Influence of levothyroxine treatment on serum levels of soluble Fas (CD95) and Fas Ligand (CD95L) in chronic autoimmune hypothyroidism

Fas/FasL-mediated apoptosis results in the destruction of thyrocytes in chronic autoimmune hypothyroidism (CAIH). In this study, we examined the serum levels of soluble Fas (sFas) and soluble sFas ligand (sFasL) in euthyroid patients with chronic autoimmune hypothyroidism, who were taking levothyroxine (euthyroid, LT4-CAIH), to investigate the possible role of thyroid hormone therapy in down-regulation of apoptotic factors. Fifty euthyroid patients with CAIH on levothyroxine (median of duration 36 months, range 6-228 months) were compared with 75 age- and sex-matched healthy individuals. Serum levels of soluble Fas and soluble Fas Ligand, autoantibodies to thyroid peroxide and thyroglobulin were measured using ELISA. Serum levels of sFas were significantly higher in the euthyroid, LT4-CAIH group [median 9.12 ng/ml, interquartile range (7.86-10.72 ng/ml)] than in the controls [6.11 ng/ml (5.60-6.81 ng/ml)] (P < 0.0001). Compared with controls [80.33 pg/ml (68.22-103.70 pg/ml)], the euthyroid, LT4-CAIH group [125.71 pg/ml (106.11-149.48 pg/ml)] had significantly higher levels of sFasL (P < 0.0001). In a chronological study, there was no significant correlation between sFas, sFasL, and the duration of levothyroxine therapy. In conclusion, normalization of serum sFas and sFasL levels cannot be achieved during levothyroxine treatment in patients with CAIH. It appears that levothyroxine therapy has no important effect on down-regulation of apoptotic factors in CAIH. Thus, like thyroid autoantibodies, monitoring of serum levels of sFas/sFasL is not indicated during thyroid hormone therapy.     

Defective CD95/APO-1/Fas signal complex formation in the human autoimmune lymphoproliferative syndrome, type Ia

Heterozygous mutations in the CD95 (APO-1/Fas) receptor occur in most individuals with autoimmune lymphoproliferative syndrome (ALPS) and dominantly interfere with apoptosis by an unknown mechanism. We show that local or global alterations in the structure of the cytoplasmic death domain from nine independent ALPS CD95 death-domain mutations result in a failure to bind the FADD/MORT1 signaling protein. Despite heterozygosity for the abnormal allele, lymphocytes from ALPS patients showed markedly decreased FADD association and a loss of caspase recruitment and activation after CD95 crosslinking. These data suggest that intracytoplasmic CD95 mutations in ALPS impair apoptosis chiefly by disrupting death-domain interactions with the signaling protein FADD/MORT1.     

Fas (CD95) expression is up-regulated on papillary thyroid carcinoma

Thyrocyte apoptosis signaled through the Fas receptor has been proposed as a mechanism for the cytotoxicity observed in thyroiditis, but the role the Fas pathway plays in thyroid cancer is not known. We examined Fas expression in thyroid tissue derived from patients with papillary carcinoma and follicular cancer. More intense immunohistological staining for the Fas protein was observed on papillary cancer cells as compared with adjacent normal follicles. To further characterize the expression of Fas in papillary cancer, paired normal and cancerous thyroid tissues were obtained at thyroidectomy from several donors, digested, and placed into cell culture. Messenger RNA was analyzed by ribonuclease protection assays, and protein was identified by flow cytometry. Fas expression was detected at levels up to 3-fold higher in cancerous thyrocytes compared with paired normal cells. To determine whether the expressed Fas antigen was functional, thyrocytes were treated with a monoclonal IgM anti-Fas antibody (clone CH11; Upstate Biotechnology, Inc., Lake Placid, NY) in the presence of interferon-gamma and cycloheximide. Whereas both normal and cancerous thyrocytes were induced to die after this treatment, the cancerous thyrocytes were more sensitive to anti-Fas antibody. This work demonstrates that the Fas antigen is expressed and functional on papillary thyroid cancer cells and this may have potential therapeutic significance.     

