isolation-primary-rat-hepatocytes-with-multiparameter-perfusion

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control

Primary hepatocytes are widely used in basic research on liver diseases and for toxicity testing in vitro. The two-step collagenase perfusion procedure ...

National University of Singapore

The release of proinflammatory mediators such as tumor necrosis factor-alpha (TNF-alpha) and nitric oxide by microglia has been implicated in neurotoxicity in chronic neurodegenerative diseases such as Alzheimer's disease. As all-trans-retinoic acid (RA) has been reported to exert anti-inflammatory actions in various cell types, we have examined its effects on the expression of TNF-alpha and inducible nitric oxide synthase (iNOS) in microglia activated by beta-amyloid peptide (Abeta) and lipopolysaccharide (LPS). Exposure of primary cultures of rat microglial cells to Abeta or LPS stimulated the mRNA expression level of TNF-alpha (6-116-fold) and iNOS (8-500-fold) significantly. RA acted in a dose-dependent manner (0.1-10 microM) by attenuating both TNF-alpha (29-97%) and iNOS (61-96%) mRNA expression in microglia exposed to Abeta or LPS. RA-induced inhibition of TNF-alpha and iNOS mRNA expression in activated microglia was accompanied by the concomitant reduction in release of iNOS and TNF-alpha proteins as revealed by nitrite assay and ELISA, respectively. The anti-inflammatory effects of RA were correlated with the enhanced expression of retinoic acid receptor-beta, and transforming growth factor-beta1 as well as the inhibition of NF-kappaB translocation. These results suggest that RA may inhibit the neurotoxic effect of activated microglia by suppressing the production of inflammatory cytokines and cytotoxic molecules.

Retinoic acid inhibits expression of TNF-alpha and iNOS in activated rat microglia.

Amoeboid microglial cells (AMC) which transiently exist in the corpus callosum in the postnatal rat brain expressed endothelins (ETs), specifically endothelin-1 (ET-1) and ET3 as revealed by real time RT-PCR. ET immunoreactive AMC occurred in large numbers at birth, but were progressively reduced with age and were undetected in 14 days. In rats subjected to hypoxia exposure, ET immunoexpression in AMC was reduced but the incidence of apoptotic cells was not increased when compared with the control suggesting that this was due to its downregulation that may help regulate the constriction of blood vessels bearing ET-A receptor. AMC were endowed ET-B receptor indicating that ET released by the cells may also act via an autocrine manner. In microglia activated by lipopolysaccharide (LPS), ET-1 mNA expression coupled with that of monocyte chemoattractant protein (MCP-1) and stromal derived factor-1 (SDF-1) was markedly increased; ET-3 mRNA, however, remained unaffected. AMC exposed to oxygen glucose deprivation (OGD) in vitro resulted in increase in both ET-1 and ET-3 mRNA expression. It is suggested that the downregulated ETs expression in vivo of AMC subjected to hypoxia as opposed to its upregulated expression in vitro may be due to the complexity of the brain tissue. Furthermore, the differential ET-1 and ET-3 mRNA expression in LPS and OGD treatments may be due to different signaling pathways independently regulating the two isoforms. The present novel finding has added microglia as a new cellular source of ET that may take part in multiple functions including regulating vascular constriction and chemokines release.

Transient expression of endothelins in the amoeboid microglial cells in the developing rat brain.

Microglial cells release monocyte chemoattractant protein-1 (MCP-1) which amplifies the inflammation process by promoting recruitment of macrophages and microglia to inflammatory sites in several neurological diseases. In the present study, dexamethasone (Dex), an anti-inflammatory and immunosuppressive drug has been shown to suppress the mRNA and protein expression of MCP-1 in activated microglia resulting in inhibition of microglial migration. This has been further confirmed by the chemotaxis assay which showed that Dex or MCP-1 neutralization with its antibody inhibits the microglial recruitment towards the conditioned medium of lipopolysaccharide (LPS)-treated microglial culture. This study also revealed that the down-regulation of the MCP-1 mRNA expression by Dex in activated microglial cells was mediated via mitogen-activated protein kinase (MAPK) pathways. It has been demonstrated that Dex inhibited the phosphorylation of Jun N-terminal kinase (JNK) and p38 MAP kinases as well as c-jun, the JNK substrate in microglia treated with LPS. The involvement of JNK and p38 MAPK pathways in induction of MCP-1 production in activated microglial cells was confirmed as there was an attenuation of MCP-1 protein release when microglial cells were treated with inhibitors of JNK and p38. In addition, Dex induced the expression of MAP kinase phosphatase-1 (MKP-1), the negative regulator of JNK and p38 MAP kinases in microglial cells exposed to LPS. Blockade of MKP-1 expression by triptolide enhanced the phosphorylation of JNK and p38 MAPK pathways and the mRNA expression of MCP-1 in activated microglial cells treated with Dex. In summary, Dex inhibits the MCP-1 production and subsequent microglial cells migration to the inflammatory site by regulating MKP-1 expression and the p38 and JNK MAPK pathways. This study reveals that the MKP-1 and MCP-1 as novel mediators of biological effects of Dex may help developing better therapeutic strategies for the treatment of patients with neuroinflammatory diseases.

