In vivo diffusion analysis with quantum dots and dextrans predicts the width of brain extracellular space

Diffusion within the extracellular space (ECS) of the brain is necessary for chemical signaling and for neurons and glia to access nutrients and therapeutics; however, the width of the ECS in living tissue remains unknown. We used integrative optical imaging to show that dextrans and water-soluble quantum dots with Stokes-Einstein diameters as large as 35 nm diffuse within the ECS of adult rat neocortex in vivo. Modeling the ECS as fluid-filled "pores" predicts a normal width of 38-64 nm, at least 2-fold greater than estimates from EM of fixed tissue. ECS width falls below 10 nm after terminal ischemia, a likely explanation for the small ECS visualized in electron micrographs. Our results will improve modeling of neurotransmitter spread after spillover and ectopic release and establish size limits for diffusion of drug delivery vectors such as viruses, liposomes, and nanoparticles in brain ECS.     

Adhesion to the extracellular matrix is positively regulated by retinoic acid in HepG2 cells.

Aims: In this work, we aimed to investigate the possible modulation of cell-matrix interactions by retinoic acid (RA), in view of the well-known role of the extracellular matrix (ECM) and integrins in hepatocyte differentiation and proliferation. For this purpose, we analysed the adhesion ability of HepG2 cells on different substrates in the presence and absence of RA evaluating both the expression and cellular localisation of major proteins involved in focal contacts, using Western blot and confocal microscopy. RESULTS: A positive and substrate-dependent effect of RA on cell-matrix adhesion was observed after long-term culture. The increased adhesiveness in the treated cells was accompanied by an enhanced expression of beta1 and alpha3 integrin subunits, together with a redistribution of beta1 receptors clustered at the basal surface. In contrast, the levels of focal adhesion kinase (FAK), paxillin and alpha-actinin were unchanged, as was the phosphorylation state of FAK. Nonetheless, a stronger association between beta1 integrin and intracytoplasmatic proteins of focal contacts was observed in coimmunoprecipitation experiments after RA treatment, suggesting improved connection with the actin cytoskeleton. These results are consistent with previously described antiproliferative and differentiative effects of RA on transformed hepatocytes, and confirm the hypothesis of a direct influence of RA on specific adhesion molecules.     

引经药柴胡对胰腺纤维化大鼠细胞外基质合成调控增效的作用

[目的]观察引经药柴胡对大黄丹参调控胰腺纤维化大鼠细胞外基质(ECM)合成增效的作用。[方法]50只雄性SD大鼠随机分为假手术组、模型组、柴胡组、大黄丹参组和柴胡大黄丹参组,每组10只。假手术组开腹后关腹,其他各组通过结扎胰胆管联合腹腔注射雨蛙素3d建立胰腺纤维化模型;柴胡组、大黄丹参组、柴胡大黄丹参组分别于术前、后3d用对应药物灌胃(剂量分别为1.38g.kg-1.d-1、3.04g.kg-1.d-1、4.11g.kg-1.d-1)。术后第4天取材用苏木精-伊红染色观察胰腺病理学变化,免疫组化检测胰腺α-平滑肌肌动蛋白及Ⅰ型胶原表达,ELISA测定血清纤维连接蛋白(FN)、层结合蛋白(LN)含量。[结果]与模型组相比,柴胡组、大黄丹参组和柴胡大黄丹参组均可改善胰腺病理变化,降低α-平滑肌肌动蛋白、Ⅰ型胶原表达,降低血清LN及FN含量,均P0.01;其中柴胡大黄丹参组FN含量降低更显著。[结论]柴胡、大黄丹参、柴胡大黄丹参均可通过抑制ECM合成来发挥抗胰腺纤维化的作用,其中柴胡大黄丹参在降低血清FN含量方面效果最优,提示柴胡在抑制胰腺ECM合成方面对大黄丹参存在一定的增效作用。     

Disturbances of T-cell interactions with endothelium and the extracellular matrix proteins in a patient with Takayasu arteritis

Abstract. T-cell interactions with endothelium and the extracellular matrix proteins were studied in vitro in a patient with Takayasu arterisis. Markedly enhanced spontaneous adhesiveness to cultured endothelium and collagen IV and fibronectin were found to be paralleled by abolished responses to the costimulating action of collagens I and IV and fibronectin. Those abnormalities were partly corrected by immunosuppressive therapy. Aberrant interactions of T cells with endothelium and the extracellular matrix proteins may underlay the pathogenesis of some forms of vasculitis.     

Diffuse expression of heparan sulfate proteoglycan and connective tissue growth factor in fibrous septa with many mast cells relate to unresolving hepatic fibrosis of congenital hepatic fibrosis.

