人参皂甙Rg1对老年大鼠免疫功能的调节作用

已知老年机体免疫功能的降低与淋巴细胞增殖能力的减弱和白细胞介素-2(IL-2)产生减少有密切关系。以老年大鼠免疫功能为主要研究对象,首次发现人参皂甙Rg1无论体内给药还是体外实验均能选择性增强老年大鼠脾淋巴细胞增殖能力和IL-2的产生与释放,采用Northern和Western印迹分析法证明,Rg₁可明显促进IL-2基因和蛋白的表达,表现在IL-2mRNA和IL-2蛋白含量的显著增加。值得注意的是,在同样的条件下,Rg₁对青年大鼠免疫功能的影响并不显著,由此可以认为Rg1一种“免疫调节剂”,而并非单纯的“免疫增强剂”。     

Very efficient myoblast allotransplantation in mice under FK506 immunosuppression

Transgenic CD1 mice expressing β-galactosidase were used as myoblast donors. The myoblasts were injected in normal or mdx muscles previously irradiated and injected with notexin. Twenty-eight days after myoblast transplantation, the percentage of muscle fibers β-galactosidase-positive was low in mice not immunosuppressed but was high (80%) in those treated with FK506. In mdx mice, muscle fibers expressing β-galactosidase were also dystrophin positive. Most of the mice not treated with FK506 produced antibodies against the donor myoblasts. These results indicate that FK506 is a very useful immunosuppressive drug for myoblast transplantation in mice. Irradiation and notexin injection used in our experiments are, however, not feasible in humans. Other manipulations capable of increasing the participation of donor myoblasts to regeneration will therefore have to be identified before new clinical trials are attempted. © 1994 John Wiley & Sons, Inc.     

Markers of oxidative stress and antioxidant activity in plasma and erythrocytes in neonatal respiratory distress syndrome

Markers of oxidative stress and antioxidant activity in plasma and erythrocytes were studied for 14 d after birth in infants with neonatal respiratory distress syndrome ( n = 9) and controls ( n = 36). In plasma, the total radical trapping antioxidant capacity and the chain-breaking antioxidants vitamin C, sulfhydryl groups and bilirubin were similar. The differences in uric acid levels were not consistent, but vitamin E levels and vitamin E/total-lipid ratio were lower in the neonatal respiratory distress group ( p < 0.01). In erythrocytes, the antioxidant enzymes glutathione peroxidase, glutathione reductase and superoxide dismutase did not differ postnatally. Indicators of oxidative damage in plasma (sulfhydryl/protein ratio and thiobarbituric acid reactive substances) showed the same postnatal course in both groups and were not influenced by oxygen therapy. In erythrocytes the reduced/oxidized glutathione ratio showed no consistent differences. In conclusion, this study, using erythrocytes and plasma, does not provide convincing evidence of oxidative damage and diminished antioxidant defenses in preterm infants with neonatal respiratory distress syndrome.     

Cloning, functional activities and in vivo tissue distribution of rat NKR-P1+ TCR alpha beta + cells

We have successfully cloned nine NKR-P1+ TCR alpha beta + cells from PVG rat spleens, utilizing murine macrophage inflammatory protein-1 alpha (MIP-1 alpha) and IL-2. These clones are either double negative (DN, CD4-CD8-), which included clones 3.31, 3.71, 4.19, 4.59 and 4.65, or single positive (SP, CD4+CD8-), which included clones 1.64, 3.8, 3.76 and 3.78. No CD8+ clone was recovered. All nine clones are restricted in terms of their expression of the V beta antigens, since they express V beta 8.2 but not V beta 8.5, V beta 10 or V beta 16. These clones are agranular and they fall to generate NK or LAK activity upon incubation with IL-2, IL-12 or their combination. On the basis of their production of intracellular cytokines they can be divided into three categories: (I) SP clones (1.64, 3.8, 3.76 and 3.78) do not produce IL-2 or IL-4, but produce IFN-gamma and IL-12, and they vary in their production of IL-1, RANTES or tumor necrosis factor (TNF)-alpha; (II) DN clones 4.59 and 4.65 produce IL-1 alpha and IFN-gamma only, and fall to produce other cytokines; and (III) DN clones 3.31, 3.71 and 4.19 produce IL-1 alpha, IL-1 beta, IL-2, IL-12, IFN-gamma, RANTES and TNF-alpha. From all the clones examined only DN clones 3.31 and to a lesser degree 4.19 produce IL-4. In vivo tissue localization of clones 3.8, 3.31 and 4.59 shows that these cells distribute into the liver and bone marrow 24 h post i.v. administration. Their accumulation in the liver and bone marrow along with their ability to secrete various cytokines suggest that these cells may influence the generation, differentiation or apoptosis of immune or hematopoietic cells.      

