[Establishment and optimization of hepatocyte steatosis model].

Objective: To establish a new model of hepatic steatosis cells by optimizing the original ethanol or high fat, the present study proposed an in vitro hepatocyte steatosis model for the study of fatty liver. Methods: Oil red O staining was used to observe the effects of fetal bovine serum, oleic acid and ethanol on lipid accumulation in human liver cell line L02 in a concentration- and time-dependent manner. RT-PCR was used to detect the mRNA expression levels of PPAR-γ and AP-2, and the suitable conditions for the establishment of hepatocyte steatosis model were screened out. A t-test was used for comparison between the two groups, and one-way Analysis of Variance (ANOVA) was used in more than three groups. Results: Oil red O staining showed the number of reddish-orange lipid droplets in L02 cells gradually increased with the increase of fetal bovine serum, oleic acid and ethanol in a concentration - and time-dependent manner. Compared with 0.00% oleic acid and 2% ethanol, the count value of red particle was 100.00% ± 17.63% at the beginning and after 24 h, 0.003% oleic acid and 2% ethanol jointly acted in L02 cells. After incubation for 48 hours with 2% ethanol and serum-free DMEM medium, the accumulation of lipid droplets was the highest with a count value of 802.38%+71.06%(t = 42.36, P < 0.001). RT-PCR analysis showed the lipid accumulation induced by this method was positively correlated with the mRNA expression of PPAR-γ and AP-2. Conclusion: L02 cells were successfully exposed to high fat and ethanol, and the hepatocyte steatosis model was established and optimized, suggesting that the occurrence of hepatic cell steatosis was related to the up-regulation of PPAR-γ and AP-2.

Analysis of immunity index and immunopathogenesis pattern of lupus nephritis patients.

Joint detection of anti-dsDNA antibodies, anti-U1RNP, anti-SM antibodies, anti-SSA antibodies, anti-ribosomal P protein antibodies, anti-nucleosome antivodies (Anua), anti-histone antibodies (AHA) and antinuclear antibodies brings to the early diagnosis of systemic lupus erythematosus (SLE) and speculation of renal lesion degree of lupus nephritis patients in order to choose a specific therapeutic schedule. This paper analyzed the abnormal immunology features and connections of each pathological pattern of LN renal biopsy and probed into the essence in order to provide basis for diagnosis, treatment, pathological pattern speculation and forward assessment of LN. We chose 97 cases, treated them with renal biopsy and pathological pattern classification, analyzed pathological pattern distribution, different pathological patterns and the correlation of immunity index with anti-dsDNA antibodies, anti-U1RNP, anti-Sm antibodies, anti-SSA antibodies, anti-ribosomal P protein antibodies, Anua, AHA and ANA of the first renal biopsy were taken as the experiment index. The results showed that the morbidity of the male was distinctly lower than the female and the age of onset was much lower (P < 0.05); pattern I, pattern II, pattern III, pattern IV, pattern V, and pattern VI accounted for 1.0%, 3.1%, 12.4%, 47.4%,16.5%, 15.5%, 4.1%, 0%,respectively; among all the LN patients, there were respectively 59, 43, 28, 52, 51, 48, 36 and 93 cases in which anti-dsDNA antibody, anti-U1RNP antibody, anti-Sm antibody, anti-SSA antibody, anti-ribosomal P protein antibodies, Anua, AHA and ANA had increased and the positive rate was 60.8%, 44.3%, 28.9%, 53.6%, 52.6%, 49.5%, 37.1% and 95.9%, respectively. In conclusion, pattern IV is the most common of all pathological patterns of LN. Among the immunity index, anti- U1RNP antibodies and anti-SSA antibodies are positively correlated with anti-dsDNA antibodies; Anua is positively correlated with anti-dsDNA antibodies and AHA; anti-dsDNA antibodies, anti U1RNP antibodies, anti-Sm antibodies, anti SSA antibodies, AHA, anti-ribosomal P protein antibodies and ANA have no obvious correlation with LN renal lesions degree; Anua level of serum is positively correlated with LN renal lesions degree.

