Interleukin 2, but not other common gamma chain-binding cytokines, can reverse the defect in generation of CD4 effector T cells from naive T cells of aged mice.

Development of effectors from naive CD4 cells occurs in two stages. The early stage involves activation and limited proliferation in response to T cell receptor (TCR) stimulation by antigen and costimulatory antigen presenting cells, whereas the later stage involves proliferation and differentiation in response to growth factors. Using a TCR-transgenic (Tg(+)) model, we have examined the effect of aging on effector generation and studied the ability of gamma(c) signaling cytokines to reverse this effect. Our results indicate that responding naive CD4 cells from aged mice, compared with cells from young mice, make less interleukin (IL)-2, expand poorly between days 3 to 5, and give rise to fewer effectors with a less activated phenotype and reduced ability to produce cytokines. When exogenous IL-2 or other gamma(c) signaling cytokines are added during effector generation, the Tg(+) cells from both young and aged mice proliferate vigorously. However, IL-4, IL-7, and IL-15 all fail to restore efficient effector production. Only effectors from aged mice generated in the presence of IL-2 are able to produce IL-2 in amounts equivalent to those produced by effectors generated from young mice, suggesting that the effect of aging on IL-2 production is reversible only in the presence of exogenous IL-2.     

Early T cell receptor beta gene expression is regulated by the pre-T cell receptor-CD3 complex.

We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR.     

Activation of the COOH-terminal Src kinase (Csk) by cAMP-dependent protein kinase inhibits signaling through the T cell receptor

In T cells, cAMP-dependent protein kinase (PKA) type I colocalizes with the T cell receptor-CD3 complex (TCR/CD3) and inhibits T cell function via a previously unknown proximal target. Here we examine the mechanism for this PKA-mediated immunomodulation. cAMP treatment of Jurkat and normal T cells reduces Lck-mediated tyrosine phosphorylation of the TCR/CD3 zeta chain after T cell activation, and decreases Lck activity. Phosphorylation of residue Y505 in Lck by COOH-terminal Src kinase (Csk), which negatively regulates Lck, is essential for the inhibitory effect of cAMP on zeta chain phosphorylation. PKA phosphorylates Csk at S364 in vitro and in vivo leading to a two- to fourfold increase in Csk activity that is necessary for cAMP-mediated inhibition of TCR-induced interleukin 2 secretion. Both PKA type I and Csk are targeted to lipid rafts where proximal T cell activation occurs, and phosphorylation of raft-associated Lck by Csk is increased in cells treated with forskolin. We propose a mechanism whereby PKA through activation of Csk intersects signaling by Src kinases and inhibits T cell activation.     

Dopamine receptor D2 regulates GLUA1-containing AMPA receptor trafficking and central sensitization through the PI3K signaling pathway in a male rat model of chronic migraine

