Pre-emption of human cell-mediated lympholysis by a suppressive mechanism activated in mixed lymphocyte cultures

The regulation of B-cell and T-cell immune responses has been extensively examined and in the experimental animal appears to involve regulatory or “suppressor” T cells (1-4). The limitations of in vitro experimentation have made comparable study of nonpathological human suppression quite difficult (5). We report here an in vitro method that generates and quantitates suppressor activity in man after antigen-specific activation in mixed leukocyte culture (MLC). The one-way MLC induces both a proliferative response (6) and the generation of cytotoxic T lymphocytes (CTLs) (7). Both of these responses are mediated by antigen-specific T-cell subpopulations (8,9) and have been correlated with recognitive and destructive phases of allograft rejection. Recent reports have examined the antigen reactivity of mouse (10,11), rat (12), or human (13,14) lymphocytes obtained after proliferation in MLC. In all cases, after the primary MLC proliferative peak, the recovered lymphocytes rapidly differentiate upon re-exposure to the initial stimulating population, but do so only weakly when exposed to a presumably noncross-reactive third-party stimulating population. Velocity sedimentation separation studies have shown that the blast cells produced in a primary MLC revert to small lymphocytes that rapidly differentiate into proliferating and/or cytotoxic T lymphocytes upon restimulation with the initial antigen (15). These findings demonstrate that positive selection for the responding population in primary MLC does exist and may account for at least part of the specificity of the secondary response. However, this positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection does not preclude possible involvement of a suppressor mechanism. In fact we have detected suppressor activity in primary MLC sensitization cultures at a time when the proliferation responsible for positive selection in not yet significant, suggesting that suppression may be overriding importance in the specificity of MLC-activated secondary responses.     

外周血Th细胞因子水平与褪黑素变化在自身免疫性肝炎大鼠的表现

目的：褪黑素是机体神经内分泌免疫调节网络中的重要成员，已证实其对四氯化碳致大鼠肝损伤具有保护作用。实验观察褪黑素对自身免疫性肝炎大鼠外周血Th细胞因子水平的影响。方法：实验于2004—10／2006—10在解放军南京军区肝病中心实验室完成。①实验动物：雄性3月龄Wistar大鼠80只，随机数字表法分为4组：正常对照组、模型对照组、褪黑素治疗组、猪促肝细胞生长素治疗组，20只，组。另取同系Wjstar大鼠10只用于提取肝细胞特异性脂蛋白抗原。（爹实验方法：除正常对照组外，其余各组均采用弗氏完全佐剂＋肝细胞特异性脂蛋白建立自身免疫性肝炎模型。褪黑素用含0．01％乙醇的生理盐水配制成1g／L的溶液，褪黑素治疗组腹腔注射2mg／kg；猪促肝细胞生长素用含0．01％乙醇的生理盐水配制成2g／L的溶液，猪促肝细胞生长素治疗组腹腔注射2mg／kg；正常对照组和模型对照组均腹腔注射含0．01％乙醇的生理盐水，1次，d，连续60d。（彭实验评估：检测各组大鼠外周血Th细胞因子的水平。结果：80只大鼠均进入结果分析。①褪黑素对Th1细胞因子水平的影响：与模型对照组比较，褪黑素治疗组、猪促肝细胞生长素治疗组白细胞介素2和干扰素γ水平均显著升高（P〈0．05），且褪黑素治疗组调节Thl细胞因子水平的作用强于猪促肝细胞生长素治疗组（P〈0．01）。②褪黑素对Th2细胞因子水平的影响：与模型对照组比较，褪黑素治疗组、猪促肝细胞生长素治疗组白细胞介素4，6水平均显著降低（P〈0．05），且褪黑素治疗组调节Th1细胞因子水平的作用强于猪促肝细胞生长素治疗组（P〈0．01）。结论：褪黑素能够刺激自身免疫性肝炎大鼠外周血Th1细胞因子表达，并抑制Th2细胞因子水平，从而改善肝组织病变程度。     

Biochemical correlates of the differential sensitivity of subtypes of human leukemia to deoxyadenosine and deoxycoformycin