A nonionic amphiphile agent promotes gene delivery in vivo to skeletal and cardiac muscles

Direct injection of naked DNA into skeletal or cardiac muscle induces detectable gene expression. Although this provides a practical system for transgene expression, the reported efficacy is too low to confer a therapeutic benefit. By following a rational strategy based on the supramolecular structures adopted by active complexes, we have discovered a novel nonionic amphiphile synthetic agent [poly(ethyleneoxide)(13)-poly(propyleneoxide)(30)-poly(ethyleneoxide)(13) block copolymer; PE6400] that enables gene expression in up to 35% of muscle fibers from mouse tibial cranial muscle. PE6400 abolishes the ceiling effect on transgene expression of increasing amounts of naked DNA and permits long-term expression of the beta-galactosidase reporter gene in immunologically tolerant transgenic rats. This improvement in gene expression over naked DNA was observed irrespective of the reporter gene, ranging from 0.7 to 3.4 kb, and of the animal model used. In skeletal muscle, the PE6400 formulation led to a level of transfection efficiency similar to that obtained by electrotransfer. PE6400 also promotes high transgene expression in cardiac muscle. In contrast, PE6400-DNA formulations were inefficient in vitro in established cell lines and in isolated cardiomyocytes. When microinjected into the cell cytoplasm, PE6400 promotes DNA trafficking into the nucleus and induces gene expression. PE6400 provides a simple gene delivery system for skeletal and myocardial gene transfer. We propose that the PE6400 formulation could serve for the treatment of diseases primarily affecting muscle or for the expression of therapeutic proteins for local or systemic benefit.     

Epstein–Barr virus in the pathogenesis of oral cancers

Epstein–Barr virus (EBV) is a ubiquitous gamma‐herpesvirus that establishes a lifelong persistent infection in the oral cavity and is intermittently shed in the saliva. EBV exhibits a biphasic life cycle, supported by its dual tropism for B lymphocytes and epithelial cells, which allows the virus to be transmitted within oral lymphoid tissues. While infection is often benign, EBV is associated with a number of lymphomas and carcinomas that arise in the oral cavity and at other anatomical sites. Incomplete association of EBV in cancer has questioned if EBV is merely a passenger or a driver of the tumorigenic process. However, the ability of EBV to immortalize B cells and its prevalence in a subset of cancers has implicated EBV as a carcinogenic cofactor in cellular contexts where the viral life cycle is altered. In many cases, EBV likely acts as an agent of tumor progression rather than tumor initiation, conferring malignant phenotypes observed in EBV‐positive cancers. Given that the oral cavity serves as the main site of EBV residence and transmission, here we review the prevalence of EBV in oral malignancies and the mechanisms by which EBV acts as an agent of tumor progression.     

Myoblasts and Embryonic Stem Cells Differentially Engraft in a Mouse Model of Genetic Dilated Cardiomyopathy

