The role of microparticles in the generation of immune complexes in murine lupus

Systemic lupus erythematosus is a systemic inflammatory disease characterized by antibodies to nuclear molecules in association with immune complex deposition. As shown previously, microparticles (MPs), which are small membrane-bound vesicles released from dying and activated cells, contain nucleic acids and can form immune complexes found in patient blood. To assess the role of MPs in murine lupus, we used flow cytometry to measure the presence of MPs with bound IgG in the blood of MRL-lpr/lpr and NZB/W mice. These studies showed much higher numbers of MPs with bound IgG in the blood of MRL lpr/lpr compared to NZB/W mice. Furthermore, these studies showed that antibodies from MRL-lpr/lpr mice bound better to MPs from apoptotic cells than those from NZB/W mice. Together, these studies indicate important differences in the serological features of the two strains as reflected by the capacity of antibodies to bind to MPs.     

Hypogammaglobulinaemia occurs in Fas-deficient MRL-lpr mice following deletion of MHC class II molecules

Fas (CD95)-mediated apoptosis in B and T cells is deficient in both human autoimmune lymphoproliferative syndrome and in MRL-lpr mice, a model for systemic lupus erythematosis (SLE). Autoimmune disease in these mice is associated with polyclonal B cell activation, increased serum immunoglobulin and autoantibodies. In non-autoimmune mice MHC class II is not required for normal serum immunoglobulin expression, and previously we have shown using MHC class II-deficient MRL-lpr mice (MRL-lpr Ab−/−) that generation of specific antibodies to DNA requires MHC class II-directed T cell help. In contrast, in the present study we demonstrate that MRL-lpr Ab−/− mice also have a profound reduction of total serum immunoglobulin levels, suggesting abnormal polyclonal regulation of B cells by MHC class II-directed T cells occurs in the autoimmune MRL-lpr strain. This abrogation of immunoglobulin production does not occur in MHC class II-deficient non-obese diabetic (NOD) mice, nor in MHC class I-deficient NOD or MRL-lpr mice. Reduced immunoglobulin levels in MRL-lpr Ab−/− mice were not due to a lack of B cells or to an increased loss of circulating immunoglobulin, but were associated with reduced numbers of surface IgG-positive B cells. These results define a general abnormal regulation of B cells in MRL-lpr mice through a process requiring MHC class II, and suggest that Fas deficiency may allow expansion of totally T-dependent B cells.     

Up-regulation of CC chemokine receptor 6 on tonsillar T cells and its induction by in vitro stimulation with α-streptococci in patients with pustulosis palmaris et plantaris

Pustulosis palmaris et plantaris (PPP) is a tonsil-related disease; tonsillectomy is somewhat effective in treating the condition. However, the aetiological association between the tonsils and PPP has not yet been elucidated fully. Recently, some chemokines and chemokine receptors, including CC chemokine receptor (CCR) 4, CCR6 and CX chemokine receptor (CXCR) 3, have been reported to play important roles in the development of psoriasis, a disease related closely to PPP. In this study, we found that CCR6 expression on both tonsillar and peripheral blood T cells was up-regulated more intensively in PPP patients than in non-PPP patients (P < 0·001 for both), but CCR4 and CXCR3 expressions were not. In vitro stimulation with α-streptococcal antigen enhanced CCR6 expression significantly on tonsillar T cells in PPP patients (P < 0·05), but this was not observed in non-PPP patients. The chemotactic response of tonsillar T cells to the CCR6 ligand CC chemokine ligand (CCL) 20 was significantly higher in PPP patients than in non-PPP patients (P < 0·05). The percentage of CCR6-positive peripheral blood T cells decreased after tonsillectomy in PPP patients (P < 0·01); this decrease correlated with an improvement of skin lesions (P < 0·05, r = −0·63). The numbers of CCR6-positive cells and the expression of CCL20 were increased significantly in pathological lesions compared with non-pathological lesions in PPP skin (P < 0·01, P < 0·05 respectively). These results suggest that a novel immune response to α-streptococci may enhance CCR6 expression on T cells in tonsils and that CCR6-positive T cells may move to peripheral blood circulation, resulting in recruitment to target skin lesions expressing CCL20 in PPP patients. This may be one of the key roles in pathogenesis of the tonsil-related disease PPP.     

