银屑病中Fas、FasL和穿孔素的检测

目的：通过对银屑病中Fas、FasL和穿孔素的检测,探讨细胞凋亡和银屑病的关系。方法：采用免疫组化ABC法对银屑病皮损中Fas、FasL和穿孔素进行检测。结果：在银屑病组织中Fas、FasL和穿孔素均阳性表达。结论：在银屑病中,细胞毒性T淋巴细胞可能通过FasL和穿孔素两条途径引起细胞凋亡,细胞凋亡参与了银屑病的发病。     

红斑狼疮皮损中CD4~+、CD8~+ T细胞表达及其与Bcl-2关系的研究

目的:确定Bcl-2及CD4 、CD8 T细胞在红斑狼疮皮损区的表达并探讨其与红斑狼疮发病的关系.方法:采用免疫组化法对30例红斑狼疮皮损区T细胞亚群CD4 、CD8 T细胞及淋巴细胞Bcl-2进行检测,并与17例正常皮肤作对照.结果:红斑狼疮患者皮损中Bcl-2淋巴细胞和CD4 T细胞的表达明显高于正常对照组,而CD8 T细胞表达明显低于正常对照组;Bcl-2的表达与CD4 T细胞表达呈正相关,与CD8 T细胞表达呈负相关.结论:红斑狼疮皮损区淋巴细胞Bcl-2的表达增强可能会导致CD4 和CD8 T细胞凋亡异常,从而导致免疫紊乱而发病.     

T淋巴细胞亚群与斑秃发病关系的研究

目的:探讨T细胞亚群(CD3 、CD4 、CD8 T)在斑秃发病中所起的作用。方法:用流式细胞仪分别检测斑秃患者PBMC中CD3 、CD4 、CD8 T淋巴细胞的表达水平。结果:重型斑秃、轻型斑秃的CD4 T淋巴细胞表达高于正常对照组,且差异有统计学意义;重型斑秃、轻型斑秃的CD8 T淋巴细胞表达亦高于正常对照组,且差异有统计学意义;CD3 T淋巴细胞表达、CD4 /CD8 比值较正常对照升高,差异无统计学意义。结论:提示了由T淋巴细胞介导的斑秃的发病机理,CD8 和CD4 T淋巴细胞在其发病过程中起到重要作用,两者协同作用导致毛囊受损。     

红斑狼疮皮损中CD4^＋、CD8^＋T细胞表达及其与Bcl-2关系的研究

目的：确定Bcl-2及CD4^＋、CD8^＋T细胞在红斑狼疮皮损区的表达并探讨其与红斑狼疮发病的关系。方法：采用免疫组化法对30例红斑狼疮皮损区T细胞亚群CD4^＋、CD8^＋T细胞及淋巴细胞Bcl-2进行检测，并与17例正常皮肤作对照。结果：红斑狼疮患者皮损中Bcl-2淋巴细胞和CD4^＋T细胞的表达明显高于正常对照组，而CD8^＋T细胞表达明显低于正常对照组；Bcl-2的表达与CD4^＋T细胞表达呈正相关，与CD8^＋T细胞表达呈负相关。结论：红斑狼疮皮损区淋巴细胞Bcl-2的表达增强可能会导致CD4^＋和CD8^＋T细胞凋亡异常，从而导致免疫紊乱而发病。     

寻常性银屑病皮损和外周血淋巴细胞中穿孔素及颗粒酶B的表达

细胞毒性T淋巴细胞(CTL)和天然杀伤细胞(NK)主要通过穿孔素/颗粒酶途径和Fas/FasL途径杀伤靶细胞.穿孔素(perforin)能够在靶细胞表面形成孔道,破坏细胞膜的完整性,颗粒酶(granzyme)作为一种丝氨酸蛋白酶,由此孔道进入靶细胞,导致DNA裂解,引起细胞凋亡.已经证实,银屑病是T淋巴细胞介导的免疫异常性皮肤病.我们用逆转录聚合酶链法检测寻常性银屑病患者的皮损和外周血淋巴细胞中的穿孔素和颗粒酶B的表达水平,以探讨其在该病发病机制中的作用.     

