core symptoms only, whereas Class 3 (n = 21, 22.6%) showed high levels of both core and noncore symptoms except motor retardation. CONCLUSIONS: Clinical care for delirium in advanced cancer patients may benefit from consideration of the core and noncore symptom factor structure and the three distinct phenomenological profiles of delirium observed in the present study.

Saliva Diagnosis as a Disease Predictor.

BACKGROUND: Saliva, the most readily available body fluid, is the product of genes which are in constant activity throughout life. Measurement of saliva can predict the onset of some diseases years before their accumulation in vulnerable tissues causes clinical signs to appear. The purpose of this study was is to demonstrate current applications of saliva analysis and to predict and prevent disease progression. METHODS: We measured levels of Abeta42, C-reactive proteins (CRPs), and tumornecrosis factors (TNFs) in saliva from both healthy and fatal diseased cases such as cancer, Alzheimer's disease (AD), and coronary heart disease by ELISA-mediated techniques. We also immunostained human tissue sections with antibodies specific to these proteins to demonstrate the data are comparable. RESULTS: We found all the proteins expressed constantly in saliva from healthy controls but increased in diseased cases. This was accompanied by data from immunohistochemistry. It was also found that these proteins wereexpressed in high amounts in some healthy controls, which reflects high risk for the onset of diseases such as AD and heart diseases. CONCLUSIONS: It is concluded that measuring changes in essential gene products in saliva can predict onset of fatal diseases and open the door to effective protection measures, thus preventing premature death.

Feasibility of two-dimensional dose distribution deconvolution using convolution neural networks.

PURPOSE: The purpose of this study was to investigate the feasibility of two-dimensional (2D) dose distribution deconvolution using convolutional neural networks (CNNs) instead of an analytical approach for an in-house scintillation detector that has a detector-interface artifact in the penumbra region. METHODS: Datasets of 2D dose distributions were acquired from a medical linear accelerator of Novalis Tx. The datasets comprise two different sizes of square radiation fields and 13 clinical intensity-modulated radiation treatment (IMRT) plans. These datasets were divided into two datasets (training and test) to train and validate the developed network, called PenumbraNet, which is a shallow linear CNN. The PenumbraNet was trained to transform the measured dose distribution [M(x, y)] to calculated distribution [D(x, y)] by the treatment planning system. After training of the PenumbraNet was completed, the performance was evaluated using test data, which were 10 × 10 cm2 open field and ten clinical IMRT cases. The corrected dose distribution [C(x, y)] was evaluated against D(x, y) with 2%/2 mm and 3%/3 mm criteria of the gamma index for each field. The M(x, y) and deconvolved dose distribution with the analytically obtained kernel using Wiener filtering [A(x, y)] were also evaluated for comparison. In addition, we compared the performance of the shallow depth of linear PenumbraNet with that of nonlinear PenumbraNet and a deep nonlinear PenumbraNet within the same training epoch. RESULTS: The mean gamma passing rates were 84.77% and 95.81% with 3%/3 mm gamma criteria for A(x, y) and C(x, y) of the PenumbraNet, respectively. The mean gamma pass rates of nonlinear PenumbraNet and the deep depth of nonlinear PenumbraNet were 96.62%, 93.42% with 3%/3 mm gamma criteria, respectively. CONCLUSIONS: We demonstrated the feasibility of the PenumbraNets for 2D dose distribution deconvolution. The nonlinear PenumbraNet which has the best performance improved the gamma passing rate by 11.85% from the M(x, y) at 3%/3 mm gamma criteria.

BioPATH: A Biomarker Study in Asian Patients with HER2+ Advanced Breast Cancer Treated with Lapatinib and Other Anti-HER2 Therapy.