Self-Reactivity and the Expression of Memory Markers Vary Independently in MRL-Mp+/+ and MRL-Mp-lpr/lpr Mice

MRL-Mp-lpr/lpr mice contain phenotypically abnormal populations of T cells, and exhibit an SLE-like autoimmune disease in which autoantibodies are a prominent feature. We analyzed the phenotype and T-cell receptor Vß expression pattern in CD4⁺ T cells of this mutant mouse strain to detect abnormalities that could explain the autoimmunity. The CD4⁺ T cells contain two distinct abnormal populations. One of these expresses B220 and HSA, and in these and other respects closely resembles the accumulating CD4^–CD8^– population. The other expresses a high level of CD44 (Pgp-1), and a high level of the 16A epitope of CD45, and so resembles post-activation T cells. Both of these cell types are exclusive to MRL-Mp-lpr/lpr. We also identified V ß5- and V ß11-positive CD4+ T cells, in both MRL-Mp-lpr/lpr and MRL-Mp-+/+ mice. We conclude that autoimmune T cells can be detected in these mice, but that they are not the cause of the accumulation of abnormal CD4⁺ and CD4^–CD8^–cells.     

Heat shock protein 90 inhibition by 17-DMAG lessens disease in the MRL/lpr mouse model of systemic lupus erythematosus

Elevated expression of heat shock protein 90 (HSP90) has been found in kidneys and serum of systemic lupus erythematosus (SLE) patients and MRL/Mp-Fas^lpr/Fas^lpr (MRL/lpr) autoimmune mice. We investigated if inhibition of HSP90 would reduce disease in MRL/lpr mice. In vitro, pretreatment of mesangial cells with HSP90 inhibitor Geldanamycin prior to immune-stimulation showed reduced expression of IL-6, IL-12 and NO. In vivo, we found HSP90 expression was elevated in MRL/lpr kidneys when compared to C57BL/6 mice and MRL/lpr mice treated with HSP90 inhibitor 17-DMAG. MRL/lpr mice treated with 17-DMAG showed decreased proteinuria and reduced serum anti-dsDNA antibody production. Glomerulonephritis and glomerular IgG and C3 were not significantly affected by administration of 17-DMAG in MRL/lpr. 17-DMAG increased CD8⁺ T cells, reduced double-negative T cells, decreased the CD4/CD8 ratio and reduced follicular B cells. These studies suggest that HSP90 may play a role in regulating T-cell differentiation and activation and that HSP90 inhibition may reduce inflammation in lupus.     

Abnormal Complement-Mediated Immune Clearance in MRL-lpr/+ Heterozygote Mice: Dose-Dependent Effect of theFasAntigen Mutation

We have previously demonstrated decreased complement-mediated clearance of IgG-opsonized erythrocytes in mice homozygous for thelprmutation of thefasgene (BALB/c-lpr/lpr,C57BL/6-lpr/lpr,and MRL-lpr/lpr). To further test the hypothesis that thelprmutation leads to a series of events resulting in selectively decreased complement-mediated immune clearance,in vivoclearance rate data were obtained from MRL-lpr/+ F₁, F₂, and reciprocal backcross mice. Southern analysis of genomic DNA extracted from F₂and backcross mice was used to correlate the presence of the normalfasgene or thelprmutation with normal or decreased complement-mediated clearance, respectively. Mean clearance rate constants for complement-dependent sequestration and phagocytosis were significantly decreased in the group of F₁, F₂, and backcross mice compared to control BALB/c mice (P< 0.0001). Data correlating clearance rate parameters from F₂and backcross mice with their respective genotype demonstrated a dose effect of thelprmutation on abnormal complement-dependent sequestration and phagocytosis (r> 0.98), with heterozygote mice expressing mean values approximately half of those observed in +/+ homozygotes. These data demonstrate that the presence of thelprmutation of thefasgene is strongly correlated in a dose-dependent manner with abnormal complement-mediated immune clearance. This clearance defect may be one mechanism through which thelprmutation acts as an enhancer of autoimmune disease. Reduced clearance of immune complexes would increase the likelihood of tissue deposition and immune-mediated damage.     

