Effects of rapid antigen degradation and VEE glycoprotein specificity on immune responses induced by a VEE replicon vaccine

Genetic vaccines are engineered to produce immunogens de novo in the cells of the host for stimulation of a protective immune response. In some of these systems, antigens engineered for rapid degradation have produced an enhanced cellular immune response by more efficient entry into pathways for processing and presentation of MHC class I peptides. VEE replicon particles (VRP), single cycle vaccine vectors derived from Venezuelan equine encephalitis virus (VEE), are examined here for the effect of an increased rate of immunogen degradation on VRP vaccine efficacy. VRP expressing the matrix capsid (MA/CA) portion of SIV Gag were altered to promote rapid degradation of MA/CA by various linkages to co-translated ubiquitin or by destabilizing mutations and were used to immunize BALB/c mice for quantitation of anti-MA/CA cellular and humoral immune responses. Rapid degradation by the N-end rule correlated with a dampened immune response relative to unmodified MA/CA when the VRP carried a glycoprotein spike from an attenuated strain of VEE. In contrast, statistically equivalent numbers of IFNgamma(+)T-cells resulted when VRP expressing unstable MA/CA were packaged with the wild-type VEE glycoproteins. These results suggest that the cell types targeted in vivo by VRP carrying mutant or wild type glycoprotein spikes are functionally different, and are consistent with previous findings suggesting that wild-type VEE glycoproteins preferentially target professional antigen presenting cells that use peptides generated from the degraded antigen for direct presentation on MHC.     

颗粒性HBV多CTL表位基因诱导BALB/c小鼠的免疫应答研究

目的：研究含HBV多CTL表位的杂合HBc基因免疫BALB/c小鼠所诱导的免疫应答.方法：构建以HBV多CTL表位取代MIR区基因的杂合HBc基因疫苗（pHBcMep）并以肌肉注射方式免疫BALB/c小鼠,ELISA、流式细胞术、LDH释放法等分别检测血清特异性抗体与淋巴细胞分泌的IFN-γ水平、CD4^＋/CD8^＋T细胞比例变化及特异性CTLs活性等免疫应答指标.结果：颗粒性杂合HBc基因疫苗pHBcMep免疫BALB/c小鼠诱导明显抗体应答,末次免疫后2周,特异性抗preS2抗体阳性率达100%（12/12）,最高效价1∶1 000,同时诱导淋巴细胞分泌IFN-γ能力增强和刺激CTLs活化,其杀伤活性达16 IU30,CD4^＋/CD8^＋T细胞比例明显升高,且诱导明显回忆反应.结论：颗粒性HBV多CTL表位基因疫苗pHBcMep具有良好免疫原性,能迅速诱导高活性体液和细胞免疫应答及回忆反应.     

两株登革2型病毒E基因重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 用含登革2型病毒(Dengue type 2 virus,DEN2)B株和NGC株E基因部分序列pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 用两株含DEN2 E基因部分序列(1～476 bp)的pcDNA3.1重组质粒与含有佐剂的重组质粒共同免疫BALB/c小鼠,初次免疫后第14天、28天分别加强免疫1次,共免疫3次.收集初次免疫后第14、28、42、70和98天外周血标本,间接ELISA法测定小鼠血浆特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异性抗体水平.结果 不同DEN2毒株E基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类抗体的产生存在差异,B株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DEN2两毒株E基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.     

Effects of histamine on the circulatory system

Summary Experiments have been made in anaesthetised cats and dogs and in healthy, human volunteers to compare the changes in blood pressure and heart rate during systemic administration of histamine.Histamine, 1×10^–9 to 1×10^–7 mol/kg/min, lowered blood pressure in a similar dose-dependent fashion in all three species. In man and the cat this was accompanied by clear dose-dependent tachycardia whereas in the dog heart rate changes were minimal.Pharmacological analysis of the depressor responses to histamine in all three species and the reduction in total peripheral resistance in the cat and dog showed that the immediate responses to histamine in all three species involved H₁-receptors and that sustained responses involved H₂-receptors. Abolition of responses to histamine throughout infusions required H₁- and H₂-receptor blockade.Histamine antagonists, used in doses which cause abolition of cardiovascular responses to large doses of histamine, do not cause any significant change in the resting cardiovascular system.     