BackgroundThe pathogenesis of chronic migraine remains unresolved. Recent studies have affirmed the contribution of GLUA1-containing AMPA receptors to chronic migraine. The dopamine D2 receptor, a member of G protein-coupled receptor superfamily, has been proven to have an analgesic effect on pathological headaches. The present work investigated the exact role of the dopamine D2 receptor in chronic migraine and its effect on GLUA1-containing AMPA receptor trafficking.MethodsA chronic migraine model was established by repeated inflammatory soup stimulation. Mechanical, periorbital, and thermal pain thresholds were assessed by the application of von Frey filaments and radiant heat. The mRNA and protein expression levels of the dopamine D2 receptor were analyzed by qRT‒PCR and western blotting. Colocalization of the dopamine D2 receptor and the GLUA1-containing AMPAR was observed by immunofluorescence. A dopamine D2 receptor agonist (quinpirole) and antagonist (sulpiride), a PI3K inhibitor (LY294002), a PI3K pathway agonist (740YP), and a GLUA1-containing AMPAR antagonist (NASPM) were administered to confirm the effects of the dopamine D2 receptor, the PI3K pathway and GULA1 on central sensitization and the GLUA1-containing AMPAR trafficking. Transmission electron microscopy and Golgi-Cox staining were applied to assess the impact of the dopamine D2 receptor and PI3K pathway on synaptic morphology. Fluo-4-AM was used to clarify the role of the dopamine D2 receptor and PI3K signaling on neuronal calcium influx. The Src family kinase (SFK) inhibitor PP2 was used to explore the effect of Src kinase on GLUA1-containing AMPAR trafficking and the PI3K signaling pathway.ResultsInflammatory soup stimulation significantly reduced pain thresholds in rats, accompanied by an increase in PI3K-P110β subunit expression, loss of dopamine receptor D2 expression, and enhanced GLUA1-containing AMPA receptor trafficking in the trigeminal nucleus caudalis (TNC). The dopamine D2 receptor colocalized with the GLUA1-containing AMPA receptor in the TNC; quinpirole, LY294002, and NASPM alleviated pain hypersensitivity and reduced GLUA1-containing AMPA receptor trafficking in chronic migraine rats. Sulpiride aggravated pain hypersensitivity and enhanced GLUA1 trafficking in CM rats. Importantly, the anti-injury and central sensitization-mitigating effects of quinpirole were reversed by 740YP. Both quinpirole and LY294002 inhibited calcium influx to neurons and modulated the synaptic morphology in the TNC. Additional results suggested that DRD2 may regulate PI3K signaling through Src family kinases.ConclusionModulation of GLUA1-containing AMPA receptor trafficking and central sensitization by the dopamine D2 receptor via the PI3K signaling pathway may contribute to the pathogenesis of chronic migraine in rats, and the dopamine D2 receptor could be a valuable candidate for chronic migraine treatment.Supplementary InformationThe online version contains supplementary material available at 10.1186/s10194-022-01469-x.     

腹腔感染性脓毒症可溶性髓样细胞触发受体(sTREM-1)与Janus激酶-信号转导和转录激活因子(JAK/STAT)通路的关系

目的 通过动物脓毒症模型探讨可溶性髓样细胞触发受体(sTREM-1)与Janus激酶-信号转导和转录激活因子(JAK/STAT)通路的关系.方法 采用盲肠结扎穿孔(CLP)法制作大鼠脓毒症模型,大鼠随机(随机数字法)分为正常对照组(n=6)、假手术组(n=24)、CLP组(n=48)、JAK 2抑制剂(AG 490)组(n=48)和STAT3抑制剂(雷帕霉素,RPM)组(n=48).留取外周血行流式细胞仪分析CD4+ CD25+ Treg细胞/CD4+T细胞比值.采用逆转录多聚酶链式反应(RT-PCR)测定肝组织sTREM-1 mRNA表达水平.结果 CLP后6h肝sTREM-1 mRNA表达即高于对照组和假手术组,且随时间变化逐渐升高.AG490组肝组织sTREM-1 mRNA的表达在术后6h、24 h(1.572±0.051,2.063±0.025)较同时间点CLP组(1.592±0.036,2.082±0.021)差异无统计学意义(P＜0.05),而在48 h和72 h AG490组肝组织sTREM-1 mRNA的表达(2.522±0.083,3.153±0.021)低于同时间点CLP组(2.592±0.055,3.204±0.013) (P＜0.05).术后6 h RPM组肝组织sTREM-1mRNA的表达(1.581±0.017)较CLP组差异无统计学意义(P＜0.05),而在术后24、48、72 h RPM组肝组织sTREM-1 mRNA的表达(1.486±0.019,1.263±0.011,1.115±0.022)显著低于同时间点CLP组肝组织sTREM-1 mRNA的表达(P＜0.05).结论 sTREM-1因子与JAK/STAT通路有关,阻断JAK/STAT通路能够抑制sTREM-1 mRNA的表达,减缓脓毒症炎症反应的进展.     

