Structural requirements of DNA used in the Farr assay to detect antibodies directed against double-stranded DNA

Summary The measurement of antibodies to DNA in SLE requires the use of double-stranded DNA (dsDNA), demonstrably free of single-stranded regions. Such dsDNA preparations can, however, contain other structural components. In this study DNA preparations with defined structure, both secondary (single- and double-stranded and random base-paired) and tertiary (superhelical and open circular), were used in the Farr assay to measure the DNA binding of sera from patients with SLE and related connective-tissue diseases. The presence of true single-stranded DNA regions in denatured DNA, native DNA, and dsDNA containing single-stranded regions increased the DNA binding measured in all sera. DsDNA, whether intact or containing small regions of random base-pairing, was bound by sera from the majority of patients with SLE but not by non-SLE sera. Superhelical dsDNA from bacteriophage PM2 was bound by SLE sera to a greater extent than linear dsDNA was. Inhibition experiments suggested that this difference in binding to DNA according to tertiary, as opposed to secondary, structure is because there are fewer available binding sites on super-helical dsDNA. DNA binding, as measured by the Farr assay, can thus be influenced by both secondary and tertiary DNA structure. Using superhelical DNA, advantage can be taken of the dsDNA form plus tertiary structure to enhance DNA binding of SLE sera beyond the levels achieved using linear dsDNA.     

B cell activation in patients with rheumatic disease: association with an increased ratio of Th2: Th1 cytokine secreting cells

Abstract

This minireview examines the pattern of T_h1 and T_h2 cytokine-secreting cells present in the peripheral blood of patients with rheumatic diseases including lupus, myositis, Sjögren’s syndrome and rheumatoid arthritis. The general conclusion is reached that diseases marked by increased B cell activation are characterized by a relative increase in the frequency of peripheral blood mononuclear cells secreting T_h2 versus T_h1 cytokines.     

B cell activation in patients with rheumatic disease: association with an increased ratio of Th2: Th1 cytokine secreting cells

This minireview examines the pattern of T_h1 and T_h2 cytokine-secreting cells present in the peripheral blood of patients with rheumatic diseases including lupus, myositis, Sjögren’s syndrome and rheumatoid arthritis. The general conclusion is reached that diseases marked by increased B cell activation are characterized by a relative increase in the frequency of peripheral blood mononuclear cells secreting T_h2 versus T_h1 cytokines.     

Fork rotation and DNA precatenation are restricted during DNA replication to prevent chromosomal instability

