OBJECTIVE:To characterize ossified bone marrow blood vessels and confirm the presence of ossified particles (OSP) in humans and rodents.METHODS:Human bone marrow blood vessels were processed for scanning and transmission electron microscopy. Whole blood samples were collected from younger (26-39 years; n = 6) and older (55-63 years; n = 6) volunteers and male Fischer-344 rats (1 month, n = 7; 6 months, n = 7; 12 months, n = 7; 18-months, n = 6; 24 months, n = 8). OSP in the whole blood samples were sorted and imaged with microscopy to determine diameter, circularity, and solidity. Additionally, the chemical composition of OSP was determined via elemental analysis.RESULTS:SEM revealed two types of ossified bone marrow blood vessels: that is, "transitioning" and "ossified." OSP were adhered to the surface of transitioning vessels and theoretically gain access to and circulate within the blood. The majority of OSP were ≤15 μm in diameter, but many were of sufficient size to serve as emboli (ie, >15 μm).OSP were predominately oblong in shape and several had jagged tips and edges.CONCLUSIONS:We introduce a novel, bone-like blood particle that may be diagnostic of bone marrow blood vessel ossification. Further, OSP may associate with several disease states (eg, atherosclerosis).

Mycoplasmas are atypical bacteria that disrupt the immune response to promote respiratory tract infections and secondary complications. However, not every immunologic response that protects or damages the host during mycoplasma infection is known.

BACKGROUND:Delivery of PLGA (poly [D, L-lactide-co-glycolide])-based biodegradable nanoparticles (NPs) to antigen presenting cells, particularly dendritic cells, has potential for cancer immunotherapy.MATERIALS & METHODS:Using a PLGA NP vaccine construct CpG-NP-Tag (CpG-ODN-coated tumor antigen [Tag] encapsulating NP) prepared using solvent evaporation technique we tested the efficacy of ex vivo and in vivo use of this construct as a feasible platform for immune-based therapy.RESULTS:CpG-NP-Tag NPs were avidly endocytosed and localized in the endosomal compartment of bone marrow-derived dendritic cells. Bone marrow-derived dendritic cells exposed to CpG-NP-Tag NPs exhibited an increased maturation (higher CD80/86 expression) and activation status (enhanced IL-12 secretion levels). In vivo results demonstrated attenuation of tumor growth and angiogenesis as well as induction of potent cytotoxic T-lymphocyte responses.CONCLUSION:Collectively, results validate dendritic cells stimulatory response to CpG-NP-Tag NPs (ex vivo) and CpG-NP-Tag NPs' tumor inhibitory potential (in vivo) for therapeutic applications, respectively.

6-Thiopurine (6-TP) prodrugs include 6-thioguanine and azathioprine. Both are widely used to treat autoimmune disorders and certain cancers. This study showed that a 6-thioguanosine triphosphate (6-TGTP), converted in T-cells from 6-TP, targets Rac1 to form a disulfide adduct between 6-TGTP and the redox-sensitive GXXXXGK(S/T)C motif of Rac1. This study also showed that, despite the conservation of the catalytic activity of RhoGAP (Rho-specific GAP) on the 6-TGTP-Rac1 adduct to produce the biologically inactive 6-thioguanosine diphosphate (6-TGDP)-Rac1 adduct, RhoGEF (Rho-specific GEF) cannot exchange the 6-TGDP adducted on Rac1 with free guanine nucleotide. The biologically inactive 6-TGDP-Rac1 adduct accumulates in cells because of the ongoing combined actions of RhoGEF and RhoGAP. Because other Rho GTPases, such as RhoA and Cdc42, also possess the GXXXXGK(S/T)C motif, the proposed mechanism for the inactivation of Rac1 also applies to RhoA and Cdc42. However, previous studies have shown that CD3/CD28-stimulated T-cells contain more activated Rac1 than other Rho GTPases such as RhoA and Cdc42. Accordingly, Rac1 is the main target of 6-TP in activated T-cells. This explains the T-cell-specific Rac1-targeting therapeutic action of 6-TP that suppresses the immune response. This proposed mechanism for the action of 6-TP on Rac1 performs a critical role in demonstrating the capability to design a Rac1-targeting chemotherapeutic agent(s) for autoimmune disorders. Nevertheless, the results also suggest that the targeting action of other Rho GTPases in other organ cells, such as RhoA in vascular cells, may be linked to cytotoxicities because RhoA plays a key role in vasculature functions.

