Novel heat shock protein Hsp70L1 activates dendritic cells and acts as a Th1 polarizing adjuvant

Heat shock proteins (HSPs) are reported to act as effective adjuvants to elicit anti-tumor and anti-infection immunity. Here, we report that Hsp70-like protein 1 (Hsp70L1), a novel HSP derived from human dendritic cells (DCs), has potent adjuvant effects that polarize responses toward Th1. With a calculated molecular weight of 54.8 kDa, Hsp70L1 is smaller in size than Hsp70 but resembles it both structurally and functionally. Hsp70L1 shares common receptors on DCs with Hsp70 and can interact with DCs, promoting DC maturation and stimulating secretion of the proinflammatory cytokines interleukin 12p70 (IL-12p70), IL-1beta, tumor necrosis factor-alpha (TNF-alpha), and the chemokines IP-10, macrophage inflammatory protein-1alpha (MIP-1alpha), MIP-1beta, and normal T cell expressed and secreted (RANTES). The induction of interferon-gamma-inducible protein 10 (IP-10) secretion by Hsp70L1 is not shared by Hsp70, and other functional differences include more potent stimulation of DC IL-12p70, CC-chemokine, and CCR7 and CXCR4 expression by Hsp70L1. Immunization of mice with the hybrid peptide Hsp70L1-ovalbumin(OVA)(257-264) induces an OVA(257-264)-specific Th1 response and cytotoxic T lymphocyte (CTL) that results in significant inhibition of E.G7-OVA tumor growth. The ability of Hsp70L1 to activate DCs indicates its potential as a novel adjuvant for use with peptide immunizations; the Hsp70L1 antigen peptide hybrid may serve as a more effective vaccine for the control of cancer and infectious diseases.

The 9L^(LUC)/Wistar rat glioma model is not suitable for immunotherapy

The availability of a well-characterized animal brain tumor model will play an important role in identifying treatments for human brain tumors. Wistar rats bearing 9L glioma cells can develop solid, well-circumcised tumors, and may be a useful animal model for the evaluation of various therapeutic approaches for gliosarcomas. In this study, the 9L/Wistar rat glioma model was produced by intracerebral implantation of 9L^LUC glioma cells syngenic to Fischer 344 （F344） rats. Bioluminescence imaging showed that tumors progressively grew from day 7 to day 21 in 9L^LUC/F344 rats, and tumor regression was found in some 9L^LUC/Wistar rats. Hematoxylin-eosin staining verified that intracranial tumors were gliomas. Immunohistochemistry results demonstrated that no CD4- and CD8-positive cells were found in the syngeneic 9L^LUC/F344 model. However, many infiltrating CD4- and CD8-positive cells were observed within the tumors of the 9L^LUC/Wistar model. Our data suggests that compared with 9L/F344 rats, 9L glioma Wistar rats may not be suitable for evaluating brain glioma immunotherapies, even though the model induced an immune response and exhibited tumor regression.

Novel conductive polypyrrole/silk fibroin scaffold for neural tissue repair

Three dimensional（3D） bioprinting, which involves depositing bioinks（mixed biomaterials） layer by layer to form computer-aided designs, is an ideal method for fabricating complex 3D biological structures. However, it remains challenging to prepare biomaterials with micro-nanostructures that accurately mimic the nanostructural features of natural tissues. A novel nanotechnological tool, electrospinning, permits the processing and modification of proper nanoscale biomaterials to enhance neural cell adhesion, migration, proliferation, differentiation, and subsequent nerve regeneration. The composite scaffold was prepared by combining 3D bioprinting with subsequent electrochemical deposition of polypyrrole and electrospinning of silk fibroin to form a composite polypyrrole/silk fibroin scaffold. Fourier transform infrared spectroscopy was used to analyze scaffold composition. The surface morphology of the scaffold was observed by light microscopy and scanning electron microscopy. A digital multimeter was used to measure the resistivity of prepared scaffolds. Light microscopy was applied to observe the surface morphology of scaffolds immersed in water or Dulbecco＇s Modified Eagle＇s Medium at 37℃ for 30 days to assess stability. Results showed characteristic peaks of polypyrrole and silk fibroin in the synthesized conductive polypyrrole/silk fibroin scaffold, as well as the structure of the electrospun nanofiber layer on the surface. The electrical conductivity was 1 × 10^-5–1 × 10^-3 S/cm, while stability was 66.67%. A 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl-2-H-tetrazolium bromide assay was employed to measure scaffold cytotoxicity in vitro. Fluorescence microscopy was used to observe Ed U-labeled Schwann cells to quantify cell proliferation. Immunohistochemistry was utilized to detect S100β immunoreactivity, while scanning electron microscopy was applied to observe the morphology of adherent Schwann cells. Results demonstrated that the polypyrrole/silk fibroin scaffold was not cytotoxic and did not affect Schwann cell proliferation. Moreover, filopodia formed on the scaffold and Schwann cells were regularly arranged. Our findings verified that the composite polypyrrole/silk fibroin scaffold has good biocompatibility and may be a suitable material for neural tissue engineering.

