大鼠延髓内脏带IFN-γ分布免疫组织化学法研究

The distribution of Interferon-γ-immunoreactive(IFN-γ-IR)in neurons and fibers in the medullary visceral zone(MVZ)of the rat were studied by using the streptavidin-perosidase(SP) immunohistochemical method.The results show that IFN-γ-IR positive neurons and fibers were densely distributed in the rat MVZ.Some INF-γ-IR positive neurons and fibers were densely stained,and some were sparsely stained.Some were large,and some small.Morphological variation also existed among them.IFN-γ-IR positive neurons and fibers mainly occurred in the ambiguous nucleus(nA),ventrolateral reticular nucleus(nVL).dorsal motor nucleus of the vagus nerve(dmnX),nucleus tractus solitarus(NTS) and intermediate(IRT) parts.IFN-γ-IR positive fibers could be divided into 3 kinds:net shaped.thin-long and dot fibers.     

Experimental study of rat beta islet cells cultured under simulated microgravity conditions

To observe the effects of simulated microgravity on beta islet cell culture, we have compared the survival rates and the insulin levels of the isolated rat islet cells cultured at micro- and normal gravity conditions. The survival rates of the cells cultured were determined by acridine orange-propidium iodide double-staining on day 3, 7 and 14. The morphology of the cells was observed by electron microscopy. Insulin levels were measured by radio immuno assays. Our results show that the cell number cultured under the microgravity condition is significantly higher than that under the routine condition (P<0.01). Some tubular structure shown by transmission electron microscopy, possibly for the transport of nutrients, were formed intercellularly in the microgravity cultured group on day 7. There were also abundant secretion particles and mitochondria in the cytoplasm of the cells. Scanning electron microscopy showed that there were holes formed between each islet, possibly connecting with the nutrient trans     

Neural stem cells isolated from amyloid precursor proteinmutated mice for drug discovery

AIM: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer’s disease and for testing new molecules.METHODS: Neural stem cells(NSCs) were isolated from the subventricular zone of Wild type(Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro(DIVs) in mitogen withdrawal conditions,spontaneous differentiation was studied using specific neural markers(MAP2 and TuJ-1 for neurons, GFAP forastroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software.RESULTS: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells.The gene expression neurogenesis pathway revealed11 altered genes in Tg2576 NSCs compared to Wt.CONCLUSION: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.     

The kinetics of IL—4 and IFN—γ gene expression in Mice after Trichosansin immunization

Trichosanthin (TCS) is a potent allergen to mice.According to our previous experiments,it could bring out the IgE response to ovabumin (OVA) if TCS was given one day before OVA immunization,while OVA alone could not induce IgE to it.In this work,the kinetics of interleukin 4(IL-4) and interferon γ(IFN-γ) gene expression in the mesenteric lymph node (MLN) of TCS-immunized mice was investigated using a semi-quantitative RT-PCR method.It indicated that TCS induced significant IL-4 gene expression and the peaks of IL4 gene expression were on day one after TCS immunization in both primary and secondary response.In contrast,the IFN-γ gene expression was suppressed.Furthermor,the IL-4 gene expression in the secondary response was lower than that in the primary response.Thus the presence of IgE memory B cells were studied.Results showed that the amount of mature IgE mRNA arose significantly and rapidly one day after TCS restimulation,while in the MLN of the mice primed 30 days before and without boost,it was almost as the same amount of the unimmunized control.These findings suggest the existence of the IgE memory B cells in the mice after the primary TCS immunization.     

Peripheral CD4+ T cell signatures in predicting the responses to anti-PD-1/PD-L1 monotherapy for Chinese advanced non-small cell lung cancer

Limited benefit population of immune checkpoint inhibitors makes it urgent to screen predictive biomarkers for stratifying the patients. Herein, we have investigated peripheral CD4~+T cell signatures in advanced non-small cell lung cancer(NSCLC)patients receiving anti-PD-1/PD-L1 treatments. It was found that the percentages of IFN-γ and IL-17 A secreting na?ve CD4+T cells(Tn), and memory CD4+T cells(Tm) expressing PD-1, PD-L1 and CTLA-4 were significantly higher in responder(R) than non-responder(Non R) NSCLC patients associated with a longer progression free survival(PFS). Logistic regression analysis revealed that the baseline IFN-γ-producing CD4+Tn cells and PD-1+CD4~+ Tm cells were the most significant signatures with the area under curve(AUC) value reaching 0.849. This was further validated in another anti-PD-1 monotherapy cohort. Conversely,high percentage of CTLA-4~+CD4~+Tm cells was associated with a shorter PFS in patients receiving anti-PD-L1 monotherapy.Our study therefore elucidates the significance of functional CD4+Tn and Tm subpopulations before the treatment in predicting the responses to anti-PD-1 treatment in Chinese NSCLC patients. The fact that there display distinct CD4~+ Tcell signatures in the prediction to anti-PD-1 and anti-PD-L1 monotherapy from our study provides preliminary evidence on the feasibility of anti-PD-1 and anti-PD-L1 combination therapy for advanced NSCLC patients.     

