Erythropoietin signaling in peripheral macrophages is required for systemic ß-amyloid clearance.

Impaired clearance of beta-amyloid (Aß) is a primary cause of sporadic Alzheimer's disease (AD). Aß clearance in the periphery contributes to reducing brain Aß levels and preventing Alzheimer's disease pathogenesis. We show here that erythropoietin (EPO) increases phagocytic activity, levels of Aß-degrading enzymes, and Aß clearance in peripheral macrophages via PPARγ. Erythropoietin is also shown to suppress Aß-induced inflammatory responses. Deletion of EPO receptor in peripheral macrophages leads to increased peripheral and brain Aß levels and exacerbates Alzheimer's-associated brain pathologies and behavioral deficits in AD-model mice. Moreover, erythropoietin signaling is impaired in peripheral macrophages of old AD-model mice. Exogenous erythropoietin normalizes impaired EPO signaling and dysregulated functions of peripheral macrophages in old AD-model mice, promotes systemic Aß clearance, and alleviates disease progression. Erythropoietin treatment may represent a potential therapeutic approach for Alzheimer's disease.

Inhibition of Smad3 in macrophages promotes Aß efflux from the brain and thereby ameliorates Alzheimer's pathology.

Impaired amyloid-ß (Aß) clearance is believed to be a primary cause of Alzheimer's disease (AD), and peripheral abnormalities in Aß clearance have recently been linked to AD pathogenesis and progression. Data from recent genome-wide association studies have linked genetic risk factors associated with altered functions of more immune cells to AD pathology. Here, we first identified correlations of Smad3 signaling activation in peripheral macrophages with AD progression and phagocytosis of Aß. Then, manipulating the Smad3 signaling regulated macrophage phagocytosis of Aß and induced switch of macrophage inflammatory phenotypes in our cell cultures. In our mouse models, flag-tagged or fluorescent-dye conjugated Aß was injected into the lateral ventricles or tail veins, and traced. Interestingly, blocking Smad3 signaling efficiently increased Aß clearance by macrophages, reduced Aß in the periphery and thereby enhanced Aß efflux from the brain. Moreover, in our APP/PS1 transgenic AD model mice, Smad3 inhibition significantly attenuated Aß deposition and neuroinflammation, and ameliorated cognitive deficits, probably by enhancing the peripheral clearance of Aß. In conclusion, enhancing Aß clearance by peripheral macrophages through Smad3 inhibition attenuated AD-related pathology and cognitive deficits, which may provide a new perspective for understanding AD and finding novel therapeutic approaches.

Zerumbone ameliorates behavioral impairments and neuropathology in transgenic APP/PS1 mice by suppressing MAPK signaling.

BACKGROUND: Alzheimer's disease (AD) is a major clinical problem, but there is a distinct lack of effective therapeutic drugs for this disease. We investigated the potential therapeutic effects of zerumbone, a subtropical ginger sesquiterpene, in transgenic APP/PS1 mice, rodent models of AD which exhibit cerebral amyloidosis and neuroinflammation. METHODS: The N9 microglial cell line and primary microglial cells were cultured to investigate the effects of zerumbone on microglia. APP/PS1 mice were treated with zerumbone, and non-cognitive and cognitive behavioral impairments were assessed and compared between the treatment and control groups. The animals were then sacrificed, and tissues were collected for further analysis. The potential therapeutic mechanism of zerumbone and the signaling pathways involved were also investigated by RT-PCR, western blot, nitric oxide detection, enzyme-linked immunosorbent assay, immunohistochemistry, immunofluorescence, and flow cytometry analysis. RESULTS: Zerumbone suppressed the expression of pro-inflammatory cytokines and induced a switch in microglial phenotype from the classic inflammatory phenotype to the alternative anti-inflammatory phenotype by inhibiting the mitogen-activated protein kinase (MAPK)/nuclear factor-kappa B signaling pathway in vitro. After a treatment period of 20 days, zerumbone significantly ameliorated deficits in both non-cognitive and cognitive behaviors in transgenic APP/PS1 mice. Zerumbone significantly reduced ß-amyloid deposition and attenuated pro-inflammatory microglial activation in the cortex and hippocampus. Interestingly, zerumbone significantly increased the proportion of anti-inflammatory microglia among all activated microglia, potentially contributing to reduced ß-amyloid deposition by enhancing phagocytosis. Meanwhile, zerumbone also reduced the expression of key molecules of the MAPK pathway, such as p38 and extracellular signal-regulated kinase. CONCLUSIONS: Overall, zerumbone effectively ameliorated behavioral impairments, attenuated neuroinflammation, and reduced ß-amyloid deposition in transgenic APP/PS1 mice. Zerumbone exhibited substantial anti-inflammatory activity in microglial cells and induced a phenotypic switch in microglia from the pro-inflammatory phenotype to the anti-inflammatory phenotype by inhibiting the MAPK signaling pathway, which may play an important role in its neuroprotective effects. Our results suggest that zerumbone is a potential therapeutic agent for human neuroinflammatory and neurodegenerative diseases, in particular AD.

