Static and dynamic brain morphological changes in isolated REM sleep behavior disorder compared to normal aging.

Objective/background: To assess whether cerebral structural alterations in isolated rapid eye movement sleep behavior disorder (iRBD) are progressive and differ from those of normal aging and whether they are related to clinical symptoms. Patients/methods: In a longitudinal study of 18 patients with iRBD (age, 66.1 ± 5.7 years; 13 males; follow-up, 1.6 ± 0.6 years) and 24 age-matched healthy controls (age, 67.0 ± 4.9 years; 12 males; follow-up, 2.0 ± 0.9 years), all participants underwent multiple extensive clinical examinations, neuropsychological tests, and magnetic resonance imaging at baseline and follow-up. Surface-based cortical reconstruction and automated subcortical structural segmentation were performed on T1-weighted images. We used mixed-effects models to examine the differences between the groups and the differences in anatomical changes over time. Results: None of the patients with iRBD demonstrated phenoconversion during the follow-up. Patients with iRBD had thinner cortices in the frontal, occipital, and temporal regions, and more caudate atrophy, compared to that in controls. In similar regions, group-by-age interaction analysis revealed that patients with iRBD demonstrated significantly slower decreases in cortical thickness and caudate volume with aging than that observed in controls. Patients with iRBD had lower scores on the Korean version of the Mini-Mental Status Examination (p = 0.037) and frontal and executive functions (p = 0.049) at baseline than those in controls; however, no significant group-by-age interaction was identified. Conclusion: Patients with iRBD show brain atrophy in the regions that are overlapped with the areas that have been documented to be affected in early stages of Parkinson's disease. Such atrophy in iRBD may not be progressive but may be slower than that in normal aging. Cognitive impairment in iRBD is not progressive.

Subclinical variations on ECG and their associations with structural brain aging networks.

Impaired cardiac function is associated with cognitive impairment and brain imaging features of aging. Cardiac arrhythmias, including atrial fibrillation, are implicated in clinical and subclinical brain injuries. Even in the absence of a clinical diagnosis, subclinical or prodromal substrates of arrhythmias, including an abnormally long or short P-wave duration (PWD), a measure associated with atrial abnormalities, have been associated with stroke and cognitive decline. However, the extent to which PWD has subclinical influences on overall aging patterns of the brain is not clearly understood. Here, using neuroimaging and ECG data from the UK Biobank, we use a novel regional "brain age" method to identify the brain aging networks associated with abnormal PWD. We find that PWD is inversely associated with accelerated brain aging in the sensorimotor, frontoparietal, ventral attention, and dorsal attention networks, even in the absence of overt cardiac diseases. These findings suggest that detrimental aging outcomes may result from subclinically abnormal PWD.

Association of Brain Age, Lesion Volume, and Functional Outcome in Patients With Stroke.

BACKGROUND AND OBJECTIVES: Functional outcomes after stroke are strongly related to focal injury measures. However, the role of global brain health is less clear. In this study, we examined the impact of brain age, a measure of neurobiological aging derived from whole-brain structural neuroimaging, on poststroke outcomes, with a focus on sensorimotor performance. We hypothesized that more lesion damage would result in older brain age, which would in turn be associated with poorer outcomes. Related, we expected that brain age would mediate the relationship between lesion damage and outcomes. Finally, we hypothesized that structural brain resilience, which we define in the context of stroke as younger brain age given matched lesion damage, would differentiate people with good vs poor outcomes. METHODS: We conducted a cross-sectional observational study using a multisite dataset of 3-dimensional brain structural MRIs and clinical measures from the ENIGMA Stroke Recovery. Brain age was calculated from 77 neuroanatomical features using a ridge regression model trained and validated on 4,314 healthy controls. We performed a 3-step mediation analysis with robust mixed-effects linear regression models to examine relationships between brain age, lesion damage, and stroke outcomes. We used propensity score matching and logistic regression to examine whether brain resilience predicts good vs poor outcomes in patients with matched lesion damage. RESULTS: We examined 963 patients across 38 cohorts. Greater lesion damage was associated with older brain age (ß = 0.21; 95% CI 0.04-0.38, p = 0.015), which in turn was associated with poorer outcomes, both in the sensorimotor domain (ß = -0.28; 95% CI -0.41 to -0.15, p < 0.001) and across multiple domains of function (ß = -0.14; 95% CI -0.22 to -0.06, p < 0.001). Brain age mediated 15% of the impact of lesion damage on sensorimotor performance (95% CI 3%-58%, p = 0.01). Greater brain resilience explained why people have better outcomes, given matched lesion damage (odds ratio 1.04, 95% CI 1.01-1.08, p = 0.004). DISCUSSION: We provide evidence that younger brain age is associated with superior poststroke outcomes and modifies the impact of focal damage. The inclusion of imaging-based assessments of brain age and brain resilience may improve the prediction of poststroke outcomes compared with focal injury measures alone, opening new possibilities for potential therapeutic targets.

