Microglia-derived exosomes modulate myelin regeneration via miR-615-5p/MYRF axis.

Demyelination and failure of remyelination in the central nervous system (CNS) characterize a number of neurological disorders. Spontaneous remyelination in demyelinating diseases is limited, as oligodendrocyte precursor cells (OPCs), which are often present in demyelinated lesions in abundance, mostly fail to differentiate into oligodendrocytes, the myelinating cells in the CNS. In addition to OPCs, the lesions are assembled numbers of activated resident microglia/infiltrated macrophages; however, the mechanisms and potential role of interactions between the microglia/macrophages and OPCs are poorly understood. Here, we generated a transcriptional profile of exosomes from activated microglia, and found that miR-615-5p was elevated. miR-615-5p bound to 3'UTR of myelin regulator factor (MYRF), a crucial myelination transcription factor expressed in oligodendrocyte lineage cells. Mechanistically, exosomes from activated microglia transferred miR-615-5p to OPCs, which directly bound to MYRF and inhibited OPC maturation. Furthermore, an effect of AAV expressing miR-615-5p sponge in microglia was tested in experimental autoimmune encephalomyelitis (EAE) and cuprizone (CPZ)-induced demyelination model, the classical mouse models of multiple sclerosis. miR-615-5p sponge effectively alleviated disease progression and promoted remyelination. This study identifies miR-615-5p/MYRF as a new target for the therapy of demyelinating diseases.

Water-Soluble Coenzyme Q10 Reduces Rotenone-Induced Mitochondrial Fission.

Parkinson's disease is a neurodegenerative disorder characterized by mitochondrial dysfunction and oxidative stress. It is usually accompanied by an imbalance in mitochondrial dynamics and changes in mitochondrial morphology that are associated with impaired function. The objectives of this study were to identify the effects of rotenone, a drug known to mimic the pathophysiology of Parkinson's disease, on mitochondrial dynamics. Additionally, this study explored the protective effects of water-soluble Coenzyme Q10 (CoQ10) against rotenone-induced cytotoxicity in murine neuronal HT22 cells. Our results demonstrate that rotenone elevates protein expression of mitochondrial fission markers, Drp1 and Fis1, and causes an increase in mitochondrial fragmentation as evidenced through mitochondrial staining and morphological analysis. Water-soluble CoQ10 prevented mitochondrial dynamic imbalance by reducing Drp1 and Fis1 protein expression to pre-rotenone levels, as well as reducing rotenone treatment-associated mitochondrial fragmentation. Hence, water-soluble CoQ10 may have therapeutic potential in treating patients with Parkinson's disease.

Engineered PDGFA-ligand-modified exosomes delivery T3 for demyelinating disease targeted therapy.

Demyelination is a proper syndrome in plenty of central nervous system (CNS) diseases, which is the main obstacle to recovery and still lacks an effective treatment. To overcome the limitations of the brain-blood barrier on drug permeability, we modified an exosome secreted by neural stem cells (NSCs), which had transfected with lentivirus armed with platelet-derived growth factors A (PDGFA)-ligand. Through the in vivo and in vitro exosomes targeting test, the migration ability to the lesion areas and OPCs significantly improved after ligand modification. Furthermore, the targeted exosomes loaded with 3,5, 30-L-triiodothyronine (T3) have a critical myelination ability in CNS development, administrated to the cuprizone animal model treatment. The data shows that the novel drug vector loaded with T3 significantly promotes remyelination compared with T3 alone. At the same time, it improved the CNS microenvironment by reducing astrogliosis, inhibiting pro-inflammatory microglia, and alleviating axon damage. This investigation provides a straightforward strategy to produce a targeting exosome and indicates a possible therapeutic manner for demyelinating disease.

Olig2-enriched exosomes: A novel therapeutic approach for cuprizone-induced demyelination.