Faithful genome duplication and inheritance require the complete resolution of all intertwines within the parental DNA duplex. This is achieved by topoisomerase action ahead of the replication fork or by fork rotation and subsequent resolution of the DNA precatenation formed. Although fork rotation predominates at replication termination, in vitro studies have suggested that it also occurs frequently during elongation. However, the factors that influence fork rotation and how rotation and precatenation may influence other replication-associated processes are unknown. Here we analyze the causes and consequences of fork rotation in budding yeast. We find that fork rotation and precatenation preferentially occur in contexts that inhibit topoisomerase action ahead of the fork, including stable protein–DNA fragile sites and termination. However, generally, fork rotation and precatenation are actively inhibited by Timeless/Tof1 and Tipin/Csm3. In the absence of Tof1/Timeless, excessive fork rotation and precatenation cause extensive DNA damage following DNA replication. With Tof1, damage related to precatenation is focused on the fragile protein–DNA sites where fork rotation is induced. We conclude that although fork rotation and precatenation facilitate unwinding in hard-to-replicate contexts, they intrinsically disrupt normal chromosome duplication and are therefore restricted by Timeless/Tipin.During DNA replication, it is essential to completely unwind and remove all of the intertwining between the two strands of the template DNA double helix. This is achieved by the combined action of replicative helicases and topoisomerases. During elongation, replicative helicases force the strands apart, generating compensatory topological overwinding stress in the unreplicated region ahead of the fork. If overwinding accumulates, it prevents further DNA replication (1, 2). Relaxation of the stress is achieved either by topoisomerase action ahead of the fork, directly on the overwound region, or by coupling helicase action with rotation of the whole fork relative to the unreplicated DNA (Fig. S1). This latter pathway relaxes topological stress ahead of the fork at the expense of generating double-stranded intertwines behind the fork, often referred to as DNA precatenanes (3, 4). These intertwines are subsequently resolved by the action of type II topoisomerases. If type II topoisomerases do not completely resolve either the precatenanes or the full DNA catenanes formed at the completion of replication, the unresolved intertwines will cause chromosome bridging, nondisjunction, and aneuploidy (5). Fork rotation and DNA precatenation appear to be the primary pathway of unlinking when forks come together at the termination of DNA replication (6, 7). In addition, fork rotation appears to be a frequent event during elongation in vitro; it can support ongoing replication, and extensive precatenation is observed behind elongating forks (8–10). Therefore, the prevailing view is that the topological stress caused by DNA unwinding is resolved stochastically during elongation by both topoisomerase action ahead of the fork and fork rotation and DNA decatenation behind the fork (3, 11). At termination, the diminishing distance between converging replisomes is thought to prevent topoisomerase action ahead of the fork, leaving fork rotation as the primary pathway for DNA unwinding in this context. However, unlike viral replisomes and replication complexes established in in vitro systems, the eukaryotic replisome holoenzyme is composed of a far greater number of proteins and activities. These factors are thought to facilitate replication through the highly variable eukaryotic genomic landscape and coordinate DNA synthesis with other chromatid maturation processes such as chromatin assembly and cohesion establishment (5). These latter processes act on the same newly replicated DNA potentially braided by precatenation. How these activities occurring in the wake of the fork may be impeded by precatenation is unexplored (5).Open in a separate windowFig. S1.Model of topological stress generation and relaxation during elongation of DNA replication. (A) During elongation, unwinding of the parental template separates the parental strands but does not resolve the linkages that exist between the two strands. (B) The linkages between the strands are displaced into the region ahead of the fork, leading to this becoming overwound, that is, positively supercoiled. (C and D) This tension is normally resolved by the action of either topoisomerase I (Top1) or topoisomerase II (Top2) (C), which act effectively as “swivelases” ahead of the fork to generate a relaxed replication region (D). (E) However, Champoux and Been (4) proposed a second mode of topological stress unwinding where the helical tension is relaxed by rotation of the fork to generate DNA precatenation behind the fork. Although these linkages should not arrest forward elongation of replication, it is essential that the type II topoisomerase resolve all DNA catenation before the completion of cell division.To study these events in vivo, we have directly examined fork rotation and precatenation in budding yeast. We show that fork rotation and DNA precatenation are not stochastic but rather are actively restricted to distinct contexts by the evolutionarily conserved homologs of Timeless/Tipin, Tof1/Csm3. Failure to regulate fork rotation leads to significantly elevated levels of DNA damage, particularly at known fragile sites. Therefore, the eukaryotic replisome appears to minimize rotation and precatenation, so that they are used to unwind DNA only when absolutely necessary to maintain genome stability.     

DNA methylation in inflammatory bowel disease and beyond

Inflammatory bowel disease(IBD)is a consequence of the complex,dysregulated interplay between genetic predisposition,environmental factors,and microbial composition in the intestine.Despite a great advancement in identifying host-susceptibility genes using genome-wide association studies(GWAS),the majority of IBD cases are still underrepresented.The immediate challenge in post-GWAS era is to identify other causative genetic factors of IBD.DNA methylation has received increasing attention for its mechanistical role in IBD pathogenesis.This stable,yet dynamic DNA modification,can directly affect gene expression that have important implications in IBD development.The alterations in DNA methylation associated with IBD are likely to outset as early as embryogenesis all the way until old-age.In this review,we will discuss the recent advancement in understanding how DNA methylation alterations can contribute to the development of IBD.     