B7H costimulates clonal expansion of, and cognate destruction of tumor cells by, CD8(+) T lymphocytes in vivo.

B7H/B7RP (hereby called B7H) is a new member of the B7 family of costimulatory molecules and interacts with inducible costimulatory molecule (ICOS). Its function for CD8 T cells has not been reported. We report here that expression of B7H on the tumor cells reduced tumorigenicity and induced immunity to subsequent challenge with parental tumor cells. The immune protection correlates with an enhanced cytotoxic T lymphocyte (CTL) response against P1A, the major tumor antigen expressed in the J558 tumor. To understand the mechanism of immune protection, we adoptively transferred transgenic T cells specific for tumor antigen P1A into mice that bore P1A-expressing tumors. We found that while the transgenic T cells divided faster in mice bearing the B7H(+) tumors, optimal B7H-induced clonal expansion of P1CTL required costimulation by B7-1 and B7-2 on the endogenous host antigen-presenting cells (APCs). Interestingly, when B7H(+) and B7H(-) tumors were coinjected, P1CTL selectively eliminated the B7H(+) tumor cells. Moreover, B7H expressed on the tumor cells made them highly susceptible to destruction by CTL in vivo, even if the CTL was administrated into mice with large tumor burdens. Tumors that recurred in the P1CTL-treated mice lost transfected B7H and/or H-2L(d), the class I molecule that presents the P1A peptide. Taken together, our results reveal that B7H costimulates clonal expansion of, and cognate destruction by CD8(+) T lymphocytes in vivo.

Regulation of fas ligand expression during activation-induced cell death in T cells by p38 mitogen-activated protein kinase and c-Jun NH2-terminal kinase.

Activation-induced cell death (AICD) is a mechanism of peripheral T cell tolerance that depends upon an interaction between Fas and Fas ligand (FasL). Although c-Jun NH2-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) may be involved in apoptosis in various cell types, the mode of regulation of FasL expression during AICD in T cells by these two MAPKs is incompletely understood. To investigate the regulatory roles of these two MAPKs, we analyzed the kinetics of TCR-induced p38 MAPK and JNK activity and their regulation of FasL expression and AICD. We report that both JNK and p38 MAPK regulate AICD in T cells. Our data suggest a novel model of T cell AICD in which p38 MAPK acts early to initiate FasL expression and the Fas-mediated activation of caspases. Subsequently, caspases stimulate JNK to further upregulate FasL expression. Thus, p38 MAPK and downstream JNK converge to regulate FasL expression at different times after T cell receptor stimulation to elicit maximum AICD.

A statistical approach for intensity loss compensation of confocal microscopy images.

In this paper, a probabilistic technique for compensation of intensity loss in confocal microscopy images is presented. For single-colour-labelled specimen, confocal microscopy images are modelled as a mixture of two Gaussian probability distribution functions, one representing the background and another corresponding to the foreground. Images are segmented into foreground and background by applying Expectation Maximization algorithm to the mixture. Final intensity compensation is carried out by scaling and shifting the original intensities with the help of parameters estimated for the foreground. Since foreground is separated to calculate the compensation parameters, the method is effective even when image structure changes from frame to frame. As intensity decay function is not used, complexity associated with estimation of the intensity decay function parameters is eliminated. In addition, images can be compensated out of order, as only information from the reference image is required for the compensation of any image. These properties make our method an ideal tool for intensity compensation of confocal microscopy images that suffer intensity loss due to absorption/scattering of light as well as photobleaching and the image can change structure from optical/temporal section-to-section due to changes in the depth of specimen or due to a live specimen. The proposed method was tested with a number of confocal microscopy image stacks and results are presented to demonstrate the effectiveness of the method.

On the site and mode of antigen presentation for the initiation of clonal expansion of CD8 T cells specific for a natural tumor antigen.