Dexamethasone suppresses monocyte chemoattractant protein-1 production via mitogen activated protein kinase phosphatase-1 dependent inhibition of Jun N-terminal kinase and p38 mitogen-activated protein kinase in activated rat microglia.

A microwave-digestion system with a closed PTFE vessel was used to improve the leaching of inorganic constituents from biological samples with tetramethylammonium hydroxide (TMAH) and ethylenediaminetetraacetic acid (EDTA). The effects of microwave parameter settings and the quantities of TMAH and EDTA used on leaching efficiency were evaluated. This new digestion method has been applied to the standard reference materials NIST SRM 1577 B Bovine Liver 1515 Apple Leaves and NIES CRM No. 1 Pepperbush, No. 3 Chorella, No. 6 Mussel and No. 7 Tea Leaves. The major and minor elements in the digests were analyzed by flame atomic absorption spectrometry or graphite furnace atomic absorption spectrometry. Good agreement of the analytical results with the certified values was obtained.

Microwave digestion of biological samples with tetramethylammonium hydroxide and ethylenediaminetetraacetic acid for element determination.

Of eight leprosy susceptibility loci identified by genome-wide association studies, five have been implicated in Crohn disease, suggesting a common genetic fingerprint between leprosy and inflammatory bowel disease (IBD). Here, we conducted a multiple-stage genetic association study of 133 IBD susceptibility loci in multiple leprosy samples (totaling 4,971 leprosy cases and 5,503 controls) from a Chinese population and discovered two associations at rs2058660 on 2q12.1 (p = 4.57 × 10(-19); odds ratio [OR] = 1.30) and rs6871626 on 5q33.3 (p = 3.95 × 10(-18); OR = 0.75), implicating IL18RAP/IL18R1 and IL12B as susceptibility genes for leprosy. Our study reveals the important role of IL12/IL18-mediated transcriptional regulation of IFN-γ production in leprosy, and together with previous findings, it demonstrates the shared genetic susceptibility between infectious and inflammatory diseases.

Identification of IL18RAP/IL18R1 and IL12B as leprosy risk genes demonstrates shared pathogenesis between inflammation and infectious diseases.

BackgroundPrevious genome-wide association studies (GWAS) identified multiple susceptibility loci that have highlighted the important role of TLR (Toll-like Receptor) and CARD (caspase recruitment domain) genes in leprosy.MethodsA large three-stage candidate gene-based association study of 30 TLR and 47 CARD genes was performed in the leprosy samples of Chinese Han.ResultsOf 4,363 SNPs investigated, eight SNPs showed suggestive association (Pâ€‰<â€‰0.01) in our previously published GWAS datasets (Stage 1). Of the eight SNPs, rs2735591 and rs4889841 showed significant association (Pâ€‰<â€‰0.001) in an independent series of 1,504 cases and 1,502 controls (Stage II), but only rs2735591 (next to BCL10) showed significant association in the second independent series of 938 cases and 5,827 controls (Stage III). Rs2735591 showed consistent association across the three stages (Pâ€‰>â€‰0.05 for heterogeneity test), significant association in the combined validation samples (Pcorrectedâ€‰=â€‰5.54â€‰Ã—â€‰10(-4) after correction for 4,363 SNPs tested), and genome-wide significance in the whole GWAS and validation samples (Pâ€‰=â€‰1.03â€‰Ã—â€‰10(-9), ORâ€‰=â€‰1.24). In addition, we demonstrated the lower expression of BCL10 in leprosy lesions than normal skins and a significant gene connection between BCL10 and the eight previously identified leprosy loci that are associated with NFÎºB, a major regulator of downstream inflammatory responses, which provides further biological evidence for the association.ConclusionWe have discovered a novel susceptibility locus on 1p22, which implicates BCL10 as a new susceptibility gene for leprosy. Our finding highlights the important role of both innate and adaptive immune responses in leprosy.