白癜风患者皮损中CD4~+,CD8~+T淋巴细胞及朗格汉斯细胞的检测

目的探讨白癜风患者皮损中CD4+,CD8+T淋巴细胞、朗格汉斯细胞(LC)及黑素细胞(MC)在白癜风发病中的作用。方法采用Envision免疫组化染色法,对12例白癜风进展期患者和9例稳定期患者皮损处CD4+,CD8+T淋巴细胞、LC及MC进行检测,并与10例正常人皮肤进行对照。结果白癜风患者皮损中CD4+,CD8+T淋巴细胞、LC表达较对照组显著增多(P<0.01),而MC表达较对照组显著减少(P<0.01)。结论白癜风患者皮损中LC,CD4+,CD8+T淋巴细胞异常表达可能参与白癜风的发病,其作用模式可能是LC抗原递呈,CD4+,CD8+T淋巴细胞浸润破坏或攻击MC,从而引起白癜风患者表皮基底层的MC减少或消失,导致白癜风的发生。     

银屑病皮损细胞凋亡的研究

目的：通过对银屑病皮损细胞凋亡以及凋亡相关基因bcl-2、Fas、bax和Fas-L、穿孔素的检测，探讨细胞凋亡与银屑病的关系。方法：采用原位末端标记及免疫组化ABC法、SABC法。结果：银屑病中细胞凋亡较正常人增多。抑凋亡基因bcl．2几乎不表达：而促凋亡基因Fas、bax阳性表达。细胞毒性T淋巴细胞可能通过Fas-L和穿孔素两条途径起作用。结论：细胞凋亡在银屑病发病机理中有十分重要的意义。     

茶多酚对CD8+T细胞杀伤白癜风患者黑素细胞的保护作用研究

目的 探讨茶多酚对CD8+T细胞杀伤白癜风患者黑素细胞的保护作用.方法 取白癜风患者白斑边缘处皮片组织,培养CD8+T淋巴细胞,细胞纯度用流式细胞仪进行鉴定,通过和黑素细胞共培养验证其针对黑素细胞的杀伤作用.将不同浓度茶多酚加入CD8+T细胞和黑素细胞共培养的体系,用流式细胞仪检测黑素细胞的凋亡.结果 取白癜风皮损边缘表皮组织中成功培养CD8+T淋巴细胞,纯度达90％以上,高表达活化抗原CD137和CD69.CD8+T细胞和黑素细胞共培养后,可明显诱导黑素细胞凋亡.200 μg/ml和400 μg/ml浓度茶多酚处理均可以降低黑素细胞的凋亡率.结论 白癜风皮损边缘组织来源的CD8+T细胞对白癜风患者黑素细胞有明显杀伤作用.茶多酚对抗CD8+T细胞杀伤黑素细胞,从而对黑素细胞有一定的保护作用.     

斑秃患者皮损中CD4+和CD8+T细胞的检测

斑秃病因未明．细胞免疫可能参与了斑秃的发病．CD4^ 和CD8^ T细胞是两种重要的免疫细胞，在免疫调节中起重要作用。为了研究这两种细胞在斑秃发病中的作用．笔者采用免疫组化的方法对斑秃应损和正常头皮中CD4^ 和CD8^ T细胞的分布情况进行了检测．现报告如下。     

慢性荨麻疹患者外周血CD4+CD25+调节性T细胞的表达

目的:探索调节性T细胞(regulatory T cells,Tregs)在慢性荨麻疹(CU)发病中的作用.方法:采用流式细胞术分别检测CU患者和健康人外周血CD3+、CD4+、CD8+、CD4+CD25+T细胞的表达水平.结果:与健康对照组比较,CU患者外周血CD3+T细胞比例无明显变化,CD4+T细胞增高(P ＜ 0.05),CD4+CD25+T细胞表达水平显著增高(P ＜ 0.01),CD8+T细胞数量降低(P ＜ 0.05),CD4+/ CD8+比值明显升高(P ＜ 0.05).自身血清皮肤试验(ASST)阳性组与阴性组的CU患者CD4+CD25+Tregs 细胞的数量无统计学差异.结论:CU患者外周血存在Treg细胞及其他T淋巴细胞亚群的数量异常和分化失衡,这一异常在CU发病中的作用值得进一步研究.     

