An update of aurones: food resource, health benefit, biosynthesis and application.

Aurones are a subclass of active flavonoids characterized with a scaffold of 2-benzylidene-3(2H)-benzofuranone. This type of chemicals are widely distributed in fruit, vegetable and flower, and contribute to human health. In this review, we summarize the natural aurones isolated from dietary plants. Their positive effects on immunomodulation, antioxidation, cancer prevention as well as maintaining the health status of cardiovascular, nervous system and liver organs are highlighted. The biosynthesis strategies of plant-derived aurones are elaborated to provide solutions for their limited natural abundance. The potential application of natural aurones in food coloration are also discussed. This paper combines the up-to-date information and gives a full image of dietary aurones.

The molecular mechanism for inhibiting the growth of nasopharyngeal carcinoma cells using polymethoxyflavonoids purified from pericarp of Citrus reticulata 'Chachi' via HSCCC.

Polymethoxyflavonoids (PMFs), the main bioactive compounds naturally occurring in the pericarp of Citrus reticulata 'Chachi' (CRCP), possess significant antitumor action. However, the action of PMFs in nasopharyngeal carcinoma (NPC) is currently unknown. The present research study was conducted to investigate the inhibitory mechanisms of PMFs from CRCP on NPC growth in vivo and in vitro. In our research, we used high-speed counter-current chromatography (HSCCC) to separate four PMFs (nobiletin (NOB), 3,5,6,7,8,3',4'-heptamethoxyflavone (HMF), tangeretin (TGN), and 5-hydroxy-6,7,8,3',4'-pentamethoxyflavone (5-HPMF)) from CRCP. CCK-8 assay was used to preliminarily screen cell viability following exposure to the four PMFs. Colony formation, Hoechst-33258 staining, transwell, and wound scratch assays were performed to assess the anti-proliferation, invasion, migration, and apoptosis-inducing effects of HMF on NPC cells. NPC tumors in xenograft tumor transplantation experiments were also established to explore the effect of HMF (100 and 150 mg/kg/day) on NPC. The histopathological changes in the treated rats were observed by H&E staining and Ki-67 detection by immunohistochemical techniques. The expressions of P70S6K, p-P70S6K, S6, p-S6, COX-2, p53, and p-p53 were measured by Western blot. The four PMFs were obtained with high purity (>95.0%). The results of the preliminary screening by CCK-8 assay suggested that HMF had the strongest inhibitory effect on NPC cell growth. The results of the colony formation, Hoechst-33258 staining, transwell, and wound scratch assays indicated that HMF had significant anti-proliferation, invasion, migration, and apoptosis-inducing ability in NPC cells. Moreover, HMF suppressed NPC tumor growth in xenograft tumor transplantation experiments. Further investigation suggested that HMF regulated NPC cells proliferation, apoptosis, migration, and invasion by activating AMPK-dependent signaling pathways. In conclusion, HMF-induced AMPK activation inhibited NPC cell growth, invasion, and metastatic potency by downregulating the activation of the mTOR signaling pathway and COX-2 protein levels, as well as enhancing the p53 phosphorylation level. Our study provides a crucial experimental basis for the clinical treatment of NPC, as well as the development and utilization of PMFs from CRCP.

Association between low levels of anti-inflammatory cytokines during pregnancy and postpartum depression.

AIM: Previous studies based on a relatively limited number of subjects have indicated potential associations between plasma cytokine concentrations in perinatal women and postpartum depression (PPD). This report aimed to examine alterations in cytokine levels during pregnancy and after delivery by measuring nine cytokines in prenatal and postnatal plasma samples in a large cohort. METHODS: A nested, case-control study was conducted using plasma samples from 247 women with PPD (Edinburgh Postnatal Depression Scale: EPDS ≥9) and 243 age-matched control (EPDS ≤2) women from among perinatal women who participated in the Tohoku Medical Megabank three-generation cohort. Concentrations of nine plasma cytokines (IFN-γ, IL-1ß, IL-4, IL-6, IL-10, IL-12p40, IL-12p70, IL-13, and TNF-α) in plasma collected at the time of enrollment during pregnancy and 1 month after delivery were determined using an immunoassay kit. RESULTS: Cross-sectional comparisons of cytokine levels during pregnancy and after delivery indicated that the PPD group maintained significantly lower plasma IL-4 levels during pregnancy and after delivery than the control group, and that plasma IL-4 levels decreased significantly during pregnancy regardless of PPD status. Plasma IL-10 levels were significantly higher during pregnancy than after delivery only among healthy controls, and plasma IL-10 levels were significantly higher in the control group than in the PPD group. Moreover, IFN-γ, IL-6, IL-12p40, and TNF-α levels were significantly lower during pregnancy compared with after delivery regardless of PPD status. CONCLUSIONS: These results suggest a potential protective effect of the anti-inflammatory cytokines IL-4 and IL-10 during pregnancy against the development of PPD.