Because of the low frequency of antigen-specific T cells, early events in the activation of tumor-specific T cells in vivo have not been well characterized. There is still no direct documentation on where the clonal expansion begins and how tumor antigens are presented to the host CD8 T cells to initiate it. Here we used transgenic T cells specific for a natural tumor antigen P1A to evaluate the kinetics, location, and modes of antigen presentation for initiating CTL response in vivo. Our results demonstrate that the initial activation of P1A-specific T cells takes place in the lymphoid organs. The activated T cells then migrate into tumors, where they undergo accelerated division and acquire distinct activation markers. The site of initiation cannot be altered by either local expression of costimulatory molecules or by intratumor injection of naïve T cells. Moreover, using genetic models that allow only one mode of antigen presentation, we show here that both cross-presentation of P1A by the host antigen-presenting cells, and direct antigen presentation and costimulation by the tumor cells are sufficient to initiate rapid T cell-clonal expansion in the lymphoid organ. These results provide direct evidence for two fundamental assumptions on the mechanisms of T-cell activation in vivo.

Absolute thickness measurement of pyrolytic graphite spheroids by STEM-EELS.

This paper studies the absolute thickness measurement of pyrolytic graphite spheroids (GSs) by using STEM-EELS mode with log-ratio method and Kramers-Kroning (K-K) method, taking the measured thickness from TEM image as reference that is the diameter of GSs ranging from 60 to 250nm. The effect of collection semi-angle (ß) on thickness measurement has been investigated. It is found that in general the thickness obtained by K-K analysis with surface effect corrected shows the best accuracy, followed by K-K sum rule and then log-ratio method for the three different collection semi-angles of 12.4, 17.3 and 21.1mrad applied. Of these angles, the smallest one gives an overestimated result and the largest one gives an underestimated result, whereas between the two, the angle of 17.3mrad that is about 2x convergence semi-angle (9.0mrad) is identified as more appropriate for K-K analysis. The surface-scattering correction, inelastic mean free path of GS and effect of refractive index n on thickness measurement for different ß angles are also investigated. Moreover, the optical property deduced from the data collected at the center of graphite spheroid, which is related to its microstructure, is characterized by K-K analysis.

Electron microscopic appearance of the chronic Campylobacter jejuni enteritis of mice.

Campylobacter jejuni is a major cause of human enteritis which mimics the inflammatory bowel disease (IBD). In this study, microstructural changes on the surfaces of the murine gastrointestinal tract persistently colonized by Campylobacter jejuni, strain GJ-S131, were investigated by using scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The results revealed that the appearance of the gastrointestinal mucosa in both BALB/C and KM mice resembled that in human with inflammatory bowel disease. Under SEM, the mucosa of the jejunum and ileum, with broken or distorted villi had a "worm eaten" look; crypts were irregular in shape and size, and the mucosa showed atrophy, especially in the colon. Epithelial junctions demonstrated furrows, clefts or deep crevasses, with exudates containing a large number of leukocytes. Cytologic appearances were characterized by microvilli dysplasia and/or atrophy, patchy erosions or necrosis and pelade-like appearance due to absence of microvilli, which were similar to the findings under TEM.

[Effects of stimulation of dorso-medial area of nucleus facialis on respiration related units in ventro-lateral region of nucleus tractus solitaris in rabbits].

In urethane-anesthetized, vagotomized and paralyzed rabbits, effects of electrical stimulation of the dorso-medial area of the nucleus facialis (DMNF) on the respiration-related units (RRUs) in ventro-lateral region of nucleus tractus solitaris (VLNTS) were observed. The experimental results showed that during electrical stimulation of DMNF the majority of the inspiratory (I) neurons (64.4%) were increased in frequency and duration of discharge, some to a marked extent. During electrical stimulation of DMNF the expiratory neurons (35%) were decreased in their frequency and duration of discharge, some to a marked extent too. The responses of RRUs in ipsilateral and contralateral VLNTS to stimulation of DMNF was not statistically significant (P greater than 0.05). It is suggested that DMNF may have a facilitating effect on the inspiratory neurons and an inhibiting effect on the expiratory neurons in VLNTS.