Applications of stem cells and bioprinting for potential treatment of diabetes

Currently, there does not exist a strategy that can reduce diabetes and scientists are working towards a cure and innovative approaches by employing stem cellbased therapies. On the other hand, bioprinting technology is a novel therapeutic approach that aims to replace the diseased or lost β-cells, insulin-secreting cells in the pancreas, which can potentially regenerate damaged organs such as the pancreas. Stem cells have the ability to differentiate into various cell lines including insulinproducing cells. However, there are still barriers that hamper the successful differentiation of stem cells into β-cells. In this review, we focus on the potential applications of stem cell research and bioprinting that may be targeted towards replacing the β-cells in the pancreas and may offer approaches towards treatment of diabetes. This review emphasizes on the applicability of employing both stem cells and other cells in 3 D bioprinting to generate substitutes for diseased β-cells and recover lost pancreatic functions. The article then proceeds to discuss the overall research done in the field of stem cell-based bioprinting and provides future directions for improving the same for potential applications in diabetic research.     

Complete nucleotide sequence of a novel porcine circovirus-like agent and its infectivity in vitro

A novel agent (hence termed as P2) was isolated from pig sera in China, which contained covalently bound circular genomic DNAs of 993 nucleotides. Sequence analyses indicated that the agent was closely related to the porcine circovirus (PCV). The molecular clone of P2 was constructed subsequently and used for the following studies. Intracytoplasmic inclusions and intranuclear inclusions were only found in PK-15 cells transfected with the tandem dimer of P2 molecular DNA clone. Intracytoplasmic inclusions were round or irregular in shape and 0.1-0.4 μm in diameter, and intranuclear inclusions were electronically denser than intracytoplasmic inclusions and had two general shapes: round/small (0.1 μm in diameter) and hexagonal/large (0.5―1.4 μm in diameter). The inclusions were not membranously bound. The cells transfected with the tandem dimer of P2 molecular DNA clone were tested positive for P2 DNA at passages 5. The P2 antigen could be detected in both transfected and passaged PK-15 cells. This is the first report regarding the complete nucleotide sequence of a small DNA genome in a circovirus-like infectious agent in vitro.     

Cell-specific regulation of APOBEC3F by interferons

Human cytidine deaminase APOBEC3F(A3F)has broad anti-viral activity against hepatitis Bvirus and retroviruses including human immunodeficiency virus type 1.However,its regulation in viralnatural target cells such CD4~ T lymphocytes,macrophages,and primary liver cells has not been wellstudied.Here we showed that A3F was up-regulated by interferon(IFN)-α in primary hepatocytes andmultiple liver cell lines as well as macrophages.Although the IFN-α signaling pathway was active in Tlymphoid cells and induction of other IFN stimulated genes such as PKR was detected,A3F and APOBEC3G(A3G)were not induced by IFN-α in these cells.Thus,additional factors other than known IFN-stimulatedgenes also regulated IFN-α-induced A3F expression distinctly.A3F and A3G expression levels in primaryhepatocytes,especially after IFN-α stimulation,were comparable to those in CD4~ T lymphocytes in someindividuals.Significant variations of A3F and A3G expression in primary hepatocytes from various subjectswere observed.Individual variations in A3F and/or A3G regulation and expression might influence the clinicaloutcomes of hepatitis B infection.     

WJSC 6th Anniversary Special Issues（2）:Mesenchymal stem cells Mesenchymal stem cells help pancreatic islet transplantation to control type 1 diabetes

Islet cell transplantation has therapeutic potential to treat type 1 diabetes,which is characterized by autoimmune destruction of insulin-producing pancreatic isletβcells.It represents a minimal invasive approach forβcell replacement,but long-term blood control is still largely unachievable.This phenomenon can be attributed to the lack of islet vasculature and hypoxic environment in the immediate post-transplantation period that contributes to the acute loss of islets by ischemia.Moreover,graft failures continue to occur because of immunological rejection,despite the use of potent immunosuppressive agents.Mesenchymal stem cells(MSCs)have the potential to enhance islet transplantation by suppressing inflammatory damage and immune mediated rejection.In this review we discuss the impact of MSCs on islet transplantation and focus on the potential role of MSCs in protecting islet grafts from early graft failure and from autoimmune attack.     

