三七总苷对大鼠自身免疫性睾丸炎的拮抗作用

目的:研究三七总苷(PNS)对自身免疫性睾丸炎模型鼠睾丸组织形态及血清肿瘤坏死因子α(TNF-α)和睾酮(T)含量的影响,以探讨PNS对睾丸炎的组织结构拮抗及作用机制。方法:建立大鼠睾丸炎模型,随机分为模型组和PNS拮抗组(200、400和800mg/kg),镜下观察各组睾丸组织结构变化。结果:800mg/kg PNS剂量组睾丸系数、生精小管直径、血清TNF-α和T含量与模型组比较,差异有统计学意义,光镜所见睾丸组织结构接近正常对照组形态;200mg/kg PNS剂量组和模型组生精上皮坏死、脱落,睾丸间质水肿,不同程度炎性细胞增生浸润。结论:400、800mg/kg PNS剂量组对大鼠睾丸炎症损伤具有拮抗作用。     

Effect of testicular macrophage conditioned media from rats with autoimmune orchitis on Leydig cell function

PROBLEM: The aim of this study was to investigate the influence of immune-activated testicular macrophages obtained from rats with autoimmune orchitis (EAO) on Leydig cell steroidogenesis. METHOD OF STUDY: EAO was induced in rats by active immunization with testis homogenate and adjuvants. Testicular and peritoneal macrophages from rats with EAO were isolated and cultured for 24 hr. Testosterone (T) production by purified Leydig cells incubated in vitro with macrophage-conditioned media (CM) from rats with EAO or control rats was measured. RESULTS: An increase in T production by Leydig cells incubated with CM from testicular, but not peritoneal, macrophages of rats with EAO was observed. This increase was dose-dependent up to a concentration of 30% CM; proportions higher than 35% exhibited an inhibitory effect. CONCLUSIONS: Immune-activated testicular macrophages obtained from rats with EAO induced both stimulatory and inhibiting steroidogenic effects on Leydig cells in vitro and not the exclusively inhibitory action that has widely been attributed to activated macrophages. This dual effect probably depends on the ability of these cells to synthesize different molecules that may exert opposite effects.     

Germ cell apoptosis in autoimmune orchitis: involvement of the Fas-FasL system

PROBLEM: The aim of this study was to determine the mechanism of germ cell death in experimental autoimmune orchitis (EAO) and the involvement of the Fas-FasL system in this process. METHOD OF STUDY: The EAO was induced in rats by immunization with testis homogenate and adjuvants. Apoptosis was studied by light microscopy, in situ end labeling of apoptotic DNA and DNA fragmentation techniques. Fas, FasL and caspase 3 expression was detected by immunohistochemistry. RESULTS: In rats with orchitis the number of Fas+ and FasL+ apoptotic germ cells increased from day 50, when the lesion develops, to 150 days, and correlates with the degree of testicular damage. Most spermatocytes expressing Fas were apoptotic. Many Fas+ germ cells were also immunoreactive for FasL. Moreover, these cells also expressed caspase 3. CONCLUSIONS: In rats with EAO germ cell death occurs through an apoptotic mechanism preceding germ cell sloughing. Immunohistochemical data suggest that the Fas-FasL system mediates germ cell apoptosis in an autocrine and/or paracrine way.     

Melanocytic lesions in lymph nodes associated with congenital naevus

Two cases of melanocytic lesions in lymph node associated with congenital naevus are presented. The first was a 30-year-old man with a nodular melanoma arising in a small congenital naevus. The second was a 2-year-old male infant with a giant congenital naevus. In both cases, naevus cell aggregates were observed in the capsule, trabeculae, perisinusoidal areas and lymphatic vessels surrounding the nodes. In the first case, clusters of large atypical melanocytes were present amongst naevus cell aggregates in the perisinusoidal areas as well as in the lymphoid parenchyma. Between the naevus cells and large atypical melanocytes, transitional forms were observed which supports the idea that the presence of large atypical melanocytes is indicative of benign naevus cells. In the second case, marginal sinuses were packed with clusters of large melanin-rich cells. Immunohistochemically, these cells were S-100 protein negative, but ultrastructural studies proved them to be melanocytes. They were considered indicative of spread of benign naevus cells via lymphatic vessels. Arrested migration of naevus cells during embryogenesis and benign spread of naevus cells are possible explanations for the histogenesis of naevus cell aggregates in lymph nodes associated with congenital naevus.     