Progress of research on the role of active ingredients of Citri Reticulatae Pericarpium in liver injury.

BACKGROUND: Liver is a vital organ responsible for metabolizing and detoxifying both endogenous and exogenous substances in the body. However, it is susceptible to damage from chemical and natural toxins. The high incidence and mortality rates of liver disease and its associated complications impose a significant economic burden and survival pressure on patients and their families. Various liver diseases exist, including cholestasis, viral and non-viral hepatitis, fatty liver disease, drug-induced liver injury, alcoholic liver injury, and severe end-stage liver diseases such as cirrhosis, hepatocellular carcinoma (HCC), and cholangiocellular carcinoma (CCA). Recent research has shown that flavonoids found in Citri Reticulatae Pericarpium (CRP) have the potential to normalize blood glucose, cholesterol levels, and liver lipid levels. Additionally, these flavonoids exhibit anti-inflammatory properties, prevent oxidation and lipid peroxidation, and reduce liver toxicity, thereby preventing liver injury. Given these promising findings, it is essential to explore the potential of active components in CRP for developing new drugs to treat liver diseases. OBJECTIVE: Recent studies have revealed that flavonoids, including hesperidin (HD), hesperetin (HT), naringenin (NIN), nobiletin (NOB), naringin (NRG), tangerine (TN), and erodcyol (ED), are the primary bioactive components in CRP. These flavonoids exhibit various therapeutic effects on liver injury, including anti-oxidative stress, anti-cytotoxicity, anti-inflammatory, anti-fibrosis, and anti-tumor mechanisms. In this review, we have summarized the research progress on the hepatoprotective effects of HD, HT, NIN, NOB, NRG, TN, ED and limonene (LIM), highlighting their underlying molecular mechanisms. Despite their promising effects, the current clinical application of these active ingredients in CRP has some limitations. Therefore, further studies are needed to explore the full potential of these flavonoids and develop new therapeutic strategies for liver diseases. METHODS: For this review, we conducted a systematic search of three databases (ScienceNet, PubMed, and Science Direct) up to July 2022, using the search terms "CRP active ingredient," "liver injury," and "flavonoids." The search data followed the PRISMA standard. RESULTS: Our findings indicate that flavonoids found in CRP can effectively reduce drug-induced liver injury, alcoholic liver injury, and non-alcoholic liver injury. These therapeutic effects are mainly attributed to the ability of flavonoids to improve liver resistance to oxidative stress and inflammation while normalizing cholesterol and liver lipid levels by exhibiting anti-free radical and anti-lipid peroxidation properties. CONCLUSION: Our review provides new insights into the potential of active components in CRP for preventing and treating liver injury by regulating various molecular targets within different cell signaling pathways. This information can aid in the development of novel therapeutic strategies for liver disease.

N-Acetylcysteine Suppresses Microglial Inflammation and Induces Mortality Dose-Dependently via Tumor Necrosis Factor-α Signaling.

N-acetylcysteine (NAC) is an antioxidant that prevents tumor necrosis factor (TNF)-α-induced cell death, but it also acts as a pro-oxidant, promoting reactive oxygen species independent apoptosis. Although there is plausible preclinical evidence for the use of NAC in the treatment of psychiatric disorders, deleterious side effects are still of concern. Microglia, key innate immune cells in the brain, play an important role in inflammation in psychiatric disorders. This study aimed to investigate the beneficial and deleterious effects of NAC on microglia and stress-induced behavior abnormalities in mice, and its association with microglial TNF-α and nitric oxide (NO) production. The microglial cell line MG6 was stimulated by Escherichia coli lipopolysaccharide (LPS) using NAC at varying concentrations for 24 h. NAC inhibited LPS-induced TNF-α and NO synthesis, whereas high concentrations (≥30 mM) caused MG6 mortality. Intraperitoneal injections of NAC did not ameliorate stress-induced behavioral abnormalities in mice, but high-doses induced microglial mortality. Furthermore, NAC-induced mortality was alleviated in microglial TNF-α-deficient mice and human primary M2 microglia. Our findings provide ample evidence for the use of NAC as a modulating agent of inflammation in the brain. The risk of side effects from NAC on TNF-α remains unclear and merits further mechanistic investigations.

