Induction of the anti-ergotypic response

The injection of syngeneic activated T cells into rodents caninduce a T cell response against activation markers of the Tcells, ergotopes. The responding antl-ergotypic T cells havebeen shown to suppress experimental autoimmune encephalomyelitis(EAE). This paper reports the characteristics of the antl-ergotypicresponse. It was found that irradiated activated T cells wereas good as untreated living activated T cells in inducing anti-ergotypiccells in vivo. Glutardialdehyde-fixed (0.3%) cells were poorstimulators in vivo and non-stimulatory in vitro. Dilution ofglutardialdehyde to 0.003% before fixation preserved the stimulatorycapacity in vitro. Fixation or irradiation of T cells at differenttimes after activation showed that the stimulatory ergotopeappears only after more than 12 h of activation. This ergotopeis not secreted by activated T cells, but is a structural componentof the activated T cell. Injection of solubilized proteins fromactivated T cells, but not of supernatants from activated Tcells, was able to induce an anti-ergotypic response in vivo.In vitro supernatants from activated T cells also were not stimulatoryto anti-ergotypic T cells. The anti-ergotypic response couldbe measured in draining lymph nodes 3 days after injection,reached a maximum after 7–10 days and subsided thereafter.It was earlier and stronger than the anti-ldiotypic response.Induction of the response was dose dependent. As few as 100cells were able to induce a marked anti-ergotypic response.The ease of the induction and the strength of the anti-ergotypicresponse suggest a physiological role in immunoregulatlon.     

Immune Abnormalities Induced by Human Endogenous Retroviral Peptides: With Reference to the Pathogenesis of Systemic Lupus Erythematosus

P15E is a specific sequence among the envelope gene (env)-encoded transmembrane proteins of exogenous and endogenous retroviruses. A synthetic peptide (CKS-17) that shows homology to this p15E region in several species of retrovirus is known to induce immune abnormalities. In this study, we examined the effect of a synthetic peptide derived from a region of human endogenous retrovirus (HERV) clone 4-1 ( 4-1) similar to sequences of CKS-17 on the induction of systemic lupus erythematosus (SLE)-related immune abnormalities. Our results indicated that this peptide could induce T-cell activation and anergy in normal peripheral blood mononuclear cells, and the peptide could also promote the production of interleukins IL-6 and IL-16. These phenomena are representative immune abnormalities observed in SLE patients. Thus, our findings support the possibility that HERV acts as a pathogen in human SLE.     

Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of β-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities. Received: 9 January / 5 March 1998     

DAF与CD3协同刺激人外周血T细胞活化的实验研究

目的：探讨人促衰变加速因子（decay-accelerating factor,DAF)作为共刺激分子参与T细胞活化的作用机制。方法：观察3株针对DAF不同SCR的单抗单独使用或者与抗人CD3单抗联合使用时，对人外周血T细胞增殖、IL－2分泌以及胞浆Ca^2 水平的影响。结果：所用3株抗人DAF单抗不能活化T细胞，而抗人DAF单抗与抗人CD3单抗可以协同刺激T细胞增殖，促进IL－2分泌以及提高胞浆Ca^2 水平。结论：抗人DAF单抗与抗人CD3单抗可协同刺激人外周血T细胞活化。     

Interferon-α activates cytotoxic function but inhibits interleukin-2-mediated proliferation and tumor necrosis factor-α secretion by immature human natural killer cells

