CD2-mediated signaling in T cells lacking the ζ-chain-specific immune receptor tyrosine-based activation (ITAM) motif

T lymphocytes can be activated via the T cell receptor (TCR) or by triggering through a number of other surface structures, including the CD2 co-receptor molecule. Signaling through the CD2 molecule was shown previously to be dependent on the TCR-associated ζ-chain. Here, we show that CD2-induced activation also functions in T cells which express ζ-chains lacking a functional immune-receptor tyrosine-based activation motif (ITAM). TCR-positive T cells that express only the transmembrane part of the ζ-chain protein and thus lack a functional ζ-derived ITAM readily produce interleukin (IL)-2 when cross-linked with CD2-specific monoclonal antibodies (mAb). TCR-negative T cell hybridomas expressing minimal receptors consisting of an extracellular CD25 and an intracellular ζ-chain-derived segment were effectively stimulated via CD2-specific mAb. For CD2-mediated co-stimulation of TCR-negative cells, two ζ-chain-derived ITAM were sufficient to induce IL-2 when the CD2 molecules were co-cross-linked with the chimeric CD25-ζ molecules. Taken together, our results show that CD2-induced signaling does not necessarily employ the ζ-chain in TCR-positive cells and that CD2-dependent co-stimulation in TCR-negative cells can be mediated via two functional ζ-chain-derived ITAM.     

Mouse blastocysts release a lipid which activates anandamide hydrolase in intact uterus

Anandamide (N-arachidonoylethanolamine, AEA) is a major endocannabinoid, known to impair mouse pregnancy and embryo development and to induce apoptosis in blastocysts. Here we show that mouse blastocysts rapidly (within 30 min of culture) release a soluble compound, that increases by approximately 2.5-fold the activity of AEA hydrolase (fatty acid amide hydrolase, FAAH) present in the mouse uterus, without affecting FAAH gene expression at the translational level. This "FAAH activator" was produced by both trophoblast and inner cell mass cells, and its initial biochemical characterization showed that it was fully neutralized by adding lipase to the blastocyst-conditioned medium (BCM), and was potentiated by adding trypsin to BCM. Other proteases, phospholipases A(2), C or D, DNAse I or RNAse A were ineffective. BCM did not affect the AEA-synthesizing phospholipase D, the AEA-binding cannabinoid receptors, or the selective AEA membrane transporter in mouse uterus. The FAAH activator was absent in uterine fluid from pregnant mice and could not be identified with any factor known to be released by blastocysts. In fact, platelet-activating factor inhibited non-competitively FAAH in mouse uterus extracts, but not in intact uterine horns, whereas leukotriene B(4) or prostaglandins E(2) and F(2)alpha had no effect. Overall, it can be suggested that blastocysts may protect themselves against the noxious effects of uterine endocannabinoids by locally releasing a lipid able to cross the cell membranes and to activate FAAH. The precise molecular identity of this activator, the first ever reported for FAAH, remains to be elucidated.     

米诺环素对小鼠T淋巴细胞体外活化和增殖的影响

研究米诺环素(MNC)对刀豆蛋白A(ConA)刺激的小鼠T细胞体外活化和增殖的影响,并对其免疫调节作用进行初步探讨。以ConA刺激小鼠淋巴结来源的淋巴细胞,以不同终浓度的MNC与T细胞共培养,荧光抗体染色结合流式细胞术,检测T细胞早期活化抗原CD69的表达;用羧基荧光素双醋酸盐琥珀酰(CFDA-SE)染色结合流式细胞术,并用ModFit软件分析T细胞增殖相关指数。结果:终浓度为10、25和50μmol/L的MNC显著抑制ConA诱导的T细胞CD69的表达,由ConA组的71.20%±1.08%,分别降低为64.43%±1.39%、53.34%±1.26%和33.65%±1.91%;CFDA-SE染色结果显示,培养48 h和72 h的ConA组T细胞的增殖指数(PI)分别为1.57±0.03和2.06±0.02,各浓度的MNC对ConA刺激的T细胞增殖具有明显的抑制作用,以50μmol/L的MNC抑制作用最明显,48 h和72 h的PI分别为1.01±0.02和1.03±0.01,与相应时间的对照组比较,均有统计学意义(P<0.01)。MNC能有效地抑制小鼠T细胞的体外活化和增殖。具有独立于其抗菌活性的抗炎作用。     