Fas (APO-1/CD95) mRNA is down-regulated in liver regeneration after hepatectomy in rats

The interaction of Fas (APO-1/CD95) and the Fas ligand system induces apoptosis. However, the role of the Fas/Fas ligand system in normal physiological liver cell death remains to be determined. Using northern blotting analysis, we investigated the role of Fas and Fas ligand mRNA expression in liver regeneration in rats until 14 days after partial hepatectomy. Partial hepatectomy led to a significant decrease in Fas mRNA levels at 2 h, with a further gradual reduction until 18 h. The minimum Fas mRNA levels persisted until 5 days after the surgery. Fas mRNA levels then increased markedly at 6 days, and gradually increased to the initial levels by 14 days after the surgery. However, partial hepatectomy did not affect the expression of Fas ligand mRNA levels. These findings suggest that Fas mRNA is down-regulated in liver regeneration after partial hepatectomy, and that when the original liver mass is gradually regenerating, Fas mRNA is again up-regulated to the initial levels. Received: March 4, 1999 / Accepted: July 23, 1999     

The induction of Bim expression in human T-cell blasts is dependent on nonapoptotic Fas/CD95 signaling

The BH3-only protein Bim is required for maintaining the homeostasis of the immune system, since Bim regulates the down-modulation of T-cell responses, mainly through cytokine deprivation. Using T-cell blasts from healthy donors and also from patients with autoimmune lymphoproliferative syndromes (ALPSs) due to homozygous loss-of-function mutation of FasL (ALPS-Ic) or heterozygous mutation in the Fas/CD95 death domain (ALPS-Ia), it is shown that the induction of Bim expression during the process of human T-cell blast generation is strictly dependent on FasL/Fas-mediated signaling. The main pathway by which Fas signaling regulates the levels of Bim expression in human T-cell blasts is the death-domain- and caspase-independent generation of discrete levels of H2O2, which results in the net increase of Foxo3a levels. The present results connect the 2 main pathways described until the moment for the control of T-cell responses: death receptor-mediated activation-induced cell death and apoptosis by cytokine deprivation.     

Homing defect of cultured human hematopoietic cells in the NOD/SCID mouse is mediated by Fas/CD95

OBJECTIVE: To determine the bone marrow homing efficiency (20 hours) of cultured compared to noncultured umbilical cord blood (UCB)-derived human hematopoietic cells in the nonobese diabetic/severe combined immunodeficient (NOD/SCID) mouse, and to explain the difference in homing between these populations. METHODS: Human UCB CD34+ cells were cultured for up to 5 days, reselected, and used for transplantation, phenotype analysis, and functional studies, including adhesion and trans-endothelial migration assays. Seeding of CD34+ cells was measured after labeling of cells with 111-Indium, while homing of colony-forming cells (CFC) and SCID-repopulating (SRC) cells was determined using functional assays. RESULTS: Short-term culture was associated with a decrease in the 20-hour homing of CD34+ cells, CFC, and SRC to the BM. Although cultured compared to noncultured cells showed increased expression and function (adhesion/migration) of several cell adhesion molecules described to play a role in homing and engraftment, culture also induced expression of Fas/CD95 and rendered cells more susceptible to apoptosis. Finally, we demonstrate that the level of Fas/CD95 on cultured cells was inversely related to the ability of CFC to home to the BM, and that the homing of cultured CFC could be restored by incubating cells prior to transplantation with Fas/CD95-blocking mAb ZB4. CONCLUSION: These data implicate Fas/CD95 in the homing defect of cultured human hematopoietic cells in the NOD/SCID transplant model and suggest that prevention of apoptosis may be an important strategy to improve engraftment of ex vivo-manipulated HSC in a clinical setting.     

Analysis of apoptotic markers Fas/FasL (CD95/CD95L) expression on the lymphocytes in patients with acute coronary syndrome

BACKGROUND: Acute coronary syndromes are caused by the rupture or erosion of an atherosclerotic plaque which by secreting a variety of proteases is capable of degrading pericellular matrix components induces death of endothelial cells. This mechanism plays the main role in apoptosis. AIM: To estimate expression of apoptotic Fas/FasL (CD95/CD95L) on lymphocytes in the peripheral blood. METHODS: We examined patients with acute myocardial infarction (n=18, mean age 62+/-8 years), in unstable angina pectoris (n=31, mean age 62+/-10 years) and in a control group (n=20, mean age 62+/-9 years) without coronary risk factors and inflammatory condition. All investigations of Fas/FasL were performed by flow cytometry. Inflammatory parameters and standard risk factors were investigated by standard methods (ELISA). RESULTS: The analysis revealed a higher expression of Fas and FasL molecules on the lymphocytes from patients with acute myocardial infarction (p<0.001, p<0.002) and unstable angina (p<0.01, p<0.02) compared to the control group. Moreover we found a statistically significant positive correlation between the level of LDL cholesterol and hypertension and prevalence of CD95 (p<0.001, p<0.01) and CD95L (p<0.02, p<0.03) in patients with acute myocardial infarction. CONCLUSIONS: A higher expression of apoptotic molecules (Fas and FasL) on lymphocytes occurs before the onset of acute ischaemia and contributes to the plaque rupture and acute coronary syndrome. Furthermore, antiapoptotic therapy leads to plaque stabilisation.     