Natural killer (NK) cells play an important role in host defense mechanisms against infection and neoplasia. Interferon- (IFN-) has been shown to activate NK cells and to augment their cytotoxic activity, albeit its role in the maturation pathway of NK cells has not been elucidated. The present study examined whether IFN- activates the immature NK subset (Free cells) to become cytotoxic and also ascertained whether IFN- uses the same pathway of activation as that mediated by interleukin-2 (IL-2). Incubation of sorted Free cells overnight with IFN- resulted in augmentation of their cytotoxic function against NK sensitive target cells. The enhanced cytotoxic activity was not accompanied by a new recruitment of NK-target binder cells but by an increase in the frequency of killer cells in the conjugate fraction. Activation of the Free subset by IFN- resulted in upregulation of CD69, CD11b, and CD2 surface expression and stimulated secretion of IFN-. Unlike IL-2, IFN- did not stimulate the Free cells to proliferate or secrete TNF- and activation of cytotoxicity and modulation of surface antigens by IFN- were independent of TNF-. The failure of IFN- to stimulate secretion and proliferation by Free cells appeared to be mediated by negative signals. This was corroborated in experiments demonstrating that when Free cells were cultured with both IFN- and IL-2, a significant inhibition was observed for both the IL-2 dependent secretion of TNF- and proliferation. These results demonstrate that IFN- serves as both an activator and a regulator of NK function. Further, activation of the immature Free NK cells by IL-2 and IFN- proceeds by TNF--dependent and independent pathways, respectively. The findings also support our contention that the mechanism of activation of the cytotoxic machinery of NK cells is not linked to the mechanism of activation of cytokine secretion and/or proliferation.Abbreviations used IFN interferon - IL interleukin - PBL peripheral blood leukocytes - PE phycoerythrin - PE-GAM PE-conjugated Fab2 goat anti-mouse IgG - NK natural killer - NRS normal rabbit serum - TNF tumor necrosis factor - FCS fetal calf serum - FITC fluorescein isothiocyanate - PBS phosphate-buffered saline - MACS magnetic cell sorting - ELISA enzyme-linked immunosorbent assay - BSA bovine serum albumin - PKC protein kinase C - mAb monoclonal antibody - PBMC peripheral blood mononuclear cells - BCLL B-chronic lymphocytic leukemia - E effector - T target     

Induction of nitric oxide (NO) synthesis in murine macrophages requires potassium channel activity

The activation of macrophages for antimicrobial responses is a multistage event involving numerous intracellular signalling cascades that makes possible target cell destruction by these effector cells. This study examined the effects of different potassium channel inhibitors and activators on the NO production of murine macrophage-like cell lines P388D.1 and B10-4(S). We found that the potassium channel inhibitors tetraethylammonium, 4-aminopyridine, and quinine caused dose-dependent reductions in the NO production of macrophages, and that the potassium channel activator, minoxidol, caused a dose-dependent enhancement of NO production. The inhibition of NO production was due to involvement of potassium channels in the priming stage of macrophage activation, since pretreatment with the priming agent interferon-gamma partially restored the NO response of the macrophages. The results of this study demonstrate a link between potassium channel activity and the activation of anitimicrobial functions of murine macrophages.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

Identification of two distinct CD5- B cell subsets from human tonsils with different responses to CD40 monoclonal antibody

This study investigated the response of different CD5^? B cell subsets to CD40 monoclonal antibody (mAb) in various combinations with interleukin (IL)-4 or rabbit anti-human μ chain antibody (a-μ-Ab). The different CD5 B cell subsets were isolated from tonsillar B cell suspensions depleted of CD5⁺ B cells and subsequently fractionated on Percoll density gradients. While resting CD5⁺ B cells proliferated and produced IgM molecules in response to a-μ-Ab, IL-4 and CD40 mAb as well as to Staphylococcus aureus Cowan strain I (SAC) and IL-2, resting CD5^? B cells, which were co-purified in the same 60% Percoll fractions, consistently failed to respond. These cells were, however, activated by the stimuli employed, as demonstrated by their capacity to express the surface activation markers CD69, CD25 and CD71. Resting CD5⁺ B cells had the typical phenotype of mantle zone B cells (IgM⁺ IgD⁺ CD39+ CD38^? CD10^? CDw75^dim), whereas resting CD5^? B cells were CD38^? CD39^? CD 10^? CDw75 intermediate and expressed surface IgM but relatively little surface IgD and could not be classified as mantle zone or germinal center cells. The finding that purified germinal center cells (CD38⁺ CD10⁺ CD39^? CDw75^bright, IgG⁺) responded to CD40 mAb and IL-4 and also to SAC plus IL-2 further underlined the differences to resting CD5^? B cells. However, some of the data collected suggest possible relationships between CD5^? B cells and germinal center cells. The CD5^? B cells isolated from the 50 % Percoll fraction proliferated in response to a-μ-Ab, CD40 mAb and IL-4 as well as to SAC and IL-2. These cells had the same mantle zone B cell phenotype as the CD5⁺ B cells, but their capacity to respond to the stimuli in vitro was unrelated to a possible contamination with CD5⁺ B cells, as documented by the appropriate controls. Furthermore, upon exposure to SAC or phorbol esters, the large majority of CD5^? B cells from the 50 % Percoll fraction did not express surface CD5 and there was very little if any accumulation of CD5 mRNA. Finally, most of the cycling cells in the stimulated CD5^? B cells did not express CD5. The CD5^? B cells from the 50 % Percoll fraction were comprised of a consistent proportion of cells that expressed surface activation markers. The removal of these cells abrogated the capacity of the suspensions to respond to the stimuli in vitro, possibly suggesting that these cells additional activation signals in vivo which were essential to acquire the capacity to respond and that could not be reproduced in vitro. The present study underlines the phenotypic and functional heterogeneity of CD5^? B cells and contributes to the identification of two subsets of these cells which differ in phenotype, tissue distribution and in vitro responses to different stimuli.     