[Identification of Zaocys type II collagen and its effect on arthritis in mice with collagen-induced arthritis].

OBJECTIVE: To analyze the homology of Zaocys type 1I collagen ( ZC II ) with the C II collagen from other species, and to investigate the effect of ZC II on arthritis in mice with collagen-induced arthritis (CIA). METHODS: ZC II was purified with restriction pepsin digestion. Then SDS-PAGE gel electrophoresis and UV spectrophotometry were used to identify the protein,the homology of the ZC II peptide was analyzed with Mass Spectrometry. The model of CIA mice were induced by subcutaneous injection of Chicken C II into male C57BL/6 mice from the base of the tails. After immunization,ZC II [H,M,L:40,20 and 10 µg/(kgd) ]was administered orally to mice from day 21 to 28 accordingly. The severity of the arthritis in each limb was evaluated using a macroscopic scoring system, and his- topathological change of joint was observed by light microscope with HE staining. RESULTS: The molecular weight of ZC II protein deter- mined by SDS-PAGE gel electrophoresis was between 110 kD and 140 kD, and UV absorption peak appeared at around 230 nm in wave- length. The peptide mass fingerprinting(PMF) of the purified protein by Mass Spectrometry analysis showed that it had at least 4 peptides matched with other species,and the protein score was greater than 95%. Compared with normal group,the CIA model group had significantly higher scores for arthritis and histopathological changes (P <0. 05). Meanwhile,the same scores for the ZC II peptide-treated mice with CIA were significantly lower than the mice from CIA model group(P <0. 05). CONCLUSION: Results of Mass Spectrometry analysis demonstrate that ZC II has high homology with the C II from other species. Oral administration of ZC II can suppress arthritis in mice with CIA and ameliorate the histopathological changes of the joint.

[The effect of the type II collagen protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis].

OBJECTIVE: To investigate the effect of the type II collagen (C II) protein from Zaocys on cytokines production by synoviocytes in rats with adjuvant arthritis (AA). METHODS: Type II collagen protein was abstracted and purificated from Zaocys. Adjuvant arthritis (AA) was induced by a single intradermal injection of 0.1 mL of complete Freund's adjuvant into the left hind paw. Synoviocytes' supernatants were harvested and synoviocytes-Peyer's Patches (PP) cells coculture system were developed. Tumor necrosis factor-alpha (TNF-alpha) activity was measured by L929 cytotoxicity bioassay and Interleukin (IL)-1beta activity was measured by MTT dye reduction. The synoviocytes' supernatants cytokines' levels were detected by ELISA. RESULTS: Each concentration of C II from Zaocys had no effect on IL-1beta and TNF-alpha production by synoviocytes in vitro. Middle concentration of C II suppressed the activity of IL-1beta and TNF-alpha production by synoviocytes-PP cells coculture system (P < 0.05). Treating with low and high dose of C II suppressed the activity of TNF-alpha and IL-1beta producing by synoviocyte (P < 0.05), significantly suppressed in the group of AA rats treated with middle dose of C II (P < 0.01). Treating with middle and high dose of C II decreased the level of synoviocytes' supernatants TNF-alpha (P < 0.05), the level of synoviocytes' supernatants IL-1beta decreased in all treating groups (P < 0.05). Treating with middle dose of C II increased the level of serum TGF-beta (P < 0.05). Middle concentration of C II suppressed the activity of IL-1 and TNF production by synoviocytes-PP cells coculture system (P < 0.05). CONCLUSIONS: C II from Zaocys has no direct effect on the activity of IL-1beta and TNF production by synoviocytes in vitro. Oral administration of type II collagen protein from Zaocys can effectively suppressed the activity and level of the cytokines production by synoviocytes in rats with adjuvant arthritis (AA).

Spontaneous HBeAg seroconversion and loss of hepatitis B virus DNA after acute flare due to development of drug resistant mutants during entecavir monotherapy.

AIMS: Patients with chronic hepatitis B virus (HBV) infection under entecavir (ETV) treatment develop resistant mutants with viral rebound. Here, we report an interesting case of spontaneous loss of HBV-DNA and seroconversion following an acute flare after the development of ETV-resistant mutants. This patient received ETV after lamivudine breakthrough. METHODS: Cloning and sequence analysis of the HBV reverse transcriptase (RT) region were performed with seven samples during ETV therapy. In addition, two full-length HBV genomes derived from samples before and after the emergence of ETV resistance were sequenced. RESULTS: ETV resistant mutants appeared at week 228, with virological and biochemical rebound at the same time. Unexpectedly, HBeAg seroconversion occurred 8 weeks later. The viral load decreased and became undetectable from week 252. Analysis of HBV isolates in the patient at week 124 revealed that wild-type HBV was predominant at that time and ETV resistant mutants were not found among 20 clones. Interestingly, a new mutant type with rtL180M+rtT184L was found alongside rtL180M+rtT184L+rtM204V/I at week 228 and appeared to develop independently, according to the sequence analysis. In contrast to the previously identified ETV resistant mutants, it did not carry the rtM204V/I mutations. CONCLUSION: The data presented here indicates that the flare following the emergence of ETV resistant mutants may reflect immune-mediated control of HBV infection, leading to a spontaneous loss of HBV-DNA and seroconversion.