PURPOSE: BioPATH is a non-interventional study evaluating the relationship of molecular biomarkers (PTEN deletion/downregulation, PIK3CA mutation, truncated HER2 receptor [p95HER2], and tumor HER2 mRNA levels) to treatment responses in Asian patients with HER2+ advanced breast cancer treated with lapatinib and other HER2-targeted agents. MATERIALS AND METHODS: Female Asian HER2+ breast cancer patients (n=154) who were candidates for lapatinib-based treatment following metastasis and having an available primary tumor biopsy specimen were included. The primary endpoint was progression-free survival (PFS). Secondary endpoints were response rate, overall survival on lapatinib, correlation between biomarker status and PFS for any previous trastuzumab-based treatment, and conversion/conservation rates of the biomarker status between tissue samples collected at primary diagnosis and at recurrence/metastasis. Potential relationships between tumor mRNA levels of HER2 and response to lapatinib-based therapy were also explored. RESULTS: p95HER2, PTEN deletion/downregulation, and PIK3CA mutation did not demonstrate any significant co-occurrence pattern and were not predictive of clinical outcomes on either lapatinib-based treatment or any previous trastuzumab-based therapy in the metastatic setting. Proportions of tumors positive for p95HER2 expression, PIK3CA mutation, and PTEN deletion/down-regulation at primary diagnosis were 32%, 31.2%, and 56.2%, respectively. Despite limited availability of paired samples, biomarker status patterns were conserved in most samples. HER2 mRNA levels were not predictive of PFS on lapatinib. CONCLUSION: The prevalence of p95HER2 expression, PIK3CA mutation, and PTEN deletion/downregulation at primary diagnosis were similar to previous reports. Importantly, no difference was observed in clinical outcome based on the status of these biomarkers, consistent with reports from other studies.

Relationship between physicians' perceived stigma toward depression and physician referral to psycho-oncology services on an oncology/hematology ward.

OBJECTIVE: This study was performed to identify relationships between physicians' perceived stigma toward depression and psycho-oncology service utilization on an oncology/hematology ward. METHODS: The study participants were 235 patients in an oncology/hematology ward and 14 physicians undergoing an internal medicine residency training program in Inha University Hospital (Incheon, South Korea). Patients completed the Patient Health Questionnaire-9 (PHQ-9), and residents completed the Perceived Devaluation-Discrimination scale that evaluates perceived stigma toward depression. A total PHQ-9 score of ≥5 was defined as clinically significant depression. Physicians decided on referral on the basis of their opinions and those of their patients. The correlates of physicians' recommendation for referral to psycho-oncology services and real referrals psycho-oncology services were examined. RESULTS: Of the 235 patients, 143 had PHQ-9 determined depression, and of these 143 patients, 61 received psycho-oncology services. Physicians recommended that 87 patients consult psycho-oncology services. Multivariate analyses showed that lower physicians' perceived stigma regarding depression was significantly associated with physicians' recommendation for referral, and that real referral to psycho-oncology services was significantly associated with presence of a hematologic malignancy and lower physicians' perceived stigma toward depression. CONCLUSION: Physicians' perceived stigma toward depression was found to be associated with real referral to psycho-oncology services and with physician recommendation for referral to psycho-oncology services. Further investigations will be needed to examine how to reduce physicians' perceived stigma toward depression.

Evidence that the Human Innate Immune Peptide LL-37 may be a Binding Partner of Amyloid-ß and Inhibitor of Fibril Assembly.

BACKGROUND: Identifying physiologically relevant binding partners of amyloid-ß (Aß) that modulate in vivo fibril formation may yield new insights into Alzheimer's disease (AD) etiology. Human cathelicidin peptide, LL-37, is an innate immune effector and modulator, ubiquitous in human tissues and expressed in myriad cell types. OBJECTIVE: We present in vitro experimental evidence and discuss findings supporting a novel hypothesis that LL-37 binds to Aß42 and can modulate Aß fibril formation. METHODS: Specific interactions between LL-37 and Aß (with Aß in different aggregation states, assessed by capillary electrophoresis) were demonstrated by surface plasmon resonance imaging (SPRi). Morphological and structural changes were investigated by transmission electron microscopy (TEM) and circular dichroism (CD) spectroscopy. Neuroinflammatory and cytotoxic effects of LL-37 alone, Aß42 alone, and LL-37/Aß complexes were evaluated in human microglia and neuroblastoma cell lines (SH-SY5Y). RESULTS: SPRi shows binding specificity between LL-37 and Aß, while TEM shows that LL-37 inhibits Aß42 fibril formation, particularly Aß's ability to form long, straight fibrils characteristic of AD. CD reveals that LL-37 prevents Aß42 from adopting its typical ß-type secondary structure. Microglia-mediated toxicities of LL-37 and Aß42 to neurons are greatly attenuated when the two peptides are co-incubated prior to addition. We discuss the complementary biophysical characteristics and AD-related biological activities of these two peptides. CONCLUSION: Based on this body of evidence, we propose that LL-37 and Aß42 may be natural binding partners, which implies that balanced (or unbalanced) spatiotemporal expression of the two peptides could impact AD initiation and progression.