Sex-Specific Differences in the Transcriptome of the Human Dorsolateral Prefrontal Cortex in Schizophrenia.

Schizophrenia presents clinical and biological differences between males and females. This study investigated transcriptional profiles in the dorsolateral prefrontal cortex (DLPFC) using postmortem data from the largest RNA-sequencing (RNA-seq) database on schizophrenic cases and controls. Data for 154 male and 113 female controls and 160 male and 93 female schizophrenic cases were obtained from the CommonMind Consortium. In the RNA-seq database, the principal component analysis showed that sex effects were small in schizophrenia. After we analyzed the impact of sex-specific differences on gene expression, the female group showed more significantly changed genes compared with the male group. Based on the gene ontology analysis, the female sex-specific genes that changed were overrepresented in the mitochondrion, ATP (phosphocreatine and adenosine triphosphate)-, and metal ion-binding relevant biological processes. An ingenuity pathway analysis revealed that the differentially expressed genes related to schizophrenia in the female group were involved in midbrain dopaminergic and γ-aminobutyric acid (GABA)-ergic neurons and microglia. We used methylated DNA-binding domain-sequencing analyses and microarray to investigate the DNA methylation that potentially impacts the sex differences in gene transcription using a maternal immune activation (MIA) murine model. Among the sex-specific positional genes related to schizophrenia in the PFC of female offspring from MIA, the changes in the methylation and transcriptional expression of loci ACSBG1 were validated in the females with schizophrenia in independent postmortem samples by real-time PCR and pyrosequencing. Our results reveal potential genetic risks in the DLPFC for the sex-dependent prevalence and symptomology of schizophrenia.

Use of Specific Duodenal Dose Constraints During Treatment Planning Reduces Toxicity After Definitive Paraaortic Radiation Therapy for Cervical Cancer.

PURPOSE: This study aimed to validate the safety of paraaortic nodal (PAN) radiation therapy (RT) for patients with cervical cancer when the duodenal dose is limited to V55 < 15 cm3 and V60 < 2 cm3. METHODS AND MATERIALS: A total of 97 patients who were treated with RT for cervical cancer between 2010 and 2018 received at least 56 Gy to grossly involved PANs. Patients were treated with concurrent chemoradiation (n = 88; 91%), with 93% of patients (n = 90) receiving intensity modulated RT to the initial PAN field and 98% (n = 95) receiving intensity modulated RT to a sequential PAN boost. The V55 < 15 cm3 and V60 <2 cm3 criteria were implemented in 2014. Normal tissues were contoured on computed tomography (CT) simulation data sets, and the duodenum was contoured from the gastric outlet to the duodenojejunal flexure. Sixty-six patients (68%) had a resimulation scan after approximately 20 fractions. Composite duodenal doses were calculated using the initial CT scan for 50 patients (52%) and the resimulation CT scan for 47 patients (48%) depending on the anatomic changes throughout treatment. RESULTS: The median duodenal V55 was 3.5 cm3 (interquartile range [IQR], 0.2-8.1 cm3) and the median V60 was 0.3 cm3 (IQR, 0.0-1.8). Constraints were exceeded in 18 patients, of whom 16 patients (89%) had been treated before 2014. Treatment for the 2 patients treated after 2014 was complicated by significant weight loss and reduced anterior-posterior diameter, which likely overestimated the true dose on the composite plan. Only 1 patient experienced grade 3 duodenal toxicity (stricture requiring endoscopic balloon dilation 3 months after treatment); however, the stricture was outside of the high-dose boost volume, and the patient had a history of gastritis. Six patients (6%) had a first recurrence within the PAN region. CONCLUSIONS: Limiting the duodenal dose to V55 < 15 cm3 and V60 < 2 cm3 for patients with cervical cancer and PAN involvement is feasible, and minimizes duodenal toxicity while maintaining acceptable local control rates.

Definitive radiotherapy in lieu of systemic therapy for oligometastatic renal cell carcinoma: a single-arm, single-centre, feasibility, phase 2 trial.