Coexistence of hepatitis B surface antigen (HBsAg) and heterologous subtype-specific antibodies to HBsAg among patients with chronic hepatitis B virus infection.

BACKGROUND: The coexistence of hepatitis B surface antigen (HBsAg) and antibodies to HBsAg (anti-HBs) in patients with chronic hepatitis B virus (HBV) infection has been explained by the presence of viral escape mutants. Yet, no systematic analysis of such patients has been performed. We analyzed both the HBV strains and the nature of anti-HBs in such patients. METHODS: Four hundred eleven patients with chronic HBV infection were tested for the presence of anti-HBs. The sequences of the HBsAg coding region were analyzed. Anti-HBs were purified and examined in commercial assays alone and with 3 different HBsAg subtypes. RESULTS: Twenty patients had positive results for anti-HBs. This serological status remained stable for 12 months (as tested thus far). Amino acid substitutions and/or variations on HBsAg were found in 13 patients, and the HBV isolates from 4 others were wild types. Importantly, no significant difference in the occurrence of amino acid substitutions within the HBsAg was found in HBV isolates from patients with and without anti-HBs. Purified immunoglobulin fractions from serum samples from patients were reactive to HBsAg but had a lower specific activity, compared with those taken from immunized persons. Anti-HBs in patients were directed to the HBsAg subtypes other than the coexisting one. No circulating immune complex could be detected in these patients. CONCLUSION: HBsAg and anti-HBs with an unmatched specificity coexisted in 4.9% of patients. The presence of anti-HBs was not associated with the appearance of specific HBV mutants in patients with chronic infection. Apparently, the presence of anti-HBs in patients with chronic HBV infection did not lead to a selection of HBV escape mutants.

[The rate of hepatitis B virus resistance to adefovir dipivoxil (ADV) and the evolution of hepatitis B virus in lamivudine-resistant chronic hepatitis B patients with ADV monotherapy].

OBJECTIVE: To study the resistant rate of hepatitis B virus (HBV) to ADV and the dynamic evolution of HBV in lamivudine (Lam)-resistant chronic hepatitis B (CHB) patients. METHODS: Twenty-three Lam-resistant CHB patients were assigned to a 10mg/d ADV monotherapy for 68-116 weeks. The baseline and different time point blood samples after ADV monotherapy were analyzed for ADV-resistant mutations using direct sequencing of PCR products; the evolution of HBV mutations was examined by clonal analysis of serial samples from one patient infected with ADV-associated resistant HBV strains. RESULTS: The cumulative incidence of genotypic ADV resistance at weeks 48 and 96 was 4.3% and 10.5% respectively respectively. The evolution analysis of HBV mutant strains in an ADV-resistant CHB patient showed that the proportion of YMDD mutants gradually decreased with rtA181S mutants increasing over time after ADV monotherapy, and that rtA181S+N236T mutants became the predominant strains during prolonged ADV monotherapy. The addition of Lam to the ongoing ADV treatment had poorer antiviral response in the patient with rtA181S or rtA181S+N236T mutant infection; one clone with multi-drug resistant mutations was selected during Lam and ADV combination therapy. CONCLUSION: Increased risk of adefovir resistance and selection of multi-drug resistant mutations are associated with long-term ADV monotherapy in patients with Lam-resistant chronic hepatitis B.

[Character of denitrifying phosphorus removal using MUCT process: operation at low C/N rate].

The effect of phosphorus uptake in the anaerobic zone of MUCT process fed with low C/N rate wastewater was studied. The experimental results shows that: (1) Because of the concentration of the COD is relative higher, nitrate is firstly used by the traditional denitrifying bacteria in the cycle sludge, and can not act as the electron acceptor for DPB. So the main action in the first anaerobic zone is phosphate release; (2) The DPB utilizes the PHB as the carbon stored in the anaerobic zone, and nitrate as electron acceptor to uptake phosphorus and the amount increases daily from 0.93 mg/L to 18 mg/L, and the anaerobic phosphorus rate stabilizes to 40 percent finally in the second anaerobic zone; (3) The ineffective phosphorus release process occurs in the third anaerobic zone because the lower nitrate and COD concentration,and phosphate release amount is from 0.27 to 3 mg/L; (4) The COD, TN, TP removal rates are higher and the TP and TN concentration in effluent is below 10 mg/L and 0.9 mg/L respectively.