Resistance to alopecia areata in C3H/HeJ mice is associated with increased expression of regulatory cytokines and a failure to recruit CD4+ and CD8+ cells

Grafting alopecia areata affected C3H/HeJ mouse skin to littermates induces alopecia areata, but high dietary soy oil reduces alopecia areata susceptibility. Alopecia areata affected and resistant mice were characterized to evaluate possible mechanisms involved in alopecia areata resistance. Of 44 mice that received alopecia areata affected skin grafts but failed to develop alopecia areata, only two of 22 receiving further alopecia areata affected skin grafts developed alopecia areata, whereas 39 of 44 controls developed alopecia areata. Alopecia areata affected skin contained increased numbers of CD4+ and CD8+ cells, increases in pro inflammatory T helper 1 and T helper 2 type cytokines, and upregulation of CD28, CD40L, and their ligands. In draining lymph nodes, a relatively high number of antigen-presenting cells was recovered, whereas several CD44v variants were downregulated. In contrast, alopecia areata resistant mouse skin did not display increased numbers of CD4+ and CD8+ cells, whereas counter-regulatory cytokines interleukins 4 and 10 were upregulated. High expression of CD28, CD80, CD86, CD40, CTLA4, CD44v variants, and FasL occurred in alopecia areata resistant mouse spleens. In vitro, lymph node cells of susceptible and resistant mice responded equally to a mitogenic stimulus, but only lymph node cells from alopecia areata affected mice displayed an increased response with T cell receptor stimulation via anti-CD3 cross-linking. These results suggest alopecia areata is a cell-mediated autoimmune disease, but alopecia areata affected skin graft hosts may resist alopecia areata onset through active counter-regulatory mechanisms. Because alopecia areata resistant mice showed unimpaired responsiveness and a transient inflammatory response towards the graft, it is suggested that alopecia areata develops as a consequence of an inappropriate immune response regulation.     

Analysis of the expression of cutaneous lymphocyte-associated antigen on the peripheral blood and cutaneous lymphocytes of alopecia areata patients

Alopecia areata has been reported to be accompanied by abnormal autoimmune dysfunction. We examined the expression of cutaneous lymphocyte-associated antigen (CLA), which is a skin-specific lymphocyte homing receptor, in the peripheral blood lymphocytes and skin of patients with alopecia areata. In the patients' peripheral blood, the percentage of CLA-positive CD4+ or CD8+ lymphocytes, was significantly higher than that of normal controls. The patients with severe or progressive alopecia areata showed a much higher CLA-positivity compared to patients recovering from the disease. A chronological study showed that the percentage of CLA-positive peripheral blood lymphocytes, CD4 + or CD8 + lymphocytes decreased in parallel with the patients' good clinical course. The CLA-positivity in peripheral blood lymphocytes, CD4+ or CD8+ lymphocytes of patients with alopecia areata who did not respond to oral corticosteroid therapy remained higher than in those who responded well to the treatment. In the affected scalp skin, many infiltrating lymphocytes around the hair follicles, which were CD4+ or CD8+ lymphocytes, expressed CLA. These findings suggest that the CLA-positivity correlates with clinical activity and that CLA-positive CD4+ or CD8+ lymphocytes may play an important role in alopecia areata.     

Strong expression of CD40, CD54 and HLA-DR antigen and lack of evidence for direct cellular cytotoxicity are unique immunohistopathological features in alopecia areata

The pathological role played by T cells infiltrating hair follicles in lesions of alopecia areata (AA) is unknown. We examined the expression in cryostat sections of scalp skin obtained from a total of 28 patients with AA and from five normal control subjects of (1) molecules related to the induction of cell death including Fas, Fas ligand (FasL), perforin, granzyme B (GB), and TIA-1, (2) molecules related to antigen presentation including CD1a, CD40, CD54, CD80, and CD86, and (3) molecules induced by interferon gamma (IFN-gamma) including CD40, CD54, Fas, and HLA-DR. CD3(+) T cells infiltrated perifollicularly, perivascularly and in the hair structure and there was a predominance of CD4(+) over CD8(+) cells. Antigen-presenting cells expressing CD1a, CD40, CD54, or HLA-DR were also seen. Expression of CD40, CD54, HLA-DR and CD95 was also seen in the hair structure including the dermal papilla. Consistent with these observations, IFN-gamma-producing cells were also detected in the perifollicular infiltrate. In contrast, few Fas-L(+), perforin(+), GB(+) or TIA-1(+) cells were found adjacent to the follicles. Apoptotic cells were recognized only in the outer root sheath of catagen hairs. These findings suggest that infiltrating T cells interact with perifollicular or follicular antigen-presenting cells to produce IFN-gamma, which deprives dermal papilla cells of their ability to maintain anagen hair growth.     