Protective effects of docosahexaenoic acid against non-alcoholic hepatic steatosis through activating of JAK2/STAT3 signaling pathway

Non-alcoholic fatty liver disease is the most common cause of hepatic dysfunction.In the present study,human normal hepatocyte L02 cells were treated with 50%fetal bovine serum to induce the formation of hepatic steatosis in vitro,and then the cells were treated with docosahexaenoic acid to investigate its protective effect on Non-alcoholic fatty liver disease.Our results showed that 50%of fetal bovine serum significantly induced intracellular lipid accumulation and hepatocyte fatty degeneration within 48 h.The expression level of adipose formation-related genes was significantly up-regulated,such as PPARγ,C/EBPαand SREBP-1;meanwhile,the content of cellular total lipid,total cholesterol and triglycerides were significantly increased after 50%fetal bovine serum treatment.Interestingly,docosahexaenoic acid treatment could inhibit FBS-induced intracellular lipid accumulation in L02 cells and the expression of lipogenic genes.Moreover,docosahexaenoic acid treatment could reduce hepatic steatosis-induced oxidative stress and endoplasmic reticulum stress response,and these responses were shown by the modification of antioxidant enzyme activities and GRP78,CHOP expression.In addition,the results showed that docosahexaenoic acid can activate the JAK2/STAT3 signaling pathway in fatty liver L02 cell;inhibition of JAK2/STAT3 signaling pathway by WP1066 abolished the beneficial effects of docosahexaenoic acid on hepatic steatosis accompanied with the increased expression of lipogenic genes and endoplasmic reticulum stress response.Above all,the present study showed that docosahexaenoic acid can alleviate non-alcoholic hepatic steatosis by activating JAK2/STAT3 signaling pathway.

Expression changes of nerve cell adhesion molecules L1 and semaphorin 3A after peripheral nerve injury

The expression of nerve cell adhesion molecule L1 in the neuronal growth cone of the central nervous system is strongly associated with the direction of growth of the axon, but its role in the regeneration of the peripheral nerve is still unknown. This study explored the problem in a femoral nerve section model in rats. L1 and semaphorin 3A m RNA and protein expressions were measured over the 4-week recovery period. Quantitative polymerase chain reaction showed that nerve cell adhesion molecule L1 expression was higher in the sensory nerves than in motor nerves at 2 weeks after injury, but vice versa for the expression of semaphorin 3A. Western blot assay results demonstrated that nerve cell adhesion molecule L1 expression was higher in motor nerves than in the sensory nerves at the proximal end after injury, but its expression was greater in the sensory nerves at 2 weeks. Semaphorin 3A expression was higher in the motor nerves than in the sensory nerves at 3 days and 1 week after injury. Nerve cell adhesion molecule L1 and semaphorin 3A expressions at the distal end were higher in the motor nerves than in the sensory nerves at 3 days, 1 and 2 weeks. Immunohistochemical staining results showed that nerve cell adhesion molecule L1 expression at the proximal end was greater in the sensory nerves than in the motor nerves; semaphorin 3A expression was higher in the motor nerves than in the sensory nerves at 2 weeks after injury. Taken together, these results indicated that nerve cell adhesion molecules L1 and semaphorin 3A exhibited different expression patterns at the proximal and distal ends of sensory and motor nerves, and play a coordinating role in neural chemotaxis regeneration.