The research aims to study the therapeutic impact of HEK293-XPack-Olig2 cell-derived exosomes on remyelination of the corpus callosum in a cuprizone-induced demyelinating disease model. A lentiviral vector expressing Olig2 was constructed using XPack technology. The highly abundant Olig2 exosomes (ExoOs) were isolated by centrifugation for subsequent experiments. Western blot, nanoparticle tracking analysis (NTA), and electron microscopy showed no significant difference in particle size and morphology between Exos and ExoOs, and a high level of Olig2 expression could be detected in ExoOs, indicating that exosome modification by XPack technology was successful. The Black Gold/Fluromyelin staining analysis showed that the ExoOs group significantly reduced the demyelination area in the corpus callosum compared to the PBS and Exos groups. Additionally, the PDGFRα/APC staining of the demyelinating region revealed an increase in APC+ oligodendrocytes and a decrease in PDGFRα+ oligodendrocyte progenitor cells (OPCs) in the ExoOs group. Furthermore, there was evident myelin regeneration in the demyelinated areas after ExoOs treatment, with better g-ratio and a higher number of intact myelin compared to the other treatment groups. The level of Sox10 expression in the brain tissue of the ExoOs group were higher compared to those of the PBS and Exos groups. The demyelination process can be significantly slowed down by the XPack-modified exosomes, the differentiation of OPCs promoted, and myelin regeneration accelerated under pathological conditions. This process is presumed to be achieved by changing the expression level of intracellular differentiation-related genes after exosomes transport Olig2 enriched into oligodendrocyte progenitors.

A sophisticated fracture classification system of the proximal femur trochanteric region (AO/OTA-31A) based on 3D-CT images.

Objective: Fracture classification evolves dynamically with new and enhanced imaging modalities. This paper aims to introduce a novel hypothesis of a sophisticated fracture classification system for the proximal femur trochanteric region (AO/OTA-31A) based on 3D-CT images and accommodate the clinical requirement of the worldwide outbreak of geriatric hip fractures with large amounts of surgical operations. Methods: In the current practice of widely preoperative 3D-CT application and cephalomedullary nailing, we attempt to propose a new comprehensive classification system to describe the fracture characteristics in a more detailed and sophisticated architecture, and pay the most important concern to the parameters that contribute to fracture stability reconstruction in osteosynthesis. Results: The new four-by-four comprehensive classification system, followed the structure of the AO/OTA system, incorporates many fracture characteristics as dividing indexes into multiple grade levels, such as fracture line direction, the number of fragments, the lesser trochanter fragment and its distal extension (>2 cm), the posterior coronal fragment and its anterior expansion (to the entry portal of head-neck implant at the lateral cortex), the lateral wall and anterior cortex fracture, and the anteromedial inferior corner comminution. From a panoramic perspective, there are four types and each type has four subtypes. A1 is simple two-part fractures (20%), A2 is characterized by lesser trochanter fragment and posterior coronal fractures (62.5%), A3 is reverse obliquity and transverse fractures with complete lateral wall broken (15.5%), and A4 is medial wall comminution which further lacks anteromedial cortex transmission of compression force (2%). For subtypes, A2.2 is with a banana-like posterior coronal fragment, A2.4 is with distal cortex extension >2 cm of the lesser trochanter and anterior expansion of the posterior coronal fragment(s) to the entry portal of head-neck implants, A3.4 is a primary pantrochanteric fracture, and A4.4 is a concomitant ipsilateral segmental fracture of the neck and trochanter region. Conclusion: Classification represents diversity under consistency. The four-by-four sophisticated classification system delineates fracture characteristics in more detail. It is applicable in the time of rapid outbreak of trochanteric fractures in the older population, the large amounts of surgical operations, and incorporates various rare and/or more complicated subtypes which is unclassifiable before.

Encapsulation of bryostatin-1 by targeted exosomes enhances remyelination and neuroprotection effects in the cuprizone-induced demyelinating animal model of multiple sclerosis.