ITM2A is induced during thymocyte selection and T cell activation and causes downregulation of CD8 when overexpressed in CD4(+)CD8(+) double positive thymocytes.

To identify novel genes that are involved in positive selection of thymocytes, we performed polymerase chain reaction (PCR)-based subtractive hybridization between selecting and nonselecting thymi. OT-1 T cell receptor (TCR) transgenic thymocytes on a recombination activating gene (RAG) null background are efficiently selected into the CD8 lineage in H-2(b) mice (RAG-2(-/-)OT-1, selecting thymi), but are not selected on a transporter associated with antigen processing (TAP) null background (RAG-2(-/-)TAP-1(-/-)OT-1, nonselecting thymi). We report here our studies of one gene, ITM2A, whose expression is dramatically higher in T cells in the selecting thymus. The expression pattern of ITM2A in thymocyte subsets correlates with upregulation during positive selection. In addition, ITM2A expression is higher in the thymus than in either the spleen or lymph nodes, but can be upregulated in peripheral T cells upon activation. ITM2A expression was also induced in RAG-2(-/-) thymocytes in vivo upon CD3 cross-linking. We demonstrate that ITM2A is a type II membrane glycoprotein that exists as two species with apparent M(r) of 45 and 43 kD and appears to localize primarily to large cytoplasmic vesicles and the Golgi apparatus, but is also expressed on the cell surface. Expression on the surface of EL4 cells increases with activation by phorbol myristate acetate (PMA) and ionomycin. Finally, overexpression of ITM2A under control of the lck proximal promoter in mice results in partial downregulation of CD8 in CD4(+)CD8(+) double positive (DP) thymocytes, and a corresponding increase in the number of CD4(+)CD8(lo) thymocytes. Possible roles for this novel activation marker in thymocyte development are discussed.     

Selective Expression and Functions of Interleukin 18 Receptor on T Helper (Th) Type 1 but not Th2 Cells

Interleukin (IL)-18 induces interferon (IFN)-γ synthesis and synergizes with IL-12 in T helper type 1 (Th1) but not Th2 cell development. We report here that IL-18 receptor (IL-18R) is selectively expressed on murine Th1 but not Th2 cells. IL-18R mRNA was expressed constitutively and consistently in long-term cultured clones, as well as on newly polarized Th1 but not Th2 cells. IL-18 sustained the expression of IL-12Rβ2 mRNA, indicating that IL-18R transmits signals that maintain Th1 development through the IL-12R complex. In turn, IL-12 upregulated IL-18R mRNA. Antibody against an IL-18R–derived peptide bound Th1 but not Th2 clones. It also labeled polarized Th1 but not Th2 cells derived from naive ovalbumin–T cell antigen receptor-αβ transgenic mice (D011.10). Anti–IL-18R antibody inhibited IL-18– induced IFN-γ production by Th1 clones in vitro. In vivo, anti–IL-18R antibody reduced local inflammation and lipopolysaccharide-induced mortality in mice. This was accompanied by shifting the balance from Th1 to Th2 responses, manifest as decreased IFN-γ and proinflammatory cytokine production and increased IL-4 and IL-5 synthesis. Therefore, these data provide a direct mechanism for the selective effect of IL-18 on Th1 but not Th2 cells. They also show that the synergistic effect of IL-12 and IL-18 on Th1 development may be due to the reciprocal upregulation of their receptors. Furthermore, IL-18R is a cell surface marker distinguishing Th1 from Th2 cells and may be a therapeutic target.     