BACKGROUND: The role of radiotherapy in metastatic renal cell carcinoma is controversial. We prospectively tested the feasibility and efficacy of radiotherapy to defer systemic therapy for patients with oligometastatic renal cell carcinoma. METHODS: This single-arm, phase 2, feasibility trial was done at one centre in the USA (The MD Anderson Cancer Center, Houston, TX, USA). Patients (aged ≥18 years) with five or fewer metastatic lesions, an Eastern Cooperative Oncology Group status of 0-2, and no more than one previous systemic therapy (if this therapy was stopped at least 1 month before enrolment) without limitations on renal cell carcinoma histology were eligible for inclusion. Patients were treated with stereotactic body radiotherapy (defined as ≤5 fractions with ≥7 Gy per fraction) to all lesions and maintained off systemic therapy. When lesion location precluded safe stereotactic body radiotherapy, patients were treated with hypofractionated intensity-modulated radiotherapy regimes consisting of 60-70 Gy in ten fractions or 52·5-67·5 Gy in 15 fractions. Additional rounds of radiotherapy were allowed to treat subsequent sites of progression. Co-primary endpoints were feasibility (defined as all planned radiotherapy completed with <7 days unplanned breaks) and progression-free survival. All efficacy analyses were intention-to-treat. Safety was analysed in the as-treated population. A second cohort, with the aim of assessing the feasibility of sequential stereotactic body radiotherapy alone in patients with low-volume metastatic disease, was initiated and will be reported separately. This study is registered with ClinicalTrials.gov, NCT03575611. FINDINGS: 30 patients (six [20%] women) were enrolled from July 13, 2018, to Sept 18, 2020. All patients had clear cell histology and had a nephrectomy before enrolment. All patients completed at least one round of radiotherapy with less than 7 days of unplanned breaks. At a median follow-up of 17·5 months (IQR 13·2-24·6), median progression-free survival was 22·7 months (95% CI 10·4-not reached; 1-year progression-free survival 64% [95% CI 48-85]). Three (10%) patients had severe adverse events: two grade 3 (back pain and muscle weakness) and one grade 4 (hyperglycaemia) adverse events were observed. There were no treatment-related deaths. INTERPRETATION: Sequential radiotherapy might facilitate deferral of systemic therapy initiation and could allow sustained systemic therapy breaks for select patients with oligometastatic renal cell carcinoma. FUNDING: Anna Fuller Foundation, the Cancer Prevention and Research Institute of Texas (CPRIT), and the National Cancer Institute.

Tumor necrosis factor α negatively regulates the retrieval and reconsolidation of hippocampus-dependent memory.

Neural inflammation is associated with cognitive decline, especially learning and memory. Tumor necrosis factor α (TNFα) is a major cytokine generated during neuroinflammation. Previous studies indicated that TNFα impairs hippocampus-dependent memory including contextual fear and spatial memories. However, it is unknown which memory processes are impaired by TNFα. Here, we show that TNFα blocked the retrieval and reconsolidation of contextual fear and spatial memories. Micro-infusion of TNFα into the dorsal hippocampus at 6-18 h before retrieval impaired the retrieval of contextual fear memory, although micro-infusion before contextual fear conditioning had no effect on memory formation. Interestingly, hippocampal TNFα micro-infusion before memory retrieval decreased freezing responses, even at 24 h after retrieval, suggesting that TNFα impairs the reconsolidation of contextual fear memory. Similarly, hippocampal TNFα micro-infusion impaired the retrieval and reconsolidation of spatial memory in the Morris water maze. Consistent with these observations, hippocampal TNFα micro-infusion before retrieval blocked the induction of c-fos expression in the hippocampus, which is a marker of neural activation, in response to the retrieval of contextual fear memory. Collectively, our findings indicate that TNFα negatively regulates the retrieval and reconsolidation of hippocampus-dependent memory.

Automatic Segmentation Using Deep Learning to Enable Online Dose Optimization During Adaptive Radiation Therapy of Cervical Cancer.