Differential Proteomics in Malignant and Normal Liver Cell Lines

Objective： To detect differential protein expression in malignant and normal liver cell lines in vitro using the SELDI ProteinChip platform, for investigating the pathogenesis of liver cancer. Methods： Two cell lines, human normal liver cell line L02 and hepatoma cell line SMMC-7721 were cultured routinely, harvested in good condition and lysed. After quantification, the supernatant of the lysate was tested by IMAC3 （Immobilized Mental Affinity Capture） and WCX2 （Weak Cation Exchange） chips on the SELDI-TOF-MS ProteinChip reader. Results： Protein expression differed between the malignant and normal liver cell lines. A total of 20 differentially expressed proteins were found, among which, 7 were captured by the IMAC3 chip and 14 by the WCX2 chip. Peaks at 5,419, 7,979 and 11,265 Da were higher and at 8,103, 8,492, 10,160 and 11,304 Da lower in SMMC-7721 cells by the IMAC3 chip; peaks at 7,517, 7,945 and 7,979 Da were higher and at 5,061, 5,551, 5,818, 7,439, 9,401,10,100, 10,312, 11,621, 11,662, 11,830 and 12,772 Da lower in SMMC-7721 cells by the WCX2 chip. Interestingly, both chips captured the 7,979 Da peak. In addition, the 11,081 Da peak corresponded precisely with the molecular mass of the calcium binding protein S100A10, which may participate in the formation of liver cancer in association with p36. Conclusion： Detecting differential protein expression in malignant and normal liver cell lines using the SELDI ProteinChip platform was simple, sensitive and repeatable. The results we obtained can serve as a basis for investigating the pathogenesis of liver cancer and aid the discovery of new therapeutic targets.

EXPERIMENTAL RESEARCH ON DIRECTIONAL SOLIDIFICATION OF Al－4．5wt％Cu ALLOY BY ACRT METHOD

Accelerated crucible rotation technique(ACRT) has been used for the directional solidification of Al-4.5wt% Cu binary alloy.By rotating the crucible at varying rate and direction,forced liquid flows are aroused These flows include Ekman flow,Couette flow and Spiral Shear flow.Especially,Ekman flow acts directly at the L/S interface,changes diffusion and heat exchange conditions and has strong influences on the morphology of L/S interface.Experimental results show that,compared with normal Bridgman specimens,the solidification region is much narrower and the cell spacing is much smaller in ACRT specimens.These influences become much stronger when the accelerating rate is increased.

Gene-guided OX40L anchoring to tumor cells for synergetic tumor“self-killing”immunotherapy

The low objective response rates and severe side effects largely limit the clinical outcomes of immune checkpoint blockade(ICB)therapy.Here,a tumor“self-killing”therapy based on gene-guided OX40L anchoring to tumor cell membrane was reported to boost ICB therapy.We developed a highly efficient delivery system HA/PEI-KT(HKT)to co-deliver the OX40L plasmids and unmethylated CG-enriched oligodeoxynucleotide(CpG).On the one hand,CpG induced the expression of OX40 on T cells within tumors.On the other hand,OX40L plasmids achieved the OX40L anchoring on the tumor cell membrane to next promote T cells responses via OX40/OX40L axis.Such synergistic tumor“self-killing”strategy finally turned“cold”tumors to“hot”,to sensitize tumors to programmed cell death protein 1/programmed cell death ligand 1(PD-1/PD-L1)blockade therapy,and promoted an immune-mediated tumor regression in both B16F10 and 4T1 tumor models,with prevention of tumor recurrence and metastasis.To avoid the side effects,the gene-guided OX40L anchoring and PD-L1 silencing was proposed to replace the existing antibody therapy,which showed negligible toxicity in vivo.Our work provided a new possibility for tumor“self-killing”immunotherapy to treated various solid tumors.

Quantum dots enhance Cu^(2+)-induced hepatic L02 cells toxicity

As a new class of xenogenous nanoparticle, quantum dots （QDs） possess the potential to co-exist with Cu^2＋ in human liver. The combined toxicity is thus concerned. Considering QDs and Cu^2＋ are known ROS （reactive oxygen species） inducer, we investigated the combined oxidative stress and corresponding protective strategy using human hepatic L02 cells. The results demonstrated that the presence of a small amount of MPA-CdTe QDs （2 μg/mL） in a Cu^2＋ solution （2.5-20 μg/mL） resulted in a higher toxicity with up to 8-fold cell viability decrease, which was accompanied by cell morphology changes. The combined toxicity was then confirmed as ROS associated oxidative stress with up to 300% and 35% increase of the intracellular ROS level and glutathione S-transferase （GST） activity, respectively. N-acetylcysteine （NAC） can also provide almost complete protection against the induced toxicity. Therefore, the ROS associated oxidant injury might be responsible for the QDs-Cu^2＋/Cu^2＋ induced toxicity and could be balanced through cytoprotective antioxidant enzyme GST.