Demyelination is a critical neurological disease, and there is still a lack of effective treatment methods. In the past two decades, stem cells have emerged as a novel therapeutic effector for neural regeneration. However, owing to the existence of the blood-brain barrier (BBB) and the complex microenvironment, targeted therapy still faces multiple challenges. Targeted exosome carriers for drug delivery may be considered a promising therapeutic method. Exosomes were isolated from mice neural stem cells. To develop targeting exosomes, we generated a lentivirus armed PDGFRα ligand that could anchor the membrane. Exosome targeting tests were carried out in vitro and in vivo. The modified exosomes showed an apparent ability to target OPCs in the lesion area. Next, the exosomes were loaded with Bryostatin-1 (Bryo), and the cuprizone-fed mice were administered with the targeting exosomes. The data show that Bryo exhibits a powerful therapeutic effect compared with Bryo alone after exosome encapsulation. Specifically, this novel exosome-based targeting delivery of Bryo significantly improves the protection ability of the myelin sheath and promotes remyelination. Moreover, it blocks astrogliosis and axon damage, and also has an inhibitory effect on pro-inflammatory microglia. The results of this investigation provide a straightforward strategy to produce targeting exosomes and indicate a potential therapeutic approach for demyelinating disease.

[TLR9 mRNA expression and tumor necrosis factor-alpha/interleukin-6 secretion in murine microglia by hepatitis C virus stimulation].

OBJECTIVE: To analyze the expression of toll-like receptor 9 mRNA, TNF (tumor necrosis factor)-α and IL (interleukin)-6 after the stimulation of murine microglia (BV2) by hepatitis C virus (HCV) so as to elucidate the immune pathogenesis of HCV in the injury of central nervous system (CNS). METHODS: BV2 cells were cultured in vitro and divided into 3 groups: HCV-positive serum group, normal serum group (negative control) and blank control group. Each group had 20 samples. The cells and supernatant were collected at 4, 12, 20, 36 h. And TLR9 mRNA was detected by RT-PCR (reverse transcription-polymerase chain reaction) and the levels of TNF-α and IL-6 were detected by ELISA (enzyme-linked immunosorbent assay). Comparison analyses were performed by SPSS13.0 statistical package. RESULTS: (1) RT-PCR showed that there was the expression of TLR9 mRNA in BV2 cells. And the level of TLR9 mRNA in HCV-positive serum group (0.85 ± 0.11, 0.55 ± 0.08, 0.58 ± 0.09, 0.61 ± 0.07) was higher than that in the other groups. The difference had statistical significance (F = 17.258, P < 0.05). It was the highest at 4 h (0.85 ± 0.11). And the difference had statistical significance (F = 7.427, P < 0.05); but there were no statistical significance between normal serum group and control group (P > 0.05); no significant difference existed among 12, 20 and 36 h (P > 0.05). (2) The levels of TNF-α [(926 ± 133), (327 ± 59), (367 ± 69), (354 ± 71) pg/ml] and IL-6 [(125 ± 34), (84 ± 20), (82 ± 24), (87 ± 18) pg/ml] in the HCV-positive serum group were higher than those in the other groups (F = 18.263 - 27.469, P < 0.05); in HCV-positive serum group, the levels of TLR9 mRNA and TNF-α (r = 0.425, P < 0.01)/IL-6 (r = 0.489, P < 0.01) were positively correlated. CONCLUSION: The expression of TLR9 mRNA is elevated after the stimulation of BV2 cells by HCV-positive serum. It peak comes at 4 h. And it may induce the secretions of TNF-α and IL-6. Thus TLR9 may increase the secretions of Th1 and Th2 cytokines so as to participate in the early inherent immune response during the infection of CNS by HCV.

[Expression of protein gene product 9.5, vasoactive intestinal peptide and neuropeptide Y in the mucosa of the neovagina after sigmoid colon vaginoplasty].