Modulation of interferon-γ receptor during human T lymphocyte alloactivation

Previous work has shown that neutralization of physiologically secreted interferon(IFN)-γ or blockade of its receptor during T lymphocyte activation inhibits both proliferation and cytotoxic T lymphocyte generation, suggesting that IFN-γ plays a crucial role in T lymphocyte induction and differentiation. In this study, the kinetics of the surface expression of the 90-kDa IFN-γ receptor (IFN-γR) was followed during human mixed lymphocyte reaction (MLR) to alloantigens. IFN-γR mRNA is constitutively expressed on resting peripheral blood lymphocytes emerging from nylon wood column (NW-PBL) and its expression increases two- to threefold on alloactivated NW-PBL. IFN-γR protein is poorly expressed on the membrane of resting CD3⁺ cells, but up-modulates after 3-day MLR and sharply down-modulates at day 6. Both the p55 and the p75 chains of interleukin-2 receptor (IL-2R) were shown to up-modulate in parallel with IFN-γR, whereas they were still highly expressed at day 6. After alloactivation, IFN-γ and IL-2 secretion starts at 24 h, peaks at day 3 and decreases just when IFN-γR and IL-2R begin to up-modulate. Proliferation peaks at day 6. Lastly, stimulation with distinct cell populations showed that the intensity of lymphocyte proliferation, IFN-γR membrane up-modulation, and IFN-γ and IL-2 secretion are regulated in a parallel manner, thus suggesting that they are interrelated. Taken as whole these results demonstrate that increased expression of IFN-γR on T lymphocytes can be a critical event during their activation, and strongly support the hypothesis that IFN-γ/IFN-γR interaction provides a signal for its progression.     

Changes in isometric force- and relaxation-time,electromyographic and muscle fibre characteristics of human skeletal muscle during strength training and detraining

HÄKKINEN, K., ALÉN, M. & KOMI, P.V. 1985. Changes in isometric force- and relaxation-time, electromyographic and muscle fibre characteristics of human skeletal muscle during strength training and detraining. Acta Physiol Scand 125, 573–585. Received 26 January 1985, accepted 9 May 1985. ISSN 0001–6772. Department of Biology of Physical Activity and Department of Health Sciences, University of Jyväskylä, Jyväskylä, Finland. Eleven male subjects (20–32 years) accustomed to strength training went through progressive, high-load strength training for 24 weeks with intensities ranging variably between 70 and 120% during each month. This training was also followed by a 12-week detraining period. An increase of 26.8% (P < 0.001) in maximal isometric strength took place during the training. The increase in strength correlated (P < 0.05) with significant (P < 0.05–0.01) increases in the neural activation (IEMG) of the leg extensor muscles during the most intensive training months. During the lower-intensity training, maximum IEMG decreased (P < 0.05). Enlargements of muscle-fibre areas, especially of fast-twitch type (P < 0.001), took place during the first 12 weeks of training. No hypertrophic changes were noted during the latter half of training. After initial improvements (P < 0.05) no changes or even slight worsening were noted in selected force-time parameters during later strength training. During detraining a great (P < 0.01) decrease in maximal strength was correlated (P < 0.05) with the decrease (P < 0.05) in the maximum IEMGs of the leg extensors. This period resulted also in decreases (P < 0.05) of the mean muscle-fibre areas of both fibre types. It was concluded that improvement in strength may be accounted for by neural factors during the course of very intensive strength training. Selective training-induced hypertrophy also contributed to strength development but muscle hypertrophy may have some limitations during long-lasting strength training, especially in highly trained subjects.