Silver-loaded spherical activated carbon (Ag-SAC) was synthesized by liquid phase ion exchange using phenolic weak acid cation exchange resin, carbonization and activation with CO2. The adsorptive capacity and selectivity of Ag-SAC for thiophene (T), 3-methylthiophene (3-MT) and 2,5-dimethylthiophene (2,5-DMT) were investigated by the fixed bed adsorption experiments under ambient conditions. The results showed that the saturation capacities of Ag-SAC for T, 3-MT and 2,5-DMT were 0.733, 0.925 and 1.143 mg-S/g-A, respectively and the adsorptive selectivity of the adsorbent for different sulfur compounds followed the order: 2,5-DMT > 3-MT > T. The effects of toluene and cyclohexene on the adsorptive performance were also studied. The results indicated that the presence of toluene and cyclohexene led to a reduction of total saturation capacity by 57 and 33 %, respectively.

Activated carbon was modified with nitric acid.The properties of activated carbon before and after modification were characterized. The adsorption equilibrium and kinetics of benzothiophene on the original activated carbon and its modified form were studied,in which the adsorption equilibrium data were fitted with Freundlich,Langmuir,Sips and BET adsorption models and the kinetic data were fitted with pseudo-first-order,pseudo-second-order,mixed-order and modified pseudo-n-order rate equations.The results showed that BET and Sips models fitted best to the adsorption equilibrium of benzothiophene on the original activated carbon,Sips and Freundlich models fitted best to the kinetics of benzothiophene on the modified activated carbon. The adsorption of benzothiophene on activated carbon before and after modification was mainly physical,and the degree of heterogeneity of the surface and the affinity with benzothiophene for the modified activated carbon were enhanced,resulting in 33.7%increase of absorption capacity.The density of oxygen containing functional groups on the surface of activated carbon was the main factor to determine the adsorption capacity.

Phenolic weak acid cation exchange resin was chosen as carbon precursor to prepare spherical activated carbon(SAC) by carbonization and activation with CO2.The product was characterized by infrared spectrum analysis and N2 adsorption,and the results showed that the specific surface area and pore volume were 799.567 m2/g and 0.969 cm3/g,respectively.The saturation capacity for benzothiophene was10.3 mg /g.The equilibrium experimental data were fitted to the Langmuir and Freundlich models,and Freundlich model gave a better correlation,which meants there was heterogeneity in the sites of the SAC adsorbents.Pseudo-first-order(PFO),pseudo-second-order(PSO),modified pseudo-n-order(MPnO)and mixed-order rate equation(MOE) were used to fit the kinetics experimental data.The results indicated that MPnO was the best model to describe the kinetics of adsorption of benzothiophene on SAC.

Mycoplasmas cause chronic respiratory diseases in animals and humans, and to date, development of vaccines have been problematic. Using a murine model of mycoplasma pneumonia, lymphocyte responses, specifically T cells, were shown to confer protection as well as promote immunopathology in mycoplasma disease. Because T cells play such a critical role, it is important to define the role of antigen presenting cells (APC) as these cells may influence either exacerbation of mycoplasma disease pathogenesis or enhancement of protective immunity. The roles of APC, such as dendritic cells and/or macrophages, and their ability to modulate adaptive immunity in mycoplasma disease are currently unknown. Therefore, the purpose of this study was to identify individual pulmonary APC populations that may contribute to the activation of T cell responses during mycoplasma disease pathogenesis. The present study indeed demonstrates increasing numbers of CD11c(-) F4/80(+) cells, which contain macrophages, and more mature/activated CD11c(+) F4/80(-) cells, containing DC, in the lungs after infection. CD11c(-) F4/80(+) macrophage-enriched cells and CD11c(+) F4/80(-) dendritic cell-enriched populations showed different patterns of cytokine mRNA expression, supporting the idea that these cells have different impacts on immunity in response to infection. In fact, DC containing CD11c(+) F4/80(-) cell populations from the lungs of infected mice were most capable of stimulating mycoplasma-specific CD4(+) Th cell responses in vitro. In vivo, these CD11c(+) F4/80(-) cells were co-localized with CD4(+) Th cells in inflammatory infiltrates in the lungs of mycoplasma-infected mice. Thus, CD11c(+)F4/80(-) dendritic cells appear to be the major APC population responsible for pulmonary T cell stimulation in mycoplasma-infected mice, and these dendritic cells likely contribute to responses impacting disease pathogenesis.

