Fas-mediated apoptosis of melanoma cells and infiltrating lymphocytes in human malignant melanomas.

In a rodent system, melanoma cells expressing Fas ligand (FasL) could kill Fas-positive lymphocytes, suggesting that FasL expression was an essential factor for melanoma cell survival in vivo. These findings led us to investigate apoptosis, and to histochemically analyze involvement of Fas and FasL in the induction of apoptosis, in human malignant melanoma tissues. The percentages of terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick end-labeling (TUNEL)-positive melanoma cells and of proliferating cell nuclear antigen (PCNA)-positive melanoma cells in melanoma tissues (n = 22) were greater than those in melanocytes in uninvolved skin (n = 6) and nevus cells in nevi tissues (n = 9). The infiltrating lymphocytes around melanomas were also TUNEL positive. Immunohistochemistry revealed expression of Fas and FasL in melanoma cells and lymphocytes, whereas no Fas or FasL expression was detected in normal skin melanocytes and nevus cells. There was significant correlation between Fas-positive indices and TUNEL indices in melanoma tissues. Moreover, TUNEL-, Fas-, and FasL-positive indices of melanoma cells from patients with Stage 3 melanomas were significantly lower than those with Stage 2 melanomas. The PCNA index of Stage 1 melanoma was significantly lower than that of the other stages, although the difference of PCNA index was insignificant among Stages 2 to 4. Among Stages 1 to 4, there was no difference in the PCNA, TUNEL-, and Fas-positive indices of lymphocytes, although the FasL-positive index of lymphocytes from Stage 3 melanomas was significantly lower than in that from Stage 2. These data reveal that melanoma cells and infiltrating lymphocytes have the potential to induce their own apoptosis regulated by Fas and FasL in an autocrine and/or paracrine fashion and that the decline of Fas-mediated apoptosis of melanoma cells, rather than the apoptosis of infiltrating lymphocytes, may affect the prognosis of melanoma patients, possibly through the accumulation of more aberrant cells acquiring metastatic activity.     

Immunodetection of histone epitopes correlates with early stages of apoptosis in activated human peripheral T lymphocytes.

By coupling intracellular staining with terminal deoxynucleotidyl transferase (TdT)-mediated labeling of internucleosomal DNA strand breaks in a flow cytometric assay, we observed a strong correlation between apoptosis-associated DNA strand breaks and immunoreactivity with the monoclonal antibody (MAb) B-F6 in activated human peripheral blood T lymphocytes (PBTs). Although MAb B-F6 has been reported to be specific for the cytokine interleukin-6, Western blot analysis of activated PBT lysates revealed that the predominant protein band detected by this MAb was 17 kd (p17), distinct from the 23-kd core protein and 26- to 30-kd mature glycosylated forms of interleukin-6. Immunoaffinity isolation and amino-terminal amino acid sequence analysis of p17 revealed identity with the histone H2B, a finding confirmed by Western blot analysis of purified histones and by similar staining of activated PBTs with an unrelated anti-histone MAb. Neither histone staining nor DNA strand breakage was observed in freshly isolated PBTs; however, after T cell activation, histone immunoreactivity appeared to precede the appearance of DNA strand breaks, with both increasing to a maximal level by day 3 after activation. Two-parameter confocal immunofluorescence microscopy of histone and DNA staining confirmed a lack of histone immunoreactivity in viable cells and demonstrated co-localization of histone epitopes with abnormally clumped chromatin in apoptotic cells. These data indicate that alteration of histone epitope accessibility is a marker of early apoptosis and suggest that multiparameter flow cytometric analysis of intracellular epitopes may be a powerful tool in the elucidation of intracellular mechanisms of apoptosis.     