两株登革2型病毒NS1基因真核重组质粒免疫BALB/c小鼠产生特异性抗体水平的比较

目的 利用登革2型病毒(dengue type 2 virus,DENV2)M株和NGC株NS1基因部分扇列PcDNA3.1重组质粒初次和加强免疫BALB/c小鼠,观察免疫小鼠体液免疫应答的差异.方法 分别构建两株DENV2 NS1基因部分序列(1-413 bp)的PcDNA3.1真核重组质粒和pET28a(+)质粒,进行原核蛋白的表达、鉴定、纯化和定量;并用pcDNA3.1重组质粒免疫BALB/c小鼠,初次免疫及第7天、14天分别加强免疫1次,共免疫3次.收集初次免疫后第7、14、28和56天外周血标本,间接ELISA法测定小鼠血清特异性IgM/IgG类抗体水平,细胞病变抑制法检测特异保护性抗体水平.结果 构建了pET28a(+)-NS1m/pET28a(+)-NS1n原核表达重组质粒,SDS-PAGE分析表明,NS1基因部分序列获得表达,其相对分子质量均约22.3×103;Western blot表明该目的 蛋白可与抗His标签单克隆抗体结合;经Ni柱亲和层析法得到纯度达92%的表达蛋白,对C6/36细胞有毒性,并可用于ELASA检测.不同DENV2毒株NS1基因部分序列的pcDNA3.1重组质粒初次和加强免疫BALB/c小鼠诱导特异性IgM、IgG类和中和抗体的产生存在差异,M株重组质粒加强免疫小鼠后特异性抗体效价水平较高并持续较长时间.结论 DENV2两毒株NS1基因部分序列重组质粒免疫小鼠后诱生的特异性抗体类别、水平存在差异.

Abstract：

Objective To compare the humoral immune response of BALB/c mice immunized by recombinant plasmids PeDNA3.1-M-NS1 and pcDNA3.1-N-NS1.Methods Dengue type 2 virus(DENV2)NS1 gene were constructed two partial sequences(1-413 bp)of the pcDNA3.1 eukaryotic plasmids and pET28a(+)plasmid for prokaryotic expression,identification,purification and quantification.The BALB/c mice were immunized by pcDNA3.1-M-NS1,pcDNA3.1-N-NS1 recombinant plasmids with adjuvant.Each animal received a primary inoculation and two boosts at 1-week intervals.Then the blood samples of BALB/c mice were collected from different experiment groups at day 7,14,28 and 56,respectively after first immunization.The specific IgM/IgG antibodies for NS1 protein in serum were confirmed by indirect ELISA.And then the activities of the specific protective antibody were determined by cytopathic effect inhibition(CPEI).Results Construction of the pET28a(+)-NS1 m/pET28a(+)-NS1n prokaryotic expression plasmid,SDS-PAGE analysis showed that,NS1 gene partial sequence was expressed,both the relative molecular weight of about 22.3×103:Western blot showed that the protein can bind anti-His tag monoclonal antibody;byNi affinity chromatographywith apurity of 92% protein,on the C6/36 cell toxicity,and can be used ELASA detection.The results showed that the levels of specific IgM/IgG antibody and neutralizing antibody activities were increased in pcDNA3.1-M-NS1 booster immunization group than other groups.The result had been observed longer duration of antibody level in peDNA3.1-M-NS1 booster immunization group.Conclusion Humoral immune response were significantly different between pcDNA3.1-M-NS1 and pcDNA3.1-N-NS1 recombinant plasmid immunized mice groups.     