PURPOSE: This study investigated deep learning models for automatic segmentation to support the development of daily online dose optimization strategies, eliminating the need for internal target volume expansions and thereby reducing toxicity events of intensity modulated radiation therapy for cervical cancer. METHODS AND MATERIALS: The cervix-uterus, vagina, parametrium, bladder, rectum, sigmoid, femoral heads, kidneys, spinal cord, and bowel bag were delineated on 408 computed tomography (CT) scans from patients treated at MD Anderson Cancer Center (n = 214), Polyclinique Bordeaux Nord Aquitaine (n = 30), and enrolled in a Medical Image Computing & Computer Assisted Intervention challenge (n = 3). The data were divided into 255 training, 61 validation, 62 internal test, and 30 external test CT scans. Two models were investigated: the 2-dimensional (2D) DeepLabV3+ (Google) and 3-dimensional (3D) Unet in RayStation (RaySearch Laboratories). Three intensity modulated radiation therapy plans were generated on each CT of the internal and external test sets using either the manual, 2D model, or 3D model segmentations. The dose constraints followed the External beam radiochemotherapy and MRI based adaptive BRAchytherapy in locally advanced CErvical cancer (EMBRACE) II protocol, with reduced margins of 5 and 3 mm for the target and nodal planning target volume. Geometric discrepancies between the manual and predicted contours were assessed using the Dice similarity coefficient (DSC), distance-to-agreement, and Hausdorff distance. Dosimetric discrepancies between the manual and model doses were assessed using clinical indices on the manual contours and the gamma index. Interobserver variability was assessed for the cervix-uterus, parametrium, and vagina for the definition of the primary clinical target volume (CTVT) on the external test set. RESULTS: Average DSCs across all organs were 0.67 to 0.96, 0.71 to 0.97, and 0.42 to 0.92 for the 2D model and 0.66 to 0.96, 0.70 to 0.97, and 0.37 to 0.93 for the 3D model on the validation, internal, and external test sets. Average DSCs of the CTVT were 0.88 and 0.81 for the 2D model and 0.87 and 0.82 for the 3D model on the internal and external test sets. Interobserver variability of the CTVT corresponded to a mean (range) DSC of 0.85 (0.77-0.90) on the external test set. On the internal test set, the doses from the 2D and 3D model contours provided a CTVT V42.75 Gy >98% for 98% and 91% of the CT scans, respectively. On the external test set, these percentages were increased to 100% and 93% for the 2D and 3D models, respectively. CONCLUSIONS: The investigated models provided auto-segmentation of the cervix anatomy with similar performances on 2 institutional data sets and reasonable dosimetric accuracies using small planning target volume margins, paving the way to automatic online dose optimization for advanced adaptive radiation therapy strategies.

Dynamics of Platelet Behaviors as Defenders and Guardians: Accumulations in Liver, Lung, and Spleen in Mice.

Systemic platelet behaviors in experimental animals are often assessed by infusion of isotope-labeled platelets and measuring them under anesthesia. However, such procedures alter, therefore may not reveal, real-life platelet behaviors. 5-Hydroxytryptamine (5HT or serotonin) is present within limited cell-types, including platelets. In our studies, by measuring 5HT as a platelet-marker in non-anesthetized mice, we identified stimulation- and time-dependent accumulations in liver, lung, and/or spleen as important systemic platelet behaviors. For example, intravenous, intraperitoneal, or intragingival injection of lipopolysaccharide (LPS, a cell-wall component of Gram-negative bacteria), interleukin (IL)-1, or tumor necrosis factor (TNF)-α induced hepatic platelet accumulation (HPA) and platelet translocation into the sinusoidal and perisinusoidal spaces or hepatocytes themselves. These events occurred "within a few hours" of the injection, caused hypoglycemia, and exhibited protective or causal effects on hepatitis. Intravenous injection of larger doses of LPS into normal mice, or intravenous antigen-challenge to sensitized mice, induced pulmonary platelet accumulation (PPA), as well as HPA. These reactions occurred "within a few min" of the LPS injection or antigen challenge and resulted in shock. Intravenous injection of 5HT or a catecholamine induced a rapid PPA "within 6 s." Intravenous LPS injection, within a minute, increased the pulmonary catecholamines that mediate the LPS-induced PPA. Macrophage-depletion from liver and spleen induced "day-scale" splenic platelet accumulation, suggesting the spleen is involved in clearing senescent platelets. These findings indicate the usefulness of 5HT as a marker of platelet behaviors, and provide a basis for a discussion of the roles of platelets as both "defenders" and "guardians."

The VEGF gene polymorphism impacts brain volume and arterial blood volume.

Vascular endothelial growth factor (VEGF) plays a critical role in the angiogenesis and proliferation of various types of cells such as neurons, astroglia, and endothelial cells in the brain. A common polymorphism in the VEGF gene (-2578 C/A) is associated with circulating VEGF levels, cancers and Alzheimer's disease. Nonetheless, the effects of this polymorphism on normal human brain volume, arterial blood volume, and blood supply remain unclear. In this study, the effects of this polymorphism on the total gray matter volume (TGMV) and total white matter volume (TWMV) using T1-weighted structural images and the total arterial blood volume (TABV) and mean cerebral blood flow (mCBF) during rest using arterial spin labeling (ASL) in 765 young adult humans were investigated. Voxel-by-voxel whole-brain analyses of these measures were also performed. Multiple regression analyses with age and sex as covariates revealed that the VEGF genotype (number of C alleles) was significantly and positively correlated with TGMV, TWMV, and TABV as well as with regional gray and white matter volumes in widespread areas and regional arterial blood volume in some areas with high arterial blood volume. However, these regional associations were not seen when the corresponding global signal was included as a covariate in the multiple regression analyses, indicating that we failed to obtain evidence of region-specific associations between these brain measures and the genotype. The results suggest that the VEGF-2578C allele, is associated with changes in the vascular system that lead to increased blood volume and larger brain volume. Hum Brain Mapp 38:3516-3526, 2017. © 2017 Wiley Periodicals, Inc.