Homo-succinic acid production by metabolically engineered Mannheimia succiniciproducens.

Succinic acid (SA) is a four carbon dicarboxylic acid of great industrial interest that can be produced by microbial fermentation. Here we report development of a high-yield homo-SA producing Mannheimia succiniciproducens strain by metabolic engineering. The PALFK strain (ldhA-, pta-, ackA-, fruA-) was developed based on optimization of carbon flux towards SA production while minimizing byproducts formation through the integrated application of in silico genome-scale metabolic flux analysis, omics analyses, and reconstruction of central carbon metabolism. Based on in silico simulation, utilization of sucrose would enhance the SA production and cell growth rates, while consumption of glycerol would reduce the byproduct formation rates. Thus, sucrose and glycerol were selected as dual carbon sources to improve the SA yield and productivity, while deregulation of catabolite-repression was also performed in engineered M. succiniciproducens. Fed-batch fermentations of PALFK with low- and medium-density (OD600 of 0.4 and 9.0, respectively) inocula produced 69.2 and 78.4g/L of homo-SA with yields of 1.56 and 1.64mol/mol glucose equivalent and overall volumetric SA productivities of 2.50 and 6.02g/L/h, respectively, using sucrose and glycerol as dual carbon sources. The SA productivity could be further increased to 38.6g/L/h by employing a membrane cell recycle bioreactor system. The systems metabolic engineering strategies employed here for achieving homo-SA production with the highest overall performance indices reported to date will be generally applicable for developing superior industrial microorganisms and competitive processes for the bio-based production of other chemicals as well.

Quercetin, not caffeine, is a major neuroprotective component in coffee.

Epidemiologic studies indicate that coffee consumption reduces the risk of Parkinson's disease and Alzheimer's disease. To determine the factors involved, we examined the protective effects of coffee components. The test involved prevention of neurotoxicity to SH-SY5Y cells that was induced by lipopolysaccharide plus interferon-γ or interferon-γ released from activated microglia and astrocytes. We found that quercetin, flavones, chlorogenic acid, and caffeine protected SH-SY5Y cells from these toxins. They also reduced the release of tumor necrosis factor-α and interleukin-6 from the activated microglia and astrocytes and attenuated the activation of proteins from P38 mitogen-activated protein kinase (MAPK) and nuclear factor kappa light chain enhancer of activated B cells (NFκB). After exposure to toxin containing glial-stimulated conditioned medium, we also found that quercetin reduced oxidative/nitrative damage to DNA, as well as to the lipids and proteins of SH-SY5Y cells. There was a resultant increase in [GSH]i in SH-SY5Y cells. The data indicate that quercetin is the major neuroprotective component in coffee against Parkinson's disease and Alzheimer's disease.

Highly selective production of succinic acid by metabolically engineered Mannheimia succiniciproducens and its efficient purification.