Previous studies showed that the endothelin B receptor (ETB) expression was upregulated and played a key role in neurodegeneration in rodent models of glaucoma. However, the mechanisms underlying upregulation of ETB receptor expression remain largely unknown. Using promoter-reporter assays, the 1258 bp upstream the human ETB promoter region was found to be essential for constitutive expression of ETB receptor gene in human non-pigmented ciliary epithelial cells (HNPE). The −300 to −1 bp and −1258 to −600 bp upstream promoter regions of the ETB receptor appeared to be the key binding regions for transcription factors. In addition, the crucial AP-1 binding site located at −615 to −624 bp upstream promoter was confirmed by luciferase assays and CHIP assays which were performed following overexpression of c-Jun in HNPE cells. Overexpression of either c-Jun or C/EBPβ enhanced the ETB receptor promoter activity, which was reflected in increased mRNA and protein levels of ETB receptor. Furthermore, knock-down of either c-Jun or C/EBPβ in HNPE cells was significantly correlated to decreased mRNA levels of both ETB and ETA receptor. These observations suggest that c-Jun and C/EBPβ are important for regulated expression of the ETB receptor in HNPE cells. In separate experiments, intraocular pressure (IOP) was elevated in one eye of Brown Norway rats while the corresponding contralateral eye served as control. Two weeks of IOP elevation produced increased expression of c-Jun and C/EBPβ in the retinal ganglion cell (RGC) layer from IOP-elevated eyes. The mRNA levels of c-Jun, ETA and ETB receptor were upregulated by 2.2-, 3.1- and 4.4-fold in RGC layers obtained by laser capture microdissection from retinas of eyes with elevated IOP, compared to those from contralateral eyes. Taken together, these data suggest that transcription factor AP-1 plays a key role in elevation of ETB receptor in a rodent model of ocular hypertension.

Phenolic weak acid cation exchange resin was used as carbon precursor to prepare spherical activated carbon supported copper catalyst by ion exchange,carbonization and activation with CO2.The catalyst was characterized by SEM,XRD,AAS and BET.The adsorptive capacities for different sulfur-containing compounds were evaluated by dynamic adsorption experiments and the competitive effects of toluene and cyclohexene were investigated.The mechanism of adsorptive desulfurization was primarily studied as well.The results showed that metals in the spherical activated carbon existed in elemental form and improved the adsorption performance through direct coordination with sulfur atoms.The saturation capacities for thiophene(T),3-methylthiophene(MT),2,5-dimethylthiophene(DMT) and benzothiophene(BT) were 0.938,1.230,1.581 and 3.744 mg/g,respectively.The adsorptive selectivity increased in the order of TMTDMTBT.After toluene and cyclohexene were added to the model gasoline,the saturation capacity decreased 77% and 56%,respectively,which might be resulted by competitive adsorption through the interaction between copper and π electron of toluene and cyclohexene.

For vaccine development, it is critical to understand the regulatory mechanisms determining resistance and immunopathology against mycoplasma respiratory diseases. The present study evaluated the contribution of the polarizing cytokines interferon gamma (IFN-gamma) and interleukin 4 (IL-4) in the regulation of mycoplasma-specific immunity. The absence of a single cytokine (either IFN-gamma or IL-4) uniquely altered the expression of multiple chemokines and cytokines in the lungs of uninfected mice and influenced responses to mycoplasma infection. Most importantly, prior nasal-pulmonary immunization of IFN-gamma(-/-) mice led to exacerbated mycoplasma disease, whereas immunized IL-4(-/-) mice were dramatically more resistant than wild-type mice. Helper T cell type 2 responses in IFN-gamma(-/-) mice corresponded to immunopathologic reactions that developed after mycoplasma infection or immunization. Thus, adaptive immunity clearly can independently promote either protection or immunopathology against mycoplasma infection, and optimal vaccination appears to be dependent on promoting protective IFN-gamma-dependent networks (perhaps helper T cell type 1 responses) while minimizing the effect of IL-4-mediated responses, which dampen the generation of protective immunity.