Identification of immunodominant autoantigens in rat autoimmune orchitis

Infection and inflammation of the genital tract are amongst the leading causes of male infertility. Experimental autoimmune orchitis (EAO) in the rat serves as a model for the investigation of inflammatory testicular impairment. In this study, experiments were conducted to identify the molecules that are responsible for eliciting the autoimmune attack on the testis. EAO was induced in in-bred Wistar rats by active immunization with testis homogenates (EAO group I). Development of disease was observed using histological techniques and a new non-invasive three-dimensional (3D) imaging technology for in vivo monitoring, termed flat-panel volumetric computed tomography (fpvCT). Examination of control and EAO testes demonstrated the superior image quality of high-resolution fpvCT. A proteomics approach using 2D SDS-PAGE and immunoblotting analysis with EAO sera identified 12 spots. Seven were subsequently identified by mass spectrometry as heat shock proteins 60 (Hsp60) and 70 (Hsp70), disulphide isomerase ER-60, alpha-1-anti-trypsin, heterogeneous nuclear ribonucleoprotein H1 (hnRNP H1), sperm outer dense fibre major protein 2 (ODF-2), and phosphoglycerate kinase 1. Hsp70, ODF-2, hnRNP H1, and ER-60 were identified by all EAO sera studied. To test the capacity of the identified proteins to elicit testicular autoimmune disease, recombinant proteins were used either individually or in combination to immunize rats (EAO group II). In all groups, the incidence of EAO was 25%. Inflammatory-type (ED1+) and resident (ED2+) macrophages, lymphocytes (CD45RA+), and dendritic cells (Ox-62+) were strongly increased in EAO group II animals, comparable to the testes of EAO I rats. Pre-immunization with a low dose of recombinant Hsp 70, hnRNP H1 or ODF-2 before induction of EAO with testis homogenate significantly delayed the onset of EAO but could not prevent disease. The identification of testicular autoantigens will allow a better understanding of disease pathogenesis and could provide a basis for the development of novel therapies for inflammation-based male infertility.     

Lymphocyte subsets recirculate from blood to lymph at different rates in conscious sheep

Lymphocytes migrate and recirculate continuously between blood and lymph. The current study compared the migratory properties of lymphocyte subsets by labelling lymphocyte surface antigens and analysing simultaneous samples of mesenteric lymph, systemic arterial blood and mesenteric venous blood from conscious sheep. The lymphocyte output in lymph averaged 5.33±0.89×10⁶ min^–1 (lymph flow rate of 92±13 l min^–1, lymphocyte count 61.9±10.7×10⁹ l^–1, n=10) and the proportion of cells which carried the CD4 antigen (CD4⁺) was considerably higher in lymph (46±3 %) than in arterial blood (23±2 %; p<0.01, n=9). This increase was paralleled by a small but statistically significant decrease in the percentage of CD4⁺ cells in mesenteric venous blood(20±2 %), as compared with systemic arterial blood (26±2 %; p<0.05, n=6), which is consistent with the hypothesis that CD4⁺ cells migrate out of the blood vessels, and recirculate through the lymphatics, more readily than other lymphocyte subsets.     

Hyperplastic mesothelial cells in subpleural lymph nodes mimicking metastatic carcinoma

We describe a patient with a history of poorly differentiated adenocarcinoma of the stomach in whom hyperplastic mesothelial cell inclusions were present in subpleural lymph nodes. The involved lymph nodes were embedded in a lipoma in the parietal pleura. The role of histochemistry and immunohistochemistry in the differentiation of these inclusions from metastatic carcinoma is discussed.     

