Anti-double-stranded DNA antibodies in the healthy elderly: Prevalence and characteristics

UsingCrithidia luciliae fluorescent assay a significant prevalence (7.6%;P<0.006) of anti-double-stranded DNA antibodies was found in a healthy old population. A negative enzyme-linked immunosorbent assay for anti-total histone antibodies excluded a false-positive reaction. Anti-double-stranded DNA antibodies in the aged differed from those found in patients with systemic lupus erythematosus and were characterized by a low titer (95.6% of cases), belonging to the IgA class alone (95.6%), no complement-fixing ability (100%), and negativity to Farr assay (100%). It is concluded that, in elderly subjects without signs and symptoms of disease, including systemic lupus erythematosus, such a peculiar anti-double-stranded DNA antibody may be detected.     

Fibulin-5 Contributes to Microfibril Assembly in Human Periodontal Ligament Cells

The elastic system fibers comprise oxytalan, elaunin and elastic fibers, which differ in their relative microfibril and elastin content. Human periodontal ligaments (PDL) contain only oxytalan fibers (pure microfibrils) among them. Since fibulin-5 regulates the organization of elastic fibers to link the fibers to cells, we hypothesized that fibulin-5 may contribute to the formation of oxytalan fibers. We used siRNA for fibulin-5 in PDL cell culture to examine the extracellular deposition of fibrillin-1 and -2, which are the major components of microfibrils. Fibulin-5 was labeled on microfibrils positive for fibrillin-1 and -2. Fibulin-5 suppression reduced the level of fibrillin-1 and -2 deposition to 60% of the control level. These results suggest that fibulin-5 may control the formation of oxytalan fibers, and play a role in the homeostasis of oxytalan fibers.     

DNA聚合酶Pol(t)在紫外损伤诱导基因组高突变中的作用

目的 研究DNA聚合酶Polt在紫外损伤耐受及基因组高突变产生中的生物学作用.方法 紫外线照射稳定高表达DNA聚合酶iota(DNA polymerase iota,Pol(t))的HEK293T细胞系(Pol(t)-HEK293T),MTT法检测紫外损伤后细胞的存活率;supF报告基因体内突变检测系统检测基因的突变频率.结果 MTT法结果提示紫外照射后稳定高表达Pol(t)的细胞系与对照组相比细胞存活率没有显著差异;supF报告基因结果显示稳定高表达Pol(t)的细胞系其基因突变频率高于对照组细胞.结论 DNA聚合酶Pol(t)的高表达对紫外损伤后细胞的存活率没有显著影响,不能提高细胞对紫外损伤的耐受,在紫外损伤中pol(t)主要参与诱发体内基因组高突变的产生.     

Interleukin-1 Contributes to High Level IgG Production in the Murine MRL/Ipr Lupus Model

The autoimmune syndrome in MRL/lpr mice resembles human lupus, both in its serologic and immunopathologic characteristics. The contribution of IL-1 to high-level Ig production in the MRL/lpr model is poorly understood. We investigated the effect of treating B-cell-enriched, or, B plus T cell suspensions derived from either pre-disease or diseased lupus-prone MRL/lpr mice with IL-1ß or IL-1 receptor antagonist (IL-1Ra). Disparate patterns of IgG production by B cells and B plus T cells derived from diseased versus pre-diseased MRL/lpr mice was observed following treatment with IL-1ß. Remarkably, IL-1ß caused significant suppression in IgG production by B cells derived from diseased MRL/lpr mice as compared to B cells derived from pre-disease mice. In mix-and-match experiments with B plus T cells from pre-disease and diseased MRL/lpr mice, both T cell help and B cell hyperactivity, originating in diseased MRL/lpr mice were found to be important factors in high-level IgG production in diseased MRL/lpr mice. Furthermore, IL-1Ra treatment of B plus T cell co-cultures derived from diseased MRL/lpr mice was able to significantly suppress IgG production, whereas, IL-1Ra treatment of B plus T cell co-cultures derived from pre-disease MRL/lpr mice demonstrated virtually no suppression in IgG production. Collectively, these results indicate a potentially important but complex role for IL-1 in influencing high-level IgG production in MRL/lpr mice with established disease.     