An MRL/MpJ-lpr/lpr substrain with a limited expansion of lpr double-negative T cells and a reduced autoimmune syndrome

The autosomal recessive mutant gene, lpr, has been shown toaccelerate the progression of lupus-like autoimmune disease,which is associated with a massive expansion of a unique CD4^–CD8^–double-negative T cell subset, in MRL/MpJ mice. Here we reporta substrain of MRL/MpJ-lpr/lpr (MRL-lpr) mice which live almosttwice as long with delayed development of glomerulonephritis,compared with conventional MRL-lpr mice. This substrain, termedMRL-lpr.II (II for long-lived), develops generalized lymphadenopathycharacteristically seen in MRL-lpr mice. However, the expansionof a double negative lpr T cell subset is markedly limited witha mean value of 15% in their lymph nodes compared to about 70%in conventional MRL-lpr mice. Overall production of autoantibodies,such as anti-DNA and rheumatoid factors, does not significantlydiffer between the two MRL-lpr mice. However, serum levels ofcryoglobulins, whose major component is lgG3, are markedly diminishedin MRL-lpr.ll mice with a parallel decrease in lgG3. Since MRL-lpr.llmice still carry the lpr mutation, as documented by the presenceof defects in the Fas antigen, a possible new mutation in thissubstrain may play a significant role in the pathogenesls oflupus-like autoimmune syndrome.     

The basis of autoimmunity in MRL-lpr/lpr mice: a role for self Ia-reactive T cells

Murine models of systemic lupus eRythematosus (SLE) have significantly contributed to our understanding of human autoimmunity. One such strain, the MRL-lpr/lpr, spontaneously develops an autoimmune disease manifested clinically by arthritis, vasculitis, immune-complex glomerulonephritis and autoantibody production^1–3. In this article Yvonne Rosenberg and her colleagues suggest a theoretical basis for the development of autoimmunity in MRL-lpr/lpr mice.     

Bead-size directed distribution of Pseudomonas aeruginosa results in distinct inflammatory response in a mouse model of chronic lung infection

Chronic Pseudomonas aeruginosa lung infection in cystic fibrosis (CF) patients is characterized by biofilms, tolerant to antibiotics and host responses. Instead, immune responses contribute to the tissue damage. However, this may depend on localization of infection in the upper conductive or in the peripheral respiratory zone. To study this we produced two distinct sizes of small alginate beads (SB) and large beads (LB) containing P. aeruginosa. In total, 175 BALB/c mice were infected with either SB or LB. At day 1 the quantitative bacteriology was higher in the SB group compared to the LB group (P < 0·003). For all time-points smaller biofilms were identified by Alcian blue staining in the SB group (P < 0·003). Similarly, the area of the airways in which biofilms were identified were smaller (P < 0·0001). A shift from exclusively endobronchial to both parenchymal and endobronchial localization of inflammation from day 1 to days 2/3 (P < 0·05), as well as a faster resolution of inflammation at days 5/6, was observed in the SB group (P < 0·03). Finally, both the polymorphonuclear neutrophil leucocyte (PMN) mobilizer granulocyte colony-stimulating factor (G-CSF) and chemoattractant macrophage inflammatory protein-2 (MIP-2) were increased at day 1 in the SB group (P < 0·0001). In conclusion, we have established a model enabling studies of host responses in different pulmonary zones. An effective recognition of and a more pronounced host response to infection in the peripheral zones, indicating that increased lung damage was demonstrated. Therefore, treatment of the chronic P. aeruginosa lung infection should be directed primarily at the peripheral lung zone by combined intravenous and inhalation antibiotic treatment.     