Abnormally high expression of BAFF on T lymphocytes from lung cancer-associated pleural effusions and its potent anti-tumor effect

In the present study, the expressions of B cell activating factor belonging to the tumor necrosis factor family （BAFF） and its receptors （BAFF-R and TACI） on T lymphocytes from malignant pleural effusion （MPE） were examined by fluorescence-activated cell sorting （FACS） analysis, and compared with those on the T lymphocytes from non-malignant pleural effusion （NMPE） and healthy controls. It was found that CD3 positive T lymphocytes （including CD4, CD8, and part of CD25 and CD69 positive cells） of MPE in lung cancer highly and consistently expressed the BAFF molecule, while high expressions of BAFF could only be found in phytohemagglutinin （PHA） or interleukin 2 （IL-2） induced T lymphocytes from NMPE or healthy controls. These results were consistent with the results from BAFF mRNA detection by real-time PCR. In addition, T lymphocytes from MPE expressed significantly more BAFF-R than those from NMPE or healthy controls, while the expression of TACI was increased on CD4＋ T cells but decreased on CD8＋ T cells when compared with controls. The Annexin/PI assay suggested that recombinant human BAFF （rhBAFF） could promote the survival rate of T lymphocytes from MPE, while the decoy receptor TACI-Fc fusion protein could promote the apoptosis rate of T lymphocytes. Cytokines in the supernatant detected by ELISA assay showed that rhBAFF could significantly upregulate the secretion of IFN-γ in vitro, and the IFN-γ level in the TACI-Fc-treated group resembled that of the control groups. All of these results indicated that the abnormally high expression of BAFF on T lymphocytes from MPE may play a role of antitumor effect.     

Inflammatory bowel disease requires the interplay between innate and adaptive immune signals

Inflammatory bowl disease （IBD） is a type 1 T helper cell （Th1）-mediated autoimmune disease. Various studies have revealed that environmental pathogens also play a significant role in the initiation and progression of this disease. Interestingly, the pathogenesis of IBD has been shown to be related to nitric oxide （NO） released from innate immune cells. Although NO is known to be highly toxic to the gut epithelia, there is very little information about the regulation of NO production, One major question in the etiology of IBD is how Thl cells and pathogens interact in the induction of IBD. In present study, we focused on the regulation of NO. We show that macrophages require both interferon-γ, （IFN-γ）-mediated and TLR4-mediated signals for the production of NO, which causes inflammation in the intestine and subsequently IBD. Thus, IBD is the result of concerted actions of innate immune signals, such as the binding of LPS to TLR-4, and adaptive immune signals, such as IFN-γ produced by Thl cells.     

Pyrrolidine dithiocarbamate protects pancreatic β-cells from oxidative damage through regulation of FoxO1 activity in type 2 diabetes rats

Pyrrolidine dithiocarbamate （PDTC） can lower the bloot glucose level and improve the insulin sensitivity in diabeti, rats. However, the mechanisms underlying this effect o PDTC treatment in diabetic rats remained uncertain, h this study, we evaluated the mechanisms by which PDT（ conferred protection against oxidative damage to pancreat ic islet β-cells in rats with experimental type 2 diabete mellitus （DM）. DM in the rats was elicited by long-tern high-fat diet accompanied with a single intraperitonea （i.p.） injection of a low dose of streptozotocin. After a 7-da1 administration of PDTC （50 mg/kg/day i.p.）, blood glucos levels were measured and pancreatic tissues were collecte / for the determination of various biochemical and enzyma 1 ic activities using immunohistochemistry, immunofluoresI cence, and western blot techniques. The percentage o 1 apoptotic pancreatic islet β-cells was detected by flow cyto metry. The results showed that diabetic rats had elevate blood glucose levels and insulin resistance, accompanieq with an increase in malondialdehyde content, nitrotyrosin production, and inducible nitric oxide synthase expression A decrease in superoxide dismutase and glutathione pero idase activities was also observed in DM rats, culminatin with elevated β-cell apoptosis. PDTC treatment significantl reduced the oxidative damage and the β-cell apoptosi and also increased the insulin production through down-reg lating FoxO1 acetylation and up-regulating nuclear PDX- level. These data suggested that PDTC can protect islet βcells from oxidative damage and improve insulin productio through regulation of PDX-1 and FoxO1 in a DM rat model.     

Functional Analysis of Multiple Transcription Factor Sites in a Regulatory Element of Human ε-Globin Gene

The human β-like globin genes are arranged as a clusterof five genes (ε, Gγ, Aγ, δ and β) in the order of theirtemporal expression. The human embryonic ε-globin geneis expressed in the blood island of the embryonic yolk sacand is silenced completel