Succinic acid (SA) is one of the fermentative products of anaerobic metabolism, and an important industrial chemical that has been much studied for its bio-based production. The key to the economically viable bio-based SA production is to develop an SA producer capable of producing SA with high yield and productivity without byproducts. Mannheimia succiniciproducens is a capnophilic rumen bacterium capable of efficiently producing SA. In this study, in silico genome-scale metabolic simulations were performed to identify gene targets to be engineered, and the PALK strain (ΔldhA and Δpta-ackA) was constructed. Fed-batch culture of PALK on glucose and glycerol as carbon sources resulted in the production of 66.14 g/L of SA with the yield and overall productivity of 1.34 mol/mol glucose equivalent and 3.39 g/L/h, respectively. SA production could be further increased to 90.68 g/L with the yield and overall productivity of 1.15 mol/mol glucose equivalent and 3.49 g/L/h, respectively, by utilizing a mixture of magnesium hydroxide and ammonia solution as a pH controlling solution. Furthermore, formation of byproducts was drastically reduced, resulting in almost homo-fermentative SA production. This allowed the recovery and purification of SA to a high purity (99.997%) with a high recovery yield (74.65%) through simple downstream processes composed of decolorization, vacuum distillation, and crystallization. The SA producer and processes developed in this study will allow economical production of SA in an industrial-scale. Biotechnol. Bioeng. 2016;113: 2168-2177. © 2016 Wiley Periodicals, Inc.

Sodium thiosulfate attenuates glial-mediated neuroinflammation in degenerative neurological diseases.

BACKGROUND: Sodium thiosulfate (STS) is an industrial chemical which has also been approved for the treatment of certain rare medical conditions. These include cyanide poisoning and calciphylaxis in hemodialysis patients with end-stage kidney disease. Here, we investigated the anti-inflammatory activity of STS in our glial-mediated neuroinflammatory model. METHODS: Firstly, we measured glutathione (GSH) and hydrogen sulfide (H2S, SH(-)) levels in glial cells after treatment with sodium hydrosulfide (NaSH) or STS. We also measured released levels of tumor necrosis factor-α (TNFα) and interleukin-6 (IL-6) from them. We used two cell viability assays, MTT and lactate dehydrogenase (LDH) release assays, to investigate glial-mediated neurotoxicity and anti-inflammatory effects of NaSH or STS. We also employed Western blot to examine activation of intracellular inflammatory pathways. RESULTS: We found that STS increases H2S and GSH expression in human microglia and astrocytes. When human microglia and astrocytes are activated by lipopolysaccharide (LPS)/interferon-γ (IFNγ) or IFNγ, they release materials that are toxic to differentiated SH-SY5Y cells. When the glial cells were treated with NaSH or STS, there was a significant enhancement of neuroprotection. The effect was concentration-dependent and incubation time-dependent. Such treatment reduced the release of TNFα and IL-6 and also attenuated activation of P38 MAPK and NFκB proteins. The compounds tested were not harmful when applied directly to all the cell types. CONCLUSIONS: Although NaSH was somewhat more powerful than STS in these in vitro assays, STS has already been approved as an orally available treatment. STS may therefore be a candidate for treating neurodegenerative disorders that have a prominent neuroinflammatory component.

Phase II Trial of Nilotinib in Patients With Metastatic Malignant Melanoma Harboring KIT Gene Aberration: A Multicenter Trial of Korean Cancer Study Group (UN10-06).

BACKGROUND: KIT has been suggested to be a potential therapeutic target for malignant melanoma. We evaluated the antitumor activity and safety of the KIT inhibitor nilotinib in metastatic melanoma patients harboring KIT gene mutations or amplifications. METHODS: We conducted a phase II multicenter trial of nilotinib in metastatic malignant melanoma with KIT mutations or amplifications. Patients received 400 mg oral nilotinib twice daily. The primary endpoint was response rate, and if seven or more responders were observed from the cumulative 36 patients, nilotinib would be considered worthy of further testing in this study population. RESULTS: Between October 2009 and June 2013, 176 patients underwent molecular screening for KIT gene aberrations, and 42 patients harboring KIT gene mutations and/or amplification were enrolled in the study. Overall, 25 (59.5%), 15 (35.7%), and 2 (4.8%) patients had KIT mutations, KIT amplifications, and both KIT mutations and amplification, respectively. Of the 42 enrolled patients, 1 patient achieved complete response, 6 patients achieved partial response, and 17 patients achieved stable disease, resulting in an overall response rate of 16.7% (95% confidence interval [CI]: 5.4%-28.0%) and a disease control rate of 57.1% (95% CI: 42.1%-72.1%). The median duration of response was 34 weeks (range: 5-55 weeks). Of the 7 responders, 6 patients had KIT mutations (exon 11: 5 patients; exon 17: 1 patient), and 1 patient had KIT amplification only. CONCLUSION: Although this study did not meet its primary endpoint of response rate, nilotinib showed durable response in a subset of metastatic melanoma patients with specific KIT mutations. IMPLICATIONS FOR PRACTICE: KIT aberration can be detected in a subset of metastatic melanoma patients. This phase II trial showed that nilotinib demonstrates durable response in a subset of patients with KIT mutations. The safety profile was very tolerable. This study suggests that a KIT inhibitor may benefit a small subset of metastatic melanoma patients with KIT mutations.