Lymphocyte subsets alteration in patients with argentine hemorrhagic fever

Peripheral blood lymphocyte subpopulations were studied in 15 patients with Argentine Hemorrhagic Fever (AHF), during the acute period of the disease and in early convalescence. Anti-human Ig antibodies were used to identify B cells and monoclonal antibodies to assess T4 and T8 subsets. During the acute period of the disease, significant alterations were found in B, T4, and T8 lymphocytes (P less than .001), as well as in T4/T8 ratios (P less than .001). These abnormalities disappeared in early convalescence, around 30 days after the clinical onset. Diminished numbers of T4 lymphocytes are interpreted as relevant to the immunodepression that characterizes the acute phase of AHF.     

Death receptor and mitochondrial pathways are involved in germ cell apoptosis in an experimental model of autoimmune orchitis

BACKGROUND: Studies on experimental autoimmune orchitis (EAO) have helped to elucidate immunological mechanisms involved in testicular damage. We previously demonstrated that EAO is characterized by lymphomononuclear cell infiltrates and apoptosis of spermatocytes and spermatids expressing Fas and TNFR1. The aim of this work was to characterize the pathways involved in germ cell apoptosis in EAO and to determine the involvement of the Bcl-2 protein family in this process. METHODS AND RESULTS: EAO was induced in rats by immunization with testicular homogenate (TH) and adjuvants, whereas control (C) rats were injected with saline solution and adjuvants. Testis of EAO rats showed procaspase 8 cleavage products (western blot) with high caspase 8 activity. Cytochrome c content increased in the cytosol and decreased in the mitochondrial fraction of testis from EAO rats compared with C, concomitant with increased caspase 9 activity. Bax was mainly expressed in spermatocytes and spermatids and Bcl-2 in basal germ cells (immunohistochemistry). Baxbeta isoform content increased in EAO rat testis compared with C, whereas content of Baxalpha remained unchanged (western blot). However, Baxalpha content decreased in the cytosol and increased in the mitochondrial and endoplasmic reticulum (ER)-enriched fractions of testis from EAO rats compared with C (western blot). Bcl-2 content also increased in the testes of EAO rats. CONCLUSIONS: Our results demonstrated that extrinsic, mitochondrial and possibly ER pathways are inducers of germ cell apoptosis in EAO and that Bax and Bcl-2 proteins modulate this process.     

引流肺淋巴的淋巴结内灰尘颗粒的分布和结构变化

目的 :研究灰尘颗粒在引流肺淋巴的淋巴结内的分布和淋巴结的组织变化。方法 :取成人和儿童肺门淋巴结、气管支气管淋巴结和气管旁淋巴结 ,石蜡切片。取肺切除术患者的肺门淋巴结作超薄切片。结果 :尘细胞和灰尘颗粒分布于淋巴结被膜、淋巴窦和髓索内。肺门淋巴结和气管支气管淋巴结的光密度比气管旁淋巴结高 ,成人肺门淋巴结、气管支气管淋巴结和气管旁淋巴结的光密度高于儿童的淋巴结。成人淋巴结的血管密度与儿童淋巴结之间存在着显著性差异。间质胶原纤维增生。结论 :在引流肺淋巴的淋巴结内 ,肺门淋巴结和气管支气管淋巴结内的灰尘颗粒明显多于气管旁淋巴结 ,成人淋巴结灰尘颗粒比儿童多。在成人 ,灰尘颗粒引起淋巴结的淋巴组织减少 ,纤维组织和血管增生     

Lymphocyte subsets and mitogen stimulation of blood lymphocytes in preeclampsia.