The prognostic value of P-cadherin in non-muscle-invasive bladder cancer.

OBJECTIVES: This research aims to specify the prognostic value of P-cadherin on recurrence and progression in non-muscle-invasive bladder cancers (NMIBC). METHODS: A total of 110 NMIBC cases were collected and P-cadherin protein was assessed by immunohistochemical test in these samples. Correlations between P-cadherin expression and clinicopathologic features were analyzed. For recurrence-free and progression-free survival, Kaplan-Meier log-rank test was used. Then Cox univariate and multivariate analyses were further performed. RESULTS: P-cadherin high expression correlated with tumor progression (P = 0.031). Kaplan-Meier results showed that patients with high P-cadherin expression had worse progression-free survival (P = 0.034) but not recurrence-free survival (P = 0.133) than low-expression patients. Cox regression results showed P-cadherin expression was an independent predictor for progression (P = 0.042) but not recurrence (P = 0.139) in NMIBC. CONCLUSIONS: Our results demonstrated that P-cadherin expression correlated with tumor progression and could be taken as an independent predictor for progression in NMIBC.

ERalpha signaling through slug regulates E-cadherin and EMT.

The ERalpha signaling pathway is one of the most important and most studied pathways in human breast cancer, yet numerous questions still exist such as how hormonally responsive cancers progress to a more aggressive and hormonally independent phenotype. We have noted that human breast cancers exhibit a strong direct correlation between ERalpha and E-cadherin expression by immunohistochemistry, suggesting that ERalpha signaling might regulate E-cadherin and implying that this regulation might influence epithelial-mesenchymal transition (EMT) and tumor progression. To investigate this hypothesis and the mechanisms behind it, we studied the effects of ERalpha signaling in ERalpha-transfected ERalpha-negative breast carcinoma cell lines, the MDA-MB-468 and the MDA-MB-231 and the effects of ERalpha knockdown in naturally expressing ERalpha-positive lines, MCF-7 and T47D. When ERalpha was overexpressed in the ERalpha-negative lines, 17beta-estradiol (E2) decreased slug and increased E-cadherin. Clones maximally exhibiting these changes grew more in clumps and became less invasive in Matrigel. When ERalpha was knocked down in the ERalpha-positive lines, slug increased, E-cadherin decreased, cells became spindly and exhibited increased Matrigel invasion. ERalpha signaling decreased slug expression by two different mechanisms: directly, by repression of slug transcription by the formation of a corepressor complex of ligand-activated ERalpha, HDAC inhibitor (HDAC1), and nuclear receptor corepressor (N-CoR) that bound the slug promoter in three half-site estrogen response elements (EREs); indirectly by phosphorylation and inactivation of GSK-3beta through phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt). The GSK-3beta inactivation, in turn, repressed slug expression and increased E-cadherin. In human breast cancer cases, there was a strong inverse correlation between slug and ERalpha and E-cadherin immunoreactivity. Our findings indicate that ERalpha signaling through slug regulates E-cadherin and EMT.

Altered thymic selection by overexpressing cellular FLICE inhibitory protein in T cells causes lupus-like syndrome in a BALB/c but not C57BL/6 strain.