Leukemic cells incubated in vitro with 2'-deoxyadenosine (dAdo) plus an inhibitor of adenosine deaminase, 2'-deoxy-coformycin (DCF), show different metabolic responses depending on the histologic and immunologic type of the leukemia. Leukemic cells were obtained from 54 patients with acute lymphoblastic leukemia (ALL), 9 with myeloid or nonlymphoblastic leukemia, 3 with chronic lymphocytic leukemia (CLL), and 3 with lymphoma. There was a wide variation in the LD50, the concentration of dAdo that caused 50% inhibition of the incorporation of 3H-thymidine into cells in the presence of 20 microM DCF. T-cell leukemia specimens were much more sensitive to dAdo than were specimens of pre-B-ALL and null-ALL. In leukemic cells that had been incubated with 14C-dAdo plus DCF, a good correlation was observed between the LD50 and the ratio of 14C-deoxyATP to ATP (correlation coefficient for the fit to a hyperbola = 0.853). The accumulation of deoxyATP by the leukemic cell specimens was correlated best with the activity of ecto- ATPase, less well with cytoplasmic 5'-nucleotidase and deoxyadenosine kinase, and poorly with adenosine deaminase and ecto-5'-nucleotidase. The clinical response to DCF therapy of a patient with T-ALL and another with pre-B-ALL was consistent with the in vitro metabolic response of their cells to DCF and dAdo.     

Constitutive in vivo cytokine and hematopoietic growth factor gene expression in the bone marrow and peripheral blood of healthy individuals

We investigated hematopoietic growth factor (HGF) and cytokine gene expression in the bone marrow (BM) and peripheral blood (PB) of healthy individuals as a starting point for delineating the physiologic role of cytokines in steady state hematopoiesis. BM biopsy specimens and PB samples from 7 healthy individuals were analyzed by polymerase chain reaction amplification of reverse-transcribed RNA using gene-specific primer sets. Consistent gene expression in the BM of all 7 individuals was detected for macrophage colony-stimulating factor (CSF), stem cell factor, interleukin-6 (IL-6), IL-7, erythroid-potentiating factor, erythroid-differentiating factor, and insulinlike growth factor 1, all cytokines with reported direct stimulatory effects on in vitro hematopoiesis. Of these, erythroid-potentiating factor and erythroid- differentiating factor appeared to be the only stimulating factors that were also expressed in the PB. Among the cytokines with inhibitory effects on in vitro hematopoiesis IL-4, tumor necrosis factor-alpha (TNF-alpha), TNF-beta, transforming growth factor-beta, and macrophage inflammatory protein-1 alpha were expressed in the BM of the 7 individuals. Except for TNF-alpha, the latter cytokines were also expressed in the PB. Consistent expression in the BM and PB of all tested individuals was also observed for IL-1 beta, IL-1 receptor antagonist, and IL-1 beta converting enzyme, which are all members of the IL-1 family with a possible indirect effect on hematopoiesis. Remarkably, no expression of granulocyte CSF, granulocyte-macrophage CSF, and IL-3 was found in the BM or PB of all investigated individuals (n = 15). This was also the case for IL-1 alpha, IL-2, IL-5, IL-9, IL- 12, IL-13, leukemia-inhibiting factor, interferon-gamma, and inhibin. Weak IL-8 and IL-10 expression was found in the BM and/or PB of a minority of investigated individuals. These findings provide insight into which cytokines or HGFs potentially are involved in the autocrine or paracrine regulation of in vivo steady state hematopoiesis. The absence of expression of granulocyte CSF, granulocyte-macrophage CSF, and IL-3 in the BM of healthy individuals implicates that it is highly unlikely that these HGFs are involved in the autocrine or paracrine regulation of constitutive hematopoiesis.     

HLA associations with leukemia

Frequencies of 35 HLA A, B, C, and DR antigens were determined in 1,834 leukemic Caucasoids to evaluate possible associations between HLA and leukemia. In comparison with the frequencies of HLA antigens in published controls, the frequency of Cw3 was significantly higher in patients with acute lymphoblastic leukemia (relative risk = 2.64, P less than 0.0002), acute myelogenous leukemia (relative risk = 1.92, P less than 0.0007), and chronic myelogenous leukemia (relative risk = 2.07, P less than 0.002; P values adjusted for multiple comparisons). The frequency of Cw4 was elevated in patients with acute lymphoblastic leukemia (relative risk = 2.01, P less than 0.0003), acute myelogenous leukemia (relative risk = 2.06, P less than 0.0002), and chronic myelogenous leukemia (relative risk = 2.14, P less than 0.0008). The frequency of Aw19 was significantly decreased in patients with acute myelogenous leukemia (relative risk = 0.68, P less than 0.01) and chronic myelogenous leukemia (relative risk = 0.59, P less than 0.005). None of the other 32 HLA antigens investigated had a statistically significant association with leukemia. The data suggest that Cw3 and Cw4 may be markers for leukemia susceptibility genes, while Aw19 may be a marker for decreased susceptibility to leukemia.     