OBJECTIVE: To investigate re-innervation in the neovaginal mucosa of patients underwent sigmoid colon vaginoplasty in treatment of Mayer-Rokitansky-Kistner-Hauser Syndrome (MRKHS). METHODS: Biopsies in the upper third of the posterior neovagina were taken in 20 patients treated by sigmoid colon vaginoplasty at 1, 2 and 3 years after surgery, respectively. Protein gene product 9.5 (PGP 9.5), vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) were detected by immunohistochemical method and compared with those in intact sigmoid colon mucosa. RESULTS: (1) Density of nerve fiber: abundant distribution of PGP 9.5 nerve fibers were observed in the mucosal muscle layer, submucosa, and smooth muscle layer of the neovagina. The nerve fibers of VIP and NPY immunoreactivity were mainly distributed around blood vessels and in the smooth muscles. In the neovagina, the density of nerve fibers of PGP 9.5 of 17 ± 6 were much more than VIP of 2.9 ± 1.0 and NPY of 2.5 ± 0.8 significantly (P < 0.05). (2) Expression of PGP 9.5 in neovagina: at 1 year after surgery, PGP 9.5 positive expression of 14 ± 4 was significantly lower in the neovagina than 28 ± 7 in the intact sigmoid colon (P < 0.05). However, after 2 to 3 years, its expression displayed an upgrade tendency in the neovagina and was significantly higher at the 3 year postoperatively than that at the 1 years postoperatively (22 ± 7 vs. 14 ± 4, P < 0.05). The changes were much more obvious in submucosa. (3) The expression of VIP and NPY in neovagina: at 1 year after surgery, VIP and NPY positive nerve fibers were also decreased in the neovagina when compared with those in the intact sigmoid colon (2.3 ± 0.7 vs. 5.3 ± 1.4, P < 0.05; 2.5 ± 1.1 vs. 5.5 ± 1.1, P < 0.05). At 2 to 3 years after surgery, the positive VIP fiber showed initially decreased and subsequently increased tendency. The density of VIP of 3.7 ± 0.7 in the neovagina at 3 years postoperatively was higher than 2.3 ± 0.7 at 1 years postoperatively (P < 0.05). No significant up-regulation was observed in NPY-positive expression in the neovagina within 3 years after operation. CONCLUSIONS: Distribution of sensory PGP 9.5, VIP and NPY immunoreactive nerve fibers was similar to the pattern observed within the intact sigmoid colon wall. The number of nerve fibers in the neovagina decreased after surgery and then increased subsequently within 3 years after surgery.

[Morphologic change and expression of neuronal nitric oxide synthase in neovaginal mucosa of patients undergoing vaginal construction with sigmoid colon].

OBJECTIVE: To study the changes of the histology and expression of neuronal nitric oxide synthase (nNOS) in the neovaginal mucosa of the patients undergoing vaginal construction with sigmoid colon. METHODS: Biopsy samples of upper one-third and lower one-third of the artificial vagina were obtained from 14 patients who underwent vaginal construction with sigmoid colon 9 - 48 months before and samples of normal sigmoid colon were collected from another 14 patients during vaginal construction with sigmoid colon as control group. The pathological and ultrastructural changes were comparatively observed by light microscopy and electron microscopy. At the same time, the expression of nNOS was semiquantitatively evaluated with immunohistochemical method. RESULTS: The upper one-third of the artificial vagina muscular layer of mucosa was hyperplastic and hypertrophic and the density of the submucosal nerve plexus was 3.6 +/- 1.5, significantly higher than that of the control group (1.7 +/- 0.8, P < 0.05). Squamous metaplasia was seen in the colonic mucosa from some 3 samples of lower one-third of the artificial vagina. Partial fusion of the intercellular tight junction, disappearance and fusion of mitochondrial cristae, and degranulation of rough endoplasmic reticulum could be seen in the vaginal colonic mucosa. Compared with the normal control, the expression of nNOS in the vaginal colonic mucosa was significantly decreased. CONCLUSION: Morphologic changes occur nNOS expression decreases in the colonic mucosa of the artificial vagina.

