OBJECTIVETo compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster.

METHODSAmoebic liver abscess was experimentally induced in a hamster by injecting 1 × 10(6) of axenically cultured virulent E. histolytica trophozoites (HM1-IMSS strain) into the portal vein. After a week post-inoculation, the hamster was sacrificed and the liver tissue sections were stained with H&E, PAS and IHC stains to detect the amoebic trophozoite.

RESULTSThe three stains revealed tissue necrosis and amoebic trophozoites, but with varying clarity. H&E and PAS stained the trophozoites pink and magenta, respectively, however it was difficult to differentiate the stained trophozoites from the macrophages because of their similarity in size and morphology. On the other hand, IHC stain revealed distinct brown appearance of the trophozoites in the infected liver tissues.

CONCLUSIONSIt can be concluded that out of the three stains, IHC is the best for identification of E. histolytica trophozoites in tissue sections.

Animals ; Disease Models, Animal ; Entamoeba histolytica ; cytology ; isolation & purification ; Histocytochemistry ; methods ; Immunohistochemistry ; methods ; Liver Abscess, Amebic ; diagnosis ; pathology ; Male ; Mesocricetus ; Microscopy ; Parasitology ; methods ; Staining and Labeling ; methods ; Trophozoites ; cytology

OBJECTIVETo establish a new method for the preparation of porcine acellular dermal matrix.

METHODSThe antigenicity of the porcine dermis was weakened by removing epidermal and dermal cells from the porcine skin through the digestion with low-concentration trypsin and repeated freeze-thaw cycles. Split thickness porcine skin was treated with 0.05% trypsin to remove the cells from the epidermis and dermis. Repeated freeze-thaw cycles were employed to further weed out the residual cells within the dermis. The prepared acellular dermis was then examined grossly, as well as histologically, and also by immunohistochemical method.

RESULTSNo cell could be identified in the prepared porcine acellular dermal matrix. The integral basement membrane was preserved on the surface of dermal matrix with compact dermal matrix collagen structure.

CONCLUSIONLow concentration trypsinization and repeated freeze-thaw cycles seemed to be a simple and effective method for the preparation of xenogeneic acellular dermal matrix.

Animals ; Dermis ; cytology ; transplantation ; Extracellular Matrix ; transplantation ; ultrastructure ; Freezing ; Skin Transplantation ; Swine ; Tissue Engineering ; methods ; Trypsin ; administration & dosage

Background/Aims: Depression and anxiety are associated with poorer outcomes in patients with hepatocellular carcinoma (HCC). However, the prevalence of depression and anxiety in HCC are unclear. We aimed to establish the prevalence of depression and anxiety in patients with HCC. Methods: MEDLINE and Embase were searched and original articles reporting prevalence of anxiety or depression in patients with HCC were included. A generalized linear mixed model with Clopper-Pearson intervals was used to obtain the pooled prevalence of depression and anxiety in patients with HCC. Risk factors were analyzed via a fractional-logistic regression model. Results: Seventeen articles involving 64,247 patients with HCC were included. The pooled prevalence of depression and anxiety in patients with HCC was 24.04% (95% confidence interval [CI], 13.99–38.11%) and 22.20% (95% CI, 10.07–42.09%) respectively. Subgroup analysis determined that the prevalence of depression was lowest in studies where depression was diagnosed via clinician-administered scales (16.07%;95% CI, 4.42–44.20%) and highest in self-reported scales (30.03%; 95% CI, 17.19–47.01%). Depression in patients with HCC was lowest in the Americas (16.44%; 95% CI, 6.37–36.27%) and highest in South-East Asia (66.67%; 95% CI, 56.68–75.35%). Alcohol consumption, cirrhosis, and college education significantly increased risk of depression in patients with HCC. Conclusions One in four patients with HCC have depression, while one in five have anxiety. Further studies are required to validate these findings, as seen from the wide CIs in certain subgroup analyses. Screening strategies for depression and anxiety should also be developed for patients with HCC.

OBJECTIVETo isolate and identify active neuraminidase constituents of Polygonum cuspidatum against influenza A (H1N1) influenza virus.

METHODOn the basis of the bioassay-guided fractionation,such chromatographic methods as silica gel, sephadex LH-20 and HPLC were adopted to isolate active constituents of extracts from Polygonum cuspidatum, and their molecular structures were identifiied on the basis of their spectral data such as NMR and MS and physico-chemical properties.

RESULTSeven compounds were isolated from the ethyl acetate extract of P. cuspidatum and identified as 2-methoxystypandrone (1), emodin (2), resveratrol (3), polydatin (4), emodin-8-O-beta-D-glucopyranoside (5), (E)-3, 5, 12-trihydroxystilbene-3-O-beta-D-glucopyranoside-2'-(3", 4", 5"-trihydroxybenzoate) (6) and catechin-3-O-gallate (7), respectively. Among them, the NA test showed that compounds 3, 6 and 7 had inhibitory effect against NAs activity, with IC50 values of 129.8, 44.8 and 21.3 micromol x L(-1), respectively. Moreover, the further CPE test showed compounds 6 and 7 had significant inhibitory effect against H1N influenza virus (EC50 = 5.9, 0.9 micromol x L(-1), respectively), with very low cytotoxicity to the host cells, their therapeutic selective index(SI) in MDCK cells ranged from 56 to 269.