Different effects of cardiometabolic syndrome on brain age in relation to gender and ethnicity.

BACKGROUND: A growing body of evidence shows differences in the prevalence of cardiometabolic syndrome (CMS) and dementia based on gender and ethnicity. However, there is a paucity of information about ethnic- and gender-specific CMS effects on brain age. We investigated the different effects of CMS on brain age by gender in Korean and British cognitively unimpaired (CU) populations. We also determined whether the gender-specific difference in the effects of CMS on brain age changes depending on ethnicity. METHODS: These analyses used de-identified, cross-sectional data on CU populations from Korea and United Kingdom (UK) that underwent brain MRI. After propensity score matching to balance the age and gender between the Korean and UK populations, 5759 Korean individuals (3042 males and 2717 females) and 9903 individuals from the UK (4736 males and 5167 females) were included in this study. Brain age index (BAI), calculated by the difference between the predicted brain age by the algorithm and the chronological age, was considered as main outcome and presence of CMS, including type 2 diabetes mellitus (T2DM), hypertension, obesity, and underweight was considered as a predictor. Gender (males and females) and ethnicity (Korean and UK) were considered as effect modifiers. RESULTS: The presence of T2DM and hypertension was associated with a higher BAI regardless of gender and ethnicity (p < 0.001), except for hypertension in Korean males (p = 0.309). Among Koreans, there were interaction effects of gender and the presence of T2DM (p for T2DM*gender = 0.035) and hypertension (p for hypertension*gender = 0.046) on BAI in Koreans, suggesting that T2DM and hypertension are each associated with a higher BAI in females than in males. In contrast, among individuals from the UK, there were no differences in the effects of T2DM (p for T2DM*gender = 0.098) and hypertension (p for hypertension*gender = 0.203) on BAI between males and females. CONCLUSIONS: Our results highlight gender and ethnic differences as important factors in mediating the effects of CMS on brain age. Furthermore, these results suggest that ethnic- and gender-specific prevention strategies may be needed to protect against accelerated brain aging.

Novel neuroelectrophysiological age index associated with imaging features of brain aging and sleep disorders.

Sleep architecture and microstructures alter with aging and sleep disorder-led accelerated aging. We proposed a sleep EEG based brain age prediction model using convolutional neural networks. We then associated the estimated brain age index with brain structural aging features, sleep disorders and various sleep parameters. Our model also showed a higher BAI (predicted brain age minus chronological age) is associated with cortical thinning in various functional areas. We found a higher BAI for sleep disorder groups compared to healthy sleepers, as well as significant differences in the spectral pattern of EEG among different sleep disorders (lower power in slow and ϑ waves for sleep apnea vs. higher power in ß and σ for insomnia), suggesting sleep disorder-dependent pathomechanisms of aging. Our results demonstrate that the new EEG-BAI can be a biomarker reflecting brain health in normal and various sleep disorder subjects, and may be used to assess treatment efficacy.

A large, curated, open-source stroke neuroimaging dataset to improve lesion segmentation algorithms.