Pulmonary platelet accumulation induced by catecholamines: Its involvement in lipopolysaccharide-induced anaphylaxis-like shock.

Intravenously injected lipopolysaccharides (LPS) rapidly induce pulmonary platelet accumulation (PPA) and anaphylaxis-like shock (ALS) in mice. Macrophages reportedly release catecholamines rapidly upon stimulation with LPS. Here, we examined the involvement of macrophage-derived catecholamines in LPS-induced PPA and ALS. A catecholamine or Klebsiella O3 (KO3) LPS was intravenously injected into mice, with 5-hydroxytryptamine in the lung being measured as a platelet marker. The tested catecholamines induced PPA, leading to shock. Their minimum shock-inducing doses were at the nmol/kg level. The effects of epinephrine and norepinephrine were inhibited by prazosin (α1 antagonist) and by yohimbine (α2 antagonist), while dopamine's were inhibited only by prazosin. Use of synthetic adrenergic α1- and/or α2-agonists, platelet- or macrophage-depleted mice, a complement C5 inhibitor and C5-deficient mice revealed that (a) α2-receptor-mediated PPA and shock depend on both macrophages and complements, while α1-receptor-mediated PPA and shock depend on neither macrophages nor complements, (b) the PPA and ALS induced by KO3-LPS depend on α1- and α2-receptors, macrophages, and complements, and (c) KO3-LPS-induced PPA is preceded by catecholamines decreasing in serum. Together, these results suggest the following. (i) Catecholamines may stimulate macrophages and release complement C5 via α2-receptors. (ii) Macrophage-derived catecholamines may mediate LPS-induced PPA and ALS. (iii) Moderate PPA may serve as a defense mechanism to remove excess catecholamines from the circulation by promoting their rapid uptake, thus preventing excessive systemic effects. (iv) The present findings might provide an insight into possible future pharmacological strategies against such diseases as shock and acute respiratory distress syndrome.

Microglial production of TNF-alpha is a key element of sustained fear memory.

The proinflammatory cytokine productions in the brain are altered in a process of fear memory formation, indicating a possibility that altered microglial function may contribute to fear memory formation. We aimed to investigate whether and how microglial function contributes to fear memory formation. Expression levels of M1- and M2-type microglial marker molecules in microglia isolated from each conditioned mice group were assessed by real-time PCR and immunohistochemistry. Levels of tumor necrosis factor (TNF)-α, but not of other proinflammatory cytokines produced by M1-type microglia, increased in microglia from mice representing retention of fear memory, and returned to basal levels in microglia from mice representing extinction of fear memory. Administration of inhibitors of TNF-α production facilitated extinction of fear memory. On the other hand, expression levels of M2-type microglia-specific cell adhesion molecules, CD206 and CD209, were decreased in microglia from mice representing retention of fear memory, and returned to basal levels in microglia from mice representing extinction of fear memory. Our findings indicate that microglial TNF-α is a key element of sustained fear memory and suggest that TNF-α inhibitors can be candidate molecules for mitigating posttraumatic reactions caused by persistent fear memory.

Fatty acid binding protein 7 regulates phagocytosis and cytokine production in Kupffer cells during liver injury.

Kupffer cells (KCs) are involved in the progression of liver diseases such as hepatitis and liver cancer. Several members of the fatty acid binding proteins (FABPs) are expressed by tissue macrophages, and FABP7 is localized only in KCs. To clarify the role of FABP7 in the regulation of KC function, we evaluated pathological changes of Fabp7 knockout mice during carbon tetrachloride-induced liver injury. During liver injury in Fabp7 knockout mice, serum liver enzymes were increased, cytokine expression (tumor necrosis factor-α, monocyte chemoattractant protein-1, and transforming growth factor-ß) was decreased in the liver, and the number of KCs in the liver necrotic area was significantly decreased. Interestingly, in the FABP7-deficient KCs, phagocytosis of apoptotic cells was impaired, and expression of the scavenger receptor CD36 was markedly decreased. In chronic liver injury, Fabp7 knockout mice showed less fibrogenic response to carbon tetrachloride compared with wild-type mice. Taken together, FABP7 is involved in the liver injury process through its regulation of KC phagocytic activity and cytokine production. Such modulation of KC function by FABP7 may provide a novel therapeutic approach to the treatment of liver diseases.