Correlated changes in feeding behavior on selection for large and small body size in mice

An automated method was used to record the temporal pattern of feeding of lines of mice selected over 15 generations for high and low body weight (L-mice and S-mice, respectively). Both L-mice and S-mice eat in meals concentrated during the night, and meal frequency is similar in the two lines, but L-mice consume much larger meals, each made up of many more separate feeding bouts. The outbred strain from which the selected lines were derived has a similar basic pattern of feeding in meals, which becomes like that of L-mice when the animal's thermogenic metabolic rate is high, and like that of S-mice when it is low, suggesting that the differences between the feeding patterns of the two selected lines are a secondary consequence of alterations in whole body metabolic rate.     

Changes in plasma levels of interleukin-2 receptor in relation to disease exacerbations and levels of anti-dsDNA and complement in systemic lupus erythematosus.

Interleukin-2 receptor (IL-2R) is expressed and released predominantly by activated T cells. In order to investigate whether disease exacerbations of systemic lupus erythematosus (SLE) are preceded by T cell activation, we prospectively measured levels of IL-2R once a month, from 6 months prior to exacerbations until 1 month afterwards. To assess the temporal relation between T cell activation and B cell activation, we measured, in addition, levels of anti-dsDNA, complement C3/C4, and total IgG. During a mean follow-up period of 23 months, 40 exacerbations occurred in 21 out of the 71 participating patients. For the present study one exacerbation per patient was evaluated. During exacerbation levels of IL-2R were increased in 18 out of the 21 cases and correlated with levels of anti-dsDNA (P less than 0.02), C3 (P less than 0.02), and C4 (P less than 0.01), but not with the score of the disease activity index. Levels of IL-2R rose prior to the exacerbation (P less than 0.02) and fell afterwards following treatment (P less than 0.05). Even in the absence of disease activity or during minor disease symptoms IL-2R levels were higher (P less than 0.01) than in healthy controls. Sixteen out of the 21 exacerbations (76%) were preceded by a significant increase in IL-2R. Changes in levels of anti-dsDNA and complement C3/C4 tended to precede changes in levels of IL-2R. We conclude that increased levels of IL-2R, compatible with T cell activation, are present in SLE already during inactive disease. These levels further increased prior to exacerbations of disease. As such, IL-2R is an indicator of disease activity in SLE. Serial measurement of IL-2R is a sensitive test for predicting disease exacerbations of SLE.     

Immune deficiency following thermal trauma is associated with apoptotic cell death

Thermal injury-associated specific immune deficiency occurs despite indicators of systemic activation of the lymphoid compartment. We investigated the possibility that postburn immune failure and T cell activation are causally related through activation-induced (apoptotic) cell death. The relationship between the cellular immune response and cell mortality was examined in cultures of peripheral blood mononuclear cells (PBMC) from 14 immunosuppressed patients with extensive burns (35–90% total body surface area). Impaired cellular immunity coincided with significantly reduced cell viability as ascertained by propidium iodide staining and dye reduction assays. Following stimulation with the mitogenic lectin, phytohemagglutinin (PHA), the majority of DNA in patient cultures was fragmented, suggesting the occurrence of apoptotic cell death. Even without stimulation a portion of patient cells was apoptotic as indicated by oligonucleosomal bands on agarose gel electrophoresis. Exogenous interleukin-2 or phorbol ester markedly reduced constitutive as well as PHAinduced DNA fragmentation.In situ demonstration of DNA strand breaks in freshly isolated patient PBMC, by a TdT-based labeling technique, confirmed that a larger fraction (up to 60%) of circulating lymphocytes was undergoing apoptosis on the periphery. These novel observations suggest that apoptosis may play a major role in thermal injury-related cellular immunodeficiency.     