Association study of TOLL and CARD with leprosy susceptibility in Chinese population.

BackgroundPrevious genome-wide association recens&eacute;e (GWAS) plusieurs loci de susceptibilit&eacute; qui ont mis en &eacute;vidence le r&ocirc;le important des TLR (Toll-like Receptor) et g&egrave;nes (domaine de recrutement caspase) carte de la l&egrave;pre.MethodsA grand trois &eacute;tapes candidat g&eacute;niques association &eacute;tude de TLR 30 et 47 g&egrave;nes de carte a &eacute;t&eacute; r&eacute;alis&eacute;e dans les &eacute;chantillons de la l&egrave;pre de Chinois Han.ResultsOf 4 363 SNP &eacute;tudi&eacute;, huit SNP a montr&eacute; association suggestive (P 0,01 &lt;) dans nos ensembles de donn&eacute;es GWAS publi&eacute;es ant&eacute;rieurement (&Eacute;tape 1). Des huit Snp, rs2735591 et rs4889841 ont montr&eacute; une association significative (P 0,001 &lt;) dans une s&eacute;rie ind&eacute;pendante de 1 504 cas et 1 502 t&eacute;moins (Stade II), mais seulement rs2735591 (&agrave; c&ocirc;t&eacute; de BCL10) a montr&eacute; une association significative dans la deuxi&egrave;me s&eacute;rie ind&eacute;pendante de 938 cas et t&eacute;moins 5 827 (Stade III). Rs2735591 a montr&eacute; une association coh&eacute;rente &agrave; travers les trois &eacute;tapes (P &gt; 0,05 pour test d&#39;h&eacute;t&eacute;rog&eacute;n&eacute;it&eacute;), association significative dans les &eacute;chantillons de validation combin&eacute;e (Pcorrig&eacute;e = 5.54 &times; 10(-4) apr&egrave;s correction pour les 4 363 SNP test&eacute;) et G&eacute;nome-large signification dans l&#39;ensemble GWAS et validation des &eacute;chantillons (P = 1,03 &times; 10(-9), ou = 1,24). En outre, nous avons d&eacute;montr&eacute; la plus faible expression de BCL10 dans les l&eacute;sions de la l&egrave;pre que peaux normales et une g&egrave;ne significative entre BCL10 et les huit locus de l&egrave;pre pr&eacute;c&eacute;demment identifi&eacute;s associ&eacute;s &agrave; NF&kappa;B, un principal r&eacute;gulateur des r&eacute;actions inflammatoires en aval, qui apporte de nouvelles preuves biologiques pour l&#39;association.ConclusionWe ont d&eacute;couvert un locus de susceptibilit&eacute; roman sur 1 p 22, ce qui implique le BCL10 comme un nouveau g&egrave;ne de susceptibilit&eacute; pour la l&egrave;pre. Notre d&eacute;couverte met en &eacute;vidence le r&ocirc;le important des r&eacute;ponses immunitaires inn&eacute;es et adaptatives la l&egrave;pre.

Association &eacute;tude des services interurbains et de la carte avec la susceptibilit&eacute; de la l&egrave;pre dans la population chinoise.

Aberrantly activated c-MET signaling occurs in several cancers, promoting the development of c-MET inhibitors. In this study, we found that eight of eight thyroid cancer cell lines (including six anaplastic thyroid cell lines) have prominent expression of c-MET protein. Fifty percent of the thyroid cancer cell lines (four of eight) were growth inhibited by two small molecule c-MET inhibitors (tivantinib and crizotinib) associated with apoptosis and G(2)-M cell-cycle arrest. However, crizotinib did not inhibit 50% proliferation of thyroid cancer cells (SW1736 and TL3) at a concentration at which the drug completely inhibited ligand-stimulated c-MET phosphorylation. However, tivantinib was less potent than crizotinib at inhibiting c-MET phosphorylation, but was more potent than crizotinib at decreasing cell growth. Suppressing c-MET protein expression and phosphorylation using siRNA targeting c-MET did not induce cell-cycle arrest and apoptosis. Taken together, tivantinib and crizotinib have off-target(s) activity, contributing to their antitumor activity. In vivo study showed that crizotinib markedly inhibited the growth of thyroid cancer cells (SW1736) in immunodeficient mice. In summary, c-MET inhibitors (tivantinib and crizotinib) suppress the growth of aggressive thyroid cancer cells, and this potential therapeutic benefit results from their non-MET-targeting effects.