Painful legs and moving toes syndrome responsive to pregabalin

Report three cases of painful legs and moving toes (PLMT) syndrome responsive to pregabalin along with a review of its literature. Three patients with PLMT syndrome improved with pregabalin. The first and third patient reported improvement in pain scores, quality of life, and quality of sleep sustained over time. The second and third patient had near complete remission of toe movements, but pregabalin was discontinued in the second patient due to aggravation of leg edema. PLMT is a rare and debilitating disorder characterized by lower limb pain and involuntary toes or feet movements. Its pathophysiology remains unknown and its therapy refractory to most drugs, except for pregabalin, as shown in this case series. PLMT is a rare and incapacitating syndrome due to the lack of an effective pain therapy. We report three patients with PLMT who favorable responded to pregabalin. We propose pregabalin be considered in the management of PLMT.KEY WORDS: Painful legs and moving toes syndrome, pregabalin, restless leg syndrome     

自身免疫性肝炎大鼠外周血CD4+细胞、肝细胞特异性脂蛋白抗体及Th细胞因子水平之间的相关性

目的:建立自身免疫性肝炎大鼠模型,分析其外周血CD4 细胞、肝细胞特异性脂蛋白抗体及Th细胞因子水平之间的相关性。方法:实验于2004-10/2006-10在南京军区肝病中心实验室完成。①实验动物:雄性3月龄Wistar大鼠80只,随机数字表法分为4组:正常对照组、模型对照组、褪黑素治疗组、猪促肝细胞生长素治疗组,20只/组。②实验方法:除正常对照组外,其余各组大鼠均给予弗氏完全佐剂 肝细胞特异性脂蛋白建立自身免疫性肝炎模型。褪黑素用含0.01%乙醇的生理盐水配制成1g/L的溶液,褪黑素治疗组腹腔注射2mg/kg;猪促肝细胞生长素用含0.01%乙醇的生理盐水配制成2g/L的溶液,猪促肝细胞生长素治疗组腹腔注射2mg/kg;正常对照组和模型对照组均腹腔注射含0.01%乙醇的生理盐水,1次/d,连续60d。③实验评估:检测各组大鼠外周血淋巴细胞亚群浓度、肝细胞特异性脂蛋白抗体水平及Th细胞因子的浓度,并分析三者之间的关系。结果:80只大鼠均进入结果分析。模型对照组CD4 细胞百分比、肝细胞特异性脂蛋白抗体水平、白细胞介素4,6水平均显著高于其余3组(P<0.01),白细胞介素2和干扰素γ水平均显著低于其余3组(P<0.01)。与正常对照组比较,褪黑素治疗组、猪促肝细胞生长素治疗组CD4 细胞百分比无明显变化(P>0.05),肝细胞特异性脂蛋白抗体水平显著升高(P<0.001)。与猪促肝细胞生长素治疗组比较,正常对照组、褪黑素治疗组白细胞介素2和干扰素γ水平显著升高(P<0.01),白细胞介素4,6水平均显著降低(P<0.01)。结论:①CD4 细胞百分比与肝细胞特异性脂蛋白抗体水平有关,当CD4 细胞百分比达到正常水平时肝细胞特异性脂蛋白抗体表达虽受到明显抑制却并未停止表达。②褪黑素调节Th细胞因子水平的作用强于猪促肝细胞生长素。③Th细胞因子水平变化与肝细胞特异性脂蛋白抗体水平有一定关系。     

