B cells from glatiramer acetate-treated mice suppress experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) thought to be primarily mediated by T cells. However, emerging evidence supports an important role for B cells in the pathogenesis and inhibition of MS. Glatiramer acetate (GA), a Food and Drug Administration-approved drug for the treatment of MS, has a good safety profile. But GA's mechanism of action in MS is still elusive. In this study, we showed that B cells from GA-treated mice increased production of IL-10 and reduced expression of co-stimulatory molecules viz.: CD80 and CD86. B cells from GA-treated mice also diminished proliferation of myelin oligodendrocyte glycoprotein (MOG(35-55)) specific T cells. Purified B cells transferred from GA-treated mice suppressed experimental autoimmune encephalomyelitis (EAE) in recipient mice compared with B cells transferred from mice treated with PBS or ovalbumin. The treatment effect of GA in EAE was abrogated in B cell-deficient mice. Transfer of B cells from GA-treated mice inhibited the proliferation of autoreactive T cells as well as the development of Th1 and Th17 cells but promoted IL-10 production in recipient mice. The number of peripheral CD11b(+) macrophages in recipient mice also decreased after transfer of B cells from GA-treated mice; however, the number of dendritic cells and regulatory T cells remained unaltered. These results suggest that B cells are important to the protective effects of GA in EAE.

Nicotine and inflammatory neurological disorders.

Cigarette smoke is a major health risk factor which significantly increases the incidence of diseases including lung cancer and respiratory infections. However, there is increasing evidence that smokers have a lower incidence of some inflammatory and neurodegenerative diseases. Nicotine is the main immunosuppressive constituent of cigarette smoke, which inhibits both the innate and adaptive immune responses. Unlike cigarette smoke, nicotine is not yet considered to be a carcinogen and may, in fact, have therapeutic potential as a neuroprotective and anti-inflammatory agent. This review provides a synopsis summarizing the effects of nicotine on the immune system and its (nicotine) influences on various neurological diseases.

Nicotinic attenuation of central nervous system inflammation and autoimmunity.

The expression of nicotinic acetylcholine receptors by neurons, microglia, and astrocytes suggests possibly diverse mechanisms by which natural nicotinic cholinergic signaling and exposure to nicotine could modulate immune responses within the CNS. In this study, we show that nicotine exposure significantly delays and attenuates inflammatory and autoimmune responses to myelin Ags in the mouse experimental autoimmune encephalomyelitis model. In the periphery, nicotine exposure inhibits the proliferation of autoreactive T cells and alters the cytokine profile of helper T cells. In the CNS, nicotine exposure selectively reduces numbers of CD11c(+) dendritic and CD11b(+) infiltrating monocytes and resident microglial cells and down-regulates the expression of MHC class II, CD80, and CD86 molecules on these cells. The results underscore roles of nicotinic acetylcholine receptors and nicotinic cholinergic signaling in inflammatory and immune responses and suggest novel therapeutic options for the treatment of inflammatory and autoimmune disorders, including those that affect the CNS.

Therapeutic effect of anthracene-based anticancer agent ethonafide in an animal model of multiple sclerosis.

The side effects of cancer chemotherapeutic agents such as mitoxantrone (MIT) in multiple sclerosis (MS) patients justify the search for less toxic drugs. Ethonafide is an anthracene-based antineoplastic drug similar to MIT. With reference to MIT, we examined the effect of ethonafide on experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice, an animal model of human MS. We demonstrated that ethonafide is effective in preventing development of EAE as well as in ameliorating the severity of EAE when disease is ongoing. In relatively higher dosages, the effects of ethonafide and MIT on EAE were identical, whereas in lower dosages, MIT seemed more effective. Therapeutic effects of ethonafide were associated with the initial reduction in cellular counts of CD3(+), CD4(+), CD8(+), B220(+), CD11b(+), NK cells, and NKT cells, followed by recovery of these cells from the bone marrow. Interestingly, the recovered autoreactive T cells in ethonafide-treated animals have reduced capacity to expand and produce cytokines in response to myelin Ag stimulation. Furthermore, CD4(+)CD25(+) regulatory T cells were relatively resistant to depletion and/or recovered faster than T effector cells. The ability of regulatory T cells to resist depletion and replenish quickly during cell ablation therapy may provide an opportunity to reprogram the immune system. Moreover, we provided evidences that ethonafide has less cardiac toxicity compared with MIT. The effectiveness and the low cardiotoxicity of ethonafide might make it a promising immunosuppressive agent for clinical use in treating MS patients.

[The effect of stimulation of dendritic cell on HBV-epitopic cytotoxic T-lymphocyte].