PDGF Receptor Alpha+ Mesoderm Contributes to Endothelial and Hematopoietic Cells in Mice

Background: Early mesoderm can be classified into Flk‐1+ or PDGF receptor alpha (PDGFRα)+ population, grossly representing lateral and paraxial mesoderm, respectively. It has been demonstrated that all endothelial (EC) and hematopoietic (HPC) cells are derived from Flk‐1+ cells. Although PDGFRα+ cells give rise to ECs/HPCs in in vitro ES differentiation, whether PDGFRα+ population can become hemato‐endothelial lineages has not been proved in mouse embryos. Results: Using PDGFRαMerCreMer mice, PDGFRα+ early mesoderm was shown to contribute to endothelial cells including hemogenic ECs, fetal liver B lymphocytes, and Lin‐Kit+Sca‐1+ (KSL) cells. Contribution of PDGFRα+ mesoderm into ECs and HPCs was limited until E8.5, indicating that PDGFRα+/Flk‐1+ population that exists until E8.5 may be the source for hemato‐endothelial lineages from PDGFRα+ population. The functional significance of PDGFRα+ mesoderm in vascular development and hematopoiesis was confirmed by genetic deletion of Etv2 or restoration of Runx1 in PDGFRα+ cells. Etv2 deletion and Runx1 restoration in PDGFRα+ cells resulted in abnormal vascular remodeling and rescue of fetal liver CD45+ and Lin‐Kit+Sca‐1+ (KSL) cells, respectively. Conclusions: Endothelial and hematopoietic cells can be derived from PDGFRα+ early mesoderm in mice. PDGFRα+ mesoderm is functionally significant in vascular development and hematopoiesis from phenotype analysis of genetically modified embryos. Developmental Dynamics 242:254–268, 2013. © 2013 Wiley Periodicals, Inc.     

Demonstration of Actomyosin in Mesangial Cells of the Renal Glomerulus

Antisera to human uterine actomyosin were prepared in rabbits and conjugated with fluorescein (F-AUAM). When F-AUAM was applied to frozen sections of normal human kidney which were then examined by ultraviolet light microscopy, it was observed that vascular smooth muscle, endothelium of arteries, veins, and peritubular capillaries and glomerular mesangial cells were immunofluorescent. Neither glomerular endothelium nor epithelial cells of Bowman's capsule or renal tubules were stained by F-AUAM. The specificity of antisera for actomyosin was confirmed by absorption and blocking studies, examination of a wide variety of tissues and immunodiffusion in agarose gel. It may be inferred from these data that mesangial cells are contractile. Contraction of the mesangium may play a significant role in regulating glomerular blood flow and in the reaction of the glomerulus to injury.     

细胞因子对肾小球系膜细胞合成PAF的影响

本文首先建立了血小板活化因子（ＰＡＦ）的生物活性检测法，并通过体外细胞培养技术，测定了肾小球系膜细胞在经ＬＰＳ、ＩＬ-１β、ＩＬ-６和ＴＮＦα的短暂刺激后，其培养上清中ＰＡＦ的活性。结果发现，它们均能诱导系膜细胞合成和分泌ＰＡＦ，ＩＬ-１β、ＩＬ-６和ＴＮＦα的诱生作用并呈一定的剂量依赖关系。实验提示上述因子在肾小球免疫病理损伤中，除直接作用外，也可通过诱生ＰＡＦ而间接发挥作用。应用ＰＡＦ拮抗剂来阻断这一途径，可能具有一定的临床应用价值。     

依维莫司对高糖诱导肾小球系膜细胞凋亡的保护作用

目的:观察依维莫司对高糖诱导肾小球系膜细胞(HBZY-1)凋亡的保护作用,并探讨其意义。方法:将培养HBZY-1细胞按不同葡萄糖和依维莫司浓度分组处理72h后,用磺酰罗丹明B法测量细胞OD值,计算各组细胞存活率,用Annexin V和PI双标法检测各组细胞凋亡率。结果:30mM葡萄糖处理的细胞存活率明显降低,凋亡率明显增加(均P<0.05)。不同浓度依维莫司均能提高细胞存活率(均P<0.05),其中以200ng/ml依维莫司效果最好(P<0.05);200ng/ml依维莫司具有明显抗HBZY-1细胞凋亡作用。结论:依维莫司对高糖诱导HBZY-1细胞凋亡具有保护作用,从而为其治疗糖尿病肾病提供新的研究方向。     