Blood monocyte activation in rheumatoid arthritis: increased monocyte adhesiveness, integrin expression, and cytokine release

Infiltration of the synovium by mononuclear cells, namely lymphocytes and monocytes, is one of the main features of rheumatoid arthritis (RA) and is considered to be responsible for the development of the disease. In this study in 31 consecutive patients with RA, we investigated whether peripheral blood monocytes exhibited markers of cellular activation related to cell migration. Using flow cytometry with the respective specific antibodies, we studied the expression of integrins CD11a, CD11b, CD11c, CD49d (VLA-4), and CD49e (VLA-5) on monocytes from patients with RA and from normal (N) subjects. IL-1β, IL-6, and tumour necrosis factor-alpha (TNF-α) production by cultured monocytes was measured by immunoassay. Adhesiveness of monocytes was studied on various surfaces (plastic, human fibronectin, gelatin-coated plasma, subendothelial matrix) and on cultured endothelial cells under basal conditions or after stimulation by IL-1β. An increased number of CD14⁺ monocytes (Mo) from RA patients expressed the CD11b molecule (RA Mo = 90·3%, N Mo = 83·4%, P < 0·005). The expression of CD11b on CD14⁺ monocytes was significantly increased in RA patients (median fluorescence intensity (FI): RA Mo = 145 (range 80–466) units; normal Mo = 95 (range 24–164) units; P < 0·003). Production of extracellular IL-1β and IL-6 by RA monocytes was significantly enhanced compared with monocytes from normal subjects (IL-1β: RA = 2·65 ± 0·91 ng/ml versusN = 1·35 ± 0·85 pg/ml, P < 0·05; IL-6: RA = 4·83 ± 0·90 ng/ml versusN = 2·40 ± 0·95 ng/ml, P < 0·05). Compared with normal monocytes, RA monocytes exhibited increased adhesion to the various surfaces studied (plastic, P < 0·01; fibronectin, P < 0·01; and gelatin-coated normal or RA plasma, P < 0·01) as well as to unstimulated (P < 0·01) and IL-1β-stimulated endothelial cells (IL-1β for 4 h, P < 0·05; IL-1β for 24 h, P < 0·05). In our study, blood monocytes from RA patients exhibited features of activation related to cell adhesion.     

Vaccination with autoreactive CD4Th1 clones in lupus-prone MRL/Mp-Fas mice

We studied the efficacy of T cell vaccination with CD4⁺αβTh1 clones in lupus-prone MRL/Mp-Fas^lpr/lpr (MRL/lpr) mice. CD4⁺αβ Th1 clones, dna51 (Vβ8.3) and rnp2 (Vβ14), which stimulated anti-dsDNA or U1 ribonucleoprotein (U1RNP) antibody (Ab) production respectively, were isolated from splenocytes of MRL/lpr mice. Antinuclear Ab kinetics, renal function, renal histology, survival rate, and lymphocyte subpopulations in the spleen were monitored after intravenous adoptive transfer of IL-2-stimulated (s-) or irradiated (i-) clones to 3 week-old female MRL/lpr mice. Anti-idiotypic humoral and T cell responses against the transferred autoreactive Th1 clones were determined in parallel. Compared with PBS-treated MRL/lpr mice, anti-dsDNA Ab titers, and the activity index for lupus nephritis were all decreased in MRL/lpr mice vaccinated with i-dna51 cells, whereas survival rate was not improved. The numbers of CD4⁺Vβ8.3 T cells in the spleen were also significantly decreased in these mice. Anti-idiotypic Abs recognizing a 12 amino acid sequence of clone dna51 T cell receptor Vβ8.3-complementarity-determining region (CDR) 3 were detected in the MRL/lpr mice that received i-dna51 or s-dna51 cells. These Abs suppressed dna51 cell proliferation, as well as cytotoxicity of CD8⁺T cells against dna51. The present study suggests that vaccination with CD4⁺αβTh1 clone, dna51, elicits anti-idiotypic T cell and humoral responses against dna51 in MRL/lpr mice, although the immunoregulatory effects on lupus may be limited.     