CONCLUSIONThe neuraminidase inhibitors against H1N1 anti-influenza virus isolated from extracts of P. cuspidatum on the basis of the bioassay-guided fractionation are significant in specifying their therapeutic material basis and drug R&D against influenza.

Cell Line ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Enzyme Inhibitors ; chemistry ; isolation & purification ; pharmacology ; Fallopia japonica ; chemistry ; Humans ; Influenza A Virus, H1N1 Subtype ; drug effects ; enzymology ; Influenza, Human ; virology ; Molecular Structure ; Neuraminidase ; antagonists & inhibitors

Objective: To characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters. Methods: Crude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein. Results: A ~75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively. Conclusions: This finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.

OBJECTIVETo characterize the Entamoeba histolytica (E. histolytica) antigen(s) recognized by moribound amoebic liver abscess hamsters.

METHODSCrude soluble antigen of E. histolytica was probed with sera of moribund hamsters in 1D- and 2D-Western blot analyses. The antigenic protein was then sent for tandem mass spectrometry analysis. The corresponding gene was cloned and expressed in Escherichia coli BL21-AI to produce the recombinant E. histolytica ADP-forming acetyl-CoA synthetase (EhACS) protein. A customised ELISA was developed to evaluate the sensitivity and specificity of the recombinant protein.

RESULTSA ∼75 kDa protein band with a pI value of 5.91-6.5 was found to be antigenic; and not detected by sera of hamsters in the control group. Tandem mass spectrometry analysis revealed the protein to be the 77 kDa E. histolytica ADP-forming acetyl-CoA synthetase (EhACS). The customised ELISA results revealed 100% sensitivity and 100% specificity when tested against infected (n=31) and control group hamsters (n=5) serum samples, respectively.

CONCLUSIONSThis finding suggested the significant role of EhACS as a biomarker for moribund hamsters with acute amoebic liver abscess (ALA) infection. It is deemed pertinent that future studies explore the potential roles of EhACS in better understanding the pathogenesis of ALA; and in the development of vaccine and diagnostic tests to control ALA in human populations.

OBJECTIVE: To examine the anti-inflammatory effects and potential mechanisms of polypeptide from Moschus (PPM) in lipopolysaccharide (LPS)-induced THP-1 macrophages and BALB/c mice. METHODS: The polypeptide was extracted from Moschus and analyzed by high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Subsequently, LPS was used to induce inflammation in THP-1 macrophages and BALB/c mice. In LPS-treated or untreated THP-1 macrophages, cell viability was observed by cell counting kit 8 and lactate dehydrogenase release assays; the proinflammatory cytokines and reactive oxygen species (ROS) were measured by enzyme-linked immunosorbent assay and flow cytometry, respectively; and protein and mRNA levels were measured by Western blot and real-time quantitative polymerase chain reaction (qRT-PCR), respectively. In LPS-induced BALB/c mice, the proinflammatory cytokines were measured, and lung histology and cytokines were observed by hematoxylin and eosin (HE) and immunohistochemical (IHC) staining, respectively. RESULTS: The SDS-PAGE results suggested that the molecular weight of purified PPM was in the range of 10-26 kD. In vitro, PPM reduced the production of interleukin 1β (IL-1β), IL-18, tumor necrosis factor α (TNF-α), IL-6 and ROS in LPS-induced THP-1 macrophages (P<0.01). Western blot analysis demonstrated that PPM inhibited LPS-induced nuclear factor κB (NF-κB) pathway and thioredoxin interacting protein (TXNIP)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 (NLRP3) inflammasome pathway by reducing protein expression of phospho-NF-κB p65, phospho-inhibitors of NF-κB (Iκ Bs) kinase α/β (IKKα/β), TXNIP, NLRP3, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and pro-caspase-1 (P<0.05 or P<0.01). In addition, qRT-PCR revealed the inhibitory effects of PPM on the mRNA levels of TXNIP, NLRP3, ASC, and caspase-1 (P<0.05 or P<0.01). Furthermore, in LPS-induced BALB/c mice, PPM reduced TNF-α and IL-6 levels in serum (P<0.05 or P<0.01), decreased IL-1β and IL-18 levels in the lungs (P<0.01) and alleviated pathological injury to the lungs. CONCLUSION PPM could attenuate LPS-induced inflammation by inhibiting the NF-κB-ROS/NLRP3 pathway, and may be a novel potential candidate drug for treating inflammation and inflammation-related diseases.