The Gene Locus yijP Contributes to Escherichia coli K1 Invasion of Brain Microvascular Endothelial Cells

Most cases of Escherichia coli meningitis develop as a result of hematogenous spread, but it is not clear how circulating E. coli crosses the blood-brain barrier. A TnphoA mutant of E. coli K1 RS218 was shown to be significantly less invasive than its parent strain in bovine and human brain microvascular endothelial cells (BMEC), which constitute the blood-brain barrier. More importantly, traversal of the blood-brain barrier was significantly less with this mutant than with the parent strain in newborn rats with experimental hematogenous meningitis. A DNA segment containing the TnphoA insertion site was cloned from RS218, and the cloned DNA complemented the TnphoA mutant in invasion of BMEC. Nucleotide sequence revealed a near identity to that of a hypothetical yijP gene (also called f577) in the E. coli K-12 genome. Sequence analysis indicated that the E. coli K1 yijP gene likely encodes a 66. 6-kDa membrane protein. Deletion and complementation experiments indicated that the yijP gene was involved in E. coli K1 invasion of BMEC, i.e., the invasive ability of E. coli K1 was significantly reduced after yijP was deleted and was restored by complementation with a plasmid containing the yijP open reading frame. This is the first demonstration that the yijP gene locus plays a role in the pathogenesis of E. coli K1 meningitis.     

Opacity-Associated Protein A Contributes to the Binding of Haemophilus influenzae to Chang Epithelial Cells

Opacity-associated protein A (OapA), which is responsible for the transparent-colony phenotype of Haemophilus influenzae, has been implicated in the colonization of the nasopharynx in an infant rat model of carriage. In this report, we show that OapA mediates attachment to Chang epithelial cells examined by using genetically defined type b and nontypeable H. influenzae strains with or without OapA. We also showed that OapA was conserved among H. influenzae strains by comparing deduced amino acid sequences. Both recombinant OapA and polyclonal anti-OapA antiserum blocked the binding of H. influenzae to Chang epithelial cells, suggesting that the interaction of H. influenzae is specific to OapA. Moreover, the binding of recombinant OapA to epithelial cells further provided evidence that OapA can promote attachment of H. influenzae. Expression of oapA gene in a nonadherent Escherichia coli strain significantly increased the binding to Chang epithelial cells, and disruption of the oapA gene with kanamycin resistance cassette insertion resulted in a significant loss of binding. These findings demonstrate that OapA plays a role in H. influenzae binding to human conjunctival epithelial cells.     

Switch in Chemokine Receptor Phenotype on Memory T Cells without a Change in the Cytokine Phenotype

Th1 and Th2 cells as defined by their cytokine profile are associated with the expression of the chemokine receptors CCR5 and CCR3, respectively. In committed human memory Th1 cells the cytokine profile is irreversibly expressed. However, it is not known if the chemokine receptor phenotypes of Th1 and Th2 cells are permanently associated to the cytokine profile or if it can be changed. To analyze the possibility of inducing a switch in chemokine receptor phenotype on memory Th cells we used differentiated memory Th cells isolated from synovial tissue (ST) samples of patients with rheumatoid arthritis (RA). Freshly isolated T cells, T-cell lines and T-cell clones from these tissues were manipulated with Th1 (interleukin (IL)-12 + anti IL-4) or Th2 (IL-4 + anti IL-12) inducing conditions. The surface expression of CCR5 and CCR3 was analyzed by flowcytometry and interferon (IFN)-gamma and IL-4 production by ELISA. A Th1-inducing cytokine environment increased the expression of CCR5 in Th1 cells and induced the expression of CCR5 in Th2 cells as compared to culture condition with only IL-2. Induction of CCR5 expression on Th2 clones was associated with secretion of some IFN-gamma. Moreover, the Th2-associated chemokine receptor CCR3 could be expressed on both Th1-dominant cell lines, and clones of Th1 and Th0 type after culture conditions with IL-4. This expression of CCR3 was associated with a reduced IFN-gamma production, but no IL-4 production could be induced. The IL-4-treated Th1 clones had a reduced migratory capacity against chemokines produced by ST cells compared to nonmanipulated T-cell clones. In contrast, the same IL-12-treated Th1 clones showed an increased migratory potential. Induction of the Th2-associated marker CCR3 on memory Th1 cells demonstrates that a change in chemokine receptor phenotype related to the Th2 type can be induced on terminally differentiated Th1 cells, without a change in the cytokine profile.     