Autoantibodies to the collagenous region of C1q occur in three strains of lupus-prone mice

We have developed an ELISA to measure murine autoantibodies to the collagenous region (CLR) of C1q, using the whole human C1q molecule as the solid-phase ligand, in the presence of 1 m NaCl. The assay was validated by testing positive sera from 20 mice using purified mouse C1q, and from 10 mice using purified human C1q-CLR, as the solid-phase ligands. There were highly significant correlations between results obtained with human C1q (whole molecule) and: (i) mouse C1q (r_Sp = 0.73, P < 0.001), and (ii) human C1q-CLR alone (r_Sp = 0.86, P = 0.001). Antibodies to C1q were measured in 53 MRL/lpr, 17 BXSB and 25 NZB/W lupus-prone mice. Median (range) anti-C1q (CLR) antibody levels in MRL/lpr, BXSB, and NZB/W autoimmune mice aged 3 months were 22 (16–66), 21 (17–39) and 19 (15–27) EU, respectively. The median anti-C1q antibody level in MRL/lpr mice aged 5 months was 76 (35–142) EU, significantly higher than that at 3 months (U = 558, P < 0.0005). Median anti-C1q antibody level in NZB/W mice at 8 months was 37 (13–74) EU and in BXSB mice at 11 months was 62 (31–231) EU, significantly higher than corresponding values at 3 months (U = 326, and U = 4, P < 0.001, respectively). This is the first demonstration of anti-C1q (CLR) antibodies in NZB/W and BXSB mice. The pathologic significance and the potential utility of these antibodies for monitoring disease in lupus-prone mice are under evaluation.     

Significance of IL-1β and IL-1 receptor antagonist (IL-1Ra) in bronchoalveolar lavage fluid (BALF) in patients with diffuse panbronchiolitis (DPB)

We evaluated the effect of erythromycin therapy on pulmonary function tests and the airway inflammatory response of patients with DPB. The number of neutrophils in BALF obtained from DPB patients was significantly higher than that of healthy volunteers. Treatment with erythromycin (600 mg/day for 12·9 ± 9·5 months (mean ±s.d.)) significantly reduced the total number of cells and neutrophils in the airway, and significantly improved pulmonary function tests. The levels of IL-1β and IL-8 were significantly higher in DPB compared with healthy volunteers (P < 0·05, P < 0·05, respectively). IL-1 Ra in patients is considered to have a weak inhibitory activity for IL-1β, with approximately five-fold concentration of IL-1β compared with that in healthy volunteers (approx. nine-fold concentration of IL-1β). Erythromycin therapy significantly reduced these cytokines to levels comparable to those of healthy volunteers, and produced a trend toward reduction in the level of IL-1Ra in BALF. The level of IL-1β correlated significantly with the concentration of neutrophils in BALF (r = 0·72, P < 0·01), as well as with the level of IL-1Ra (r = 0·688, P < 0·05) and IL-8 (r = 0·653, P < 0·05). A nearly significant or significant correlation was observed between the concentration of neutrophils and levels of IL-1Ra or IL-8 in BALF (r = 0·526, P = 0·053 or r = 0·776, P < 0·01, respectively). There was also a significant relationship between FEV, and the concentration of neutrophils in BALF (r = 0·524, P < 0·05). Our results suggest that the relative amounts of IL-1β and IL-1Ra or IL-8 may contribute, at least in part, to the neutrophil-mediated chronic airway inflammation in patients with chronic airway disease, and long-term erythromycin therapy may down-regulate the vigorous cycle between the cytokine network and neutrophil accumulation, with resultant reduction of neutrophil-mediated inflammatory response.     

