Both CD8+ and CD16+ human T cell clones can provide B cell help for immunoglobulin production

Summary The functional properties of two CD4⁺CD8⁻CD16⁻, five CD4⁻CD8⁺CD16⁻ and three CD4⁻CD8⁻CD16⁺ human T cell clones were compared. All CD4⁻ T cell clones displayed strong cytolytic activity in the lectin-dependent lytic assay against the P815 murine mastocytoma cell line, but only the CD4⁻CD8⁻CD16⁺ T cell clones exhibited lytic activity against the natural killer-sensitive K562 cell line. Upon activation with anti-CD3 monoclonal antibody, all T cell clones were able to support IgM and IgA synthesis in autologous B cells. Both CD4⁺ and CD4⁻ T cell clones required cell-to-cell interaction with the B cells in order to exert their helper activity for immunoglobulin production. However, unlike CD4⁺, CD4⁻CD8⁺CD16⁻ and CD4⁻CD8⁻CD16⁺ T cell clones provided helper function for immunoglobulin synthesis only when low T/B cell ratios were used in culture. At higher T/B cell ratios, there was a decline in the B cell helper activity of CD4⁻ T cell clones that was probably related to the expression of cytolytic capacity against the antigen-presenting B cell. These data support the notion that under certain experimental conditions even cytotoxic T lymphocytes and natural killer cells may provide B cell helper function.     

Expression patterns of the lipopolysaccharide receptor CD14, and the FCgamma receptors CD16 and CD64 on polymorphonuclear neutrophils: data from patients with severe bacterial infections and lipopolysaccharide-exposed cells

In polymorphonuclear neutrophils (PMN) CD14, one of the receptors for lipopolysaccharides (LPS) is stored intracellularly as a preformed protein, with only few receptors expressed on the surface. We now report that in patients with severe bacterial infections, CD14 expression is profoundly upregulated, as is CD64 (FcgammaRI), the high-affinity receptor for IgG, whereas CD16 (FcgammaRIII) was partly lost from the surface. To further analyze regulation of these receptors, PMN of healthy donors were exposed to low doses of LPS. By brief exposure (10-120 min) to LPS, CD14 was transferred to the surface in a cytochalasin B-sensitive manner, as were CD16 and CD64. Prolonged culture (up to 48 h) resulted in a further upregulation of CD14, sustained expression of CD64, and profound decline of CD16, yielding a similar pattern of receptor expression as seen in the patients. Subsequent studies revealed that LPS induced de novo synthesis of CD14: the increase of surface expression could be inhibited by cycloheximide and by interfering with a known LPS-induced signaling event, the translocation of NFkappaB. Moreover, an up to 10-fold increase of specific mRNA was seen, as was incorporation into CD14 of 35S-methionine. The de novo synthesis prolonged expression of CD14, whereas the CD16 expression declined, generating a PMN phenotype characteristic for severe infection and indicative of escape from apoptosis of a PMN subpopulation.     

深圳地区无偿献血者群体CD36抗原缺失型的表型分析

目的分析深圳地区无偿献血者群体CD36抗原缺失型的筛查和表型情况。方法应用单克隆抗体和流式细胞技术在327名献血者中检测CD36抗原缺失型个体,应用血小板单克隆抗体捕获酶联免疫技术和流式细胞术筛查献血者血浆中的血小板抗体。结果此次筛查得到的深圳献血者的CD36抗原缺失型频率为3.06%(10/327),其中Ⅰ型占0.31%(1/327),Ⅱ型占2.75%(9/327);在3名女性CD36抗原缺失型献血者的血浆中,均未检出血小板反应性抗体,包括抗-CD36。结论深圳地区献血者群体中存在CD36抗原缺失型个体,其频率与亚洲其他地区人群的频率相当;对CD36的同簇免疫应该予以重视。     

Markedly different pathogenicity of four immunoglobulin G isotype-switch variants of an antierythrocyte autoantibody is based on their capacity to interact in vivo with the low-affinity Fcgamma receptor III

Using three different Fcgamma receptor (FcgammaR)-deficient mouse strains, we examined the induction of autoimmune hemolytic anemia by each of the four immunoglobulin (Ig)G isotype-switch variants of a 4C8 IgM antierythrocyte autoantibody and its relation to the contributions of the two FcgammaR, FcgammaRI, and FcgammaRIII, operative in the phagocytosis of opsonized particles. We found that the four IgG isotypes of this antibody displayed striking differences in pathogenicity, which were related to their respective capacity to interact in vivo with the two phagocytic FcgammaRs, defined as follows: IgG2a > IgG2b > IgG3/IgG1 for FcgammaRI, and IgG2a > IgG1 > IgG2b > IgG3 for FcgammaRIII. Accordingly, the IgG2a autoantibody exhibited the highest pathogenicity, approximately 20-100-fold more potent than its IgG1 and IgG2b variants, respectively, while the IgG3 variant, which displays little interaction with these FcgammaRs, was not pathogenic at all. An unexpected critical role of the low-affinity FcgammaRIII was revealed by the use of two different IgG2a anti-red blood cell autoantibodies, which displayed a striking preferential utilization of FcgammaRIII, compared with the high-affinity FcgammaRI. This demonstration of the respective roles in vivo of four different IgG isotypes, and of two phagocytic FcgammaRs, in autoimmune hemolytic anemia highlights the major importance of the regulation of IgG isotype responses in autoantibody-mediated pathology and humoral immunity.     