Galectin-1 expression correlates with the regenerative potential of rubrospinal and spinal motoneurons

Axotomized spinal motoneurons are able to regenerate to their peripheral targets, whereas injured rubrospinal neurons that lie completely within the CNS fail to regenerate. The differing cell body reactions to axotomy of these two neuronal populations have been implicated in their disparate regenerative ability. Recently, the lectin galectin-1 has been shown to be involved in both spinal motoneurons and primary afferent regeneration. Using in situ hybridization, we compared the endogenous galectin-1 mRNA expression in spinal motoneurons and rubrospinal neurons after axotomy. We found that 7 and 14 days after axotomy, galectin-1 mRNA increased in spinal motoneurons but decreased in rubrospinal neurons. Infusion of the brain-derived neurotrophic factor into the vicinity of the injured rubrospinal nucleus, which we have previously shown to increase the regenerative capacity of rubrospinal neurons, significantly increased galectin-1 mRNA compared with uninjured control levels. Thus, the expression of galectin-1 in neurons correlates with the regenerative propensity.     

Galectin-3 expression in papillary microcarcinoma of the thyroid

AIMS: Galectin-3 is a beta-galactoside binding protein, recently recognized as a promising molecular marker of thyroid malignancy. As reported in several studies, galectin-3 is highly expressed in papillary thyroid carcinoma, but its expression has not been investigated in papillary microcarcinoma, which is a variant of papillary thyroid carcinoma. METHODS AND RESULTS: Using a monoclonal antibody to galectin-3 and the avidin-biotin-peroxidase complex (ABC) immunohistochemical technique, we analysed galectin-3 expression in 63 cases of papillary microcarcinoma. The results showed immunohistochemical reactivity for galectin-3 in 51 (80.9%) cases. Intensity of staining varied from strong or moderate to weak. Galectin-3 localization was mostly cytoplasmic, but also membranous or nuclear in some cells. Immunohistochemical expression of galectin-3 was not found in 12 (19.1%) cases. Most galectin-3 negative microcarcinomas (10/12) were of the non-classical type, i.e. without papillary architecture. Neither the frequency nor the intensity of a positive reaction was related to tumour size. CONCLUSIONS: Galectin-3 gene is expressed at the protein level in most papillary microcarcinomas, although with slightly lower frequency than that reported for clinically evident papillary thyroid carcinoma. The presence of galectin-3 in clinically silent microcarcinomas may indicate that galectin-3 is not related to growth or aggressiveness of papillary thyroid microcarcinomas but rather plays some other role in thyroid tumour biology.     

Diverse expansion potential and heterogeneous avidity in tumor-associated antigen-specific T lymphocytes from primary melanoma patients

While tumor-associated antigen (TAA)-specific CD8(+) T lymphocytes have been detected in metastatic melanoma patients, immune response in early disease phases has not yet been carefully evaluated. We looked for circulating cytotoxic T lymphocytes (CTL) directed against Melan-A / MART1, tyrosinase, gp100 and MAGE-3 antigens in patients with a diagnosis of primary cutaneous melanoma by using fluorescent HLA-A2 tetramers. In five out of six cases high numbers of CD8(+)/tetramer(+) cells could be detected by flow cytometry, and in four patients lymphocyte populations specific for two different melanoma antigens (Melan-A/MART1 and tyrosinase) were contemporaneously present. The TAA-specific cells could represent as much as 1/220 T lymphocytes in the circulating CD8(+) population. When tetramers were used to monitor the in vitro expansion of TAA-specific CTL precursors upon antigen-specific stimulation, a diverse expansion potential was evidenced in CTL from the different donors and, more strikingly, in CTL specific for the different TAA. Melan-A/MART1-specific CTL clones derived from two patients exhibited a broad range of avidity. Only the highest avidity clones, representing about 50 % of the cases analyzed, were tumor specific. By correlating tetramer staining with clone avidity, we found that tetramer fluorescence intensity could represent a good indicator of TCR affinity, but not of overall clone avidity.     