Accurate lesion segmentation is critical in stroke rehabilitation research for the quantification of lesion burden and accurate image processing. Current automated lesion segmentation methods for T1-weighted (T1w) MRIs, commonly used in stroke research, lack accuracy and reliability. Manual segmentation remains the gold standard, but it is time-consuming, subjective, and requires neuroanatomical expertise. We previously released an open-source dataset of stroke T1w MRIs and manually-segmented lesion masks (ATLAS v1.2, N = 304) to encourage the development of better algorithms. However, many methods developed with ATLAS v1.2 report low accuracy, are not publicly accessible or are improperly validated, limiting their utility to the field. Here we present ATLAS v2.0 (N = 1271), a larger dataset of T1w MRIs and manually segmented lesion masks that includes training (n = 655), test (hidden masks, n = 300), and generalizability (hidden MRIs and masks, n = 316) datasets. Algorithm development using this larger sample should lead to more robust solutions; the hidden datasets allow for unbiased performance evaluation via segmentation challenges. We anticipate that ATLAS v2.0 will lead to improved algorithms, facilitating large-scale stroke research.

Cilostazol Versus Aspirin on White Matter Changes in Cerebral Small Vessel Disease: A Randomized Controlled Trial.

BACKGROUND AND PURPOSE: Cerebral small vessel disease is characterized by progressive cerebral white matter changes (WMCs). This study aimed to compare the effects of cilostazol and aspirin on changes in WMC volume in patients with cerebral small vessel disease. METHODS: In a multicenter, double-blind, randomized controlled trial, participants with moderate or severe WMCs and at least one lacunar infarction detected on brain magnetic resonance imaging were randomly assigned to the cilostazol and aspirin groups in a 1:1 ratio. Cilostazol slow release (200 mg) or aspirin (100 mg) capsules were administered once daily for 2 years. The primary outcome was the change in WMC volume on magnetic resonance images from baseline to 2 years. Secondary imaging outcomes include changes in the number of lacunes or cerebral microbleeds, fractional anisotropy, and mean diffusivity on diffusion tensor images, and brain atrophy. Secondary clinical outcomes include all ischemic strokes, all ischemic vascular events, and changes in cognition, motor function, mood, urinary symptoms, and disability. RESULTS: Between July 2013 and August 2016, 256 participants were randomly assigned to the cilostazol (n=127) and aspirin (n=129) groups. Over 2 years, the percentage of WMC volume to total WM volume and the percentage of WMC volume to intracranial volume increased in both groups, but neither analysis showed significant differences between the groups. The peak height of the mean diffusivity histogram in normal-appearing WMs was significantly reduced in the aspirin group compared with the cilostazol group. Cilostazol significantly reduced the risk of ischemic vascular event compared with aspirin (0.5 versus 4.5 cases per 100 person-years; hazard ratio, 0.11 [95% CI, 0.02-0.89]). CONCLUSIONS: There was no significant difference between the effects of cilostazol and aspirin on WMC progression in patients with cerebral small vessel disease. Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT01932203.

Optimal Method for Fetal Brain Age Prediction Using Multiplanar Slices From Structural Magnetic Resonance Imaging.

The accurate prediction of fetal brain age using magnetic resonance imaging (MRI) may contribute to the identification of brain abnormalities and the risk of adverse developmental outcomes. This study aimed to propose a method for predicting fetal brain age using MRIs from 220 healthy fetuses between 15.9 and 38.7 weeks of gestational age (GA). We built a 2D single-channel convolutional neural network (CNN) with multiplanar MRI slices in different orthogonal planes without correction for interslice motion. In each fetus, multiple age predictions from different slices were generated, and the brain age was obtained using the mode that determined the most frequent value among the multiple predictions from the 2D single-channel CNN. We obtained a mean absolute error (MAE) of 0.125 weeks (0.875 days) between the GA and brain age across the fetuses. The use of multiplanar slices achieved significantly lower prediction error and its variance than the use of a single slice and a single MRI stack. Our 2D single-channel CNN with multiplanar slices yielded a significantly lower stack-wise MAE (0.304 weeks) than the 2D multi-channel (MAE = 0.979, p < 0.001) and 3D (MAE = 1.114, p < 0.001) CNNs. The saliency maps from our method indicated that the anatomical information describing the cortex and ventricles was the primary contributor to brain age prediction. With the application of the proposed method to external MRIs from 21 healthy fetuses, we obtained an MAE of 0.508 weeks. Based on the external MRIs, we found that the stack-wise MAE of the 2D single-channel CNN (0.743 weeks) was significantly lower than those of the 2D multi-channel (1.466 weeks, p < 0.001) and 3D (1.241 weeks, p < 0.001) CNNs. These results demonstrate that our method with multiplanar slices accurately predicts fetal brain age without the need for increased dimensionality or complex MRI preprocessing steps.