PROBLEM: The question of whether there are differences in systemic immune reactivity in severe preeclampsia compared with normal pregnancy was addressed. METHOD OF STUDY: During the third trimester, blood samples were taken from 12 pregnant women with severe preeclampsia. Five of the preeclamptic pregnancies were analyzed separately because they were treated with dexamethasone before the blood samples were taken. The seven dexamethasone-treated preeclamptic pregnant women were analyzed and compared with six uncomplicated pregnancies. A control group consisted of 15 nonpregnant females. Lymphocyte subsets were identified by flow cytometry. The function of peripheral blood mononuclear cells (PBMCs) was studied as proliferative responses to mitogens alone and in combination with immunomodulating drugs. RESULTS: An increased number of B lymphocytes (CD19+) (P < 0.05) and natural killer (NK) cells (P < 0.05) was noticed in severe preeclampsia compared with normal pregnancy. The proliferative response of PBMCs in phytohemagglutinin (PHA)-stimulated cultures in autologous serum from patients with severe preeclampsia was reduced (P < 0.05) compared with normal pregnancy. The addition of indomethacin and cimetidine significantly stimulated (P < 0.05) the proliferative responses. The enhancing effect of cimetidine was not found in dexamethasone-treated preeclamptic patients. CONCLUSIONS: The presence of systemic immunosuppression in severe preeclampsia is demonstrated as a reduced proliferative response of PBMCs to PHA, which could be partly restituted by indomethacin or cimetidine, indicating immunosuppressor activity that is mediated by prostaglandin and histamine. Increased levels of B lymphocytes and NK cells were also noticed.     

Role of cervical lymph nodes in autoimmune encephalomyelitis in the Lewis rat

Lymphocytes enter the central nervous system (CNS) in response to virus infections and in autoimmune diseases, such as multiple sclerosis (MS), but the origin of such lymphocytes is unclear. This study investigates the role of the cervical lymph nodes as a source of lymphocytes involved in experimental autoimmune disease of the brain. Acute active experimental autoimmune encephalomyelitis (EAE) is used as a model for the autoimmune aspects of MS and is characterized by lymphocyte and monocyte invasion and microglial activation, mainly in the spinal cord, 12–15 days post-inoculation (dpi) of antigen. Few lesions occur in the cerebral hemispheres in acute EAE, but a cryolesion to the surface of the brain 8 dpi results in a six-fold enhancement of cerebral EAE. The present study tests the hypothesis that cervical lymphadenectomy will reduce the enhancement of cerebral EAE induced by a cryolesion. Acute EAE was induced in 25 Lewis rats and a cryolesion to the brain, 8 dpi, in 16 rats was immediately followed by either cervical lymphadenectomy (n=8) or sham lymphadenectomy (n=8). The severity of EAE at 15 dpi, in the brain and spinal cord, was evaluated using immunocytochemistry for T lymphocytes (W3/13) and MHC class II expression (OX6). The results of the study showed that cervical lymphadenectomy reduced the level of cerebral EAE induced by a cryolesion by 40 per cent when compared with the sham-operated animals (P<0·01). This suggests that cervical lymph nodes play a pivotal role in the induction of EAE in the brain, possibly as a site for ‘priming’ T cells to target the brain. Investigation of the interrelationships between cervical lymph nodes and the brain in man may lead to new therapeutic strategies for multiple sclerosis. © 1997 John Wiley & Sons, Ltd.     

Development of the lymph nodes in the very young, and their evolution in the mature, nude rat

We recently revised the concepts on the morphology of the lymph nodes of the young adult athymic nude rat. The present work studied the postnatal development of its nodal structures and their evolution with aging. The structural development of the deep cortex "units" was found to progress as usual. However, while the concentration of lymphocytes appeared to develop normally in the periphery of a unit, the center of the unit remained lymphocyte-depleted. Further, the peripheral cortex failed to develop over the middle part of a unit center. With aging, the peripheral cortex over the remainder of a unit center could atrophy and disappear completely. The present findings did not yield information as to whether thymic elements are necessary to trigger the development of a unit, but they revealed that its further development is determined by stimuli. It was concluded that, in the absence of T-cells, stimuli for cellular immune responses provoke the proliferation of the reticular or interdigitating cells of a unit center. On the other hand, an increase of these stimuli was concluded to cause the peripheral cortex to fail to develop over part of a unit center and, later, to atrophy over the remainder of the unit center. The mechanisms of the phenomena are discussed.     