Effective antiretroviral therapy reduces degradation of tryptophan in patients with HIV-1 infection

Antiretroviral therapy (ART) has a significant impact on HIV-1 RNA levels, the CD4 cell count, and immune activation. We examined whether these changes are associated with a change in the rate of tryptophan degradation (expressed as the kynurenine to tryptophan ratio, kyn/trp) as an estimate for the activity of interferon-gamma inducible enzyme indoleamine (, )-dioxygenase (IDO). Plasma levels of tryptophan, kynurenine, and neopterin were measured pretherapy and 6 months postinitiation of therapy in 45 patients with HIV-1 RNA levels of less than 1000 copies/ml 6 months after initiation of ART. Before ART, the patients had decreased tryptophan and increased kynurenine concentrations compared to healthy controls. During ART, average tryptophan levels increased; in the same time kynurenine and kyn/trp decreased (P < 0.001), although not to normal levels. Since pretherapy tryptophan concentrations correlated inversely with neopterin, and kynurenine correlated with viral load and neopterin but not with CD4 cell count, the data support the view that HIV production may induce immune activation and consequently tryptophan is degraded at a higher rate. In agreement, kyn/trp positively correlated with neopterin (r(s) = 0.60, P < 0.001), with virus load (r(s) = 0.37, P = 0.013), and very weakly with CD4(+) cells counts (r(s) = 0.30, P = 0.049). The change in the kyn/trp ratio during ART correlated more strongly with the change in neopterin levels (r(s) = 0.49, P = 0.001) than with the change in HIV RNA levels and weakly with the CD4 cell count. The data underscore the fact that both neopterin production and tryptophan degradation are triggered by immune activation. Tryptophan degradation is increased in HIV infection and partially reversed under ART. The data agree with the concept that immune activation is the common background of IDO activation which may be an important factor underlying T-cell hyporesponsiveness.     

In-vitro development of mouse zygotes following reconstruction by sequential transfer of germinal vesicles and haploid pronuclei

We evaluated whether mouse oocytes reconstructed by germinal vesicle (GV) transfer can develop to blastocyst stage. The oocytes were artificially activated with sequential treatment of A23187 and anisomycin; fertilization was then established by transfer or exchange of pronuclei with those of zygotes fertilized in vivo. Type 1 zygotes were constructed by placing the male haploid pronucleus from a zygote into the cytoplasm of an oocyte that underwent GV transfer, in-vitro maturation and activation; for type 2 zygotes, the female pronucleus was removed from a zygote and replaced with the female pronucleus of an oocyte subjected to GV transfer, in-vitro maturation and activation. Karyotypes of activated oocytes and type 2 zygotes were also subjected to analysis. When cultured in human tubal fluid (HTF) medium, reconstructed oocytes matured and, following artificial activation, consistently developed a pronucleus with a haploid karyotype; the activation rate for this medium was two- to three-fold higher than that of oocytes cultured in M199 (87% versus 30% respectively). Following transfer of a male pronucleus, only 47% of the type 1 zygotes developed to morula or blastocyst stage and embryo morphology was poor. In contrast, 73% of the type 2 zygotes developed to morula or blastocyst stage, many even hatching, with few morphological anomalies. Normal karyotypes were observed in 88% of the type 2 zygotes analysed. These observations demonstrate that the nucleus of a mouse oocyte subjected to sequential nuclear transfer at GV and pronucleus stages is, nonetheless, capable of maturing meiotically, activating normally and supporting embryonic development to hatching blastocyst stage. In contrast, the developmental potential of the cytoplasm of such oocytes appears to be compromised by these procedures.     

Estimating relative physical workload using heart rate monitoring: a validation by whole-body indirect calorimetry

Measuring physical workload in occupational medicine is fundamental for risk prevention. An indirect measurement of total and relative energy expenditure (EE) from heart rate (HR) is widely used but it has never been validated. The aim of this study was to validate this HR-estimated energy expenditure (HREEE) method against whole-body indirect calorimetry. Twenty-four-hour HR and EE values were recorded continuously in a calorimetric chambers for 52 adult males and females (19–65 years). An 8-h working period was retained, comprising several exercise sessions on a cycloergometer at intensities up to 65% of the peak rate of oxygen uptake. HREEE was calculated with reference to cardiac reserve. A corrected HREEE (CHREEE) was also calculated with a modification to the lowest value of cardiac reserve. Both values were further compared to established methods: the flex-HR method, and the use of a 3rd order polynomial relationship to estimate total and relative EE. No significant difference was found in total EE when measured in a calorimetric chamber or estimated from CHREEE for the working period. A perfect linear and identity relationship was found between CHREEE and energy reserve values for intensities ranging from 15% to 65%. Relative physical workload can be accurately assessed from HR recordings when expressed in CHREEE between 15% to 65%, and EE can be accurately estimated using the CHREEE method.