Anti-nuclear antibody positivity and the use of certolizumab in inflammatory bowel disease patients who have had arthralgias or lupus-like reactions from infliximab or adalimumab

OBJECTIVE: Tumor necrosis factor (TNF) inhibitors can be used to treat inflammatory bowel disease (IBD) but may lead to anti‐nuclear antibody (ANA) positivity and lupus‐like reactions. Because of its unique structure, certolizumab has lower rates of these complications. We sought to investigate whether patients who have had lupus‐like reactions to infliximab or adalimumab would be able to tolerate certolizumab. METHODS: We performed a retrospective analysis on the 23 patients at the Roberts Inflammatory Bowel Disease Center who received certolizumab for the treatment of Crohn's disease from March 2008 to June 2009. We identified 6 patients who were switched to this drug because of lupus‐like reactions from prior anti‐TNF therapy and had documented ANA after the reaction and prior to certolizumab initiation. We then rechecked the ANA status after certolizumab initiation. RESULTS: Five out of 6 patients had a resolution of their arthralgias or lupus‐like symptoms after being switched to certolizumab (P < 0.001). Of the 4 patients who were ANA positive after receiving infliximab or adalimumab, 2 became ANA negative after induction with certolizumab (P = 0.17). There was no difference in Harvey Bradshaw index scores (10.3 vs. 9.8) pre‐certolizumab and post‐certolizumab (P = 0.73). CONCLUSION: Nearly all patients experienced relief from their lupus‐like symptoms with certolizumab and 2 out of 4 patients reversed their ANA positivity. While future investigation is warranted, patients who have lupus‐like reactions from infliximab or adalimumab may benefit from switching to certolizumab.     

Analyses of serum levels of type 1, type 2 and type 3 cytokines reveal multiple abnormalities in lupus‐prone (NZB × NZW) F1 mice

Aim: Inherent in vivo cytokine milieu may help protect normal subjects from developing clinical autoimmunity, whereas changes in cytokine milieu may contribute to the development of lupus‐like autoimmunity. Method: Here, we measured circulating levels of the three prototypic cytokines, type 1 (IFN‐γ and IL‐2), type 2 (IL‐4) and type 3 (TGFβ) in the NZB/NZW F1 model of lupus and MHC‐matched nonautoimmune mice. Results: Our results demonstrate that circulating IFN‐γ and IL‐4 levels were higher and active TGFβ levels were lower in lupus‐prone animals than in nonautoimmune mice. Conclusion: Such an in vivo cytokine milieu may contribute to the development of lupus in NZB/NZW F1 mice and to the nonautoimmune phenotype in normal animals.     

炎症性肠病患者肠道菌丛结构特征的指纹图谱分析

目的建立炎症性肠病（IBD）患者和健康人肠道细菌DNA指纹图谱以分析其肠道菌丛的整体差异。方法应用肠杆菌基因间的重复共有序列（ERIC）-PCR技术建立10例IBD患者及10例正常对照者的肠道细菌DNA指纹图谱,分析IBD患者及正常对照者的肠道菌丛的整体差异。结果IBD患者及正常对照者的肠道菌丛存在整体差异,正常对照组ERIC．PCR指纹图谱电泳DNA条带多,主带位置无统一趋势;IBD组ERIC-PCR指纹图谱电泳DNA条带较少,且所有样本主带分布非常一致,均出现在约1．1kb处。结论正常人肠道细菌种类多,IBD患者少。IBD组1．1kb处主带可能为某一种肠道细菌中特有的序列,或为不同序列或几个序列的混合物。     