Human antimicrobial peptide LL-37 induces glial-mediated neuroinflammation.

LL-37 is the sole cathelicidin-derived antimicrobial peptide found in humans. It becomes active upon C-terminal cleavage of its inactive precursor hCAP18. In addition to antimicrobial action, it also functions as an innate immune system stimulant in many tissues of the body. Here we report that hCAP18 and LL-37 are expressed in all organs of the human body that were studied with the highest basic levels being expressed in the GI tract and the brain. Its expression and functional role in the central nerve system (CNS) has not previously been reported. We found increased expression of LL-37 in IFNγ-stimulated human astrocytes and their surrogate U373 cells, as well as in LPS/IFNγ-stimulated human microglia and their surrogate monocyte-derived THP-1 cells. We found that treatment of microglia, astrocytes, THP-1 cells and U373 cells with LL-37 induced secretion of the inflammatory cytokines IL-1ß and IL-6; the chemokines IL-8 and CCL-2, and other materials toxic to human neuroblastoma SH-SY5Y cells. The mechanism of LL-37 stimulation involves activation of intracellular proinflammatory pathways involving phospho-P38 MAP kinase and phospho-NFκB proteins. We blocked the inflammatory stimulant action of LL-37 by removing it with an anti-LL-37 antibody. The inflammatory effect was also prevented by treatment with inhibitors of PKC, PI3K and MEK-1/2 as well as with the intracellular Ca(2+)-chelator, BAPTA-AM. This indicates involvement of these intracellular pathways. Our data suggest that LL-37, in addition to its established roles, may play a role in the chronic neuroinflammation which is observed in neurodegenerative diseases such as Alzheimer's and Parkinson's disease.

Activated human microglia stimulate neuroblastoma cells to upregulate production of beta amyloid protein and tau: implications for Alzheimer's disease pathogenesis.

Neuroinflammation is hypothesized to be a major driving force behind Alzheimer's disease (AD) pathogenesis. This hypothesis predicts that activated microglial cells can stimulate neurons to produce excessive amounts of ß-amyloid protein (Aß1₋42) and tau. The excess Aß1₋42 forms extracellular deposits which stimulate further microglial activation. The excess tau is partially released but also becomes phosphorylated forming intracellular neurofibrillary deposits. The end result is a positive feedback mechanism which drives the disease development. To test the viability of this hypothesis, we exposed differentiated SH-SY5Y and N-tera2/D1 (N-tera2) cells to conditioned medium (CM) from LPS/IFNγ-stimulated human microglia. We found that the CM caused a large increase in the production and release of Aß and tau. The CM also caused SH-SY5Y cells to increase their expression of amyloid precursor protein and release of its ß-secretase cleaved products (sAPPßs) as well as Aß oligomers, but the CM reduced release of its α-secretase cleaved products (sAPPαs). Direct treatment of SH-SY5Y and N-tera2 cells with the inflammatory cytokines IL-6 and IL-1ß as well as with Aß1₋42, resulted in an increase in tau messenger RNA and protein expression. Pretreatment of LPS/IFNγ-stimulated human microglia cells with the nonsteroidal anti-inflammatory drugs ibuprofen and aspirin, the antioxidant GSH, the H2S donor NaSH, and the anti-inflammatory cytokine IL-10, resulted in a CM with diminished ability to stimulate tau expression. There was no effect on the morphology of SH-SY5Y cells, or on their viability, following exposure to micromolar levels of Aß1₋42. Our data indicate that reactive microglia play an important role in governing the expression of Aß and tau, and therefore the progression of AD. They provide further evidence that appropriate anti-inflammatory treatment should be beneficial in AD.