White matter hyperintensities segmentation using the ensemble U-Net with multi-scale highlighting foregrounds.

White matter hyperintensities (WMHs) are abnormal signals within the white matter region on the human brain MRI and have been associated with aging processes, cognitive decline, and dementia. In the current study, we proposed a U-Net with multi-scale highlighting foregrounds (HF) for WMHs segmentation. Our method, U-Net with HF, is designed to improve the detection of the WMH voxels with partial volume effects. We evaluated the segmentation performance of the proposed approach using the Challenge training dataset. Then we assessed the clinical utility of the WMH volumes that were automatically computed using our method and the Alzheimer's Disease Neuroimaging Initiative database. We demonstrated that the U-Net with HF significantly improved the detection of the WMH voxels at the boundary of the WMHs or in small WMH clusters quantitatively and qualitatively. Up to date, the proposed method has achieved the best overall evaluation scores, the highest dice similarity index, and the best F1-score among 39 methods submitted on the WMH Segmentation Challenge that was initially hosted by MICCAI 2017 and is continuously accepting new challengers. The evaluation of the clinical utility showed that the WMH volume that was automatically computed using U-Net with HF was significantly associated with cognitive performance and improves the classification between cognitive normal and Alzheimer's disease subjects and between patients with mild cognitive impairment and those with Alzheimer's disease. The implementation of our proposed method is publicly available using Dockerhub (https://hub.docker.com/r/wmhchallenge/pgs).

Fetal Cortical Plate Segmentation Using Fully Convolutional Networks With Multiple Plane Aggregation.

Fetal magnetic resonance imaging (MRI) has the potential to advance our understanding of human brain development by providing quantitative information of cortical plate (CP) development in vivo. However, for a reliable quantitative analysis of cortical volume and sulcal folding, accurate and automated segmentation of the CP is crucial. In this study, we propose a fully convolutional neural network for the automatic segmentation of the CP. We developed a novel hybrid loss function to improve the segmentation accuracy and adopted multi-view (axial, coronal, and sagittal) aggregation with a test-time augmentation method to reduce errors using three-dimensional (3D) information and multiple predictions. We evaluated our proposed method using the ten-fold cross-validation of 52 fetal brain MR images (22.9-31.4 weeks of gestation). The proposed method obtained Dice coefficients of 0.907 ± 0.027 and 0.906 ± 0.031 as well as a mean surface distance error of 0.182 ± 0.058 mm and 0.185 ± 0.069 mm for the left and right, respectively. In addition, the left and right CP volumes, surface area, and global mean curvature generated by automatic segmentation showed a high correlation with the values generated by manual segmentation (R 2 > 0.941). We also demonstrated that the proposed hybrid loss function and the combination of multi-view aggregation and test-time augmentation significantly improved the CP segmentation accuracy. Our proposed segmentation method will be useful for the automatic and reliable quantification of the cortical structure in the fetal brain.

Assessment of the efficacy of an attenuated live marker classical swine fever vaccine (Flc-LOM-BErns) in pregnant sows.

Here, we constructed an attenuated live marker classical swine fever (CSF) vaccine (Flc-LOM-BErns) to eradicate CSF. This was done by taking infectious clone Flc-LOM, which is based on an attenuated live CSF vaccine virus (LOM strain), and removing the full-length classical swine fever virus (CSFV) Erns sequences and the 3' end (52 base pairs) of the CSFV capsid. These regions were substituted with the full-length bovine viral diarrhoea virus (BVDV) Erns gene sequence and the 3' end (52 base pairs) of the BVDV capsid gene. Sows were vaccinated with the Flc-LOM-BErns vaccine 3 weeks before insemination and then challenged with virulent CSFV at the early, mid- or late stages of pregnancy. We then examined transplacental transmission to the foetuses. Piglets born to sows vaccinated with Flc-LOM-BErns did not show vertical infection, regardless of challenge time. In addition, CSFV challenge did not affect the delivery date, weight or length of the foetus. Pregnant sows inoculated with the Flc-LOM-BErns vaccine were anti-CSF Erns antibody-negative and anti-BVDV Erns antibody-positive. Challenge of pregnant sows with virulent CSFV resulted in anti-CSF Erns antibody positivity. These results strongly indicate that differential diagnosis can be conducted between the Flc-LOM-BErns vaccinated animal and virulent CSFV affected animal by detecting antibody against BVDV Erns or CSF Erns gene. Therefore, the Flc-LOM-BErns vaccine may fulfil the function of differential diagnosis which required for DIVA vaccine.