The purpose of the present study was to determine the impact of NK cells on the development of protective adaptive immunity in response to nasal-pulmonary immunization against mycoplasma. Depletion of NK cells before nasal-pulmonary immunization enhanced resistance to mycoplasma respiratory infection. The effect of NK cells on the generation of protective immunity in lungs was dependent on lymphoid cells, as immunization of either SCID mice or immunocompetent mice depleted of CD4+ T cells did not demonstrate any increased resistance in the presence or absence of NK cells. The presence of NK cells at the time of nasal-pulmonary immunization modulated mycoplasma-specific cytokine responses in lungs and lower respiratory nodes. In particular, NK cells skewed the mycoplasma-specific T cell cytokine responses in the draining lymph nodes to higher IL-4, IL-13, and IL-17 while lowering IFN-γ responses. Adoptive transfer of total lung lymphocytes isolated from immunized mice into naive mice led to a significant reduction in the mycoplasma numbers in lungs, and the resistance was greater if cells were obtained from immunized mice that were depleted of NK cells. Similar results were obtained if purified B cells, T cells, or CD4+ T cells were used. Interestingly, this is the first time that a favorable role of functional CD4+ T cells in mediating protection in mycoplasma respiratory disease was demonstrated. Thus, NK cells can influence the responses of multiple lymphocyte populations capable of mediating resistance to mycoplasma infection.

Mycoplasma respiratory diseases have a significant impact on the economy, health and wildlife. The hallmark of these diseases is the persistence of the mycoplasma infections and chronic inflammatory responses associated with the airways. There is still much that needs to be understood about the immune mechanisms involved in mycoplasma disease and resistance from infection. It is clear that immune responses can contribute to the generation of inflammatory lesions in mycoplasma respiratory disease, as well as provide protection from infection and extrapulmonary dissemination of the organisms. The evolution of this lung disease is under the control innate immune mechanisms and the contrasting effects of different T cell populations. The mechanisms of immunity involved in mycoplasma diseases are multifaceted, and a fascinating story of its complexity is being uncovered. Research in mycoplasma respiratory diseases have underscored the idea that immunity along the respiratory tract against infectious agents is a dynamic process and involves a network of cellular and cytokine signals that determine the type of responses generated, and ultimately, the outcome of infection. The aim of this article is to present on overview of our work on mycoplasma disease and immunity, focusing on the interactions and regulation of T cell responses that influence disease pathogenesis. We will first provide an overview of immune mechanisms involved in controlling infection and participate in the generation of T cell responses, and the role of T cell populations in generating protection and contributing to lesion development will be discussed.

Murine mycoplasma disease progression is dependent on host immune responses. IFNγ is critical in developing beneficial innate immunity. Lack of IFNγ impairs the ability to control infection whereas loss of IL4 does not. The purpose of this study was to discern whether the loss of either IFNγ or IL4 altered, the cytokine cascade after infection & the generation of adaptive immunity against mycoplasma. Cytokine mRNA expression was monitored in M. pulmonis infected wild-type, IFNγ & IL4 KO BALB/c mice using microarrays. The lungs of naïve IFNγ & IL4 KO mice had Th2 & Th1 dominant cytokine mRNA profiles, respectively. There was a significant increase in pro-inflammatory cytokine mRNA expression in infected IFNγ KO mice compared to WT, along with severe disease. Whereas, these mRNA levels were lower in the lungs of IL4 KO mice than the WT mice. In vitro T cell responses were consistent with mRNA profiles. To examine generation of protective immunity, nasal pulmonary immunized WT & KO mice were challenged & mycoplasma numbers determined in the nasal passages (NP) & lungs. Immunized IFNγ KO mice had severe lesions & higher numbers of mycoplasma in both NP & lungs than WT. In contrast, the IL4 KO mice were significantly better protected than the immunized WT mice. In conclusion, IFNγ is critical in reducing the development of immunopathology & mediating immune protection whereas IL4 has the opposite effect.

The progression of murine mycoplasma pneumonia is dependent on T cells and other immune cells. The role of cytokines in immunity are complex, and identifying the network of cytokines produced after infection of mice is essential in dissecting the key cytokine cascades involved mycoplasma disease pathogenesis. In the present study, mRNA expression of 143 different cytokines, chemokines, or receptors were evaluated in lung tissues from both susceptible (BALB/c and C3H/HeN) and resistant (C57BL/6) mice after Mycoplasma pulmonis infection. To accomplish this, membrane-based cDNA microarrays were used to monitor changes mRNA expression in lungs. There was a clear association with disease susceptibility and development of cytokine mRNA expression. In addition to proinflammatory cytokines, mRNA expression of an anti-inflammatory cytokine, interleukin-10, increased with disease severity, suggesting an attempt to moderate the severity of the inflammatory response. Furthermore, it is clear that an array of chemokines produced in susceptible mice could contribute to the recruitment and maintenance of inflammatory cells at the site of disease. In support of this, there was an increase in macrophage inflammatory protein 1beta (MIP-1beta; CCL4) and monocyte chemoattractant protein 2 (MCP-2; CCL8) mRNA levels from mycoplasma-infected mice and a corresponding accumulation of CD4+ Th cells expressing the MIP-1beta/MCP-2 receptor, CCR5, in the lungs of mice. Furthermore, MIP-1beta- and MCP-2-producing cells and CD4+ T cells were found to be in close association in pulmonary lesions. Thus, there was a significant cytokine response associated with disease pathogenesis, and these studies provide important leads and insights into ongoing cytokine- and chemokine-mediated processes in this persistent inflammatory disease.