CT and MRI features in adult patients with orbital subperiosteal abscess secondary to paranasal sinus mucocele.

Orbital subperiosteal abscess (OSPA) secondary to paranasal sinus mucocele (PSM) is rare, and it may be misdiagnosed as PSM with orbital invasion or even as a malignant neoplasm. The present study explored the computed tomography (CT) and magnetic resonance imaging (MRI) features of OSPA. The cases of 13 patients with OSPA secondary to PSM were retrospectively reviewed. CT had been performed in 12, MRI in 7, and postcontrast MRI in 4. OSPA was revealed as a well-demarcated, spindle-shaped mass that was broad-based and located beneath the superior orbital wall (orbital roof) in 11 and at the medial wall in 2. PSM appeared as an expansile cystic lesion in the ethmofrontal sinus in 7, frontal sinus in 5, and ethmoidal sinus in 1. Because the OSPA was connected to the PSM, it looked like a single lesion involving both the orbit and the sinus. All 12 OSPAs examined on CT were low-density; 9 of the 12 PSMs were low-density and 3 were iso-density. Densities of the OSPAs and PSMs were equal in 4 and slightly different in 8. Five of the 10 OSPAs occurring beneath the orbital roof had unclear boundaries with the PSMs on CT. On MRI, although both OSPAs and PSMs mainly demonstrated hypointensity on T1-weighted images and hyperintensity on T2-weighted images, the signal intensities were slightly different, and linear-shaped hypointensity could be found between them. Postcontrast MRI revealed arch- and ring-shaped enhancement, respectively, at the edge of the OSPA and the PSM. Septal enhancement separated them more clearly. PSM is an important cause of OSPA in adults. CT and MRI can accurately display these entities' characteristic findings and their anatomic relationship, as well as playing an important role in the differential diagnosis.

Melatonin prevents sleep deprivation-associated anxiety-like behavior in rats: role of oxidative stress and balance between GABAergic and glutamatergic transmission.

Sleep deprivation (SD) has been shown to induce anxiety-like behavior. Melatonin, an endogenous potent antioxidant, protects neurons from oxidative stress in many disease models. Here we investigated the effect of melatonin against SD-induced anxiety-like behavior and attempted to define the possible mechanisms involved. SD was induced in rats using modified multiple platform model. Melatonin (15 mg/kg) was administered to the rats via intraperitoneal injection. The elevated plus maze test, open field test and light-dark exploration were used to evaluate anxiety-like behavior. Serum corticosterone was measured to determine stress level. Malondialdehyde (MDA) level and superoxide dismutase (SOD) enzyme activity of amygdala and serum were performed to determine the level of oxidative stress. Levels of protein were detected by means of Western blot. The results showed that SD induces anxiety-like behavior, while melatonin treatment prevented these changes. Serum corticosterone also increased with SD but its levels were normalized by melatonin. In addition, melatonin reversed SD-induced changes in MDA and SOD in both of amygdala and serum. The results of Western blot showed that melatonin attenuated the up-regulation of NR2B-containing N-methyl-D-aspartate receptors, GluR1 subunit of the alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor as well as phosphorylation of GluR1 at Ser831, and Ca2+/calmodulin-dependent protein kinase II-alpha in SD rats. Meanwhile, melatonin blocked the down-regulation of γ-aminobutyric acid A-alpha-2 receptor. In conclusion, our results suggest that melatonin prevents anxiety-like behavior induced by SD. The possible mechanism may be attributed to its ability to reduce oxidative stress and maintain balance between GABAergic and glutamatergic transmission.

[Clinical observation of microwave endometrial ablation for treatment of abnormal uterine bleeding].

