A transient acidification linked to intracellular Ca2 in anti-μ-stimulated human B-lymphocytes

Mitogen-induced cellular proliferation is in many cell types preceded by rapid changes in intracellular pH and free Ca²⁺ concentration. We studied the patterns of pH and Ca²⁺ changes in normal resting human B-lymphocytes after exposure to and-μ antibodies and the monoclonal antibody 1F5, reactive with the CD20 antigen, both able to activate resting B-lymphocytes to enter the G₁ phase of the cell cycle. Monitoring intracellular pH with the pH-sensitive, fluorescent dye, 2',7'-bis(carboxyethyl)-5,6-carboxy-fluorescein, we demonstrated that poly- and monoclonal anti-μ antibodies induced a rapid (maximum change within 2 min) intracellular acidification of 0.06 pH units followed by a slower (10–15 min) alkalinization towards, or slightly above, the resting pH of 6.88. The acidification response was amiloride-resistant, whereas the return to baseline was sensitive. Intracellular free Ca²⁺ was measured by using the fluorescent Ca²⁺ dye, indo-i. Exposure of cells to anti-/i resulted in a rapid increase (maximum change within 2 min) in cytoplasmic Ca²⁺ of 340 nM and a slower decline in fluorescence back to baseline of about 180 nM. In contrast to anti-μ, 1F5 caused no change in cytoplasmic Ca²⁺ and pH. However, the Ca²⁺ ionophore ionomycin at low concentrations mimicked the Ca²⁺ response as well as the pH response to anti-μ. In Ca²⁺-free solutions the intracellular Ca²⁺ stores are usually rapidly depleted and, indeed, the Ca²⁺ and pH responses to anti-μ were reduced after 5 min and almost abolished after 35 min under such conditions. Our data therefore suggest that the anti-μ-induced rapid acidification is the result of increased intracellular Ca²⁺ and dependent on it.     

Adenoviras-mediated expression of human CD59 on xenogeneic endothelial cells: Protection against human complement-mediated lysis and induction of cellular activation by adenoviral transduction

Abstract: The expression of human regulators of complement activation in endothelium of a transplanted organ is an approach to overcoming the complement (C)-mediated rejection of a xenograft. We designed a replication-deficient adenovirus (Ad) containing the human CD59 cDNA (AdCD59) in order to induce expression of human CD59 on xenogeneic endothelial cells (EC) and confer protection against human C-mediated lysis. In vitro transduction of rat EC with AdCD59 led to consistent levels of CD59 expression in all cells and significantly reduced EC lysis induced by human xenogeneic natural antibodies (XNA) binding and C activation. In addition, we analyzed whether Ad transduction had modified the phenotype of EC and the xenogeneic recognition of EC by human serum. For this, we first demonstrated that transduction of rat EC with AdCD59 or an Ad carrying the lacZ gene (AdlacZ) markedly enhanced the expression of some cell-membrane markers of EC activation, including class I MHC antigens and rat CD59, to levels comparable to those obtained with TNFα Up-regulation of class I MHC antigens was observed for both mRNA and protein expression. In contrast, AdCD59-mediated gene transfer slightly increase intercellular adhesion molecule-1 (ICAM-1) and did not modify the expression of Crry, a rat complement regulatory molecule. Second, we showed that these phenotypic modifications of EC did not affect the expression of the Galαl-3Gal epitope and the binding of human XNA. In addition, neither AdlacZ-mediated transduction, nor TNFα treatment, modified the sensitivity of xenogeneic EC to human serum-mediated lysis. In conclusion, this study demonstrated that transduction of human CD59 with an adenoviral vector conferred resistance to xenogeneic cultured EC against human C-mediated lysis but is associated with cellular activation of transduced EC. This finding may have important implications for the in vivo protocols and strategies intended to generate safer Ad vectors.     

雷公藤对致敏小鼠T细胞活化及其IL-5 mRNA表达的影响

目的　探讨致敏小鼠T淋巴细胞活化与IL 5mRNA表达间的关系及雷公藤内酯醇 (TP)的影响。方法　采用卵蛋白(OVA )致敏的方法建立模型 ;运用免疫细胞化学与原位杂交双染法 (ICC ISH)观察活化的T淋巴细胞 (CD2 5 )与IL 5mRNA表达的相关性及TP、地塞米松 (DM)的影响 ;同时就TP的作用与DM相比较。结果　致敏小鼠T淋巴细胞CD2 5与IL 5mRNA表达均显著高于正常对照组 (P <0 0 1)。CD2 5 IL 5mRNA双染结果显示 :CD2 5 + IL 5mRNA+ T淋巴细胞显著高于正常对照组 (P <0 0 1) ,CD2 5 - IL 5mRNA- T淋巴细胞显著低于正常对照组 (P <0 0 1) ,CD2 5 + IL 5mRNA- 及CD2 5 - IL 5mRNA+ T淋巴细胞与正常对照组间无显著差异(P >0 0 5 )。经TP、DM处理后 ,致敏小鼠T淋巴细胞CD2 5与IL 5mRNA表达均显著低于致敏组 (P <0 0 1)。CD2 5 IL 5mRNA双染结果显示 :CD2 5 + IL 5mRNA+ T淋巴细胞显著低于致敏组 (P <0 0 1) ,CD2 5 - IL 5mRNA- T淋巴细胞显著高于致敏组 (P <0 0 1) ,而CD2 5 + IL 5mRNA- 及CD2 5 - IL 5mRNA+ T淋巴细胞与致敏组间无显著性差异 (P >0 0 5 )。TP组与DM组间无显著性差异 (P >0 0 5 )。结论　IL 5mRNA表达与T淋巴细胞活化关系密切。TP、DM抑制T淋巴细胞活化可能是其抑制IL 5产生的机制之     