实验性自身免疫性脑脊髓炎小鼠发病高峰期外周及中枢淋巴细胞亚群的变化

目的:观察髓鞘少突胶质细胞糖蛋白MOG35-55诱导的实验性自身免疫性脑脊髓炎小鼠发病高峰期中枢及外周淋巴细胞亚群的变化,探讨EAE发病高峰期细胞与体液免疫学的变化。方法:用MOG35-55免疫诱导雌性C57BL/6小鼠制作EAE模型,记录小鼠行为学变化,HE染色观察CNS炎症组织病理变化,使用流式细胞仪检测小鼠中枢及外周脾脏淋巴细胞中CD3+CD4+、CD3+CD8+、CD4+CD25+、B220+细胞亚群变化情况。结果:EAE组小鼠中枢神经系统有CD3+CD4+、CD3+CD8+、CD4+CD25+、B220+淋巴细胞的浸润,CFA阴性对照组中枢神经系统未检测到淋巴细胞浸润。EAE组小鼠外周脾细胞中CD3+CD4+、CD3+CD8+细胞较CFA阴性对照组减少(P0.05),B220+细胞较CFA阴性对照组明显升高(P0.01),CD4+CD25+细胞较CFA阴性对照组升高但无统计学差异。结论:小鼠在EAE发病高峰期,外周脾细胞中CD3+CD4+、CD3+CD8+阳性细胞明显减少,B220+明显升高,CD4+CD25+也开始有升高趋势,表明EAE发病高峰期细胞免疫及体液免疫共同调控了EAE的病理过程,T淋巴细胞与B淋巴细胞都起了很重要的主导作用。     

Generation and characterization of a continuous line of CD8+ suppressively regulatory T lymphocytes which down-regulates experimental autoimmune orchitis (EAO) in mice.

We have previously shown that two injections with viable syngeneic testicular germ cells (TC) alone developed experimental autoimmune orchitis (EAO) in C3H/He mice, and that the induction of antigen-specific tolerance in this EAO model is associated with the generation of antigen-specific suppressively regulatory T (Ts) cells. For the elucidation of the nature of these Ts cells, a murine Ts cell line (designated Ts-A) was established. This line was generated from the spleen cells of C3H/He mice which had received three i.v. injections of a soluble (deaggregated) form of murine testicular antigen (mTA), followed by the repeated selection of these spleen lymphocytes in vitro by stimulation with mTA. Adoptive transfer of Ts-A cells into naive syngeneic mice immediately before the first TC injection was found to downgrade EAO in actively immunized recipients. The transferred Ts-A cells significantly inhibited the cellular immune response to TC in the recipients in an antigen-specific manner, but these cells had no inhibitory effect on the humoral immune response to TC. This line could also inhibit in vitro syngeneic TC-driven proliferation of orchitogenic lymphocytes. Surface phenotype of this line was CD8+, CD4-, Thy-1.2+, CD3+, and TCR alpha beta+. These findings may suggest an in vivo role for suppressively regulatory lymphocytes, capable of inhibiting helper T cells, in the regulation of EAO.     

Fine-needle aspiration of supraclavicular lymph nodes

Malignancies from many primary sites may metastasize to supraclavicular lymph nodes (SCLN). We reviewed 100 fine-needle aspirations (FNAs) of SCLNs. There were three major types of malignancy detected by this method: adenocarcinoma (n=40), squamous cell carcinoma (n=14), and other malignancies (n=29). Adenocarcinomas and other malignancies from all sites tended to metastasize to the left SCLN. Squamous cell carcinomas from all sites, however, tended to appear on the right side. For 61 patients, a previous diagnosis of malignancy had been made within 1 yr of the clinical appearance of the abnormal SCLN. For 20 patients, the primary diagnosis antedated SCLN metastasis by more than 1 yr, particularly patients with adenocarcinoma of the breast. prostate, or thyroid papillary carcinoma. Diagn Cytopathol 1996 14:216-220. © 1996 Wiley-Liss, Inc.     