IgG4 autoantibodies to DNA in systemic lupus erythematosus patients

The presence of DNA-specific IgG4 antibodies was demonstrated in the sera of patients with systemic lupus erythematosus (SLE) by a microtiter solid-phase radioimmunoassay. A patient with distal interphalangeal swelling and extensive ulcers in the oral cavity, seronegative for anti-DNA antibodies of the IgG isotype, was found to have anti-DNA autoantibodies exclusively of the IgG4 subclass. These autoantibodies directed against the dsDNA conformation cross-reacted with chondroitin sulfate, dermatan sulfate and heparin.     

IL-13抑制人肾小球系膜细胞IL-12的表达

为了探讨白细胞介素 13(IL 13)对体外培养的人肾小球系膜细胞产生白细胞介素 12 (IL 12 )的影响。我们用脂多糖(LPS 10 μg/ml) ,不同浓度的IL 13对系膜细胞培养 ,分别采用ELISA法和半定量RT PCR法检测细胞上清液的IL 12和系膜细胞IL 12p4 0mRNA表达。结果提示 5 %NCSRPMI 16 4 0基础培养条件下的系膜细胞未检测到IL 12蛋白分泌及其mRNA表达。在LPS刺激下系膜细胞的IL 12p4 0mRNA表达加强 ,并分泌出大量的IL 12。IL 13在 1～ 10 0ng/ml浓度范围内对LPS诱导的系膜细胞IL 12分泌及IL 12p4 0mRNA表达的抑制作用呈剂量依赖趋势。本研究认为IL 13可能通过抑制IL 12的产生 ,而调整了体内Th1/Th2细胞因子平衡 ,作为抗炎性细胞因子在肾小球肾炎发病机制中发挥一定作用     

The CD40-CD40L Pathway Contributes to the Proinflammatory Function of Intestinal Epithelial Cells in Inflammatory Bowel Disease