Combined treatment of murine fibrosarcoma with chemotherapy (Paclitaxel), radiotherapy, and intratumoral injection of dendritic cells.

BACKGROUND: New antitumor therapeutic strategies aim to combine different approaches that are able to induce tumor-specific effector and memory T cell responses that might control tumor growth. Dendritic cells (DCs) have the capacity to induce antigen-specific cytotoxic T lymphocytes. We have previously shown that the combined treatment of paclitaxel chemotherapy (Chemo) and injection of DCs led to complete tumor regression. OBJECTIVE: The goal of this study was to evaluate synergistic antitumor effect of a triple combination treatment comprising radiotherapy, paclitaxel Chemo and intratumoral injection of syngeneic bone marrow-derived DCs on murine fibrosarcoma, compared to other single or double combination treatments. METHODS: For the murine fibrosarcoma model, naïve C57BL/6 mice were inoculated intradermally with 2×10(3) MCA102 cells in the right upper flank. Mice were assigned to five groups (untreatedcontrol, RT alone, RT+Chemo, RT+DC, and RT+Chemo+DC), with eight mice in each group. In vitro cytotoxicity assays were performed to assess the immune activity. The persistence of tumor-specific immunity was determined by second tumor challenge in mice with complete tumor regression. RESULTS: The triple combination treatment showed a significantly enhanced therapeutic efficacy by decreasing tumor size and inducing complete tumor regression, resulting in a cure of 50% of mice. The results of in vitro cytotoxicity assays and the second tumor challenge experiment strongly indicated the induction of a tumor-specific cytotoxic T lymphocyte response and acquisition of prolonged tumor immunity. CONCLUSION: These findings suggest that the triple combination treatment can be a promising strategy for the treatment of murine fibrosarcoma.

A novel pathway links oxidative stress to loss of insulin growth factor-2 (IGF2) imprinting through NF-κB activation.

Genomic imprinting is the allele-specific expression of a gene based on parental origin. Loss of imprinting(LOI) of Insulin-like Growth Factor 2 (IGF2) during aging is important in tumorigenesis, yet the regulatory mechanisms driving this event are largely unknown. In this study oxidative stress, measured by increased NF-κB activity, induces LOI in both cancerous and noncancerous human prostate cells. Decreased expression of the enhancer-blocking element CCCTC-binding factor(CTCF) results in reduced binding of CTCF to the H19-ICR (imprint control region), a major factor in the allelic silencing of IGF2. This ICR then develops increased DNA methylation. Assays identify a recruitment of the canonical pathway proteins NF-κB p65 and p50 to the CTCF promoter associated with the co-repressor HDAC1 explaining gene repression. An IκBα super-repressor blocks oxidative stress-induced activation of NF-κB and IGF2 imprinting is maintained. In vivo experiments using IκBα mutant mice with continuous NF-κB activation demonstrate increased IGF2 LOI further confirming a central role for canonical NF-κB signaling. We conclude CTCF plays a central role in mediating the effects of NF-κB activation that result in altered imprinting both in vitro and in vivo. This novel finding connects inflammation found in aging prostate tissues with the altered epigenetic landscape.

NOSH-aspirin (NBS-1120), a novel nitric oxide and hydrogen sulfide releasing hybrid, attenuates neuroinflammation induced by microglial and astrocytic activation: a new candidate for treatment of neurodegenerative disorders.