The cellular FLICE inhibitory protein (c-FLIP) is an endogenous inhibitor of the caspase-8 proapoptotic signaling pathway downstream of death receptors. Recent evidence indicates that the long form of c-FLIP (c-FLIP(L)) is required for proliferation and effector T-cell development. However, the role of c-FLIP(L) in triggering autoimmunity has not been carefully analyzed. We now report that c-FLIP(L) transgenic (Tg) mice develop splenomegaly, lymphadenopathy, multiorgan infiltration, high titers of auto-antibodies, and proliferative glomerulonephritis with immune complex deposition in a strain-dependent manner. The development of autoimmunity requires CD4(+) T cells and may result from impaired thymic selection. At the molecular level, c-FLIP(L) overexpression inhibits the zeta chain-associated protein tyrosine kinase of 70 kDa (ZAP-70) activation, thus impairing the signaling pathway derived from ZAP-70 required for thymic selection. Therefore, we have identified c-FLIP(L) as a susceptibility factor under the influence of epistatic modifiers for the development of autoimmunity.

Energy transmission in a mechanically-linked double-wall structure coupled to an acoustic enclosure.

The energy transmission in a mechanically linked double-wall structure into an acoustic enclosure is studied in this paper. Based on a fully coupled vibro-acoustic formulation, focus is put on investigating the effect of the air gap and mechanical links between the two panels on the energy transmission and noise insulation properties of such structures. An approximate formula reflecting the gap effect on the lower-order coupled frequencies of the system is proposed. A criterion, based on the ratio between the aerostatic stiffness of the gap cavity and the stiffness of the link, is proposed to predict the dominant transmitting path, with a view to provide guidelines for the design of appropriate control strategies. Numerical results reveal the existence of three distinct zones, within which energy transmission takes place following different mechanisms and transmitting paths. Corresponding effects on noise insulation properties of the double-wall structure are also investigated.

The effect of ebselen on polymorphonuclear leukocyte and lymphocyte migration to inflammatory reactions in rats.

Ebselen, a selinyl organic compound with anti-inflammatory properties was found by us previously to inhibit in vitro human polymorphonuclear leukocyte (PMNL) adhesion to and migration through umbilical vein endothelium monolayers. Here we investigated in rats the effect of ebselen on PMNL and spleen T lymphocyte (SPLT) migration to inflamed joints induced by intra-articular (i.a) injection of recombinant murine tumor necrosis factor alpha (mTNF alpha) and to dermal inflammatory reactions. Inflammation was induced in the carpal and talar joints of rats by intra-articular (i.a.) injection (100 ng) of mTNF alpha once daily for 2 days. Corresponding joints in the opposite limb received diluent. Simultaneously, the rats were treated p.o. with either ebselen (100 mg/kg/day) or indomethacin (2 mg/kg/day) or vehicle for 2 days. Dermal inflammation was induced by intradermal injection (0.05 ml) of inflammatory stimuli. Accumulation of 51Cr-labelled rat blood PMNL, 111In-labeled SPLT, and extravasation of 125I-labelled human serum albumin (HSA) in the joints and in skin sites were measured. Treatment of rats with ebselen inhibited by 33-65% PMNL migration to the mTNF alpha inflamed joints, and to dermal inflammation induced by zymosan activated serum (ZAS; containing C5adesArg), endotoxin (LPS), mIL-1 beta and mTNF alpha. Migration of SPLT to dermal inflammation induced by interferon gamma (IFN gamma), poly-inosine-cytosine (poly I:C) and LPS was also significantly inhibited (22-33%), but SPLT migration into the inflamed joints was not effected by ebselen. Indomethacin treatment of rats also inhibited PMNL migration into the inflamed joints, but unlike ebselen, indomethacin inhibited only ZAS induced dermal PMNL accumulation. In contrast to ebselen, indomethacin inhibited SPLT migration into the inflamed joints as well as to the dermal inflammation induced by poly I:C and a delayed-type hypersensitivity reaction (DTH). In addition, treatment of rats with indomethacin significantly inhibited plasma protein (125I-HSA) extravasation in the inflamed joints and the dermal inflammatory reaction induced by ZAS, but ebselen had no such effect. In conclusion, ebselen appears to have a distinct antiinflammatory mechanism of action from indomethacin and the PMNL findings are consistent with a direct inhibitory action on PMNL activation and PMNL transendothelial migration as observed previously in vitro.