BACKGROUND AND AIMS: Congenital hepatic fibrosis (CHF) is characterized by dense portal/septal fibrosis and bile duct proliferation and tortuosity. In this study, the roles and significance of fibrosis-related cells and molecules in the process of progressive and unresolving fibrosis of CHF were examined in comparison with other fibrotic liver diseases. METHODS: Seven CHF livers were examined, and a total of 74 control livers (chronic viral hepatitis (CVH), alcoholic fibrosis/cirrhosis (F/C), extrahepatic biliary obstruction and livers showing non-specific reactive changes) were used as controls. All of these livers were wedge biopsied or surgically resected ones, and were formalin fixed and paraffin embedded. In addition to histologic observations, expression of heparan sulfate proteoglycan (HSPG), connective tissue growth factor (CTGF), mast cell-specific tryptase, alpha-smooth muscle actin for activated hepatic stellate cells (HSC) or myofibroblasts (MF) were immunohistochemically surveyed. HSPG and CTGF at mRNA were also examined by in situ hybridization. RESULTS: Portal/septal fibrosis of CHF were mature collagenous and elastic fiber poor, when compared with controls. HSPG and CTGF were diffusely abundant in fibrous portal tracts/septa in CHF, while they were more or less accentuated at periportal areas in alcoholic F/C and CVH. In CHF, the number of interface and portal/septal MF was increased from mild-to-moderate degree, while their increase was moderate to marked in alcoholic F/C and CVH, particularly F3/F4. While activated HSC were frequent in alcoholic F/C and CVH and they were continuous with interface MF, activated HSC in CHF were scanty. Instead, mast cells were increased in portal/septal fibrosis of CHF. Portal mononuclear cells and endothelial cells were positive for HSPG mRNA, and mononuclear cells for CTGF mRNA, and such cells were accentuated around proliferated bile ducts and ductules in CHF. CONCLUSIONS: Abundant CTGF retained diffusely in HSPG in the fibrous portal tracts/septa may be responsible for non-resolving hepatic fibrosis in CHF, and many mast cells and portal MF not related to HSC may causally relate to such characteristic finding in CHF.     

Bone marrow stroma‐mediated resistance to FLT3 inhibitors in FLT3‐ITD AML is mediated by persistent activation of extracellular regulated kinase

A consistent pattern of response has been observed when FMS‐like tyrosine kinase 3 (FLT3) tyrosine kinase inhibitors (TKIs) have been used as monotherapy to treat patients with relapsed or refractory FLT3‐ internal tandem duplication (ITD) acute myeloid leukaemia (AML). Circulating blasts are cleared from the peripheral blood, while bone marrow blasts are either unaffected or are cleared from the marrow at a much slower rate. We used an in vitro model of FLT3‐ITD AML blasts co‐cultured with normal human bone marrow stromal cells to investigate the basis for this dichotomous response pattern to FLT3 inhibitors. We have found that in blasts on stroma, potent FLT3 inhibition predominantly results in cell cycle arrest rather than apoptosis. The anti‐apoptotic effect is mediated through a combination of direct cell‐cell contact and soluble factors. The addition of exogenous FLT3 ligand (FL) augments the protection, primarily by shifting the 50% inhibitory concentration for FLT3 inhibition upwards. Cytokine‐activated extracellular regulated kinase (ERK), rather than STAT5, appears to be the most important downstream signalling protein mediating the protective effect, and inhibition of MEK significantly abrogates stromal‐mediated resistance. These findings explain the phenomenon of peripheral blood versus bone marrow blast responses and suggest that the combination of potent FLT3 inhibition and MEK inhibition is a promising strategy for the treatment of FLT3‐ITD AML.     

In vivo efficacy of intravenous gammaglobulins in patients with lupus anticoagulant is not mediated by an anti-idiotypic mechanism.

The authors have investigated the presence in commercially available intravenous gammaglobulins (IVIg) of anti-idiotypic antibodies directed to Lupus Anticoagulant (LA). In vitro incubation of 4 LA plasmas with increasing concentrations of IVIg (from 0 to 39 mg/ml) resulted in a dose-dependent inhibition of LA activity (the highest inhibitions ranged from 14.0 to 53.4%). Similar results were obtained when patients' plasma was substituted with total IgG (the highest inhibitions ranged from 43.0 to 55.0% and were obtained at IgG:IVIg molar ratios ranging from 1:15 to 1:50). Also the incubation of patients' F(ab')2 with F(ab')2 from IVIg produced a similar dose-dependent inhibition of LA activity. These data are suggestive of an in vitro idiotypic-anti-idiotypic interaction between LA and IVIg. However, when injected in patients with LA, IVIg do not seem to operate by this mechanism of action. In fact, reduction or disappearance of LA was only observed in 2 out of 4 patients; also the quick reappearance of LA activity was not consistent with the time course of anti-idiotypic response. Finally, this effect was reached by half the IVIg concentrations necessary to produce an appreciable inhibitory effect on LA activity in vitro. Thus, it is concluded that, even if IVIg contain anti-idiotypic antibodies reacting with LA, the clinical efficacy of IVIg treatment in patients with these autoantibodies should be attributed to other mechanisms.     

Phosphoprotein enriched in diabetes gene product (Ped/pea-15) is increased in omental adipose tissue of women with the polycystic ovary syndrome: ex vivo regulation of ped/pea-15 by glucose, insulin and metformin

Polycystic ovary syndrome (PCOS), the commonest endocrine disorder in women, is characterized by an altered steroid milieu and is associated with insulin resistance and type 2 diabetes mellitus (T2DM). Phosphoprotein enriched in diabetes gene product (Ped/pea-15) regulates glucose metabolism and is increased in T2DM. Our novel data indicate that Ped/pea-15 mRNA expression and protein levels are significantly increased in omental adipose tissue (AT) from PCOS women compared to matched controls (p < 0.01); Ped/pea-15 levels in subcutaneous AT were not significantly different. Furthermore, Ped/pea-15 mRNA expression and protein levels were higher in omental compared to subcutaneous AT in PCOS subjects (p < 0.01); however, in control subjects, this was not significant. Glucose was predictive of omental AT Ped/pea-15 mRNA expression (p = 0.045). Importantly, glucose and insulin increased whereas metformin significantly decreased Ped/pea-15 levels in human omental AT explants. Our findings should serve to promote further research on Ped/pea-15 biology.