Hydrogen sulfide (H2 S) and nitric oxide (NO) have been described as gasotransmitters. Anti-inflammatory activity in the central and peripheral nervous systems may be one of their functions. Previously we demonstrated that several SH(-) donors including H2 S-releasing aspirin (S-ASA) exhibited anti-inflammatory and neuroprotective activity in vitro against toxins released by activated microglia and astrocytes. Here we report that NOSH-ASA, an NO- and H2 S-releasing hybrid of aspirin, has a significantly greater anti-inflammatory and neuroprotective effect than S-ASA or NO-ASA. When activated by LPS/IFNγ, human microglia and THP-1 cells release materials that are toxic to differentiated SH-SY5Y cells. These phenomena also occur with IFNγ-stimulated human astroglia and U373 cells. When the cells were treated with the S-ASA or NO-ASA, there was a significant enhancement of neuroprotection. However, NOSH-ASA had significantly more potent protection properties than NO-ASA or S-ASA. The effect was concentration-dependent, as well as incubation time-dependent. Such treatment not only reduced the release of the TNFα and IL-6, but also attenuated activation of P38 MAPK and NFκB proteins. All the compounds tested were not harmful when applied directly to SH-SY5Y cells. These data suggest that NOSH-ASA has significant anti-inflammatory properties and may be a new candidate for treating neurodegenerative disorders that have a prominent neuroinflammatory component such as Alzheimer disease and Parkinson disease.

Neurotransmitters and microglial-mediated neuroinflammation.

Reciprocal interactions between cells caused by release of soluble factors are essential for brain function. So far, little attention has been paid to interactions between neurons and glia. However, in the last few decades, studies regarding such interactions have given us some important clues about possible mechanisms underlying degenerative processes in neurological diseases such as Alzheimer's disease and Parkinson's disease. Activated microglia and markers of inflammatory reactions have been consistently found in the post-mortem brains of diseased patients. But it has not been clearly understood how microglia respond to neurotransmitters released from neurons during disease progression. The main purpose of this review is to summarize studies performed on neurotransmitter receptor expression in microglia, and the effects of their activation on microglial-mediated neuroinflammation. A possible mechanism underlying transmitter-mediated modulation of microglial response is also suggested. Microglia express receptors for neurotransmitters such as ATP, adenosine, glutamate, GABA, acetylcholine, dopamine and adrenaline. Activation of GABA, cholinergic and adrenergic receptors suppresses microglial responses, whereas activation of ATP or adenosine receptors activates them. This latter effect may be due primarily to activation of a Ca(2+)-signaling pathway which, in turn, results in activation of MAP kinases and NFkB proteins with the release of proinflammatory factors. However, glutamate and dopamine are both pro- and anti-inflammatory depending on the receptor subtypes expressed in microglia. More detailed studies on downstream receptor-signaling cascades are needed to understand the roles of neurotransmitters in controlling neuron-microglia interactions during inflammatory processes in disease progression. Such knowledge may suggest new methods of treatment.

Weak BMAA toxicity compares with that of the dietary supplement ß-alanine.

ß-N-methylamino-L-alanine (BMAA) is routinely described in the literature as a potent neurotoxin and as a possible cause of neurodegenerative disorders of aging such as Alzheimer's disease, amyotrophic lateral sclerosis, and the amyotrophic lateral sclerosis/parkinsonism-dementia complex (ALS-PDC) syndrome of Guam. To test for the toxicity of BMAA against human neurons, we chose 3 standard human neuronal cell lines for examination and compared the toxicity with the muscle-building nutritional supplement ß-alanine, glutamic acid, and the established excitotoxins kainic acid, quisqualic acid, ibotenic acid, domoic acid, and quinolinic acid. Neurotoxicity was measured by the standard lactic dehydrogenase release assay after 5-day incubation of NT-2, SK-N-MC, and SH-SY5Y cells with BMAA and the comparative substances. The ED(50) of BMAA, corresponding to 50% death of neurons, varied from 1430 to 1604 µM while that of the nutritional supplement ß-alanine was almost as low, varying from 1945 to 2134 µM. The ED(50) for glutamic acid and the 5 established excitotoxins was 200- to 360-fold lower, varying from 44 to 70 µM. These in vitro data are in accord with previously published in vivo data on BMAA toxicity in which mice showed no pathological effects from oral consumption of 500 mg/kg/day for more than 10 weeks. Because there are no known natural sources of BMAA that would make consumption of such amounts possible, and because the toxicity observed was in the same range as the nutritional supplement ß-alanine, the hypothesis that BMAA is an environmental hazard and a contributor to degenerative neurological diseases becomes untenable.