Vλ-light chain genes reconstitute immune responses to defined carbohydrate antigens or haptens by utilizing different VH genes

The contribution of the λ-light chain to the development of peripheral B cell repertoire and generation of specific antibodies to haptens and polysaccharide antigens was studied in genetically manipulated kappa-deficient and λ₂-transgenic mice. The results clearly demonstrate a non-stoichiometric V_H gene family expression in the absence of k-light chain and suggest a non-stochastic pairing between V_H and V_λ genes, expressed in the peripheral B cell repertoire. A shift in V_H gene utilization in the case of Vl_λ⁺ antibodies was evident in response to β2–6 fructosan and TNP hapten. These observations demonstrate the availability of compensatory mechanisms in the absence of V_K genes and are consistent with the hypothesis that V_H gene family expression is controlled by genetic factors from inside the V_H locus. Furthermore, genetic factors from outside the V_H locus, namely restricted available light chain diversity, may lead to a shift in V_H gene utilization in the peripheral B cell repertoire.     

Effects of lactational exposure to low-dose BaP on allergic and non-allergic immune responses in mice offspring

Benzo[a]pyrene (BaP) can induce developmental and reproductive toxicity; however, the full scope of its immunotoxic effects remains unknown. This study aimed to assess effects of lactational exposure to low-dose BaP (comparable to human exposure) on potential allergicnon-allergic immune responses in murine offspring. Lactating C3H/HeJ dams were orally dosed with BaP at 0, 0.25, 5.0, or 100?pmol/animal/week) at post-natal days [PND] 1, 8, and 15. Five-weeks-old pups then received intratracheally ovalbumin (OVA) every 2 weeks for 6 weeks. Following the final exposure, mice were processed to permit analyses of bronchoalveolar lavage (BAL) fluid cell profiles as well as levels of lung inflammatory cytokines and chemokines, serum OVA-specific immunoglobulin, and mediastinal lymph node (MLN) cell activation/proliferation. In OVA-sensitized male offspring, lactational low-dose BaP exposure led to enhanced (albeit not significantly) macrophage, neutrophil, and eosinophil infiltration to, and increased T-helper (T_H)-2 cytokine production in, the lungs. In females, BaP exposure, regardless of dose, led to slightly enhanced lung levels of macrophages and eosinophils, and of inflammatory molecules. Protein levels of interleukin (IL)-33 in the OVA?+?BaP (middle dose) group, and interferon (IFN)-γ in the OVA?+?BaP (low dose) group, were higher than that of the OVA (no BaP) group. Ex vivo studies showed lactational exposure to BaP partially induced activation of T-cells and antigen-presenting cells (APCs) in the MLN cells of both male and female offspring, with or without OVA sensitization. Further, IL-4 and IFNγ levels in MLN culture supernatants were elevated even without OVA-re-stimulation in OVA?+?BaP groups. In conclusion, lactational exposure to low-dose BaP appeared to exert slight effects on later allergic and non-allergic immune responses in offspring by facilitating development of modest T_H2 responses and activating MLN cells. In addition, lactational exposures to BaP might give rise to gender differences in allergic/non-allergic immune responses of offspring.     

Activity rhythms in inbred mice. I. Genetic analysis with recombinant inbred strains

Rhythm of activity of inbred mice was recorded automatically by a set of actographs. This rhythm was characterized by six indices in the temporal and frequency domains. Two methods of genetic analysis were applied to these indices using parental strains BALB/c and C57BL/6, the reciprocal F₁'s, and the seven recombinant inbred strains (RI). Findings on the F₁'s show no maternal effect but indicate dominance and heterosis. The RI method successfully rejects the hypothesis of a monogentic correlate for all measures. In line with F₁ data, it demonstrates the presence of a polygenetic correlate: at least one other locus is involved in each of the six outcome parameters.This study was supported by CNRS (URA 1294, affilieé Inserm), MEN (UFR Biomédicale des Saints-Pères, Paris V), Foundation pour la Recherche Medicale.     