Clinical and Biomarker Characteristics According to Clinical Spectrum of Alzheimer's Disease (AD) in the Validation Cohort of Korean Brain Aging Study for the Early Diagnosis and Prediction of AD.

We aimed to present the study design of an independent validation cohort from the Korean Brain Aging Study for the Early Diagnosis and Prediction of Alzheimer's disease (AD) (KBASE-V) and to investigate the baseline characteristics of the participants according to the AD clinical spectrum. We recruited 71 cognitively normal (CN) participants, 96 with subjective cognitive decline (SCD), 72 with mild cognitive impairment (MCI), and 56 with AD dementia (ADD). The participants are followed for three years. The Consortium to Establish a Registry for AD scores was significantly different between all of the groups. The logical memory delayed recall scores were significantly different between all groups, except between the MCI and ADD groups. The Mini-Mental State Examination score, hippocampal volume, and cerebrospinal fluid (CSF) amyloid-ß42 level were significant difference among the SCD, MCI, and ADD groups. The frequencies of participants with amyloid pathology according to PET or CSF studies were 8.9%, 25.6%, 48.3%, and 90.0% in the CN, SCD, MCI, and ADD groups, respectively. According to ATN classification, A+/T+/N+ or A+/T+/N- was observed in 0%, 15.5%, 31.0%, and 78.3% in the CN, SCD, MCI, and ADD groups, respectively. The KBASE-V showed a clear difference according to the AD clinical spectrum in neuropsychological tests and AD biomarkers.

Automatic Segmentation of Corpus Callosum in Midsagittal Based on Bayesian Inference Consisting of Sparse Representation Error and Multi-Atlas Voting.

In this paper, we introduce a novel automatic method for Corpus Callosum (CC) in midsagittal plane segmentation. The robust segmentation of CC in midsagittal plane is key role for quantitative study of structural features of CC associated with various neurological disorder such as epilepsy, autism, Alzheimer's disease, and so on. Our approach is based on Bayesian inference using sparse representation and multi-atlas voting which both methods are used in various medical imaging, and show outstanding performance. Prior information in the proposed Bayesian inference is obtained from probability map generated from multi-atlas voting. The probability map contains the information of shape and location of CC of target image. Likelihood in the proposed Bayesian inference is obtained from gamma distribution function, generated from reconstruction errors (or sparse representation error), which are calculated in sparse representation of target patch using foreground dictionary and background dictionary each. Unlike the usual sparse representation method, we added gradient magnitude and gradient direction information to the patches of dictionaries and target, which had better segmentation performance than when not added. We compared three main segmentation results as follow: (1) the joint label fusion (JLF) method which is state-of-art method in multi-atlas voting based segmentation for evaluation of our method; (2) prior information estimated from multi-atlas voting only; (3) likelihood estimated from comparison of the reconstruction errors from sparse representation error only; (4) the proposed Bayesian inference. The methods were evaluated using two data sets of T1-weighted images, which one data set consists of 100 normal young subjects and the other data set consist of 25 normal old subjects and 22 old subjects with heavy drinker. In both data sets, the proposed Bayesian inference method has significantly the best segmentation performance than using each method separately.

Multi-Modality Sparse Representation for Alzheimer's Disease Classification.