Functional and phenotypic characteristics of testicular macrophages in experimental autoimmune orchitis

Testicular inflammation with compromised fertility can occur despite the fact that the testis is considered an immunoprivileged organ. Testicular macrophages have been described as cells with an immunosuppressor profile, thus contributing to the immunoprivilege of the testis. Experimental autoimmune orchitis (EAO) is a model of organ-specific autoimmunity and testicular inflammation. EAO is characterized by an interstitial inflammatory mononuclear cell infiltration, damage of the seminiferous tubules and germ cell apoptosis. Here we studied the phenotype and functions of testicular macrophages during the development of EAO. By stereological analysis, we detected an increased number of resident (ED2+) and non-resident (ED1+) macrophages in the testicular interstitium of rats with orchitis. We showed that this increase was mainly due to monocyte recruitment. The in vivo administration of liposomes containing clodronate in rats undergoing EAO led to a reduction in the number of testicular macrophages, which correlated with a decreased incidence and severity of the testicular damage and suggests a pathogenic role of macrophages in EAO. By immunohistochemistry and flow cytometry we detected an increased number of testicular macrophages expressing MHC class II, CD80 and CD86 costimulatory molecules in rats with orchitis. Also, testicular macrophages from rats with EAO showed a higher production of IFNgamma (ELISA). We conclude that testicular macrophages participate in EAO development, and the ED1+ macrophage subset is the main pathogenic subpopulation. They stimulate the immune response through the production of pro-inflammatory cytokines and antigen presentation and thus activation of T cells in the target organ.     

胸导管的引流途径及其与周围淋巴结联系的研究

在49具胎儿尸体和5只狗,用淋巴管间接注射法,研究了胸导管的引流途径及其周围淋巴结。在胎儿,57%胸导管借胸导管侧支与周围淋巴结相连,左锁骨上淋巴结的出现率为31%。胸导管侧支、淋巴结及其输出淋巴管构成胸导管的侧副淋巴回流径路。在狗,注射后2小时,部分淋巴结显色,注射剂是经胸导管顺向流入淋巴结的。     

Irgm1基因敲除对实验性自身免疫性脑脊髓炎小鼠CD4~+ T细胞亚群的影响

目的:探讨免疫相关GTP酶1(Irgm1)基因敲除对实验性自身免疫性脑脊髓炎(Experimental autoimmune encephalomyelitis,EAE)小鼠CD4+T细胞的影响。方法:C57BL/6纯系小鼠与Irgm1基因敲除杂合小鼠(Irgm1+/-)回交十代繁殖C57BL/6背景下Irgm1+/-小鼠,用C57BL/6 Irgm1+/-小鼠杂交获取Irgm1-/-、Irgm1+/-、Irgm1+/+三种基因型小鼠,PCR扩增DNA检测基因型。用髓鞘少突胶质糖蛋白(Myelin oligodendrocyte glycoprotein,MOG33-55)多肽与弗氏完全佐剂等量混合制成乳剂,免疫C57BL/6野生型(Wt.)和Irgm1基因敲除(Irgm1-/-)小鼠,建立EAE模型,并进行临床评分。MTT法测定MOG33-55免疫后7 d的EAE小鼠淋巴结细胞中MOG33-55特异性T细胞增殖分化水平。取MOG33-55免疫后14 d EAE小鼠脊髓切片,HE染色检测炎细胞浸润情况。取MOG33-55免疫后16 d的EAE-小鼠淋巴结、脊髓和脑组织,提取单个核细胞,用MOG33-55(20μg/ml)体外刺激培养7 d,收集细胞,用流式细胞仪分析EAE小鼠淋巴结、中枢神经系统浸润细胞中Th1、Th17的改变。结果:成功诱导了EAE小鼠模型;HE染色结果显示Wt.小鼠脊髓周围出现明显炎细胞浸润,而Irgm1-/-小鼠则无明显变化;MTT实验表明Irgm1-/-小鼠与Wt.小鼠相比,淋巴结中T细胞对MOG33-55特异性增殖能力降低;流式细胞分析表明相对于Wt.小鼠,Irgm1-/-小鼠EAE模型在淋巴结及中枢神经系统浸润细胞中Th1细胞亚群比例明显增高而Th17细胞亚群比例下降。结论:Irgm1基因敲除可部分保护EAE小鼠的脊髓功能及临床症状。在EAE发病早期,Irgm1可能起到了关键性的作用,因此Irgm1有可能成为EAE治疗的重要分子靶点。