Prime role of bone IL-1 in mice may lie in emergency Ca(2+)-supply to soft tissues, not in bone-remodeling.

IL-1 and TNF-α are thought to be important bone-remodeling regulators. However, mice lacking either them or their receptors reportedly grow healthily. Here, we examined the roles of IL-1 and TNF-α in bone. Although a significant IL-1 level was detected in the tibia of non-stimulated wild-type (WT) mice, no significant physicochemical, morphological, or histological defects were detected in the tibias in mice lacking IL-1 (both α and ß types) (IL-1KO) or lacking both IL-1 and TNF-α (IL-1/TNF-αKO). Injection of sub-lethal doses of lipopolysaccharide (LPS) into WT mice induced a transient hypocalcemia, increased IL-1 (in the plasma and markedly in the tibia), and increased TNF-α (markedly in the plasma, but only slightly in the tibia). LPS-induced hypocalcemia was modest in IL-1KO mice, and not detected in IL-1/TNFαKO mice. IL-1α (but not TNFα) induced hypocalcemia in both WT and IL-1KO mice. In both WT and IL-1KO mice treated with clodronate (osteoclast inhibitor), the LPS-induced hypocalcemia was markedly augmented. Nifedipine (inhibitor of both voltage-activated and capacitative Ca(2+)-entry) reduced the LPS-induced hypocalcemia. These results suggest that in mice: (i) IL-1 and TNF-α may contribute little to physiological bone-formation, and (ii) a time-lag between IL-1- and TNF-α-stimulated Ca(2+)-entry into cells throughout the body from the circulation and IL-1-stimulated Ca(2+)-release from the bone may cause the observed transient LPS-induced hypocalcemia. Thus, the prime role of bone IL-1 may reside in the supply of Ca(2+) from the bone to cells throughout the body when the need is urgent.

Roles of platelets and macrophages in the protective effects of lipopolysaccharide against concanavalin A-induced murine hepatitis.

Platelets are reportedly causal in hepatitis. We previously showed that in mice, lipopolysaccharide (LPS) induces a reversible and macrophage-dependent hepatic platelet accumulation (HPA), including translocation of platelets into Disse spaces and their entry into hepatocytes. Concanavalin A (ConA), which induces hepatitis in mice via both T cells and macrophages, also induces HPA. Here, we examined the relationship between HPA and ConA-hepatitis. ConA-hepatitis and HPA were evaluated by serum transaminases, hepatic 5-hydroxytryptamine, and/or electron microscopy. Unlike LPS-induced HPA, ConA-induced HPA was only moderately dependent on phagocytic macrophages. Against expectations, platelet-depletion significantly exacerbated ConA-hepatitis, and anti-P-selectin antibody and P-selectin receptor blockade reduced both ConA-induced HPA and hepatitis. Prior induction of HPA by pretreatment with low-dose LPS powerfully reduced ConA-hepatitis. Such protection by LPS-pretreatment was not effective in mice depleted of phagocytic macrophages. In platelet-depleted mice, LPS-pretreatment severely exacerbated ConA-hepatitis. In mice depleted of both macrophages and platelets, neither ConA nor LPS-pretreatment+ConA induced hepatitis. In mice deficient in IL-1α and IL-1ß (but not in TNFα), ConA-induced hepatitis was mild, and a protective effect of LPS was not detected. These results suggest that (i) there are causal and protective types of HPA, (ii) the causal type involves hepatic aggregation of platelets, which may be induced by platelet stimulants leaked from injured hepatocytes, (iii) the protective type is inducible by administration of prior low-dose LPS in a manner dependent on phagocytic (or F4/80-positive) macrophages, and (iv) IL-1 is involved in both the causal and protective types.

Daily alignment results of in-room computed tomography-guided stereotactic body radiation therapy for lung cancer.