Effects of histamine on hippocampal pyramidal cells of the rat in vitro

Summary The actions of bath applied histamine on CA1 pyramidal cells were investigated in hippocampal slices of the rat. Histamine caused a) a slight depolarization but no significant change in resting membrane conductance; b) an abbreviation of long afterhyperpolarizations after single action potentials, bursts of action potentials or TTX resistant spikes; c) a loss of accommodation of firing. In the presence of TEA or barium, histamine prolonged and increased the size and number of the slow TTX resistant spikes. A depolarizing plateau which follows such spikes was also increased by histamine. Evoked synaptic potentials were unaffected by histamine, but the population spike was increased. The frequency of spontaneous chloride dependent potentials, which reflect interneurone firing, was also increased. These effects considerably outlasted histamine application and were mimicked by the H₂-agonist impromidine but not the H₁-agonist thiazolethylamine, and blocked by the H₂-antagonists cimetidine and metiamide but not the H₁-antagonists mepyramine or the beta-antagonist propranolol. It is concluded that histamine, by activating H₂-receptors, antagonizes a calcium mediated potassium conductance in hippocampal pyramidal cells without affecting calcium current. By this mechanism histaminergic afferent fibres could effectively regulate cortical responsiveness by selectively potentiating large excitatory inputs of target neurones.     

Effects of adjuvants on the immune response to allergens in a murine model of allergen inhalation: cholera toxin induces a Th1-like response to Bet v 1, the major birch pollen allergen

Based on the fact that type I allergies are frequently elicited by inhalant allergens, we have established a model of aerosol inhalation leading to allergic sensitization in BALB/c mice. Using this model we studied the effects of aluminium hydroxide (Al(OH)₃), known to enhance IgE antibody responses, compared with cholera toxin (CT), a potent mucosal adjuvant, on the immune response to birch pollen (BP) and its major allergen Bet v 1. Two groups of BALB/c mice were either systemically immunized with recombinant Bet v 1 in Al(OH)₃ and subsequently aerosol exposed to BP allergen, or aerosolized with BP and CT. IgE-mediated skin reactions were only elicited in the mice which had received Bet v1/Al(OH)₃. Allergen-specific serum IgE and IgG1 antibodies dominated in the Al(OH)₃ group, IgG2a antibody levels to BP and rBet v 1 were markedly higher in the sera of mice exposed to CT with the allergen. IgA antibodies were only detected in the bronchial lavage of the CT-treated group. Moreover, the latter group displayed consistently higher T cell proliferative responses to BP and interferon-gamma production in vitro. Thus, the systemic immunization with rBet v 1 in Al(OH)₃ before inhalation of the BP extract promoted a Th2-like immune response, while CT mixed with the aerosolized BP extract rather induced a Th1-like immune response. In an attempt to reverse these ongoing immune responses we could achieve a shift towards a Th0 response. Immunization with BP extract without adjuvant treatment led to undetectable antibody or cellular immune responses. We conclude from the present study that the induction of an immune response to BP allergen after aerosol inhalation can be directed towards a Th1- or a Th2-like response. Once established, the immune response can be modulated.     

An evaluation of the effects of Lactobacillus ingluviei on body weight, the intestinal microbiome and metabolism in mice

Background

Food can modify the intestinal flora, and Lactobacillus ingluviei has been shown to cause weight gain in chicks and ducks but not in mammals.

Methodology

Female BALB/c mice were divided into a control and two experimental groups and were inoculated either once or twice with L. ingluviei or with PBS. Faecal samples were collected and tested using qPCR in order to detect and quantify Lactobacillus spp., Bacteroidetes spp. and Firmicutes spp. Gene expression was examined in liver and adipose tissue by microarray and qPCR. Metabolic indicators in the plasma were also measured.

Results

Mice that were inoculated with 4 × 10¹⁰L. ingluviei presented a significant increase in weight gain and liver weight and significant increases in Lactobacillus spp. and Firmicutes DNA copy numbers in their faeces. The mRNA levels of fatty acyl synthase (Fas), sterol regulatory element binding factor 1 (Srebp1c), tumour necrosis factor alpha (Tnf), cytochrome P450 2E1 (Cyp2e1), 3-phosphoinositide-dependent protein kinase-1 (Pdpk1), acyl-Coenzyme A dehydrogenase 11 (Acad11), ATP-binding cassette sub family member G (ABCG2) and DEAD box polypeptide 25 (Ddx25) were significantly elevated in the liver tissues of animals in the experimental group. In gonadal adipose tissue, the expression levels of leptin, peroxisome proliferator-activated receptor γ (Pparg) and Srebp1c were significantly higher in animals from the experimental group, whereas the expression of adiponectin was significantly lower in these animals.

Conclusions

The inoculation of L. ingluviei in mice resulted in alterations in the intestinal flora, increased weight gain and liver enlargement, accelerated metabolism and increased inflammation.     