Diagnostic protein expression in human muscle biopsies

Using immunohistochemistry in diagnosing neuromuscular diseases is meant to enhance the diagnostic yield in two ways. The first application aims at visualizing molecules which are developmentally, neurally, and/or immunologically regulated and not expressed by normal muscle. They are upregulated in pathological conditions and may help assign a given muscular biopsy to one of the main diagnostic entities (muscular dystrophies, inflammatory myopathy, neurogenic atrophy). In the past, muscle-specific molecules with a defined expression pattern during fetal myogenesis served as antigens, with the rationale that the developmental program was switched on in new fibers. Recently, myofibers in diseased muscle are thought of as targets of stimuli which are released by macrophages in muscular dystrophy, by lymphocytes in inflammatory myopathies, or by a lesioned peripheral nerve in neurogenic atrophies. This has somewhat blurred the borders between the diagnostic groups, for certain molecules, e.g. cytokines, may be upregulated after experimental necrotization, denervation, and also in inflammatory myopathies. In the second part of this review we summarise the experiences of a Centre in the North of England that specialises in the diagnosis and clinical support of patients with muscular dystrophy. Emphasis is placed on the use of protein expression to guide mutation analysis, particularly in the limb-girdle muscular dystrophies (a group of diseases that are very difficult to differentiate on clinical grounds alone). We confirm that genetic analysis is essential to corroborate the results of protein analysis in certain conditions (particularly in calpainopathy). However, we conclude that analysing biopsies for abnormal protein expression is very useful in aiding the decision between alternative diagnoses.     

Increased Tim-3 expression on peripheral lymphocytes from patients with rheumatoid arthritis negatively correlates with disease activity

Tim-3 has been reported as an important regulatory molecule and plays a pivotal role in several autoimmunity diseases. Here, we demonstrated the increased expression of Tim-3 on peripheral CD4⁺ T, CD8⁺ T, NKT cells and monocytes from RA patients compared to those from healthy controls. Percentage of Tim-3⁺ cells in peripheral blood mononuclear cells (PBMCs) showed an inverse correlation with disease activity score 28 (DAS28) and plasma TNF-α level. Similar negative correlations were found between disease activity and Tim-3 levels on CD4⁺ T, CD8⁺ T and NKT cells. Consistently, Tim-3 expression on CD3⁺ T cells was further increased in patients with disease remission after treatment. Tim-3 expression on CD8⁺ T and NKT cells negatively correlates with plasma TNF-α. Our results suggest that Tim-3 might participate in the proceeding of RA by its negative regulation on various T cell subsets. Tim-3 might be a potential new marker for assessing severity of RA.     

Activation of human intraepithelial lymphocytes reduces CD3 expression

The aim of this study was to examine in detail the low functional capacity of human intraepithelial lymphocytes (IELs) in response to phytohaemagglutinin (PHA) and CD3 ligation. Human IELs were extracted from jejunal mucosa obtained from patients undergoing gastric bypass operations for morbid obesity and compared to peripheral blood (PB) lymphocytes composed predominantly of CD8+ T cells. Calcium influx ([Ca2+]i) was analysed using Fura-2-loaded cells; IL-2 receptor expression was measured by immunofluorescence and flow cytometry; IL-2 binding was determined using radiolabelled IL-2; IL-2 production was quantified by ELISA; and apoptosis was detected with Apo 2.7 staining. Compared to naive PB CD8+ T lymphocytes, calcium influx by IELs was only transient with CD3 ligation and low in amplitude with PHA. IL-2 receptor expression was reduced after CD3 ligation, yet normal in numbers and affinity after PHA stimulation. Both cell types secreted similar amounts of IL-2. CD3 expression on IELs, but not PB CD8+ T cells, declined upon activation, due partly to incomplete reexpression after modulation. Little apoptosis was found. The partial activation of IELs in response to PHA and CD3 ligation, as manifested by diminished [Ca2+]i, resulted in a decline in CD3 expression.     