抗双链DNA抗体阳性肝功能异常患者临床及生化特征

目的 探讨抗双链DNA抗体阳性肝功异常患者的临床及生化特征.方法 收集肝功异常患者血清884例,采用免疫印迹法检测抗双链DNA抗体.结果 抗双链DNA抗体阳性患者16例(1.81%),女性患者14例,男性2例;平均年龄(48.94±17.57)岁(9岁～69岁).其中自身免疫性肝炎13例(81.25%).16例抗双链DNA阳性患者中肝硬化8例(50%),亚急型重型肝炎2例,急性重型肝炎1例.ALT平均值为(433.91±405.86)U/L,AST平均值为(535.05±513.92)U/L,TBil平均值(182.46±149.11)μmol/L,DB il平均值为(118.33±103.97)μmol/L,PTA平均值为(75.27±31.22)%,IgG平均值(23.48 ±13.26)g/L.16例患者ANA抗体均阳性,并伴有多种自身抗体.结论 在抗双链DNA抗体阳性的肝功异常患者中AIH发病率较高,且病情较重.对抗双链DNA抗体阳性的肝功异常患者临床应予以重视.     

Strategies to prevent type 1 diabetes

Type 1 diabetes is a chronic autoimmune condition resulting from T cell–mediated destruction of the insulin-producing cells in the islets of Langerhans. Its primary cause remains unknown, but it has been established that the clinical presentation is preceded by a long prodrome. This enables individuals at high risk of disease to be identified and offers the possibility of intervention to prevent clinical disease. Many groups are working in this field, concentrating on manipulation of environmental exposures that are potential triggers of autoimmunity and on immunomodulation strategies that aim to prevent destruction of β-cells. Some interventions have shown promising results in early trials, but effective disease prevention remains elusive. This article reviews current progress in the field.     

IDO1 scavenges reactive oxygen species in myeloid-derived suppressor cells to prevent graft-versus-host disease

Tryptophan-catabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) also has an immunological function to suppress T cell activation in inflammatory circumstances, including graft-versus-host disease (GVHD), a fatal complication after allogeneic bone marrow transplantation (allo-BMT). Although the mononuclear cell expression of IDO1 has been associated with improved outcomes in GVHD, the underlying mechanisms remain unclear. Herein, we used IDO-deficient (Ido1^−/−) BMT to understand why myeloid IDO limits the severity of GVHD. Hosts with Ido1^−/− BM exhibited increased lethality, with enhanced proinflammatory and reduced regulatory T cell responses compared with wild type (WT) allo-BMT controls. Despite the comparable expression of the myeloid-derived suppressor cell (MDSC) mediators, arginase-1, inducible nitric oxide synthase, and interleukin 10, Ido1^−/− Gr-1⁺CD11b⁺ cells from allo-BMT or in vitro BM culture showed compromised immune-suppressive functions and were skewed toward the Ly6C^lowLy6G^hi subset, compared with the WT counterparts. Importantly, Ido1^−/−Gr-1⁺CD11b⁺ cells exhibited elevated levels of reactive oxygen species (ROS) and neutrophil numbers. These characteristics were rescued by human IDO1 with intact heme-binding and catalytic activities and were recapitulated by the treatment of WT cells with the IDO1 inhibitor L1-methyl tryptophan. ROS scavenging by N-acetylcysteine reverted the Ido1^−/−Gr-1⁺CD11b⁺ composition and function to an MDSC state, as well as improved the survival of GVHD hosts with Ido1^−/− BM. In summary, myeloid-derived IDO1 enhances GVHD survival by regulating ROS levels and limiting the ability of Gr-1⁺CD11b⁺ MDSCs to differentiate into proinflammatory neutrophils. Our findings provide a mechanistic insight into the immune-regulatory roles of the metabolic enzyme IDO1.