In inflammatory bowel diseases (IBD), intestinal epithelial cells (IECs) are involved in the outbalanced immune responses toward luminal antigens. However, the signals responsible for this proinflammatory capacity of IECs in IBD remain unclear. The CD40/CD40L interaction activates various pathways in immune and nonimmune cells related to inflammation and was shown to be critical for the development of IBD. Here we demonstrate CD40 expression within IECs during active IBD. Endoscopically obtained biopsies taken from Crohn’s disease (n = 112) and ulcerative colitis patients (n = 67) consistently showed immunofluorescence staining for CD40 in IECs of inflamed ileal or colonic mucosa. In noninvolved mucosa during active disease, tissue obtained during Crohn’s disease or ulcerative colitis in remission and biopsies from healthy controls (n = 38) IECs almost entirely lacked CD40 staining. Flow cytometry and RT-PCR analysis using different intestinal epithelial cell lines (HT29, SW480, and T84) showed IFN-γ to effectively induce CD40 in IECs. Cells were virtually unresponsive to LPS or whole E. coli regarding CD40 expression. In addition, a moderate induction of CD40 was found in response to TNF-α, which exerted synergistical effects with IFN-γ. CD40 ligation by CD40L-transfected murine fibroblasts or soluble CD40L increased the secretion of IL-8 in IFN-γ pretreated HT29 cells. Our findings provide evidence for the epithelial expression and modulation of CD40 in IBD-affected mucosa and indicate its involvement in the proinflammatory function of IECs.Crohn’s disease (CD) and ulcerative colitis (UC) are common inflammatory disorders of the intestine. Inflammation in these entities of inflammatory bowel disease (IBD) results from a complex interaction of genetic, environmental, microbial, and immune factors primarily driven by defects of the mucosal barrier.^1,2 The secondary loss of tolerance toward microbiota or dietary antigens of the gut lumen leads to an inappropriate and continuous activation of the mucosal immune system.Intestinal epithelial cells (IECs) represent the critical interface between the environment and host. IECs are known to be involved in the regulation of mucosal immune responses to luminal antigens.^3,4 They function as antigen presenting cells (APCs) to different subsets of T cells^3,5,6,7 and moreover play a role in the innate immune response through secretion of a panel of cytokines and chemokines.^8,9,10,11 In this regard IECs substantially contribute to the inflammatory processes in IBD by stimulating effector T cells and the release of interleukin (IL)-1, IL-6, IL-8, or tumor necrosis factor (TNF)-α. However, the signals that drive IECs to function as a proinflammatory mediator during IBD remain incompletely understood.CD40 is a 45- to 50-kDa cell-surface glycoprotein that belongs to the TNF-receptor family and was initially described on B cells.¹² Subsequently, CD40 was identified on various cell types such as professional APCs, endothelial cells, epithelial cells of different organs, and fibroblasts.^13,14 Its ligand, CD40L, is a 39-kDa surface glycoprotein and a member of the TNF gene superfamily. CD40L is mainly expressed on activated CD4⁺ T-helper cells and platelets but could also be found on cytotoxic CD8⁺ T cells.^13,15,16 Produced as a transmembrane protein CD40L may be present as cell surface heteromultimeric complex composed of membrane bound and soluble forms (sCD40L). The interaction between CD40 and CD40L exerts pleiotropic functions.^{13,14,15,16,17} Among these the engagement of CD40 on APCs or epithelial cells is accompanied by the up-regulation of Major Histocompatability Complex (MHC) and costimulatory molecules such as CD80 or CD86. The CD40-mediated stimulation further results in the secretion of a row of cytokines/chemokines such as IL-1, IL-6, IL-8, IL-12, and TNF-α.Studies in animal models demonstrate a crucial role of the CD40-CD40L system in autoimmune diseases such as collagen-induced arthritis, experimental allergic encephalomyelitis, and lupus nephritis.^18,19,20 Remarkably, Stuber et al showed that the CD40-CD40L interaction is also an important element in the pathogenesis of colitis.²¹ Blocking the CD40-CD40L system using anti-CD40L antibodies, authors found a prevention of the TNBS-induced colitis. In addition, CD40L transgenic mice with the highest transgene copy numbers were reported to acquire a lethal bowel inflammation resembling IBD.²² The particular relevance of this system in CD or UC is further emphasized by evidence obtained from IBD patients. CD40, CD40L, and sCD40L were observed to be strongly up-regulated in course of active CD and UC, particularly within the inflamed mucosa.^23,24,25These data suggest that the CD40-CD40L system might be essentially involved in the proinflammatory action of IECs during IBD. Of note, the expression and potential function of CD40 in IECs, especially in the context of IBD, still remain to be elucidated. In this study we provide evidence for the epithelial expression of CD40 in IBD, its modulation, and functional implication in the processes of mucosal inflammation.     

Ultrastructural Phenotype of "Intestinal-Type" Cells in Columnar-Lined Esophagus

An intestinal-type epithelium is often present at columnar-lined esophagus, gastroesophageal junction or within the so-called short segment Barrett's esophagus, but ultrastructural study failed to detect enterocytes in columnar-lined esophagus. The authors have analyzed the intestinal aspects present in areas of columnar-lined esophagus in a population of patients with reflux esophagitis to better understand the morphology and histogenesis of the proliferating elements. Columnar-lined mucosa was studied in 35 patients. Columnar surface cells displayed a wide spectrum of ultrastructural features. Well-differentiated columnar secretory cells, secretory-absorptive cells, poorly differentiated columnar cells, atypical columnar cells, and goblet cells were detected. Well-differentiated absorptive cells were never found. These results demonstrate that the areas of intestinal metaplasia show a wide spectrum of ultrastructural phenotypes, ranging from poorly to well-differentiated cells. However, true enterocytes were not found and the most represented phenotype is that of secretory-absorptive cells, whose principal characteristic is the presence of secretory and absorptive aspects together. They can be described as secretory enterocytes or cells with double specialization. To the authors' knowledge, similar cells were not previously described in normal intestinal mucosa, and ultrastructural studies are consistent in describing a broad spectrum of ultrastructural features, suggesting that Barrett's specialized metaplasia is derived from cells with the capacity for a wide range of differentiation. Therefore, despite the wide use of term intestinal metaplasia in the medical literature, experimental data clearly failed to detect enterocytes in the columnar-lined esophagus, and ultrastructural data do not support the concept of intestinal metaplasia. The cellular heterogeneity seems to be the result of a "phenotypic shift" of undifferentiated elements, which show a different pattern of evolution. The result of this process is the formation of new cell types dissimilar from those normally present in esophageal, gastric, or duodenal mucosa.