Galectin-3 expression in functioning and silent ACTH-Producing adenomas

Galectin-3 (Gal-3), a β galactoside-binding protein, has been implicated in a variety of biological functions including cell growth, differentiation, tumor cell adhesion, angiogenesis, tumor progression, and metastasis. We recently reported that Gal-3 was expressed in a subset of normal pituitary cells and tumors including PRL, ACTH, and in folliculostellate (FS) cells and tumors [1,2] and that Gal-3 had an important regulatory role in pituitary cell proliferation [1]. We further investigated the expression of Gal-3 protein in ACTH- and PRL-producing tumors and the expression of various galectin mRNAs by RT-PCR in pituitary adenomas and normal pituitary. Most silent ACTH subtypes 1 and 2 adenomas were negative or only focally positive for Gal-3 expression compared to functioning ACTH tumors from patients with Cushing’s disease and Nelson’s syndrome. In the normal pituitary, Gal-3 was expressed in less than 1% of the basophil-invading cells (ACTH cells present in the posterior pituitary) and in a subset of the anterior lobe ACTH-positive cells. RT-PCR analyses showed that many members of the galectin family including galectins 1, 2, 3, 4, 5, 6, 7, 8, and 9 were expressed in normal pituitary and in functioning ACTH- and PRL-producing tumors. These results indicate that Gal-3 is associated with functioning ACTH and PRL tumors and is expressed infrequently in silent ACTH adenomas, suggesting that Gal-3 protein and/or gene is altered in non-functioning ACTH tumors. The use of ACTH and Gal-3 immunostaining should help in the diagnosis of silent ACTH adenomas.     

Expression of tumor-associated membrane antigen, RCAS1, in human colorectal carcinomas and possible role in apoptosis of tumor-infiltrating lymphocytes.

RCAS1, a novel tumor-associated antigen, is expressed in advanced human neoplasias including uterine and ovarian carcinomas. RCAS1 protein was indicated to induce cell cycle arrest and apoptosis of cultured human lymphoid and myeloid cell lines and normal lymphocytes. In the present study, we investigated the expression and prognostic value of RCAS1 in 58 patients with colorectal carcinomas. RCAS1 protein was detected by immunoperoxidase staining using a mouse monoclonal anti-RCAS1 antibody (22-1-1 antibody). Immunohistochemical examination showed expression of RCAS1 in 75% of colorectal carcinomas with lymph node metastases (n = 24), whereas it was present in only 41% of tumors without metastases (n = 34, P <.05). Patients with RCAS1-positive tumors showed a significantly poorer prognosis than those negative for RCAS1 (P <.05). Multivariate analysis using the Cox regression model indicated that RCAS1 positivity was an independent negative predictor for survival (P =.0300; risk ratio, 0.496). In addition, apoptotic cells of tumor-infiltrating lymphocytes were examined using nonradioactive in situ nick translation in paraffin-embedded sections. The proportion of apoptotic tumor-infiltrating lymphocytes was significantly higher in RCAS1-positive colorectal carcinomas (11.2 +/- 1.0) than in RCAS1-negative tumors (7.9 +/- 1.0, P <.05). Our results suggest that overexpression of RCAS1 may negatively affect the prognosis of human colorectal carcinomas and that RCAS1 may play a role in tumor immune privilege in vivo.     

Expression of γδ T lymphocytes derived from human intestinal biopsies

Although most T cells express the alpha/beta TCR, the gamma/delta TCR is expressed only on a small percentage of peripheral lymphocytes and CD3+ intestinal T cells. The most striking feature is a wide variation in the proportion of gamma/delta+ T cells in freshly isolated peripheral blood cells from normal individuals and patients with IBD. The augmentation of the gamma/delta+ T cell subpopulation derived from human intestinal biopsies after repeated stimulation with MT, even in the absence of filler cells, suggests that gamma/delta+ cells from human gut mucosa may play a role in generating a primary immune response to MT.     