PURPOSE: To determine the extent of interfractional setup errors and day-to-day organ motion errors by assessing daily bone alignment results and changes in soft tissue tumor position during hypofractionated, in-room computed tomography (CT)-guided stereotactic body radiation therapy (SBRT) of lung cancer. METHODS AND MATERIALS: Daily alignment results during SBRT were analyzed for 117 tumors in 112 patients. Patients received 40-50 Gy of SBRT in four to five fractions using an integrated CT-LINAC system. The free-breathing CT scans acquired during treatment setup were retrospectively realigned to match with each of the bony references and the gross tumor volume (GTV) defined on the reference CT by rigid-body registration, and the daily deviations were calculated. RESULTS: The mean magnitude (± SD) three-dimensional shift from the initial skin marks to the final bone-aligned positions was 9.4 ± 5.7 mm. The mean daily GTV deviation from the bone position was 0.1 ± 3.8 mm in the anterior-posterior direction, -0.01 ± 4.2 mm in the superior-inferior direction, and 0.2 ± 2.5 mm in the lateral direction. A clinically noteworthy trend (net change >5 mm in any direction) in GTV position relative to the bone was observed in 23 cases (20%). CONCLUSIONS: Soft tissue target position can change significantly beyond the motion envelope defined in the original internal target volume in four-dimensional CT-based treatment planning for SBRT of lung cancer. Additional margin should be considered for adequate coverage of interfractional changes.

Dynamics of platelet mobilisation into lungs in response to 5-hydroxytryptamine (serotonin) in mice.

In experimental animals, the lung rapidly removes intravenously injected 5-hydroxytryptamine (5HT), but the mechanism underlying this pulmonary 5HT removal (P-5HT-R) and the responsible cells remains unclear. 5HT reportedly induces rapid pulmonary platelet accumulation (P-PLT-A). Here, we examined the relationship between P-5HT-R and P-PLT-A in mice by comparing the platelet count in the blood with the endogenous 5HT in the tissues (a marker for platelets because the 5HT is largely contained within platelets). 5HT levels in murine blood and tissues were also examined after intravenous injection of 5HT. The data revealed that: (i) 5HT injection (at > or = 0.04 micromol/kg) induced a transient P-PLT-A (occurring within 6 seconds), (ii) platelets rapidly took up injected 5HT, (iii) the P-5HT-R was saturated following injection of 5HT at 1 micromol/kg, (iv) ketanserin (5HT(2)-receptor antagonist) strongly inhibited P-PLT-A, (v) under fluoxetine (5HT-uptake inhibitor), 5HT levels at 6 seconds after 5HT injection were markedly higher in blood, but significantly lower in lung (versus fluoxetine-untreated mice), (vi) P-5HT-R was barely detectable in mutant mice with platelets lacking dense bodies, and was much reduced in platelet-depleted mice, (vii) 5HT injected intravenously at 10 micromol/kg had a half-life in the lung of < 20 seconds, and (viii) unlike 5HT, injected histamine was largely excreted by the kidney. These results demonstrate that platelets rapidly translocate into the lung upon stimulation of 5HT(2) receptors, take up 5HT (and possibly swiftly metabolise it), and then return to the circulation. Hence, pulmonary platelet accumulation plays an important role in pulmonary 5HT removal in mice.

Effectiveness of using fewer implanted fiducial markers for prostate target alignment.

PURPOSE: To evaluate the impact of the number and location of intraprostatic fiducial markers on the accuracy and reproducibility of daily prostate target alignment and to evaluate the migration of such markers. METHODS AND MATERIALS: Three gold fiducial markers were implanted transrectally under ultrasound guidance near the apex, middle, and base of the prostate in 10 prostate cancer patients. The patients had pretreatment in-room computed tomography (CT) scans three times a week, for approximately 25 CT scans per patient during the 8-week treatment course. A total of 1280 alignments were performed using different alignment scenarios: whole-prostate soft tissue alignment (the gold standard), bone alignment, and seven permutations of alignments using one, two, or three fiducial markers. The results of bone alignment and fiducial alignment were compared with the results of whole-prostate alignment. Fiducial migration was also evaluated. RESULTS: Single-fiducial-marker alignment was more accurate and reproducible than bone alignment. However, due to organ deformation, single fiducial markers did not always reliably represent the position of the entire prostate. The use of two-fiducial combinations was more accurate and reproducible than single-fiducial alignment, and use of all three fiducials was the best. Use of an apex fiducial together with a base fiducial rivaled the use of all three fiducials markers together. Fiducial migration was minimal. CONCLUSIONS: The number and the location of implanted fiducial markers affect the accuracy and reliability of daily prostate target alignment. The use of two or more fiducial markers is recommended.