Transient CD44 variant isoform expression and reduction in CD4(+)/CD25(+) regulatory T cells in C3H/HeJ mice with alopecia areata

Alopecia areata, an autoimmune disease affecting anagen stage hair follicles, can be induced by grafting spontaneous alopecia areata affected skin to normal-haired C3H/HeJ mice. As the onset of alopecia areata can be significantly retarded by anti-CD44 variant isoform 10 treatment, it was interesting to explore the underlying disease mechanism. Two weeks after transplanting alopecia areata affected skin, expression of CD44 variant isoforms 3, 6, 7, and 10 was strikingly upregulated as compared with sham-grafted mice. By 6 wk after grafting, CD44 variant isoform levels had returned to normal, whereas in draining lymph nodes, CD44 variant isoform expression was slightly decreased. Leukocytes in the skin of mice with chronic alopecia areata expressed a hematopoietic isoform of CD44 and CD44 variant isoform 6 at an elevated level, but CD44 variant isoform 3 expression was reduced. Cytokine expression in leukocytes of chronic alopecia areata affected skin was higher than in normal-haired controls. Cytokine expression also increased postsurgery in sham and alopecia areata grafted mice, but remained elevated only in mice receiving alopecia areata affected skin. Finally, from the skin of mice with chronic alopecia areata and of mice transplanted with alopecia areata affected skin, an increased number of CD4(+) and CD8(+) cells, but a strongly decreased number of CD4(+)/CD25(+) regulatory T cells was recovered. Thus, expression of CD44 variant isoforms is important for the migration of leukocytes during the initial period of alopecia areata. CD44, however, is apparently not involved in the maintenance of the disease state, which is characterized by high cytokine expression levels, an increased number of CD4(+) and CD8+ cells, but a low level of CD4(+)/CD25(+) suppressor cells.     

Gene array profiling and immunomodulation studies define a cell-mediated immune response underlying the pathogenesis of alopecia areata in a mouse model and humans

Alopecia areata is a suspected autoimmune hair loss disease. In a rodent model, alopecia areata can be induced in normal haired C3H/HeJ mice by transfer of skin grafts from mice with spontaneous alopecia areata. At weeks 2, 4, 6, and 10 after surgery, grafted mice were euthanized, skin collected and processed for histology, and RNA extracted. Age-matched sham-grafted mice, and mice with and without spontaneous alopecia areata, were similarly processed. For comparison, skin biopsies from alopecia areata and androgenetic alopecia affected humans were also collected. Skin mRNA processed to cDNA was analyzed using Affymetrix mouse 11K and human 6800 gene chip(R) array technology. Microarray results indicated 42 known genes upregulated or downregulated during onset of mouse alopecia areata consistent with an inflammatory cell-mediated disease pathogenesis involving antigen presentation, costimulation, and a T helper 1 lymphocyte response. In contrast, 114 genes, many regulating immunoglobulin response, were altered late in disease development. In alopecia areata affected humans, 95 genes were significantly modulated. As confirmation of microarray analysis results, lymph node and spleen cells from alopecia areata affected mice injected into normal haired littermates transferred the alopecia areata phenotype. Alopecia areata onset could be inhibited in skin-grafted mice by modulation with B7.1- and B7.2-specific monoclonal antibodies. In addition, depletion of CD4+ CD8+ expressing cells in chronic alopecia areata affected mice using monoclonal antibodies permitted hair regrowth. The results consistently demonstrated the importance of an immune cell-mediated disease mechanism in alopecia areata pathogenesis and suggested targeting antigen-presenting cells and reactive lymphocytes may be effective in alopecia areata treatment.     