OBJECTIVE: To observe the enhancement of cytotoxic T lymphocyte responses in patients with chronic hepatitis B following vaccination with dendritic cell stimulated by Poly (I:C) in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation on Ficoll-Hypaque, the adherent cells were cultured in the medium AIM-V contained recombinant human IL-4 and recombinant human GM-CSF. On day 7, one part of wells were added with Poly (I:C). On day 9, mature DCs (mDCs) were harvested and used to phenotype analysis. Both of the immature DCs and mature DCs were concultured with the autologous T cells for another two-three days. According to the source of dendritic cells concultured with the T cells, the subjects were divided into three groups: the T cells isolated from CHB group; the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group; the T cells stimulated by dendritic cells concultured with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide group. The function and frequency of HBV specific CTL were detected by Elispot (Enzyme-linked immunospot assay, Elispot) and tetramer staining. RESULTS: The average of percentage of HBV specific CD8(+) cells of total CD8(+) cells was 0.77% (0.45% approximately 1.74%) in the T cells isolated from 22 chronic hepatitis B patients and the average of the spots of antigen-specific IFN-gamma-releasing effector cells was 16 (9 approximately 28); The percentage of HBV specific CD8(+) cells of the T cells stimulated by dendritic cells pulsed with HBcAg18-27 CTL epitope peptide group rose to 1.92% (1.36% approximately 2.65%) and the average of the spots of antigen-specific IFN-gamma-releasing effector cells was 46 (30 approximately 67); while stimulated by dendritic cells added with Poly (I:C) and pulsed with HBcAg18-27 CTL epitope peptide, the percentage of HBV specific CD8(+) cells of total CD8(+) cells rose to 3.49% (2.02% approximately 4.60%) and the average of the spots was 98 (59 approximately 130). There was statistical difference between the results of Elispot and tetramer of the three groups (P < 0.01). CONCLUSION: The T lymphocyte of patients with chronic hepatitis B stimulated by the autologous dendritic cells may result in obtaining high percentage and functional antigen-specific T lymphocyte. The addition of Poly (I:C) which was used as maturation promoting factor in DC culture can enhance the function of DC significantly and can get even higher percentage antigen-specific T lymphocyte with enhanced function.

[Study of phenotypes and functions of cord blood dendritic cells from fetuses whose mothers had chronic hepatitis B].

OBJECTIVE: To investigate the phenotypes and functions of cord blood dendritic cells of fetuses whose mothers are patients with chronic hepatitis B. METHODS: Peripheral blood and cord blood mononuclear cells (PBMC) were isolated from whole blood by density gradient centrifugation with Ficoll-Hypaque. The adherent cells were cultured in AIM-V medium containing recombinant human IL-4, TNF-alpha and GM-CSF. On day 9, mature DCs (mDC) were harvested and used for phenotype analysis. The amounts of IL-12 which dendritic cells produced were measured. The dendritic cells that were studied and compared were from cord blood of fetuses of both CHB positive and negative mothers and from CHC adult peripheral blood. RESULTS: The expression rate of CD80 and CD83 of chronic hepatitis B mother cord blood dendritic cells was low compared with that of the healthy cord blood, healthy adult peripheral blood, and chronic hepatitis B adult peripheral blood, P < 0.05. The amount of IL-12 produced by chronic hepatitis B mother cord blood dendritic cells was lower than that of healthy cord blood, healthy adult peripheral blood, chronic hepatitis B adult peripheral blood (P < 0.05). The T lymphocyte proliferation inducing ability of dendritic cells of healthy adult peripheral blood was higher in inducing cord blood T lymphocytes proliferation, which was greater than that of the healthy adult peripheral blood in inducing adult T lymphocytes and was greater than that of the healthy cord blood dendritic cells in inducing cord blood T lymphocytes, which was greater than that of the healthy cord blood in inducing adult T lymphocytes, which was greater than that of chronic hepatitis B mothers in inducing cord blood T lymphocytes, which was greater than that of chronic hepatitis B mother cord blood in inducing adult T lymphocytes. CONCLUSION: The maturation and functioning of CHB mother cord blood dendritic cells were lower than those of healthy cord blood, healthy adult peripheral blood and CHB adult peripheral blood.

[Dynamic expression of hepatitis B virus covalently closed circular DNA in 2.2.15 cell].