Off-target effects of c-MET inhibitors on thyroid cancer cells.

A linear array of periodically spaced and individually controllable skyrmions is introduced as a magnonic crystal. It is numerically demonstrated that skyrmion nucleation and annihilation can be accurately controlled by a nanosecond spin polarized current pulse through a nanocontact. Arranged in a periodic array, such nanocontacts allow the creation of a skyrmion lattice that causes a periodic modulation of the waveguide's magnetization, which can be dynamically controlled by changing either the strength of an applied external magnetic field or the density of the injected spin current through the nanocontacts. The skyrmion diameter is highly dependent on both the applied field and the injected current. This implies tunability of the lowest band gap as the skyrmion diameter directly affects the strength of the pinning potential. The calculated magnonic spectra thus exhibit tunable allowed frequency bands and forbidden frequency bandgaps analogous to that of conventional magnonic crystals where, in contrast, the periodicity is structurally induced and static. In the dynamic magnetic crystal studied here, it is possible to dynamically turn on and off the artificial periodic structure, which allows switching between full rejection and full transmission of spin waves in the waveguide. These findings should stimulate further research activities on multiple functionalities offered by magnonic crystals based on periodic skyrmion lattices. 

Skyrmion-Based Dynamic Magnonic Crystal.

Magnetic skyrmions are promising candidates as information carriers for the next-generation spintronic devices because of their small size, facile current-driven motion and topological stability. The controllable nucleation and motion of skyrmions in magnetic nanostructures will be essential in future skyrmionic devices. Here, we present the microwave assisted nucleation and motion of skyrmion-chains in magnetic nanotrack by micromagnetic simulation. A skyrmion-chain is a one-dimensional cluster of equally spaced skyrmions. A skyrmion-chain conveys an integer bit n when it consists of n skyrmions. A series of skyrmion-chains with various lengths is generated and moved in the nanotrack driven by spin-polarized current. The period, length and spacing of the skyrmion-chains can be dynamically manipulated by controlling either the frequency of the microwave field or the time dependent spin-polarized current density. A skyrmion-chain behaves as a massless particle, where it stops without delay when the current is stopped. Their velocity is found to be linearly dependent on the current density and insensitive to the frequency and amplitude of the excitation microwave field. Uniform motion of trains of skyrmion-chains in nanotrack offers a promising approach for spintronic multi-bit memories containing series of skyrmion-chains to represent data stream. 

Microwave field frequency and current density modulated skyrmion-chain in nanotrack.

Chronic infection with hepatitis B virus (HBV) progresses through different phases. The first, called the immune-tolerant phase, has been associated with a lack of disease activity. We examined HBV-DNA integration, clonal hepatocyte expansion, HBV antigen expression, and HBV-specific immune responses in patients in the immune-tolerant phase to assess whether this designation is appropriate or if there is evidence of disease activity.

HBV DNA Integration and Clonal Hepatocyte Expansion in Chronic Hepatitis B Patients Considered Immune Tolerant.

Optical microscope is one of the most widely used imaging tools for its great flexibility, reliable design, and low cost. Optical microsphere nanoscope (OMN) is invented as a method that can greatly enhance the observation power of conventional optical microscopes. In this perspective, the promising outlook for this approach is briefly discussed. There exists a great freedom to apply this method in various applications. OMN has been successfully commercialized. Our past experience and strategies are summarized in this perspective, which serves as a good reference for the future technology entrepreneurs. Based on our story and model, the factors for success are listed. It can be used to evaluate other commercialization projects and find out the directions that require further improvement.

Yan Zhou

Mechanobiology Institute

National University of Singapore

Isolation of Primary Rat Hepatocytes with Multiparameter Perfusion Control