PSD-95 is required for activity-driven synapse stabilization

The activity-dependent regulation of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)-type glutamate receptors and the stabilization of synapses are critical to synaptic development and plasticity. One candidate molecule implicated in maturation, synaptic strengthening, and plasticity is PSD-95. Here we find that acute knockdown of PSD-95 in brain slice cultures by RNAi arrests the normal development of synaptic structure and function that is driven by spontaneous activity. Surprisingly, PSD-95 is not necessary for the induction and early expression of long-term potentiation (LTP). However, knockdown of PSD-95 leads to smaller increases in spine size after chemically induced LTP. Furthermore, although at this age spine turnover is normally low and LTP produces a transient increase, in cells with reduced PSD-95 spine turnover is high and remains increased after LTP. Taken together, our data support a model in which appropriate levels of PSD-95 are required for activity-dependent synapse stabilization after initial phases of synaptic potentiation.     

Increase in CD95 (Fas/APO-1)-positive CD4+ and CD8+ T cells in peripheral blood derived from patients with autoimmune hepatitis or chronic hepatitis C with autoimmune phenomena

BACKGROUND: The expressions of CD95 (Fas/APO-1) and Bcl-2 are determinants of apoptosis in normal lymphocytes, and abnormalities in their expressions might contribute to the induction of autoimmunity. In this study, we examined the expressions of CD95 and Bcl-2 on freshly isolated T and B cells from patients with autoimmune hepatitis (AIH) or chronic hepatitis C associated with autoimmune phenomena (CH-C(AI)). METHODS: The CD95 and Bcl-2 expressions within CD4+ T, CD8+ T, and CD19+ B cell subsets were analysed by two-colour flow cytometry. RESULTS: The surface expression of CD95 was significantly high in both the CD4+ T and CD8+ T cell subsets derived from the patients with AIH and those with CH-C(AI), compared with expression in patients with CH-C and normal subjects. The increase in CD95 expression was associated with the phenotypic conversion of naive CD45RO- to primed CD45RO+ CD4+ T cells. Bcl-2 was detected in the vast majority of peripheral T and B cells. There was no significant difference in the percentage of Bcl-2-positive cells in the CD4+ T cell, CD8+ T cell and CD19+ B cell subsets among the patient groups and normal subjects. CONCLUSIONS: These results indicate that an increase in CD4+ T cells expressing CD45RO and CD95 marks an important subset of AIH and CH-C(AI) patients. These expanded CD95+ CD45RO+ primed T cells most likely reflect a continuous antigen-specific or non-specific activation of T lymphocytes, and/or the persistent presence of activated lymphocytes as a consequence of abnormalities in the peripheral deletion of activated lymphocytes. These persistently activated lymphocytes might play a role in the induction of autoimmunity in AIH and CH-C(AI).     

热休克蛋白70与慢性糜烂性胃炎胃黏膜损伤的相关性分析及其临床意义

目的探讨热休克蛋白70(HSP70)与慢性糜烂性胃炎胃黏膜损伤的相关性并分析其临床意义。方法纳入慢性糜烂性胃炎患者140例为观察组,按疾病分级分为Ⅰ级、Ⅱ级、Ⅲ级组,纳入同期健康体检者40例为对照组,对比4组血清HSP70蛋白及mRNA水平;检测观察组胃黏膜损伤指数、胃黏膜血流量、HSP70蛋白及mRNA表达水平、血清苯二醛(MDA)水平及超氧化物歧化酶(SOD)活性,分析上述指标相关性;患者予常规治疗,对比治疗前后血清HSP70蛋白及mRNA水平。结果观察组血清HSP70蛋白及mRNA水平均明显高于对照组(P0.05),观察组各亚组间HSP70蛋白及mRNA水平差异无统计学意义(P0.05);观察组胃黏膜HSP70蛋白及mRNA表达水平与胃黏膜损伤指数呈正相关、与胃黏膜血流量呈负相关、与血清SOD活性呈负相关、与血清MDA水平呈正相关(P均0.05);观察组治疗期间血清HSP70蛋白及mRNA表达水平均明显上升(P0.05)。结论 HSP70蛋白及mRNA表达水平与慢性糜烂性胃炎胃黏膜损伤程度有关,但与疾病分级无明显相关性;其可能参与慢性糜烂性胃炎胃黏膜损伤的修复,在治疗结束后水平迅速下降。     