First report on chlorophyllin to protect mammalian and fish muscle cells from pesticide toxicity via activation of p53 and PARP

Objectives:Pesticide toxicity has become one of the major environmental menaces affecting all types of life forms of the ecosystem.Pesticides get washed off from agricultural fields into nearby water bodies and enter the aquatic organisms.Their bio-accumulated form finally reaches the human race,through consumption of pesticide infested aquatic animals,causing several physiological dysfunctions.Hence it becomes necessary to find a therapeutic cure/a preventive measure to stop the health hazard issues of pesticide.With this projection a search for a phyto-based-product was made whose primary objective would be to lower the pesticidal toxicity in fish and simultaneously in the human race.Methods:In this study we tried to check whether the phyto-chemical,Chlorophyllin(CHL),known for its anti-genotoxic,anti-oxidant activities,could render any kind of protection against Cypermethrin(CM)induced-toxicity in fish model and mammalian cell line L6.Both the model L6 and fish were pre-treated with CHL prior to exposure of CM.Different scientific parameters like%cellular cytotoxicity,reactive oxygen species(ROS)generation,nuclear condensation,etc were checked to validate the possibility of CHL in protecting CM-induced toxicity.Results:The overall results revealed that pre-treatment with CHL could restrict the ROS generation leading to modulation in associated cytokine proteins expression NFkβand IFNγ.Further,CHL lowered nuclear condensation and elevated expression of DNA repair proteins p53 and PARP,showing a kind of pre-activation of signalling cascades for overall protection against the severity of pesticidal toxicity.Conclusion:Thus,this phyto-based preventive approach would possibly solve many areas of human health issues related to pesticide toxicity in future.

Synthesis and Insulin-sensitizing Activity of a Series of 2-Benzyl-1,3-dicarbonyl Derivatives

A series of 2 benzyl 1,3 dicabonyl derivatives was synthesized. Their insulin sensitizing activity was evaluated in 3T3 L1 preadipocyte cells. Compounds 3, 26 and 27 were found to possess strong insulin sensitizing activity in vitro and were selected for further hypoglycemic evaluation in vivo.

Antihyperglycemic and antihyperlipidemic activities of wild musk melon (Cucumis melo var. agrestis) in streptozotocin-nicotinamide induced diabetic rats

Objective:Wild musk melon(Cucumis melo var.agrestis,CMA)is one of the edible plants form Tamil Nadu.Traditionally,this plant was used as diabetic diet(leaves of CMA with Momordica charantia leaves),but there is no scientific report on antidiabetic action of this plant material.Hence,the current research work was designed to evaluate the antihyperglycemic and antihyperlipidemic effect of hydroalcoholic extract of CMA leaves(HALEC)in streptozotocin(STZ)-nicotinamide(NIC)-induced diabetic rats.Methods:Diabetes was induced by administration of STZ(60 mg/kg,i.p.)after 15 min of NIC(120 mg/kg i.p.)administration.The diabetic rats were treated with HALEC(300 and 600 mg/kg,p.o.,respectively)for 21 d.Results:After the management with HALEC,blood glucose,HbA1c levels,total cholesterol,LDL cholesterol,triglycerides levels,glycogen phosphorylase and glucose-6-phosphatase levels were significantly diminished in diabetic rats.However,haemoglobin level,HDL cholesterol,liver glycogen,total protein,hexokinase,glucose-6-phosphate dehydrogenase levels were significantly increased in HALEC treated diabetic rats.The histopathological studies of the pancreas in HALEC-treated diabetic rats showed almost normal appearance.L6 cell line study revealed the increased glucose uptake activity of HALEC.High performance thin layer chromatography(HPTLC)analysis confirms the presence of active principles such as rutin,gallic acid and quercetin in HALEC.Conclusion:The results indicated that HALEC possess significant antihyperglycemic and antihyperlipidemic activity in STZ-NIC-induced typeⅡdiabetic rats with protective effect.This research work will be useful for the isolation of active principles and development of herbal formulation in phytopharmaceuticals.

Dietary L-arginine attenuates expression of vascular cell adhesion molecule-1 in the aortae of hypercholesterolemic rats

Objective To determine whether the antiatherogenic effect of L arginin e is due to an inhibition of vascular cell adhesion molecule 1 (VCAM 1) expres sion in the aortae of hypercholesterolemic rats. Methods Twenty four male Wistar rats were randomized into three gr oups : Group NC with normal diet (NC, n=8), Group CC with 4% cholesterol and 1% choli c acid diet (CC, n=8), Group AC with 4% cholesterol and 1% cholic acid diet supp lemented with 3% L arginine HCl in the drinking water (AC, n=8). Eight weeks la ter, the blood samples were collected for biochemical studies, and the aortae we re harvested for RT PCR and immunohistochemical studies.Results The results showed that dietary L arginine supplementation red uced expression of VCAM 1 in protein level and mRNA level. Conclusion Inhibitory effect of dietary supplementation of L argin ine on VCAM 1 expression may be one of the mechanisms of its antiatherosclerosis.