Polymorphonuclear leucocyte migration through human dermal fibroblast monolayers is dependent on both beta 2-integrin (CD11/CD18) and beta 1-integrin (CD29) mechanisms.

Accumulation of leucocytes in inflammation involves their migration through vascular endothelium and then in the connective tissue. We investigated human polymorphonuclear leucocyte (PMNL) migration through a biological barrier of human dermal fibroblasts grown on microporous filters, as a model of PMNL migration in the connective tissue. PMNL did not migrate through a fibroblast monolayer unless a chemotactic factor, e.g. C5a, interleukin-8 (IL-8) or zymosan-activated plasma (ZAP; C5adesArg), was added. This migration was partially inhibited (35-70%, depending on the stimulus) by treatment of PMNL with monoclonal antibody (mAb) to CD18 (beta 2-integrins). Most of the CD18-independent migration was inhibited by mAb to beta 1-integrins (CD29). Inhibition by mAb to beta 1 was observed when the PMNL, but not the fibroblasts, were treated with mAb. The role of beta 1-integrins in PMNL transfibroblast migration was detectable only when the function of the CD11-CD18 complex was blocked, because mAb to beta 1-integrin alone had no significant effect on PMNL migration. Migration induced by C5a was more CD18-independent compared to IL-8 or C5adesArg. The CD18-independent migration was also inhibited by mAb to the beta 1-integrin subunits alpha 5 (of very late antigens-5; VLA-5) and alpha 6 (of VLA-6). Treatment of the fibroblasts (4 hr) with tumour necrosis factor-alpha (TNF-alpha) or IL-1 alpha enhanced C5a-induced PMNL transfibroblast migration and increased the proportion of migration utilizing the CD11-CD18 mechanism. However, TNF-alpha treatment had no effect on the degree of beta 1-integrin-dependent migration. These findings suggest that in response to the chemotactic factors C5a, IL-8 and C5adesArg, PMNL migration in the connective tissue is mediated by both CD11-CD18 (beta 2) and beta 1-integrins on the PMNL. The VLA-5 and VLA-6 members of beta 1-integrins are involved in this process. This is in contrast to PMNL migration across endothelium in this system, which is virtually all CD18 dependent with no significant role for beta 1-integrins.

The effect of ebselen on T-lymphocyte migration to arthritic joints and dermal inflammatory reactions in the rat.

We have previously observed that ebselen (PZ 51, 2-Phenyl-1,2-Bensoisoselenazol-3-(2H)-one) can inhibit human polymorphonuclear leukocyte (PMNL) transendothelial migration in vitro and PMNL migration to arthritic joints and dermal inflammatory reactions in rats. In this study, we investigated the effect of ebselen on T-lymphocyte migration to the inflamed joints in rats with adjuvant arthritis (AA) and to dermal inflammation induced by cytokines (IFN gamma, mTNF alpha), cytokine inducing stimuli (poly I:C and LPS), or a delayed type hypersensitivity (DTH) reaction. Treatment of rats with AA with ebselen (100 mg/kg/day) p.o. for three days significantly reduced accumulation of 111In-labelled spleen T-cells (SPLT) in the arthritic joints, including forepaws, carpal joints, hindpaws and talar joints, and in all the above dermal inflammatory reactions. The inhibitory effect of ebselen on SPLT cell accumulation was greater than with indomethacin (2 mg/kg/day) and was observed within 3 h of initiation of ebselen treatment. Ebselen also inhibited SPLT migration to mandibular, axillary and mesenteric lymph nodes, and to the spleen. The results suggest that not only does ebselen inhibit SPLT migration to inflamed joints and to dermal inflammation but it also may inhibit lymphocyte homing and recirculation. Whether these effects of ebselen are related to its reported inhibition of cellular activation and intracellular signalling requires further investigation. However, the inhibition of T-lymphocyte migration reported here and of PMNL migration reported previously may both be beneficial in the treatment of human arthritis.