不同剂量白细胞介素2作为乙型肝炎病毒核酸疫苗佐剂的效应

目的构建表达乙型肝炎病毒 (HBV) S蛋白的重组质粒 p CR3.1- S作为核酸疫苗 ,观察重组人白细胞介素 2(rh IL - 2 )作为佐剂对其诱导 BAL B/ c小鼠产生免疫应答的影响。方法分别以 EL ISA法、3H- Td R掺入法、Cr5 1 4h释放法等测定免疫小鼠的血清抗 HBs、淋巴细胞增殖活性、淋巴细胞杀伤效应 ,初步研究不同免疫组体液和细胞免疫应答的差异。结果血清抗 HBs EL ISA效价 ,rh IL - 2组与对照组相比较差异不显著 (P>0 .0 5 ) ;淋巴细胞增殖指数、淋巴细胞杀伤率 ,10 0 0 U (只·次 ) rh IL - 2 /组和 2 0 0 0 U (只·次 ) rh IL - 2 /组与对照组相比较差异显著 (P<0 .0 5 ) ,而 5 0 0 U (只·次 ) rh IL - 2 /组与对照组相比较无显著差异 (P>0 .0 5 )。结论一定剂量的 rh IL - 2作为佐剂可增强 HBV核酸疫苗的免疫效应     

The Xid defect determines an improved clinical course of murine leishmaniasis in susceptible mice

The course ofLeishmania majorinfection in B cell-defective BALB.Xidmice was investigated. Infected BALB.Xid mice showed a significantiyslower lesion development compared with BALB/c controls accompaniedby a 10– to 30– fold lower parasite burden in lymphaticorgans. The B cell immune response, as quantified by anti-leishmanialantibody production and B cell numbers in lymphatic organs,remained significantly lower in BALB.Xid mice as compared withBALB/c control mice. In accordance with disease development,CD4⁺ T cells from lymph nodes of infected BALB.Xid mice produced6– to 10– fold more IFN-; than the respective Tcells of BALB/c mice, when stimulated with leishmanial antigeninvitro. B cells from lymph nodes and the peritoneal cavitiesof BALB/c mice could be induced to produce 3– to 8–fold more IL-10 than the respective cells from B cell-defectiveBALB.Xid mice. The data thus indicate that the Xid mutationallows for the development of T_h1 cells which confer resistanceto infection withL. major. Moreover, the data suggest that Bcells contribute to susceptibility to L. major infection inBALB/c mice by skewing the T_h cell network towards a T_h2 phenotype.Since the difference in B cell-derived IL–10 productionbetween BALB/c and BALB.Xid mice was more prominent in peritonealB cells, the data support the notion that the skewing of theT cell response may be predominantly mediated by the B1 cellsubset.     

An engineered Plasmodium falciparum C-terminal 19-kilodalton merozoite surface protein 1 vaccine candidate induces high levels of interferon-gamma production associated with cellular immune responses to specific peptide sequences in Gambian adults naturally exposed to malaria

The 19-kDa C-terminal region of merozoite surface protein 1 (MSP1(19)), a major blood stage malaria vaccine candidate, is the target of cellular and humoral immune responses in humans naturally infected with Plasmodium falciparum. We have previously described engineered variants of this protein, designed to be better vaccine candidates, but the human immune response to these proteins has not been characterized fully. Here we have investigated the antigenicity of one such variant compared to wild-type MSP1(19)-derived protein and peptides. Gambian adults produced both high T helper type 1 (Th1) [interferon (IFN)-γ] and Th0/Th2 [interleukin (IL)-13 and sCD30] responses to the wild-type MSP1(19) and the modified protein as wells as to peptides derived from both forms. Response to the modified MSP1(19) (with three amino acid substitutions: Glu27Tyr, Leu31Arg and Glu43Leu) relative to the wild-type, included higher IFN-γ production. Interestingly, some peptides evoked different patterns of cytokine responses. Modified peptides induced higher IL-13 production than the wild-type, while the conserved peptides P16 and P19 induced the highest IFN-γ and IL-13 and/or sCD30 release, respectively. We identified P16 as the immunodominant peptide that was recognized by cells from 63% of the study population, and not restricted to any particular human leucocyte antigen D-related (HLA-DR) type. These findings provide new and very useful information for future vaccine development and formulation as well as potential Th1/Th2 immunmodulation using either wild-type or modified protein in combination with their peptides.     