BACKGROUND: Alzheimer's disease (AD) and mild cognitive impairment (MCI) are age-related neurodegenerative diseases characterized by progressive loss of memory and irreversible cognitive functions. The hippocampus, a brain area critical for learning and memory processes, is especially susceptible to damage at early stages of AD. OBJECTIVE: We aimed to develop prediction model using a multi-modality sparse representation approach. METHODS: We proposed a sparse representation approach to the hippocampus using structural T1-weighted magnetic resonance imaging (MRI) and 18-fluorodeoxyglucose-positron emission tomography (FDG-PET) to distinguish AD/MCI from healthy control subjects (HCs). We considered structural and function information for the hippocampus and applied a sparse patch-based approach to effectively reduce the dimensions of neuroimaging biomarkers. RESULTS: In experiments using Alzheimer's Disease Neuroimaging Initiative data, our proposed method demonstrated more reliable than previous classification studies. The effects of different parameters on segmentation accuracy were also evaluated. The mean classification accuracy obtained with our proposed method was 0.94 for AD/HCs, 0.82 for MCI/HCs, and 0.86 for AD/MCI. CONCLUSION: We extracted multi-modal features from automatically defined hippocampal regions of training subjects and found this method to be discriminative and robust for AD and MCI classification. The extraction of features in T1 and FDG-PET images is expected to improve classification performance due to the relationship between brain structure and function.

C-terminal substitution of HBV core proteins with those from DHBV reveals that arginine-rich 167RRRSQSPRR175 domain is critical for HBV replication.

To investigate the contributions of carboxyl-terminal nucleic acid binding domain of HBV core (C) protein for hepatitis B virus (HBV) replication, chimeric HBV C proteins were generated by substituting varying lengths of the carboxyl-terminus of duck hepatitis B virus (DHBV) C protein for the corresponding regions of HBV C protein. All chimeric C proteins formed core particles. A chimeric C protein with 221-262 amino acids of DHBV C protein, in place of 146-185 amino acids of the HBV C protein, supported HBV pregenomic RNA (pgRNA) encapsidation and DNA synthesis: 40% amino acid sequence identity or 45% homology in the nucleic-acid binding domain of HBV C protein was sufficient for pgRNA encapsidation and DNA synthesis, although we predominantly detected spliced DNA. A chimeric C protein with 221-241 and 251-262 amino acids of DHBV C, in place of HBV C 146-166 and 176-185 amino acids, respectively, could rescue full-length DNA synthesis. However, a reciprocal C chimera with 242-250 of DHBV C ((242)RAGSPLPRS(250)) introduced in place of 167-175 of HBV C ((167)RRRSQSPRR(175)) significantly decreased pgRNA encapsidation and DNA synthesis, and full-length DNA was not detected, demonstrating that the arginine-rich (167)RRRSQSPRR(175) domain may be critical for efficient viral replication. Five amino acids differing between viral species (underlined above) were tested for replication rescue; R169 and R175 were found to be important.

Establishment and characterization of an infectious cDNA clone of a classical swine fever virus LOM strain.

Classical swine fever virus (CSFV) causes a highly contagious disease among swine that has an important economic impact worldwide. CSFV strain LOM is an attenuated virus of low virulent strain of Miyagi isolated from Japan in 1956. Eight DNA fragments representing the genome of the CSFV strain LOM were obtained by RT-PCR. These were used to determine the complete nucleotide sequence and construct a full-length cDNA clone which was called Flc-LOM. Sequence analysis of the recombinant clone (Flc-LOM) revealed the presence of eight mutations, resulting in two amino acid substitutions, when compared to the parental sequence. RNA transcripts of both LOM and Flc-LOM were directly infectious in PK-15 cells. The rescued Flc-LOM virus grew more slowly than the parental virus, LOM, in the cells. Intramuscular immunization with Flc-LOM was safe and highly immunogenic in pigs; no clinical signs or virus transmission to sentinel animals were observed after 35 days. CSFV-specific neutralizing antibodies were detected 14 days post-infection. After challenge with the virulent CSFV strain SW03, pigs immunized with Flc-LOM were shown to be fully protected. Thus, our newly established infectious clone of CSFV, Flc-LOM, could serve as a vaccine candidate.

The conformation and CETP inhibitory activity of [10]-dehydrogingerdione isolated from Zingiber officinale.

In the course of searching for cholesteryl ester transfer protein (CETP) inhibitors from natural sources, a new type of CETP inhibitor, [10]-dehydrogingerdione (1), was isolated from the extract of rhizomes of Zingiber officinale Roscoe. By NMR spectroscopic analysis of its (1)HNMR, (13)C-NMR, and (1)H-(1)H COSY, HMBC, HMQC and NOESY, more precise structure, compared with its originally proposed structures, of [10]-dehydrogingerdione has been elucidated. This active compound inhibited human plasma CETP with IC(50) values of 35 µM.