Indoleamine 2,3-dioxygenase 1 (IDO1) is a heme-binding metabolic enzyme that catalyzes the conversion of tryptophan (Trp) into kynurenine (Kyn). In addition to Trp catabolism, IDO1 has long been recognized to have immune-regulatory roles, preventing excessive inflammation (1). IDO1 is up-regulated in response to inflammatory stimuli, including Toll-like receptor (TLR) and type I/II interferon (IFN) signaling (1, 2). The induction of IDO1 after TLR9 stimulation has been demonstrated to mitigate experimental colitis (3). Catalytic function blockade in mice by pharmacological inhibition or genetic ablation of IDO1 (Ido1^−/−) enhanced inflammation and aggravated autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE) (4). The enhanced immune responses induced by IDO1 deficiency were associated with increased T helper (Th)1/Th17 responses; in contrast, regulatory T cell (Treg) responses were repressed (4–6). Consistently, IDO1 inhibition enhanced antitumor immune responses (7–9). The immune-regulatory effects of IDO1 have been ascribed to the depletion of Trp (10, 11) and the production of toxic catabolites along the Kyn pathway (4, 12–14). However, it remains unclear whether additional mechanisms are involved in IDO1-mediated immune suppression.Graft-versus-host disease (GVHD) is a severe inflammatory disease for which IDO1 has been shown to play a protective role (2, 14, 15). GVHD often develops as an adverse systemic complication following allogeneic hematopoietic stem cell transplantation (allo-HSCT) and is induced by activation of donor T cells reactive to the recipient’s major histocompatibility complexes (MHCs) and/or minor histocompatibility antigens (MiHAs) (16). Allo-reactivity of the activated donor T cells promotes tissue inflammation in the host, leading to morbidity and mortality. IDO1 deficiency in the bone marrow (BM) of the donor or the recipient has been linked to increased lethality (2, 14, 15), indicating a crucial role of IDO1 expression in the parenchymal and hematopoietic compartments in preventing GVHD. Kyn produced in IDO1-expressing lung epithelial cells and tissue macrophages suppressed T cell activation by binding to and activating immunomodulatory aryl hydrocarbon receptors (AhRs), which could explain the GVHD aggravation in Ido1^−/− recipients (14). Nevertheless, the mechanisms behind GVHD exacerbation by Ido1^−/− BM transfer remain obscure. Wild-type (WT) donor antigen-presenting cells prolonged survival in GVHD regardless of epithelial cell expression of IDO1, and IDO1 up-regulation after treatment of donor BM with TLR ligands reduced GVHD severity (2). These findings suggest an important role of IDO1 expressed by donor-derived myeloid cells in preventing severe GVHD. However, the immune-regulatory roles of IDO1 expressed in myeloid cells (termed myeloid IDO1 hereafter) remain elusive.Myeloid-derived suppressor cells (MDSCs) are innate cells that have immune-suppressive functions (17). Conventionally, MDSCs are identified as Gr-1⁺CD11b⁺ cells and can be further classified into Ly6C^hiLy6G^low monocytic (M) or Ly6C^lowLy6G^hi polymorphonuclear (PMN) subsets. MDSCs produce various immune-suppressive mediators, including arginase-1 (Arg-1), inducible nitric oxide synthase (iNOS), and interleukin 10 (IL-10) (17, 18). Their ability to enhance Treg responses has also been reported (19, 20). As immature cells, MDSCs maintain the ability to differentiate into dendritic cells (DCs), macrophages, or neutrophils (21, 22). In GVHD, MDSCs derived from donor BM are the major population of myeloid cells expanding in the host (23), and along with Tregs they suppress GVHD (24–26). We previously reported that transplantation of MyD88-deficient (Myd88^−/−) BM suppressed Gr-1⁺CD11b⁺ cell expansion and polarized the differentiation of Gr-1⁺CD11b⁺ cells into DCs, aggravating GVHD (27, 28). These findings indicate that increasing the number of undifferentiated Gr-1⁺CD11b⁺ cells is essential for MDSC-mediated immune suppression in GVHD. Additionally, the finding that IDO1 expression in mononuclear cells, rather than in parenchymal cells, correlated positively with the survival of GVHD patients (29) suggested that IDOl expression in myeloid cells might be involved in the MDSC-mediated suppression of GVHD. Understanding the role of IDO1 in the function of MDSCs derived from the donor BM could lead to novel therapeutic strategies for the treatment of GVHD.In this study, we investigated the mechanisms underlying GVHD aggravation in hosts transplanted with IDO1-deficient BM. We found that IDO1 deficiency in donor BM did not affect the expansion of Gr-1⁺CD11b⁺ cells in GVHD hosts but polarized them toward a Ly6C^lowLy6G^hi phenotype, reducing their immune-regulatory potential. This phenomenon was ascribed to increased reactive oxygen species (ROS) generation in the Ido1^−/− Gr-1⁺CD11b⁺ cells and their skewing to neutrophil differentiation. Treatment of ROS-scavenging chemical reversed this phenomenon. Our findings suggest that the immune-regulatory roles of IDO1 are mediated by ROS scavenging and suppression of the differentiation of Gr-1⁺CD11b⁺ cells.     