A Network Analysis of 15O-H2O PET Reveals Deep Brain Stimulation Effects on Brain Network of Parkinson's Disease

Purpose

As Parkinson''s disease (PD) can be considered a network abnormality, the effects of deep brain stimulation (DBS) need to be investigated in the aspect of networks. This study aimed to examine how DBS of the bilateral subthalamic nucleus (STN) affects the motor networks of patients with idiopathic PD during motor performance and to show the feasibility of the network analysis using cross-sectional positron emission tomography (PET) images in DBS studies.

Materials and Methods

We obtained [¹⁵O]H₂O PET images from ten patients with PD during a sequential finger-to-thumb opposition task and during the resting state, with DBS-On and DBS-Off at STN. To identify the alteration of motor networks in PD and their changes due to STN-DBS, we applied independent component analysis (ICA) to all the cross-sectional PET images. We analysed the strength of each component according to DBS effects, task effects and interaction effects.

Results

ICA blindly decomposed components of functionally associated distributed clusters, which were comparable to the results of univariate statistical parametric mapping. ICA further revealed that STN-DBS modifies usage-strengths of components corresponding to the basal ganglia-thalamo-cortical circuits in PD patients by increasing the hypoactive basal ganglia and by suppressing the hyperactive cortical motor areas, ventrolateral thalamus and cerebellum.

Conclusion

Our results suggest that STN-DBS may affect not only the abnormal local activity, but also alter brain networks in patients with PD. This study also demonstrated the usefulness of ICA for cross-sectional PET data to reveal network modifications due to DBS, which was not observable using the subtraction method.     

Effects of vitamin K2 on the development of osteopenia in rats as the models of osteoporosis

Vitamin K2 is widely used for the treatment of osteoporosis in Japan. To understand the effects of vitamin K2 on bone mass and bone metabolism, we reviewed its effects on the development of osteopenia in rats, which characterizes models of osteoporosis. Vitamin K2 was found to attenuate the increase in bone resorption and/or maintain bone formation, reduce bone loss, protect against the loss of trabecular bone mass and its connectivity, and prevent the decrease in strength of the long bone in ovariectomized rats. However, combined treatment of bisphosphonates and vitamin K2 had an additive effect in preventing the deterioration of the trabecular bone architecture in ovariectomized rats, while the combined treatment of raloxifene and vitamin K2 improved the bone strength of the femoral neck. The use of vitamin K2 alone suppressed the increase in trabecular bone turnover and endocortical bone resorption, which attenuated the development of cancellous and cortical osteopenia in orchidectomized rats. In addition, vitamin K2 inhibited the decrease in bone formation in prednisolone-treated rats, thereby preventing cancellous and cortical osteopenia. In sciatic neurectomized rats, vitamin K2 suppressed endocortical bone resorption and stimulated bone formation, delaying the reduction of the trabecular thickness and retarding the development of cortical osteopenia. Vitamin K2 also prevented the acceleration of bone resorption and the reduction in bone formation in tail-suspended rats, which counteracted cancellous bone loss. Concomitant use of vitamin K2 with a bisphosphonate ameliorated the suppression of bone formation and more effectively prevented cancellous bone loss in tail-suspended rats. Vitamin K2 stimulated renal calcium reabsorption, retarded the increase in serum parathyroid hormone levels, and attenuated cortical bone loss primarily by suppressing bone resorption in calcium-deficient rats while maintaining the strength of the long bone in rats with magnesium deficiency. These findings suggest that vitamin K2 may not only stimulate bone formation, but may also suppress bone resorption. Thus, vitamin K2 could regulate bone metabolism in rats, which represented the various models of osteoporosis. However, the effects of vitamin K2 on bone mass and bone metabolism seem to be modest.     