OBJECTIVE:To compare humoral immune response by co-inoculating mice with antigen HPV16L1 virus-like particle (VLP) and HPV16L1 recombinant plasmids and then observing the neutralizing antibody activity in vitro.METHODS:C57BL/6 mice were injected intramuscularly/subcutaneously with pcDNA-L1 plasmids plus HPV16L1 VLP. Serum IgG levels were detected by ELISA, antibody neutralizing protective activities were determined by hemagglutination inhibition and HPV16L1 VLP binding inhibition assay.RESULTS:Serum antibody titers and neutralizing antibody activities were increased in HPV16L1 plasmids plus HPV16L1 VLP proteins in co-immunized mice when compared with controls.CONCLUSION:Co-inoculation of the HPV16L1 VLP protein can enhance production of neutralizing antibody activities against aimed antigen, which should be a more promising strategy for effective HPV16 prophylactic vaccine development.

We have investigated whether co-injection of DNA encoding the costimulatory molecule B7-2 augments immune response to the major capsid protein L1 of the high-risk human papillomavirus type 16 (HPV16L1). While immunoglobulin G (IgG) specific to HPV16L1 was detected in sera from mice injected intramuscularly with pcDNA-L1 that encodes HPV16L1, a significantly increased level of IgG was found in sera from mice immunised with pcDNA-L1 in conjunction with pLXHDmB7-2 DNA. Levels of IgG in the anti-sera were correlated with the inhibitory activity of the murine erythrocyte hemagglutination caused by the virus-like particles (VLP) and the binding of VLP to HeLa cells. Moreover, splenic cells isolated from mice co-injected with pLXHDmB7-2 had stronger proliferation and more IFN-γ producing T cells (CD4+ and CD8+) when stimulated with HPV16 VLP compared with cells from mice that had received pcDNA-L1 alone and mice of the control groups. Furthermore, in footpad swelling test, mice co-immunised with pLXHDmB7-2 had greater skin thickness over those immunised with pcDNA-L1 alone or control mice. We conclude that co-injection of DNA encoding B7-2 can enhance both humoral and cellular immune responses elicited by DNA-based vaccination against HPV16 infection in mice.

目的:以pcDNAL1质粒免疫啮齿类动物(C57BL/6)为模型,观察HPV16L1VLP细胞结合抑制实验是否可以用于检测免疫抗体的中和保护作用.方法:实验组包括Ⅰ组pcDNAL1、Ⅱ组HPV16L1 VLP;Ⅲ组pcDNA3.1.每组动物均为6只C57BL/6鼠.每组动物均肌肉注射免疫3次,间隔3 w.末次免疫后14 d眼球后取血并拉颈处死动物,进行HPV16 L1VLP结合抑制试验:免疫血清中和HPV16L1VLP;制备Hela,EJ和RLC310细胞爬片,CS1213细胞涂片;细胞免疫组化染色.结果:实验组血清中和后Hela细胞呈染色阴性,而对照组、不共育组则细胞呈棕黄色.结论:这说明实验组血清具有抑制VLP与Hela细胞粘附的活性效应.这一方法可能比HAI更能直接反映中和抗体的活性和保护作用.

Objective To compare humoral immune response by co inoculating mice with co stimulatory molecule B7 2 and HPV16L1 recombinant plasmids and observe the neutralizing antibody activity in vitro . Methods C57BL/6 mice were injected i.m with pcDNA L1+PLXHDmB7 2 plasmids. Serum IgG was detected by ELISA, Ab neutralizing protective activities were determined by Hemagglutination inhibition and HPV16 VLP binding inhibition assay. Results Titers of serum antibody and neutralizing antibody activities were increased in HPV16L1 plus B7 2 co immunized mice compared to those of control. Conclusion Co inoculation of co stimulatory molecule B7 2 enhanced production of neutralizing antibody against specific antigen, which should be a more promising strategy for effective HPV16 prophylactic vaccine development. [