Mediation of alopecia areata by cooperation between CD4+ and CD8+ T lymphocytes: transfer to human scalp explants on Prkdc(scid) mice

OBJECTIVE: To determine the role of CD4+ and CD8+ T lymphocytes in the pathogenesis of alopecia areata. DESIGN: Relapse of alopecia areata was induced in autologous human scalp grafts on Prkdc(scid) mice by injection of activated T lymphocytes derived from lesional skin. CD4+ and CD8+ T cells were separated by magnetic beads before injection. SETTING: University-based dermatology practice. PARTICIPANTS: Eleven patients with either alopecia totalis or severe alopecia areata. MAIN OUTCOME MEASURES: Hair regrowth, hair loss, and immunohistochemical findings of scalp explants. INTERVENTION: Transfer of scalp T cells to autologous lesional scalp explants on Prkdc(scid) mice. RESULTS: Injection of unseparated T cells and mixed CD4+ plus CD8+ T cells resulted in significant hair loss (P<.01) in 5 of 5 experiments. However, injection of purified CD4+ or CD8+ T cells alone did not result in reproducible hair loss. CD4+ and CD8+ T cells induced follicular expression of intercellular adhesion molecule 1 (CD54), HLA-DR, and HLA-A, HLA-B, and HLA-C after injection into scalp grafts. CONCLUSIONS: CD4+ and CD8+ T cells have a role in the pathogenesis of alopecia areata. It is hypothesized that CD8+ T cells act as the effector cells, with CD4+ T cell help. It is now necessary to look for HLA-A, HLA-B, and HLA-C associations with alopecia areata. Therapeutic manipulations that interfere with CD8+ activity should be examined.     

Acneiform primary cutaneous CD4‐positive small/medium pleomorphic T‐cell lymphoma with prominent necrosis

Primary cutaneous CD4‐positive small/medium pleomorphic T‐cell lymphoma (SMPTCL) is an indolent form of cutaneous lymphoma that usually presents in solitary fashion and is histopathologically characterized by nodular infiltration of small‐ to medium‐sized pleomorphic T‐cells. We report the case of a patient who presented with a 5‐year history of acneiform lesions on his face. Histopathologic examination of two lesions revealed a nodular infiltrate of small to medium‐sized lymphocytes with necrosis in the dermis. The proliferating cells were positive for CD2, CD3 and CD4 and negative for CD8, CD30 and CD56. They were positive for TIA‐1 and negative for perforin and granzyme B. The Ki67 proliferation index was approximately 10%. The neoplastic cells expressed programmed death‐1 and lacked expression of CXCL‐13, bcl‐6 and CD10. In situ hybridization for Epstein–Barr virus‐encoded RNA yielded a negative result. T‐cell receptor gene rearrangement showed identical T‐lymphocyte monoclonality in both lesions. In brief, we report a rare case of acneiform SMPTCL with prominent necrosis.     

斑秃患者外周血T淋巴细胞亚群及CD4~+CD25~+调节T细胞的检测

目的检测斑秃(AA)患者外周血T淋巴细胞亚群及CD4+CD25+调节性T(Tr)细胞数量变化,分析AA的可能病因。方法利用流式细胞仪和单克隆荧光抗体技术,测定重度和局限性AA各40例患者外周血中T淋巴细胞亚群占T淋巴细胞的比率及CD4+CD25+Tr细胞在CD3+CD4+T淋巴细胞中的比率。结果重度AA患者外周血中CD4+CD25+Tr细胞占CD3+CD4+T细胞的比率为(1.43±0.74)%,显著低于正常对照组(2.25±0.97)%(P<0.01),重度AA患者的CD4+T占T淋巴细胞的比率为(31.42±6.66)%,略高于正常对照组(30.69±7.47)%(P>0.05),差异无显著性,而CD8+T占T淋巴细胞的比率为(25.86±4.35)%,明显高于正常对照组(22.42±6.10)%(P<0.01);局限性AA患者的三项指标分别为(2.14±0.87)%,(32.60±10.27)%和(21.59±5.24)%,与对照组差异无显著性(P>0.05)。结论AA患者外周血中CD4+CD25+Tr明显低于正常对照组,CD8+T比率明显高于正常对照组,可能是导致重度AA发病的主要免疫机制。     

T cell repertoire in mice with alopecia areata

It has become clear that skin infiltrating autoreactive CD4+ T helper cells play a crucial role in the initiation of alopecia areata. However, the natures of the pathogenic T cell clones as well as of the skin antigen they recognize remain obscure. Here, we analyzed the T cell receptor repertoire expressed in the spleen of diseased mice. We consistently observed the dominant expansion of a limited set of T cell clones expressing Vbeta8.2/Jbeta2.5 T cell receptor rearrangement. We conclude that T cell response in mice alopecia areata is markedly oligoclonal; a feature that may permit the design of selective immunotherapy.