Measurement of basophil-activating capacity of grass pollen allergens, allergoids and hypoallergenic recombinant derivatives by flow cytometry using anti-CD203c

BACKGROUND: The assessment of the basophil-activating potential is an important aspect in the development of improved preparations for specific immunotherapy. The aim of the study was to evaluate the suitability of CD203c expression as a measure of basophil activation to compare allergoids with original allergen extracts, and recombinant hypoallergenic allergen derivatives with recombinant wild-type and natural allergens. METHODS: Heparinized whole blood samples from grass pollen allergic subjects were stimulated with grass pollen allergens and allergen derivatives followed by labelling of the basophils with PE-conjugated anti-CD203c. After lysis of the erythrocytes and fixation, the basophils were detected by flow cytometry. In some experiments, histamine release was determined simultaneously. RESULTS: Grass pollen allergoids revealed a 10-10 000-fold reduction of basophil-activating capacity measured by CD203c expression. The deletion mutant DM4 of rPhl p 5b showed stronger hypoallergenic characteristics in a range of 50-10 000-fold reduction, whereas a combination mutant of rPhl p 5b and Phl p 6 revealed less hypoallergenic features. Histamine release experiments led to a similar outcome as CD203c measurement. CONCLUSIONS: The measurement of CD203c expression on basophils by flow cytometry provides a rapid and sensitive method for the estimation of the allergic or hypoallergenic features of allergen preparations. The results demonstrated the hypoallergenicity of grass pollen allergoids and of the rPhl p 5b variant DM4, which may be a candidate in future preparations for specific immunotherapy.     

Demonstration of human motor cortex activation using SPECT

Summary Recent advances in single photon emission computed tomography (SPECT) have allowed improved image resolution with lower doses of labelled tracer. Capitalizing on these improvements, the authors have developed a new SPECT protocol for imaging neuronal activation. We outline this technique and describe how it can demonstrate increased human motor cortex activity in normal subjects performing a motor task. The ability to accurately demonstrate neuronal activation with SPECT using this method may have important scientific and clinical implications.     

急性胰腺炎腺泡细胞内胰酶激活机制的探讨

采用缺乏胆碱的乙硫酸饮食喂养幼年雌鼠诱发急性坏死性胰腺炎时，胰组织蛋白含量及淀粉酶活性升高，胰腺腺泡细胞内酶原颗粒大量积聚，胰管内均质性分泌物明显减少，表明急性胰腺炎时腺泡细胞对酶原颗粒的细胞外放作用受到抑制。腺泡细胞内大量空泡形成，通过噬分泌作用，发生酶原颗粒和溶酶体或空泡间隙融合，致使溶酶体酶对胰酶原发生水解，导致胰酶干腺泡细胞内被激活，从而引起胰腺自身消化。     

绞股蓝总皂甙刺激淋巴细胞活化及分泌IL—2

绞股蓝总皂甙在体外能刺激大鼠和小鼠脾淋巴细胞自发性3~H-TdR掺入;与次亚适量ConA(5μg/ml)配伍,能协同刺激小鼠脾淋巴细胞3~H-TdR掺入;在体外还能刺激大鼠脾淋巴细胞自发分泌白细胞介素-2(IL-2)。提示绞股蓝总皂甙具有轻度类似ConA的促有丝分裂原样作用。     

生物材料体外动态血清接触对CH50及补体的影响

选用硅橡胶管（ＳＲ）、涤纶纤维材料（ＰＥＴＦ）、聚氯乙烯管（ＰＶＣ）及用醋酸纤维素（ＣＡ）、聚醚砜（ＰＥＳ）、聚氨酯涂层的聚氯乙烯管，分别与血清在３７℃下进行循环接触，根据接触前（０ｍｉｎ）、接触后５、１５、６０、１８０ｍｉｎ取血清，用ＩＬ－Ｍｏｎａｒｃｈ７６１型生化分析仪测定Ｃ３、Ｃ４，用试管法测定ＣＨ５０，实验结果表明，ＣＡ，ＰＥＴ，ＳＲ材料接触后血清的Ｃ３、Ｃ４，ＣＨ５０明显低于接触前，其中     

Dynamics of the expression of activation marker complex during polyclonal activation of human peripheral blood CD4+ and CD8+ lymphocytesin vitro

A study of the expression of early and late activation markers on the surface of regulatory subsets of human peripheral blood T lymphocytes revealed marked differences between patients with bronchopulmonary pathology and healthy donors in the dynamics of CD25, CD71, and HLA-DR expression. The results are of significance for the evaluation of both the activation state of the cell and its functional potential in the realization of the immune response. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 11, pp. 483–485, November, 1994 Presented by R. V. Petrov, Member of the Russian Academy of Sciences