Differentiation of promyelocytic leukaemia: alterations in Fas (CD95/Apo-1) and Fas ligand (CD178) expression

The survival of leukaemic blasts contributes to the pathological mechanism of acute promyelocytic leukaemia (APL). While treatment of APL using retinoic acid (RA) is a model of differentiation therapy, little is known about possible effects of this treatment on the Fas/FasL system. Investigation of APL cells from patients undergoing differentiation therapy with RA and of promyelocytic HL-60 and monoblastic U-937 cells cultured with RA revealed a reduction of surface expression of both Fas and its ligand. Accordingly, the sensitivity of the cells to anti-Fas-induced apoptosis decreased proportionally and the reduced expression of FasL resulted in a decreased ability of the leukaemic cells to induce apoptosis in T cells. Our findings demonstrate that there are significant changes in Fas and FasL expression during RA treatment of APL, which probably have consequences for the interaction between host immune and leukaemia cells, and thus may be involved in the beneficial effects of differentiation therapy.     

Vascular injury in acute gastric mucosal damage

Recent investigations indicate that microvascular injury, leading to increased vascular permeability and capillary stasis, precede the development of chemically induced hemorrhagic mucosal lesions in the stomach. The vascular damage is more amenable to protection by prostaglandins and sulfhydryls than the diffuse surface mucosal cell injury. The vascular and mucosal lesions may be the result of direct toxicity of damaging agents (eg, ethanol, HCl, NaOH) and the release of vasoactive amines and leukotrienes. We review here our recent studies performed in rats indicating that intraarterial infusion of LTC4 or LTD4 in the stomach caused vascular injury as revealed by monastral blue. Infusion of leukotrienes alone caused no hemorrhagic mucosal lesions but aggravated the damage caused by 25, 50, or 100% ethanol and 0.2 N HCl given intragastrically. The ethanol-induced mucosal lesions were slightly diminished by the lipoxygenase inhibitor L-651,392 and markedly decreased by eicosapentaenoic acid, which competes with arachiconic acid as a substrate for 5-lipoxygenase. These results are discussed and correlated with biochemical results from other laboratories demonstrating increased levels of leukotrienes in the gastric mucosa after administration of ethanol and decreased release following pretreatment with prostaglandins or sulfhydryl-related agents. New data thus support a mediatory role for leukotrienes in the pathogenesis of vascular injury and mucosal lesions in the stomach.     

CD95 (Fas) expression is regulated by sequestration in the Golgi complex in B-cell lymphoma

The CD95 (Fas) molecule transmits apoptotic signals important in B-cell development and the genesis of B-cell lymphoma. We have investigated the surface and intracellular expression of CD95 in Burkitt's lymphoma (BL) cells, an important non-Hodgkin's lymphoma of B-cell origin. Group I BL cells did not express CD95 at the cell surface, but contained high levels of this receptor in the cytoplasm. In contrast, group III BL cells expressed CD95 intracellularly and at the cell surface. In group I and group III BL cells, cytoplasmic CD95 was localized to the Golgi complex, as assessed by confocal immunofluorescence microscopy and subcellular fractionation followed by immunoblotting. Trafficking through the Golgi complex is regulated by elements within the target protein and cellular sorting mechanisms. CD95 contains candidate signals for interaction with trafficking machinery. Group I BL cells can be induced to upregulate surface expression of CD95 following CD40 ligation and certain group I BL cell lines drift invitro to a group III phenotype, with consequent surface expression of CD95. Taken together, these observations show that CD95 can either be retained in the Golgi complex or exported to the cell surface, and suggest that membrane trafficking has an important and previously unrecognized role in regulating CD95 expression in B lymphocytes.