推移腓肠肌肌皮瓣移植修复小腿中下部软组织缺损的临床应用

目的:腓肠肌肌皮瓣因受到蒂部长度限制,只能修复小腿中上部的皮肤软组织缺损,难以修复中下部的软组织缺损。采用推移腓肠肌肌皮瓣移植的方法,观察其修复小腿中下部软组织缺损的临床效果。方法:选择2000-02/2006-10应用推移腓肠肌肌皮瓣移植修复小腿中下部软组织缺损患者14例,软组织缺损范围为12.0cm×6.5cm￣4.5cm×3.5cm,均知情同意。只保留内和/或外侧腓肠血管蒂,切开肌皮瓣周围皮肤及切断腓肠肌的起止点,形成岛状腓肠肌内和/或外侧腓肠肌肌皮瓣,在屈膝30°￣60°时,肌皮瓣能明显向远端、前端推移4￣10cm,修复至中小腿下段创面,腓肠肌肌皮瓣最大为26.0cm×8cm,最小为13.0cm×4.5cm。术后定期随访。结果:所有病例均随访半年以上,12例肌皮瓣完全成活;2例远端部分皮肤坏死,经清创及比目鱼肌肌瓣转移填塞、中厚皮植皮愈合。术后有4例皮肤裂开,2例经换药愈合;1例用局部皮瓣转移修复;1例再行取钢板及清创后伤口愈合。结论:推移腓肠肌肌皮瓣能明显延伸向小腿远、前方,达小腿下段,血运丰富,转移方便,适应症广,是移植修复小腿中下部软组织缺损的一种优良供区。     

Binding kinetics differentiates functional antagonism of orexin-2 receptor ligands

Orexin receptor antagonism represents a novel approach for the treatment of insomnia that directly targets sleep/wake regulation. Several such compounds have entered into clinical development, including the dual orexin receptor antagonists, suvorexant and almorexant. In this study, we have used equilibrium and kinetic binding studies with the orexin-2 (OX₂) selective antagonist radioligand, [³H]-EMPA, to profile several orexin receptor antagonists. Furthermore, selected compounds were studied in cell-based assays of inositol phosphate accumulation and ERK-1/2 phosphorylation in CHO cells stably expressing the OX₂ receptor that employ different agonist incubation times (30 and 5 min, respectively). EMPA, suvorexant, almorexant and TCS-OX-29 all bind to the OX₂ receptor with moderate to high affinity (pK_I values ≥ 7.5), whereas the primarily OX₁ selective antagonists SB-334867 and SB-408124 displayed low affinity (pK_I values ca. 6). Competition kinetic analysis showed that the compounds displayed a range of dissociation rates from very fast (TCS-OX2-29, k_off = 0.22 min⁻¹) to very slow (almorexant, k_off = 0.005 min⁻¹). Notably, there was a clear correlation between association rate and affinity. In the cell-based assays, fast-offset antagonists EMPA and TCS-OX2-29 displayed surmountable antagonism of orexin-A agonist activity. However, both suvorexant and particularly almorexant cause concentration-dependent depression in the maximal orexin-A response, a profile that is more evident with a shorter agonist incubation time. Analysis according to a hemi-equilibrium model suggests that antagonist dissociation is slower in a cellular system than in membrane binding; under these conditions, almorexant effectively acts as a pseudo-irreversible antagonist.Linked ArticlesThis article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-2     

Decreased lung function associated with perinatal exposure to Dutch background levels of dioxins

Perinatal exposure to Dutch background dioxin levels is rather high. Studies of calamities have shown that dioxins negatively influence the respiratory system. It was hypothesized that perinatal exposure to background dioxin levels leads to lung suboptimality, probably through developmental interference. This study aimed to assess lung function in relation to perinatal dioxin exposure. Spirometry was performed in 41 healthy children (aged 7-12 y, mean 8.2 y) with known perinatal dioxin exposure. The ratio of forced expiratory volume in 1 s to forced vital capacity (FEV 1 /FVC ratio) was determined. A complete medical history was taken. The prenatal exposure ranged from 8.74 to 88.8 (mean 34.6) ng TEQ dioxin kg fat -1 , measured in breast milk. The postnatal exposure ranged from 4.34 to 384.51 (mean 75.4) ng TEQ dioxin. Twelve children had to be excluded. significant decrease in lung function in relation to both prenatal ( p = 0.045) and postnatal ( p = 0.0002) dioxin exposure was seen in the 29 non-excluded children. A clinical association between chest congestion and perinatal dioxin exposure was seen.

Conclusion: Perinatal background dioxin exposure may be inversely associated with the FEV 1 /FVC ratio.