Toll-like receptor 4-mediated immunoregulation by the aqueous extract of Mori Fructus.

The aqueous extract of Mori Fructus (MF) exerts a change of phenotype and a cytoprotective effect in macrophages. The present study was carried out to investigate the immunomodulating activity of MF on the expression of nitric oxide (NO), tumor necrosis factor alpha (TNF-alpha), co-stimulatory molecules and also interferon-gamma (IFN-gamma) in macrophages and splenocytes. Toll-like receptor 4 (TLR4) is a promising molecular target for immune-modulating drugs. It was hypothesized that one possible upstream signaling pathway leading to immunoregulation of MF may be mediated by TLRs. Multiple signaling molecules (NF-kappaB, ERK1/2, p38 and JNK) of the TLR4 signaling pathway were also detected. It was found that MF increased NO production and TNF-alpha secretion in RAW 264.7 and peritoneal macrophages, co-stimulatory molecules expression in peritoneal macrophages and IFN-gamma expression in splenocytes. Further studies indicated that MF could significantly induce the phosphorylation of signal molecules of MAPKs and the degradation of IkappaBalpha which finally led to the activation and nuclear translocation of nuclear factor-kappaB (NF-kappaB) for the target gene expression. All those notions disclosed that the aqueous extract MF is a new TLR4 activator, which induces a Th1 immune response as a consequence of induction of cytokines secretion, especially TNF-alpha and IFN-gamma.

Modulation of hepatitis B virus replication by expression of polymerase-surface fusion protein through splicing: implications for viral persistence.

In hepatitis B virus (HBV)-infected livers and -transfected hepatoma cells, spliced transcripts are not essential for HBV replication. However, their ability to modulate HBV replication has not been clearly elucidated. In the current study, we found that the polymerase-surface (PS) fusion protein, generated from a spliced HBV transcript, colocalized with the nuclear pore complex, vimentin, microtubules, and the endoplasmic reticulum (ER) in the perinuclear region of transfected cells. We found that PLC/PRF/5-hepatoma cells expressed PS transcript and PS protein. Hepatitis B surface antigen (HBs) secretion, core particle formation, and HBV DNA synthesis were inhibited by the expression of PS transcript and PS protein, and by expression of PS transcript alone, suggesting that HBV replication is modulated by splicing. Our results suggest that splicing may be one of the outcomes of the host-virus interaction.

Secretion of biologically active recombinant human granulocyte-macrophage colony-stimulating factor by transduced gastric cancer cells.

PURPOSE: Gastric cancer has the highest incidence rate among cancers in Asia. The advanced type of signet ring cell carcinoma has poor prognosis compared to other types of gastric cancer. The immuno-gene therapy with cytokine-based tumor vaccines has not yet been investigated for gastric cancer. The granulocyte macrophage colony-stimulating factor (GM-CSF)-based tumor vaccine has been demonstrated as the most potent stimulator for specific and long-lasting systemic tumor immunity. MATERIALS AND METHODS: In the present study, KATO III cells, the human signet ring cell gastric carcinoma cell line, were genetically modified by the transduction with the human GM-CSF cDNA or the modified hGM-CSF in replication-deficient retroviruses. The genomic integrations and mRNA expressions of the transgenes were determined by Southern and Northern blot analyses. RESULTS: Wild type (wt) or modified hGM-CSF was integrated into the genome of KATO III cells. The modified hGM-CSF mRNA was more stable than that of wt. The KATO III cells with the modified hGM-CSF produced higher level of hGM-CSF (12.4-19 ng/10(6)cells/48hrs) than that with wt hGM-CSF, when determined by enzyme-linked immunosorbent assay (ELISA). The secreted recombinant hGM-CSF could support the proliferation of the GM-CSF-dependent cell line, indicating that the hGM-CSF secreted by the transduced KATO III cells has biological activities. Irradiated, transduced KATO III cells continued to secret hGM-CSF without proliferation. CONCLUSION: Our results suggest that GM-CSF secreting KATO III cells could be tested for the treatment of gastric cancer as an allogeneic tumor vaccine as a part of immunotherapeutic treatment.