Cyclooxygenase-2 expression correlates with angiogenesis and apoptosis in gastric cancer tissue

In vitro studies suggest that cyclooxygenase-2 (COX-2) induces angiogenesis by stimulating angiogenic growth factors while inhibiting apoptosis in cancer cell lines. A series of 107 gastric adenocarcinoma cases that had undergone gastrectomy was studied to determine the correlation between COX-2 expression, angiogenesis, and apoptosis in human gastric cancer tissue. COX-2, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and Bcl-2 were stained by single and dual immunoassaying methods. Microvessel density was determined by immunostaining for CD34. Apoptosis was evaluated with the TUNEL assay. COX-2 expression was positive exclusively in cancer cells in 46 cases (43%). COX-2 expression significantly correlated with VEGF and PDGF expression. Dual staining for COX-2 and VEGF showed that colocalization of these proteins was most frequent at the advancing edge of cancer cells. Microvessel density was higher in COX-2-and VEGF-positive cases than in COX-2- and VEGF-negative cases. In addition, COX-2 expression correlated with Bcl-2 expression. The apoptotic index was lower in COX-2-positive cancer cells than in COX-2-negative cases. Multivariate analysis revealed that coexpression of COX-2 and VEGF, age, lymph node status, and serosal invasion were independent prognostic factors for overall survival in gastric cancer patients. Therefore, these data suggest that COX-2 contributes to gastric cancer development by promoting angiogenesis and inhibiting apoptosis.     

Proteomic study of Galectin-1 expression in human mesenchymal stem cells

Bone marrow-derived mesenchymal stem cells (MSCs) are known to interact with hematopoietic stem cells (HSCs) and immune cells, and are of potential interest to be used as therapeutic agents for enhancing allogenic hematopoietic engraftment and preventing graft-versus-host disease (GVHD). Galectin 1 (Gal1) belongs to a family of structurally related molecules expressed in many vertebrate tissues that exert their functions both by binding to glycoconjugates, and by interaction with protein partners. In this work using a proteomic approach, we looked for the presence and the localization of Gal1 in short- and long-term culture of human (h) hMSC. We first determined, that Gal1 is one of the major proteins expressed in hMSC. We futher demonstrated that its expression is maintained when hMSC are expanded through a subculturing process up to five passages. Moreover, Gal1 is secreted and found at the cell surface of MSC, participating in extra cellular matrix (ECM)-cell interactions. Given the immunomodulatory properties of Gal1, its potential involvement in immunological functions of hMSC could be suggested.     

激活的人外周T淋巴细胞表达水通道蛋白AQP3

目的:探讨水通道蛋白(Aquaporin,AQP)在人外周T淋巴细胞的表达和生理学意义.方法:利用逆转录PCR和免疫印迹技术检测AQP0～9 mRNA和蛋白在静止的和经活化的T淋巴细胞的表达.结果:静止的淋巴细胞无AQP0-9表达,而活化的T淋巴细胞表达AQP3 mRNA和蛋白.结论:T淋巴细胞激活过程可能需要AQP3转运水和甘油的功能.     

Apoptin基因通过激活caspase-3诱导人黑色素瘤细胞A375凋亡

目的研究caspase-3在肿瘤特异性凋亡基因诱导人黑色素瘤细胞A375凋亡中的作用。方法用含有apoptin基因的真核表达载体瞬间转染体外培养的人黑色瘤细胞A375;采用RT-PCR、DNA凝胶电泳、流式细胞术检测A375细胞的凋亡;以比色法检测caspase-3的相对活性。结果Ap-optin基因瞬间转染的A375细胞可出现典型的细胞凋亡所具有的DNA梯状带;流式细胞术发现实验组细胞凋亡率明显高于其他各组(P<0.01);转染后24h实验组caspase-3的活性开始升高,72h达高峰,明显高于其他各组(P<0.01)。结论Apoptin基因可通过激活caspase-3诱导人黑色素瘤细胞A375凋亡。