OBJECTIVE: To evaluate the efficacy, indication, and complication of microwave endometrial ablation (MEA) in treating abnormal uterine bleeding (AUB). METHODS: One hundred and sixty-eight women with AUB due to benign causes received MEA treatment. Pre-operative endometrial thinning was carried out using uterine curettage. Then, the applicator radiating microwaves was moved by progressive withdrawal as well as "W" shape motion inside uterine cavity. All the patients were followed-up. The change of menstrual cycle, the amount of flow, dysmenorrhoea, anemia after treatment at 1, 3, 6, 12 and 24 months was recorded. RESULTS: The mean operating time was (286 +/- 75) seconds. Average follow-up time was (22 +/- 6, range 6-36) months. Of these patients, 156 women (92.9%) were premenopausal, 97 cases (62.2%) were amenorrhea, 56 cases (35.9%) were hypomenorrhoea or eumenorroea, and 3 cases (1.9%) had irregular bleeding. The overall satisfaction of this treatment reached 98.1% (153/156). The follow-up of 119 cases was up to 24 months after operation. The concentration of hemoglobin in 107 women with anemia increased significantly from (83 +/- 24) g/L to (117 +/- 18) g/L 3 months after operation (P < 0.01). Dysmenorrhoea was relieved in 74.5% (35/47) patients. No bleeding occurred in any one of 12 postmenopausal patients after MEA. There was no intraoperative complication in any case. The procedure was successful in all of 47 patients with severe medical disorders. After operation, 12 cases were complicated with endometritis, 2 with hematometra, and one case was performed with hysterectomy due to postablation tubal sterilization syndrome. CONCLUSIONS: MEA is a simple, safe and effective treatment of patients with abnormal uterine bleeding, especially suitable for those women associated with severe medical complications. Complete endometrial ablation is one of the most important determinants of treatment success. Stringent selection of patients may reduce the rate of complications.

Dendritic and Langerhans cells respond to Aß peptides differently: implication for AD immunotherapy.

Both wild-type and mutated beta-amyloid (Aß) peptides can elicit an immune response when delivered subcutaneously. However, only mutated forms of Aß can sensitize dendritic cells when administered intravenously or intraperitoneally. To understand the role of mutation and delivery routes in creating immune responses, and the function of dendritic cells as therapeutic agents, we used fluorescent-conjugated WT Aß1-40 (WT40) and artificially mutated Aß1-40 (22W40) peptides to treat dendritic and Langerhans cells from young and/or old mice at different time points. The cell types were analyzed by flow cytometry and confocal microscopy to identify differences in function and antigen presentation, and Luminex and Western blots for cell activation and associated mechanisms. Our results demonstrated that the artificial mutant, 22W40, enhanced dendritic cell's phagocytosis and antigen presentation better than the WT40. Interestingly, Langerhans cells were more effective at early presentation. The artificial mutant 22W40 increased CD8α+ dendritic cells, CD8+ T-cells, and IFN-γ production when co-cultured with self-lymphocytes and dendritic cells from aged mice (30-month-old). Here, the 22W40 mutant peptide has been found to be potent enough to activate DCs, and that dendritic cell-based therapy may be a more effective treatment for age-related diseases, such as Alzheimer's disease (AD).

High-throughput sequencing of 16S rDNA amplicons characterizes bacterial composition in bronchoalveolar lavage fluid in patients with ventilator-associated pneumonia.

Ventilator-associated pneumonia (VAP) is a life-threatening disease that is associated with high rates of morbidity and likely mortality, placing a heavy burden on an individual and society. Currently available diagnostic and therapeutic approaches for VAP treatment are limited, and the prognosis of VAP is poor. The present study aimed to reveal and discriminate the identification of the full spectrum of the pathogens in patients with VAP using high-throughput sequencing approach and analyze the species richness and complexity via alpha and beta diversity analysis. The bronchoalveolar lavage fluid samples were collected from 27 patients with VAP in intensive care unit. The polymerase chain reaction products of the hypervariable regions of 16S rDNA gene in these 27 samples of VAP were sequenced using the 454 GS FLX system. A total of 103,856 pyrosequencing reads and 638 operational taxonomic units were obtained from these 27 samples. There were four dominant phyla, including Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. There were 90 different genera, of which 12 genera occurred in over ten different samples. The top five dominant genera were Streptococcus, Acinetobacter, Limnohabitans, Neisseria, and Corynebacterium, and the most widely distributed genera were Streptococcus, Limnohabitans, and Acinetobacter in these 27 samples. Of note, the mixed profile of causative pathogens was observed. Taken together, the results show that the high-throughput sequencing approach facilitates the characterization of the pathogens in bronchoalveolar lavage fluid samples and the determination of the profile for bacteria in the bronchoalveolar lavage fluid samples of the patients with VAP. This study can provide useful information of pathogens in VAP and assist clinicians to make rational and effective therapeutic decisions.