C1q rs292001 polymorphism and C1q antibodies in juvenile lupus and their relation to lupus nephritis

C1q deficiency is related strongly to systemic lupus erythematosus (SLE), but very few and inconsistent studies explored the single nucleotide polymorphisms of the C1q gene in relation to juvenile SLE (jSLE) and lupus nephritis (LN). The objective of this study was to analyse whether C1q rs 292001 polymorphism is associated with SLE and disease phenotype, especially nephritis, and to investigate the relation between this polymorphism and clinical data, treatment outcome, serum level of C1q protein and antibodies. Typing of C1q rs292001 polymorphism using restriction fragment length polymorphism and measuring serum levels of C1q protein and antibodies by enzyme-linked immunosorbent assay (ELISA) were performed for 130 children with SLE and 208 healthy controls. The A allele of C1q rs292001 was associated with jSLE and LN (P = 0·005 and 0·013, respectively) and the AA genotype was associated with jSLE (P = 0·036). Low serum levels of C1q protein were found in jSLE and LN (P < 0·001 and 0·009, respectively), and these levels were increased after treatment in patients with LN (P = 0·009) and active renal disease (P = 0·027). Higher titres of C1q antibodies were found in patients with LN (P = 0·015) and correlated negatively with C1q protein level (P < 0·001) and patient age (P = 0·04). The A allele and AA genotype of C1q rs292001 can be considered a susceptibility risk factor and the GG genotype could be considered protective for jSLE and LN in the studied cohort of Egyptian children. Decreased serum levels of C1q protein and increased titres of C1q antibodies may be involved in the pathogenesis of jSLE, especially LN.     

Enhanced natural but diminished antibody-mediated cytotoxicity in the lungs of MRLlpr/lpr mice

Human systemic lupus erythematosus (SLE) patients, as well as MRLlpr/lpr mice which develop a SLE-like disease, have decreased numbers and functional activity of systemic natural killer (NK) cells. In contrast, it has been found that among lymphocytes recovered from the bronchoalveolar lavage fluid of SLE patients, NK cells were increased in number, correlating with the severity of the lung engagement. The present study was undertaken to assay the capacity for natural killing in the lung compartment of MRLlpr/lpr mice compared with healthy congenic MRL +/+ and heterozygous MRL +/lpr mice. ⁵¹Cr-labelled YAC-1 cells were injected intravenously to settle in the lungs where they were targeted for lysis by NK cells. YAC-1 cell killing inversely correlated with radioactivity remaining in the lungs after the assay, and was inhibited by antibody to the asialo-GM1 antigen expressed on NK cells. To analyse the capacity in the lung for cytolysis of non-NK cell-sensitive target cells, a similar in vivo⁵¹Cr-release assay was set up for antibody-mediated allospecific cytotoxicity. We demonstrate that MRLlpr/lpr mice throughout their lifespan display significantly increased natural cytotoxic activity in the lungs compared with MRL +/+ and MRL +/lpr mice, as demonstrated by more efficient killing of YAC-1 cells. In contrast, antibody-mediated allospecific cytotoxicity in the lungs was significantly less effective in the MRLlpr/lpr strain.     

Effect of glucagon on insulin secretion through cAMP signaling pathway in MIN6 cells