Five genetic markers in the interleukin 1 family in relation to inflammatory bowel disease

Background—An imbalance between theproinflammatory cytokine interleukin 1β (IL-1β) and theanti-inflammatory cytokine IL-1 receptor antagonist (IL-1ra) has beenpostulated as a pathogenic factor in inflammatory bowel disease (IBD).
Aims—To study allelic frequenciesof novel polymorphisms in the genes for IL-1β and IL-1ra in patientswith IBD and to assess the relation between ex vivo cytokine productionand allelic variants of the IL-1β and IL-1ra genes.
Subjects—Two hundred and seventyhealthy controls, 74 patients with ulcerative colitis (UC), 72 withCrohn's disease (CD), 40 with primary sclerosing cholangitis for theallelic frequencies, and 60 healthy individuals for the ex vivostimulation test.
Methods—Genotyping was performed bypolymerase chain reaction and subsequent cleavage with specificendonucleases (Mwo1, MspAI1, Alu1, Taq1, BsoF1) for five novelrestriction fragment length polymorphisms (RFLPs) in the genes forIL-1ra and IL-1β.
Results—No significant differences were found inthe allelic frequencies or allele carriage rates of the markers in theIL-1β and IL-1ra genes between CD, UC, and healthy controls. Noassociation between the genetic markers and cytokine production levelswas observed. Patients with UC carried the combination of both the infrequent allele of the Taq1 RFLP and the Mwo1 RFLP significantly morefrequently (35.2% in UC versus 71.1% in controls).
Conclusions—UC is associatedwith carriage of both infrequent alleles of the Taq1 and Mwo1 RFLPs.However, it could not be confirmed whether the association reflects apathogenic mechanism underlying UC.

     

靶向性沉默DNMT1基因对胰腺癌细胞DNA甲基化的影响

目的 观察DNA甲基转移酶1(DNMT1)基因沉默后对人胰腺癌PaTu8988细胞的DNMT活性及hMLH-1基因CpG岛DNA甲基化状态的影响.方法 由美国Ambion公司设计合成DNMT1siRNA和阴性对照siRNA,分别用15、30 nmol/L的浓度转染胰腺癌PaTu8988细胞,以未转染组细胞作为对照.应用实时PCR和蛋白质印迹法检测DNMT1 mRNA和蛋白的表达;用DNMT活性检测试剂盒检测其活性;用亚硫酸氢盐测序PCR(BSP)法检测hMLH-1基因CpG岛的甲基化状态;实时PCR法检测hMLH-1 mRNA的表达.结果 转染48 h后,DNMT1 siRNA15、30 nmol/L组细胞DNMT1 mRNA表达量分别为0.573±0.026和0.143±0.044,显著低于对照组的1.020±0.217及阴性siRNA 15、30 nmol/L组的0.900±0.475和0.938±0.327(P值均＜0.05);DNMT1 siRNA组的DNMT1蛋白表达亦低于对照组和阴性siRNA组.DNMT1 siRNA15、30 nmol/L组细胞DNMT活性分别为0.364±0.124和0.250±0.072,明显低于对照组的0.931±0.065及阴性siRNA 15、30 nmol/L组的0.665±0.055和0.472±0.040.DNMT活性与DNMT1 mRNA表达呈正相关(r=0.69,P＜0.01).DNMT1 RNA干扰后导致hMLH-1基因CpG位点的8个甲基化减少到1个甲基化.结论 DNMT1 siRNA能特异性抑制胰腺癌PaTu8988细胞DNMT1基因的表达,明显抑制DNMT的活性,并引起hMLH-1基因CpG岛去甲基化.     