Brucella outer membrane protein Omp25 induces microglial cells in vitro to secrete inflammatory cytokines and inhibit apoptosis.

Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection.

Coffee and caffeine potentiate the antiamyloidogenic activity of melatonin via inhibition of Aß oligomerization and modulation of the Tau-mediated pathway in N2a/APP cells.

There is an increasing prevalence of Alzheimer's disease (AD), which has become a public health issue. However, the underlying mechanisms for the pathogenesis of AD are not fully understood, and the current therapeutic drugs cannot produce acceptable efficacy in AD patients. Previous animal studies have shown that coffee (Coff), caffeine (Caff), and melatonin (Mel) have beneficial effects on AD. Disturbed circadian rhythms are observed in AD, and chronotherapy has shown promising effects on AD. In this study, we examined whether a combination of Coff or Caff plus Mel produced a synergistic/additive effect on amyloid-ß (Aß) generation in Neuro-2a (N2a)/amyloid precursor protein (APP) cells and the possible mechanisms involved. Cells were treated with Coff or Caff, with or without combined Mel, with three different chronological regimens. In regimen 1, cells were treated with Coff or Caff for 12 hours in the day, followed by Mel for 12 hours in the night. For regimen 2, cells were treated with Coff or Caff plus Mel for 24 hours, from 7 am to 7 am the next day. In regimen 3, cells were treated with Coff or Caff plus Mel with regimen 1 or 2 for 5 consecutive days. The extracellular Aß40/42 and Aß oligomer levels were determined using enzyme-linked immunosorbent assay (ELISA) kits. The expression and/or phosphorylation levels of glycogen synthase kinase 3ß (GSK3ß), Erk1/2, PI3K, Akt, Tau, Wnt3α, ß-catenin, and Nrf2 were detected by Western blot assay. The results showed that regimen 1 produced an additive antiamyloidogenic effect with significantly reduced extracellular levels of Aß40/42 and Aß42 oligomers. Regimen 2 did not result in remarkable effects, and regimen 3 showed a less antiamyloidogenic effect compared to regimen 1. Coff or Caff, plus Mel reduced oxidative stress in N2a/APP cells via the Nrf2 pathway. Coff or Caff, plus Mel inhibited GSK3ß, Akt, PI3K p55, and Tau phosphorylation but enhanced PI3K p85 and Erk1/2 phosphorylation in N2a/APP cells. Coff or Caff, plus Mel downregulated Wnt3α expression but upregulated ß-catenin. However, Coff or Caff plus Mel did not significantly alter the production of T helper cell (Th)1-related interleukin (IL)-12 and interferon (IFN)-γ and Th2-related IL-4 and IL-10 in N2a/APP cells. The autophagy of cells was not affected by the combinations. Taken together, combination of Caff or Coff, before treatment with Mel elicits an additive antiamyloidogenic effects in N2a/APP cells, probably through inhibition of Aß oligomerization and modulation of the Akt/GSK3ß/Tau signaling pathway.

Effect of mifepristone on estrogen and progesterone receptors in human endometrial and endometriotic cells in vitro.