A Trichosanthin-derived peptide suppresses type 1 immune responses by TLR2-dependent activation of CD8CD28 Tregs

A group of 15-aa-long Trichosanthin-derived peptides was synthesized and screened based on their differential abilities to induce low-responsiveness in mouse strains with high and low susceptibility. One of them was conjugated to form a homo-tetramer Tk-tPN. At concentrations of 0.1–50 μg/ml, Tk-tPN activated CD8⁺CD28⁻ Tregs in vitro to induce immune suppression as effectively as the native Trichosanthin but did not exhibit cytotoxicity. In EAE mice which were pre-treated with Tk-tPN or Tk-tPN-activated CD8⁺ T cells, a marked attenuation of clinical scores was recorded together with an expansion of the CD8⁺CD28⁻ Treg from 2.2% to 36.1% in vivo. A pull-down assay and signal transduction analyses indicated that the ability of Tk-tPN to convert the CD8⁺CD28⁻ Treg-related cytokine secretion pattern from type 1 to type 2 depends on the TLR2-initiated signaling in macrophages. The high production of IL-4/IL-10 by the Tk-tPN-activated CD8⁺CD28⁻ Treg suggests the value of using Tk-tPN as a therapeutic reagent for Th1-dominant immunological diseases.     

Prostaglandin E2, collagenase,and cell death responses depend on cyclical load magnitude in an explant model of tendinopathy

Tendinopathy is a significant clinical problem that can result from repetitive activity. While the precise etiology of this condition remains unclear, the cellular response to cyclical loading is believed to have a contributory role to the pathology of tendinopathy. This study examined the short-term biochemical response of avian flexor digitorum profundus tendon to repetitive cyclic loadings of varying magnitude. An in vitro tendon explant model was utilized to apply four levels of haversine tensile stress (peak stress of 0, 3, 12, and 18 MPa) at 1.0 Hz, 8 hr/day for 3 days. The 12 and 18 MPa levels were known to cause significant mechanical damage based on previous work. Tissue media was recovered and analyzed for prostaglandin E₂ (PGE₂), lactate dehydrogenase (LDH, measure of cell death), and collagenase levels. Tissue samples were recovered and analyzed for cell viability, total collagen, and sulfated glycosaminoglycan content. Collagenase, LDH, and PGE₂ levels were found to be influenced by loading magnitude (p < 0.05) with higher levels being present at higher load magnitudes. Varying cyclical load magnitude caused minimal compositional changes as collagen content and glycosaminoglycan did not change. These results indicate that elevated cyclical mechanical loading of tendon quickly results in altered biochemical tissue responses indicative of tissue injury. More sustained cyclical loading over time may be required for these initial responses to induce more dramatic tissue changes as observed in clinical tendinopathy.     

Neuroprotective effects of phenylenediamine derivatives independent of an antioxidant pathway in neuronal HT22 cells

Resistance to oxidative stress often determines neuronal survival in the brain. Thus, antioxidants are supposed to be promising neuroprotective compounds against neurodegenerative diseases. For example, N,N′-diphenyl-p-phenylenediamine (DPPD) reportedly exerts cytoprotective effects against oxidative stress possibly by acting as an antioxidant. DPPD can give electron(s) to free radicals and thus scavenge them, and protect the cells from oxidative stress. The antioxidative activities of DPPD are prominent at the micromolar order, but what about its effects at much lower concentrations? We concluded that DPPD has two actions on neuronal cells, antioxidant activity and an unknown neuroprotective effect, which are effective at micromolar and nanomolar levels, respectively. In the present report, we found that DPPD inhibited cell death caused by oxidative stress at nanomolar order (1/1000 lower than concentrations needed for antioxidant activity) and that the effects were independent of antioxidant activities. DPPD inhibited the oxidative glutamate toxicity but not the tumor necrosis factor α-, hydrogen peroxide-, or xanthine + xanthine oxidase-induced death of HT22 cells, a mouse neuronal cell line. DPPD and phenylenediamine derivatives protected HT22 cells against oxidative glutamate toxicity at nanomolar concentrations. By studying the structure-function relationship of these compounds, we found the structure of phenyl-amine–phenyl-amine–phenyl (or butyl) to be essential for the neuroprotective effects.