A novel method for determining functional LDL receptor activity in familial hypercholesterolemia: Application of the CD3/CD28 assay in lymphocytes

BackgroundThe objective of this study was to develop a new and simple method for measuring low-density lipoprotein receptor (LDLR) activity using peripheral lymphocytes enabling us to clinically diagnose familial hypercholesterolemia (FH) and ascertain the involved mutations (such as K790X mutation), that might not be clearly detected in the conventional method.MethodsOur method comprised the following 2 features: first, we used anti-CD3/CD28 beads to stimulate T-lymphocytes to obtain a uniform fraction of lymphocytes and maximum up-regulation of LDLR. Second, we excluded the possibility of overestimation of lymphocyte signals bound only to its surface, by adding heparin to the cultured lymphocytes used for the LDLR assay.ResultsBased on the genetic mutation, the FH subjects were divided into 2 groups, K790X, (n = 20) and P664L, (n = 5), and their LDLR activities was measured by this method, which was found to be 55.3 ± 8.9% and 63.9 ± 13.8%, respectively, of that of the control group (n = 15). In comparison, the LDLR activity was 86.1 ± 11.6% (K790X) and 73.3 ± 6.3% (P664L) of that of the control group when measured by the conventional method, indicating that impairment of LDLR function in FH K790X subjects was much more clearly differentiated with our method than with the conventional method (paired t-test, p < 0.0001). The levels of LDLR expression also showed similar tendencies, that is, 89.4 ± 13.2% (K790X) and 76.9 ± 17.4% (P664L) of that of the control group when measured by the conventional method, and 78.1 ± 9.7% (K790X) and 70.3 ± 26.5% (P664L) when measured by our new method. In addition, we confirmed that there was little influence of statin treatment on LDLR activity among the study subjects when our method was used.ConclusionThese results demonstrate that our new method is applicable for measuring LDLR activity, even in subjects with an internally defective allele, and that T-lymphocytes of the FH K790X mutation possess characteristics of that allele.     

Signal transduction by the CD2 antigen in T cells and natural killer cells: requirement for expression of a functional T cell receptor or binding of antibody Fc to the Fc receptor, Fc gamma RIIIA (CD16)

Crosslinking of CD2 antigen on T lymphocytes and natural killer (NK) cells leads to a rise in cytoplasmic-free Ca2+ concentration ([Ca2+]i). However, CD2 seems unlikely to interact directly with the second messenger pathways since signaling via CD2 is poor in T cells that lack the T cell receptor (TCR) and is absent in L cells or insect cells that express CD2. In contrast, NK cells that are also TCR- can be triggered via CD2, but it is unclear as to whether the CD16 Fc receptor (FcR) may facilitate this effect. The CD16 transmembrane molecule is expressed in a complex with the zeta homodimer or the zeta/gamma heterodimer and these dimers are also associated with the TCR complex. Thus, it seemed that zeta chains may provide the link between signaling on NK cells and T cells. This could be tested on TCR- cells since when CD16 is transfected into T cells it is expressed in a complex with TCR zeta homodimer or the zeta/gamma heterodimer. At first, potentiation of CD2 signaling was seen on TCR- Jurkat cells expressing CD16, but this was found to be dependent on trace levels (1%) of IgG in F(ab')2 antibody preparations. With pure F(ab')2, the effect was lost. Signaling on a rat NK cell line was also re-examined with F(ab')2 antibodies that had no IgG contamination, and again no signal transduction via CD2 was seen. We thus conclude that there is no clear evidence for potent signaling via CD2 on cells that lack a TCR complex and that TCR zeta chain expressed at the cell surface is not sufficient to potentiate signaling via CD2 as measured by an increase in [Ca2+]i.     

In vitro and in vivo effects of the new nonpeptide bradykinin B2 receptor antagonist, LF 16-0335C, on guinea-pig and rat kinin receptors