OBJECTIVE: To investigate the expressions of estrogen (E) receptor and progesterone (P) receptor in human eutopic and ectopic endometrium and the effect of mifepristone (RU486) on them. DESIGN: Prospective study. SETTING: University hospital. PATIENT(S): Twenty-two patients with ovarian endometriosis and 13 patients with uterine leiomyoma were recruited. INTERVENTION(S): Samples of ovarian endometrioma cyst tissue and endometrium were obtained from the 22 patients. A sample of endometrium was obtained from the 13 patients. MAIN OUTCOME MEASURE(S): Expressions of E and P receptors were determined using immunocytochemical method. RESULT(S): P receptor expression in endometrial epithelial cells with endometriosis was significantly higher than that without endometriosis in the early secretory phase. Estrogen receptor and epithelial P receptor expressions in endometriotic cells were significantly lower than those of endometrial cells during the proliferative phase, similar with the latter during the early secretory phase and significantly higher during the late secretory phase. RU486 down-regulated the expressions of E and P receptors in both the eutopic and the ectopic endometrial cells, and in some cases this down-regulating effect was more apparent when the concentration of RU486 was higher. CONCLUSION(S): Different steroid receptor expressions indicate different hormonal regulation between endometriotic and endometrial cells. The down-regulating effect on E and P receptors may be one of the therapeutic mechanisms of RU486 on endometriosis.

[TLR9 expression and its relationship with MCP-1 and TNF-α in HCV-stimulated BV2 cells].

AIM: To detect TLR9 expression and its effect on the secretion of MCP-1 and TNF-α after mouse microglia (BV2) were infected by hepatitis C virus (HCV), and to explore the mechanism of HCV-induced central nervous system (CNS) infection. METHODS: BV2 cells were cultured in vitro and divided into a HCV-positive serum group, a normal serum group (negative control) and a blank control group, with each group of 20 samples. The cells and the supernatants were collected at 12 h, detected for TLR9 expression by flow cytometry (FCM), and determined for the levels of TNF-α and MCP-1 by ELISA. Then the data was statistically analyzed by SPSS11.0. RESULTS: (1) FCM showed TLR9 expression in all three groups, with the strongest expression in the HCV-positive serum group (P<0.01), while there was no statistical significance between the normal serum group and the blank control group (P>0.05). (2) The levels of TNF-α and MCP-1 in the HCV-positive serum group were higher than those in the other groups (P<0.01), which correlated with enhanced TLR9 expression in the HCV-positive serum group. CONCLUSION: HCV-positive serum causes enhanced TLR9 expression and the secretion of MCP-1 and TNF-α. in BV2 cells, suggesting the involvement of TLR9 in the early inherent immune response triggered by HCV-induced CNS infection.

[Construction of phylogenetic tree and sequence analysis on BDV p24 gene in both livestock and host in Ningxia].

OBJECTIVE: To gain detailed insights into the Borna disease virus infection and its genetic characteristics and phylogeny in Ningxia, China. METHODS: BDV p24 segment were detected by fluorescence quantitative nested RT-PCR from peripheral blood mononuclear cells of 119 patients with viral encephalitis, 205 cattle, 978 sheep, 46 patients with cerebrovascular diseases and 13 patients with multiple sclerosis. Data from phylogenetic analysis on BDV p24 positive samples together with those from the positive gene sequences of animals and patients in the previous studies in Ningxia, were compared to the 29 sequences provided by the GenBank from 7 animals in 5 countries. Both the sequence homologous similarity of the nucleotide and amino acid sequences were analyzed and gene phylogenetic tree was reconstructed. RESULTS: Data from 23 collections of positive test samples, together with the ones from early detection of gene sequence analysis, showed that the homologous similarity sequences of both nucleotide and amino acid was between 95.3% and 100.0% and highly homophylic with HE80 that detected from ill horses in Germany. One part of nucleotide sequences formed the 'Ningxia independent branch' while the other one belonged to the 'Germany-Ningxia-Japan mixed branch'. There was a high identity within the branch. CONCLUSION: A Ningxia independent BDV strain from geographical origin might exist while the epidemic strains were imported with multiple sources.