Objective: To explore the direct regulation effects and mechanisms of glucagon in insulin secretion of MIN6 cells that in the kind of the islet β cells. Methods ICUE3 and PCDNA3.1 plasmid were transfected to the MIN6 cells by electroporation transfection, and then treated with different concentrations of glucagon (Glg) and glucose (Glu). Biosensor technology that based on the fluorescence resonance energy transfer (FRET) was used to monitor the change of cAMP quantitatively and real-time. The level of cAMP and insulin were measured by the enzyme-linked immunosorbent assay (ELISA). Results: The receptor of Glg was mainly located on the cell membrane in MIN6 cells. Compared with the 0 ng/L Glg group in the Glu-free state, the average value of CFP/YFP increased 4% ± 0.02 in the 500 ng/L Glg group, and the value in the 1000 ng/L Glg group increased 6% ± 0.03 (P > 0.05). While in the high-Glu (16.7 mmol/L) state, the value increased 11% ± 0.02 in the 500 ng/L Glg group, and increased 23% ± 0.06 in the 1000 ng/L Glg group when compared with the 0 ng/L Glg group(P < 0.01). The levels of the cAMP of 1000 ng/L and 500 ng/L Glg group were higher than those of the 100 ng/L and 0 ng/L Glg group in the condition of Glu-free (81.27±6.29, 76.73±2.10,39.45±2.83, 40.36±4.20; P < 0.01). The levels of the cAMP of 1000 ng/L, 500 ng/L and 100 ng/L Glg group were higher than those of the 0 ng/L Glg group, at the meanwhile, the levels of the cAMP of 1000 ng/L and 500 ng/L Glg group were also higher than 100 ng/L Glg group in the condition of low-Glu (2.8 mmol/L) (92.91±7.35, 90.36±3.15, 65.82±10.49, 46.73±1.05; P < 0.01). And this trend in the condition of high-Glu was almost to the low-Glu (106.75±7.26, 94.18±2.99, 83.09±1.16, 55.60±5.51, P < 0.01). The levels of the insulin of 1000 ng/L, 500 ng/L and 100 ng/L Glg group were higher than those of the 0 ng/L Glg group. While 1000 ng/L Glg group was higher than that of the 500 ng/L and 100 ng/L Glg group in the condition of Glu-free (1844.02±200.93, 1387.94±483.12, 1251.817±60.30, 787.33±81.72; P < 0.01). The levels of the insulin of 1000 ng/L and 500 ng/L Glg group were higher than those of the 100 ng/L and 0 ng/L Glg group, and the 1000 ng/L and was also higher than 500 ng/L Glg group in the condition of low-Glu (1552.31±81.20, 1285.62±131.67, 1020.85±42.60, 762.89±26.94, P < 0.01). And this trend in the condition of high-Glu was almost to the low-Glu (1898.337±169.03, 1399.30±148.66, 1061.735±9.13, 972.89±22.19; P < 0.01). The levels of cAMP and insulin secretion of MIN6 cells had a positive correlation in different Glu conditions (r² = 0.559, P < 0.01). Conclusion: Glg may stimulate insulin secretion by increasing cAMP levels in the way of concentration gradient within the islet β cell lines--MIN6 cells. And the increasing trend was Glu dependent.     

Cytolytic mechanisms and T-cell receptor Vβ usage by ex vivo generated Epstein–Barr virus-specific cytotoxic T lymphocytes

Ex-vivo-generated Epstein–Barr virus (EBV)-specific cytotoxic T lymphocytes (CTL) have been used for cellular adoptive immunotherapy of EBV-associated lymphomas. Here we investigated the phenotypes, cytolytic mechanisms, polyfunctionality and T-cell receptor (TCR) usage in growing and established CTL, generated by weekly stimulation with an EBV-transformed autologous lymphoblastoid cell line (LCL). Our results showed that phenotypically mature CTL developed within the first 4 weeks of culture, with an increase in CD45RO and CD69, and a decrease in CD45RA, CD62L, CD27 and CD28 expression. Spectratyping analysis of the variable β-chain of the TCR revealed that TCR repertoire remained diverse during the course of culture. Cytotoxicity of CTL was significantly inhibited by concanamycin A (P < 0·0001) and ethylene glycol-bis tetraacetic acid (P < 0·0001), indicating that a calcium and perforin-mediated exocytosis pathway with the release of granzyme B was the principal cytotoxic mechanism. The CTL mainly produced interferon-γ (IFN-γ) or tumour necrosis factor-α (TNF-α) upon restimulation with autologous LCL, although there were some polyfunctional cells producing IFN-γ and TNF-α. Granzyme B, perforin and Fas ligand were detected in CD8⁺ and CD4⁺ cells in all CTL; however, a greater proportion of CD8⁺ than CD4⁺ T cells expressed granzyme B (P < 0·0001) and more granzyme B was detected in CD8⁺ T cells than in CD4⁺ T cells (P = 0·001). This difference was not observed with Fas ligand or perforin expression. Our results provide insight into the basic characteristics of ex-vivo-generated CTL.