Activation of the kinin-kallikrein system and stimulation of bradykinin (BK) B2 receptors are thought to play an important role in the pathophysiology of inflammation and pain. In the present study, we report the pharmacological properties of a novel nonpeptide bradykinin B2 receptor antagonist, LF 16-0335C, (1-[[3-[(2,4-dimethylquinolin-8-yl) oxymethyl]-2,4-dichloro-phenyl]sulfonyl]-2(S)-[[4-[4- (aminoiminomethyl)-phenylcarbonyl]piperazin-1-yl]carbo nyl]pyrrolidine, 2HCl). In binding studies, LF 16-0335C competed with [3H]bradykinin giving Ki values of 1.65 +/- 0.36 nM and 2.20 +/- 0.30 nM in membrane preparations from rat uterus (RU) and guinea-pig ileum (GPI), respectively. In functional experiments, LF 16-0335C inhibited in a competitive manner BK-induced contractions of both isolated RU and GPI, leading to calculated pA2 values of 7.70 +/- 0.70 and 8.30 +/- 0.30, respectively. The inhibitory effect of LF 16-0335C was fully reversible by washing in the guinea-pig ileum. In vivo, LF 16-0335C given intravenously inhibited in a dose-dependent manner BK-induced hypotension in both animal species, although it was more potent in the guinea-pig than in the rat (ED50, 2.5 +/- 1.6 micrograms/kg versus 22.6 +/- 2.3 micrograms/kg). BK is a potent constrictor of guinea-pig airways and this effect was markedly attenuated by LF 16-0335C. In contrast, LF 16-0335C did not affect histamine- and acetylcholine-induced hypotensive response in the rat. We conclude that LF 16-0335C is a potent and selective nonpeptide B2 receptor antagonist which equally binds to the rat and guinea-pig receptor but displays a different in vivo potency in the two species. Therefore, this drug represents a useful tool to better assess the role of bradykinin in pathophysiological conditions.     

实时荧光定量PCR检测CD44和RHAMM在胃肠肿瘤中的表达研究

目的探讨CD44和透明质酸介导运动的受体（RHAMM）在胃肠肿瘤组织中的表达,研究其在肿瘤诊断中的价值。方法收集病理分期为Ⅱ～Ⅲ级胃肠道恶性肿瘤样本30例,并通过实时荧光定量聚合酶链反应（PCR）对CD44和RHAMM进行定量分析。结果CD44和RHAMM在癌组织中相对于管家基因GAPDH的表达中位数分别为1．587和1．074,正常组织中分别为0．704和0．098;肿瘤组织中,区域淋巴结转移组的CD44拷贝数／GAPDH拷贝数的中位数为1．650,未转移组相应的均值为1．431;区域淋巴结转移组的RHAMM拷贝数／GAPDH拷贝数的中位数为1．301,未转移组的中位数为1．064。区域淋巴结转移样本其CD44和RHAMM表达同步增高,肿瘤组织中CD44和RHAMM之间的相关系数（r）为0．96。结论胃肠肿瘤中的CD44和RHAMM表达高于正常组织,且两者表达具相关性。CD44和RHAMM的表达量增加的多少不足以判断肿瘤是否有区域淋巴转移,CD44和RHAMM同时增高可能预示肿瘤的淋巴转移。     

Will killing the last HIV1 particle cure AIDS patients? II: Second part. Decrease of viral load and of T-suppressor cells, and increase of the cytotoxic cells, without effect on CD4, after the use of 10 virostatics applied in 3 or 4 drug combinations of different sequences. The time for CD4 immunotherapy?

We reported in the first part of this editorial [1] and in an article on AIDS therapy with five HIV₁ virostatics applied in two then three, or initially three, or initially four agent combinations, given in 3 week sequences differing from each other due to drug rotation [2], the contrast between: a) the decrease of viral load, possibly below the detectable level, b) the absence of effect on the helper CD4⁺, the CD8⁺ C57⁻ cytotoxics and the CD8⁺ C57⁺ suppressor cells. We proposed a thesis according to which the HIV₁-AIDS complex might have another pathogenic component other than HIV₁, ie, a microchimerism graft-versus-host reaction (GvH) or an autologous GvH-like reaction [1].Shifting from five to 10 virostatics owing to the availability of lamivudine or 3TC [3], stavudine or d4T [4] and three HIV₁ protease inhibitors, saquinavir [5], ritonavir [6] and indinavir [7], applied according to the same modality, we have enhanced the reduction of viral load, and significantly decreased the CD8⁺ C57⁺ suppressor cell counts, and increased those of the CD8⁺ C57⁻ cytotoxic cells.This result which indirectly shows the role of HIV₁ in the increase of suppressor CD8⁺ cells, hence in the late loss pf immune memory and of opportunistic infections [8], reinforces the thesis of a role, in AIDS pathogenesis, of a latent GvH reaction activated by HIV, primo-infection, and its evolution from the hyperplastic phase to the hypoplastic one, which, inducing severe immune suppression, is responsible for HIV₁ active infection relapse after the so-called latent phase [1].Hence the proposition we make, of an indication of CD4 modulation with non specific immunotherapy by bestatin [9], of which we showed the effect in another population of HIV₁-AIDS complex patients [10]. Its effect can be potentiated by tuftsin [11, 12]. When the suppressor cell number goes up over that of the cytotoxic one after the HIV, active infection relapse, Interferon y could be added, which, by amplifying the CD28 pathway [13] on CD8⁺ cytotoxics, while suppressor cells lack CD28 [14], which might reestablish a ratio of suppressor over cytotoxic cells nearer to normal.It remains that the role of the five secondarily included agents in the decrease of suppressor cells will only be attributed with certainty and entirely to their virostatic effect, if it is shown that none of them exerts a selective anti-suppressor cell action.