The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.Cell therapies are progressively emerging as promising tools for the treatment of heart failure. In an attempt to achieve cardiac cell-based replacement therapy in the setting of postischemic cardiomyopathies (ICM), a variety of adult cell types have been tested up to preclinical stages in small and large animal models, including skeletal myoblasts (Mb), muscle-derived stem cells, adipose-derived stem cells, bone marrow mononuclear cells, hematopoietic stem cells, circulating endothelial progenitors, mesenchymal stem cells, smooth muscle cells, cardiac stem cells, and most of these approaches have demonstrated some degree of efficacy.^{1,2,3,4,5,6,7} Except for some specific populations of cardiac stem cells, most categories of adult stem cells show partial or complete inability to produce bona fide cardiomyocytes and to participate to true myocardial tissue formation, with respect to homogeneity of electrical conduction.⁸ Their functional benefits would be linked, essentially, to the mechanical strengthening of the scar tissue, and/or to the promotion of myocardial cell survival through paracrine synthesis of trophic factors and/or improved local angiogenesis.^{1,4,7,8,9,10,11} Indeed, phase II randomized clinical trials developed using adult stem cells have provided encouraging but still limited results.^12,13 However, the applicability and therapeutic relevance of cell therapies remain under-explored for nonischemic heart failure (dilated cardiomyopathy (DCM), myocarditis), probably due to the progressive nature of the disease and extension of fibrotic remodeling, which make the targeting of a specific area more difficult than when considering a delineated scar formed upon myocardial infarction. A few preclinical studies have been carried out using Mb,^14,15 smooth muscle cells or ventricular heart cells¹⁶ in cardiomyopathic hamsters, or mesenchymal stem cells,¹⁷ mixed mesenchymal stem cells and Mb,¹⁸ or bone marrow cells in rat models of DCM.¹⁹ Among those studies, Mb seem to have the best potential of integration in the dilated myocardium, and represent a “gold standard” for cell-based therapy, although these cells are not able to differentiate into cardiomyocyte lineage.In contrast, embryonic stem cells (ESC) are pluripotent and can be readily committed towards the cardiogenic lineage in vitro. There is also increasing evidence that cardiac-committed ESC can engraft into the scar tissue within the infarcted myocardium and differentiate into cardiomyocytes, thereby operating a regeneration of the myocardium, eliminating fibrotic scar tissue, and promoting sustained improvement of left ventricular function.^{7,8,11,20,21,22,23,24,25,26} This confers the potential ability to rebuild true cardiac tissue, to replenish areas that have been depopulated following ischemic accidents or the progression of fibrosis.⁸ In contrast, this positive benefit is limited by the formation of teratomas²⁷ or if too many cells are in uncommitted state at the time of injection.²⁴In the present study, we have compared the integration and functional efficacy of the CGR8 line of murine ESC with the D7LNB1 line of murine Mb, in the myocardium of Lmna^H222P/H222P mice. This genetic model of laminopathy reproduces a human missense mutation in the Lamin A/C gene causing Emery-Dreifuss muscular dystrophy. This model exhibits a rapidly progressive and lethal DCM,²⁸ showing pathophysiological evolution and conduction defects comparable to the human situation. Of note, these animals are immunocompetent.The CGR8 cell line of ESC was chosen because it can be grown feeder-free, and it is efficiently committed toward cardiogenic differentiation in vitro upon treatment with bone morphogenic protein 2 (BMP-2),^23,24,29,30 a treatment that indirectly lowers the risk of teratoma formation in vivo by decreasing the proportion of pluripotent cells.^24,27 The committed CGR8 cells, whether selected or not, have been previously shown to efficiently improve cardiac function following injection into the scar tissue in animal models of postischemic heart failure.^7,8,23,24,25 The time window for the addition of BMP-2 is of crucial importance,³⁰ therefore we pretreated CGR8 ESC for a short period of time, and we designed the experiments using limited amounts of cells to reach a compromise between myocardial differentiation and risk of teratoma formation (3 × 10⁵ per heart, at four different sites).The Mb have been assayed, in the present study, as a gold standard for validating the injection procedure, the efficacy of the immunosuppression regimen, the natural evolution of the implanted cells, the immunohistological procedures. Comparisons between Mb and ESC in murine models of postischemic heart failure have pinpointed important intrinsic differences in the efficacies and persistence of these two cell types, which now deserve a comparison in a DCM model.¹¹ The D7 Mb cell line was originally derived from the dy/dy mouse model of laminin-2 deficient congenital muscular dystrophy.³¹ It was engineered to express β-Galactosidase (β-Gal) constitutively and named D7LNB1. It showed no modification in its ability to form myotubes in vitro and in vivo. In addition, unlike C2 or G8 murine Mb, D7LNB1 Mb cell line did not produce carcinomas in vivo.³² CGR8 ESC and D7 Mb originate from close parental strains of 129 mice derived from the same animals in the 1970''s (129P/Ola background for ESC, 129/ReJ background for D7), and they share identical markers.³³ To avoid immune rejection triggered by murine ESC³⁴ or Mb³⁵ further expressing β-Gal in allogenic contexts, recipient mice were immunosuppressed using Tacrolimus.³⁶ We compared the engraftment of committed ESC and D7LNB1 Mb in terms of functional benefits and fate of engrafted cells in vivo. Our results suggest that cardiac-committed ESC failed to engraft into the dilated myocardium of the Lmna^H222P/H222P mice model, whereas Mb have a higher transplantation efficiency and greater functional improvement of cardiac function in this genetic model of dilated heart failure.     

Biliary stricture dilatation: multicenter review of clinical management in 73 patients

Eighty-nine biliary strictures in 73 patients who had undergone percutaneous balloon dilatation were reviewed to determine long-term patency rates and clinical management problems. The majority of dilatations were performed in patients with anastomotic strictures (n = 44), iatrogenic strictures (n = 28), and strictures associated with sclerosing cholangitis (n = 17). Patency rates after 36 months or more were 67%, 76%, and 42%, respectively. Complications, mostly minor, occurred in less than 7% of patients. Of patients with significant biliary obstruction, 15% had little or no intrahepatic biliary duct dilatation demonstrated by cross-sectional imaging and/or direct cholangiography. No definite conclusions could be drawn about the utility of long-term internal/external stenting.     

Superparamagnetic iron oxide: enhanced detection of focal splenic tumors with MR imaging

Superparamagnetic iron oxide (AMI-25), a reticuloendothelial cell-specific contrast agent for magnetic resonance (MR) imaging, was evaluated for its ability to permit detection of splenic metastases in 18 patients. Superparamagnetic iron oxide, at a dose of 30 mumol of iron per kilogram, decreased the signal intensity of spleen from 19.5 +/- 4.8 to 3.1 +/- 2.2 (spin-echo sequence, repetition time msec/echo time msec = 1,500/42; P less than .05), without changing the signal intensity of tumor. As a result, the tumor-spleen contrast-to-noise ratio increased from 0.2 (tumor isointense relative to spleen) to 18.0 (tumor hyperintense relative to spleen). As a consequence of increased contrast, splenic tumors were detected in four of 18 patients (45 individual lesions; P less than .05), whereas nonenhanced MR imaging permitted detection of splenic lesions in only two of 18 patients (four individual lesions). Maximum tumor-spleen contrast was achieved within 60 minutes after intravenous administration. These initial clinical results indicate that MR imaging with superparamagnetic iron oxide may offer improved accuracy in the diagnosis of splenic tumors.