慢性乙型肝炎患者肝组织中Toll样受体2的表达及其临床意义——附63例报告

目的:探讨Toll样受体2(Toll-like receptor 2,TLR2)在慢性乙型肝炎(chronic hepatitis B, CHB)中的表达及其临床意义.方法:取63份CHB患者的肝组织标本(CHB组)及9份移植肝常规留取的正常肝组织标本(对照组),采用免疫组织化学法检测2组TLR2的表达情况.结果:TLR2表达主要定位于肝细胞胞浆及部分胞膜上.CHB组中、重度患者肝组织上的TLR2的表达均比对照组明显增强,比较差异均有统计学意义(P＜0.05～P＜0.01);在CHB组中,中、重度患者肝组织上的TLR2的表达均比轻度患者明显增强,比较差异均有统计学意义(P＜0.05～P＜0.01);CHB组患者肝组织上的TLR2的表达程度与临床分度呈显著正相关(r=0.559,P＜0.001).结论:对于CHB,TLR2具有重要意义,提示调控或干扰TLR2有利于控制病情.     

Migraine association and linkage analyses of the human 5-hydroxytryptamine (5HT2A) receptor gene

5-Hydroxytryptamine (5HT), commonly known as serotonin, which predominantly serves as an inhibitory neurotransmitter in the brain, has long been implicated in migraine pathophysiology. This study tested an Mspl polymorphism in the human 5HT_2A receptor gene ( HTR2A ) and a closely linked microsatellite marker (D13S126), for linkage and association with common migraine. In the association analyses, no significant differences were found between the migraine and control populations for both the Mspl polymorphism and the D13S126 microsatellite marker. The linkage studies involving three families comprising 36 affected members were analysed using both parametric (FASTLINK) and non-parametric (MFLINK and APM) techniques. Significant close linkage was indicated between the Mspl polymorphism and the D13S126 microsatellite marker at a recombination fraction (θ) of zero (lod score=7.15). Linkage results for the Mspl polymorphism were not very informative in the three families, producing maximum and minimum lod scores of only 0.35 and 0.39 at recombination fractions (θ) of 0.2 and 0.00, respectively. However, linkage analysis between the D13S126 marker and migraine indicated significant non-linkage (lod2) up to a recombination fraction (θ) of 0.028. Results from this study exclude the HTR2A gene, which has been localized to chromosome 13q14-q21, for involvement with common migraine.     

儿童急性白血病T细胞受体δ、γ基因重排连接区序列的动态分析

目的 探讨儿童急性淋巴细胞白血病（ＡＬＬ）患在初诊、完全缓解（ＣＲ）及复发不同时期的Ｔ细胞３受体（ＴＣＲ）δＶ－Ｄ,γＶ－Ｊ基因重排连接区序列的差异及其意义。方法 采用Ｔ－载体分子克隆测离１限制性酶团及聚合酶链反应等技术,对３４丛ＡＬＬ患标本进行ＴＣＴδ、γ连接区序列动态分析。结果 ３４丛ＡＬＬ患标本进行ＴＣＲ－５γ连接区序列动态分析。结果 ３４份ＡＬＬ患标本的ＴＣＲ５、γ基因重排序列在初     

与人体肥胖相关的神经肽Y受体Y2基因的克隆及序列分析

目的 克隆与肥胖相关的人神经肽Y受体Y2(NPY2R)基因,并鉴定该克隆基因序列的正确性.方法 从人的脂肪组织中提取总RNA并进行反转录,以此为模板用PCR扩增NPY2R基因的eDNA,然后与pET 28a+载体重组,筛选阳性克隆和DNA序列分析鉴定,测序后与GeneBank中NPY2R基因进行同源性比较和序列分析.结果 PCR扩增出一个1100 bp左右的DNA片段,与载体重组和DNA序列分析鉴定,DNA序列分析显示克隆的DNA片段是人NPY2R基因,且所克隆的基因共编码381个氨基酸,分子量为4.2 kD,与GeneBank中NPY2R基因序列同源性达100%.结论 克隆的人NPY2R基因与GeneBank中NPY2R序列完全一致,为进一步应用分子生物学技术深入研究NPY2R与人体肥胖发生、发展及转化等相关作用机制及应用该基因进行其基因蛋白表达奠定了基础.