BACKGROUND: To determine the presence of covalently closed circular DNA (cccDNA), and to investigate the expression kinetics of HBV DNA, HBsAg and HBeAg in 2.2.15 cell. METHODS: HBV cccDNA was assessed by polymerase chain reaction, HBV DNA was measured by Taqman quantitative PCR and HBsAg and HBeAg was measured by EIA. RESULTS: HBV cccDNA was found in both intracellular and extracellular space. There was a good correlation between HBsAg, HBeAg and HBV DNA in the supernatant of 2.2.15 cell (r= 0.833, P < 0.05 and r= 0.939, P < 0.01 for HBsAg and HBeAg, respectively), whereas there was no significant correlation between intracellular HBV DNA levels and virus antigen levels (r= 0.024, P= 0.955 and r= 0.177; P= 0.625 for HBsAg and HBeAg, respectively). CONCLUSION: HBV cccDNA was detectable in the culture medium and intracellularly in 2.2.15 cells, and these data provided an indication of HBV replication in 2.2.15 cell.

Cloning, expression, and purification of HLA-A2-BSP and beta-2m in Escherichia coli.

Tetramer analysis is a novel technique in immunological research that has dramatically changed our knowledge of the immune response to pathogens, tumors and autoimmune disease. Through the formation of major histocompatibility complex (MHC)-peptide tetrameric complexes, it can provide accurate counts of antigen-specific T-cells and it allows their phenotypical and functional analysis. The tetramer is composed of the human leukocyte antigen (HLA) heavy chain, beta-2 microglobulin (beta-2m), the nominal peptide, and streptavidin. The HLA heavy chain and the beta-2m are expressed in Escherichia coli. But up to now, all laboratories have been expressing these two proteins by using isopropyl beta-d-thiogalactopyranoside IPTG. IPTG is very expensive, and it is tedious and laborious to induce expression protein. So it is difficult to scale up to express the objective protein. To address this problem, extracellular fractions of HLA-A0201 and beta-2m (absent signal peptide) genes were cloned from peripheral blood mononuclear cells (PBMCs) by RT-PCR. DNA coding for a Gly-Ser linker and a BSP (15-amino acid substrate peptide for BirA-dependent biotinylation) was added to the COOH-terminus of the extracellular fraction of HLA-A0201 by PCR, using an HLA-A0201 as the template. Then the HLA-A0201-BSP and beta-2m genes were cloned into pBV220 vector and expressed, respectively. The expressed proteins were purified and detected by ELISA and Western blot analyses. High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs.

[Construction of HLA-A2-peptide tetramer and application in HBV/HCV infection].

OBJECTIVE: To construct HBV and HCV-specific HLA-A2-peptide tetramers, and to direct clinical therapy. METHODS: Recombinant class I HLA-A2 heavy chains and beta-2 M were produced in Escherichia coli cells transformed with pBV220 vectors. Only the extracellular domain of class I heavy chain was expressed, following modification by replacement of the C-terminal domain with a substrate sequence for BirA biotinylation. HLA-A2-BSP was folded in the presence of beta-2 microglobulin and a specific peptide to form a peptide-MHC complex. The MHC complexes were biotinylated using purified BirA enzyme. Biotinylated MHC-peptide complexes were purified. Tetramers were generated by mixing biotinylated protein complex with streptavidin-PE at a molar ratio of 4:1. Then analysis of stained PBMCs was performed using FACScan and CellQuest software. RESULTS: The expression levels of pBV220-HLA-A2-BSP and beta-2M were 46% and 48% of total bacterial proteins estimated from SDS - PAGE, respectively. And they were mainly located in the insoluble fraction of the cell as inclusion bodies and the proportion were about 85% and 90%, respectively. The purity of pBV220-HLA-A2-BSP and beta-2M was above 95% analyzed by SDS-PAGE, and the concentration of pBV220-HLA-A2-BSP and beta-2M was about 1.5 g/L and 1.2 g/L, respectively. Using the constructed HLA-A2-peptide tetramer to detect the HBV/HCV-specific CTL, the HBV-specific CD8(+) frequencies were 1.84% and 0.02% - 0.68% of the total CD8(+) T cells in acute and chronic HBV hepatitis, respectively. As an additional control, an HLA-A2/HCV tetramer was tested in the acute and chronic HBV hepatitis. The frequencies never exceeded 0.02% of the total CD8(+) T cell number. Similar low levels of background staining were also detected in the HLA-A2(+) or A2(-) healthy control. The HCV-specific CD8(+) frequency was 0.02 - 0.72% of the total CD8(+) T cells in chronic HCV hepatitis. The same frequencies of control were detected. CONCLUSION: High-efficient expressions of HLA-A0201-BSP and beta-2m proteins lay a good foundation for further expression and purification in prokaryotic system and constructing MHC class I-peptide tetramer complexes to study the function of CTLs. Especially, using these two HBV and HCV-specific tetramer can detect the frequencies of the HBV/HCV-specific CD8(+) T cells directly in vitro.