Structural insight into maintenance methylation by mouse DNA methyltransferase 1 (Dnmt1)

Methylation of cytosine in DNA plays a crucial role in development through inheritable gene silencing. The DNA methyltransferase Dnmt1 is responsible for the propagation of methylation patterns to the next generation via its preferential methylation of hemimethylated CpG sites in the genome; however, how Dnmt1 maintains methylation patterns is not fully understood. Here we report the crystal structure of the large fragment (291-1620) of mouse Dnmt1 and its complexes with cofactor S-adenosyl-L-methionine and its product S-adenosyl-L-homocystein. Notably, in the absence of DNA, the N-terminal domain responsible for targeting Dnmt1 to replication foci is inserted into the DNA-binding pocket, indicating that this domain must be removed for methylation to occur. Upon binding of S-adenosyl-L-methionine, the catalytic cysteine residue undergoes a conformation transition to a catalytically competent position. For the recognition of hemimethylated DNA, Dnmt1 is expected to utilize a target recognition domain that overhangs the putative DNA-binding pocket. Taking into considerations the recent report of a shorter fragment structure of Dnmt1 that the CXXC motif positions itself in the catalytic pocket and prevents aberrant de novo methylation, we propose that maintenance methylation is a multistep process accompanied by structural changes.     

DNA甲基转移酶1在胰腺癌组织中的表达及其临床意义

目的 探讨DNA甲基转移酶1(DNMT1)在胰腺癌组织中的表达及其临床意义.方法 收集手术切除的30例胰腺癌组织和配对癌旁组织.采用实时定量PCR法检测DNMT1 mRNA的表达;免疫组织化学法检测DNMT1蛋白的表达;分析胰腺癌组织DNMT1蛋白表达强度与临床病理参数之间的关系.结果 胰腺癌组织中DNMT1 mRNA的表达量为2.32(1.17～5.17),显著高于配对癌旁组织的0.78(0.07～3.14,P＜0.05).胰腺癌组织中导管细胞DNMT1蛋白表达阳性率为(54.5±21.2)%,显著高于癌旁组织(10.9±15.0)%的表达阳性率(P＜0.01).以胰腺癌导管细胞DNMT1阳性率54.5%为界,分为高表达组(19例)和低表达组(11例).DNMT1表达强度和临床分期(x2=6.897,P=0.029)、淋巴结转移(x2=4.739,P=0.029)、神经浸润与否(x2=5.44,P=0.020)相关,而与年龄、性别、肿瘤位置、肿瘤大小、肿瘤分化、血清CEA和CA19-9浓度无关.结论 胰腺癌组织DNMT1 mRNA和蛋白表达明显增加,DNMT1蛋白表达强度与胰腺癌的侵袭力、淋巴结转移和神经浸润相关.     

RIDDLE immunodeficiency syndrome is linked to defects in 53BP1-mediated DNA damage signaling

Cellular DNA double-strand break-repair pathways have evolved to protect the integrity of the genome from a continual barrage of potentially detrimental insults. Inherited mutations in genes that control this process result in an inability to properly repair DNA damage, ultimately leading to developmental defects and also cancer predisposition. Here, we describe a patient with a previously undescribed syndrome, which we have termed RIDDLE syndrome (radiosensitivity, immunodeficiency, dysmorphic features and learning difficulties), whose cells lack an ability to recruit 53BP1 to sites of DNA double-strand breaks. As a consequence, cells derived from this patient exhibit a hypersensitivity to ionizing radiation, cell cycle checkpoint abnormalities, and impaired end-joining in the recombined switch regions. Sequencing of TP53BP1 and other genes known to regulate ionizing radiation-induced 53BP1 foci formation in this patient failed to detect any mutations. Therefore, these data indicate the existence of a DNA double-strand break-repair protein that functions upstream of 53BP1 and contributes to the normal development of the human immune system.