Use of serum-free hormonally defined media to evaluate the effects of growth factors and inhibitors on proliferation of estrogen-responsive mammary and pituitary tumor cells in culture

Summary Serum-free defined media have been developed for assay of the mitogenic effects of growth factors on human MCF-7, human T-47D, and mouse COMMA-D mammary cells as well as for identification of mitogens and inhibitors of GH₄C₁ rat pituitary tumor cell growth. These lines were shown to grow in vivo in response to a variety of hormones including estrogens and thyroid hormones. With mammary cells, complete hormonally and nutritionally defined media were established that supported continuous passage at 50 to 90% of the serum stimulated rate. The strategy used to measure mitogens for mammary cells was to identify nutritional conditions where the growth rate was reduced greatly without impairing the response to picomolar to nanomolar concentration of growth factors. The effects of polypeptide growth factors and tissue extracts were estimated by their addition to basal medium and measuring cell number increases or labeled thymidine incorporation into DNA. In a variation of this methodology, the MTW9/PL2 rat mammary cells were used to identify secreted autocrine growth factors; nutritionally defined conditions were sought for growth of these rat cells in the complete absence of exogenous growth factors. The factors secreted into the medium were detected by bioassays with COMMA-D or MCF-7 mammary cell lines. The effects of growth factors-inhibitors on pituitary cells were measured by a related method; the GH₄C₁ cells were grown at less than optimal rates in a defined medium designated TRM-1. Addition of mitogens to TRM-1 stimulated pituitary cell growth whereas addition of inhibitors caused reduced levels of growth. The methods described in this report offer new means of assaying growth factors-inhibitors for a range of mammary and pituitary tumor cells.     

Immunostaining of galectin-3 and CD44v6 using fine-needle aspiration for distinguishing follicular carcinoma from adenoma

To evaluate the clinical applicability of galectin-3 and CD44 variant 6 (CD44v6) immunostaining in fine-needle aspiration cytology (FNAC) of thyroid follicular tumors, 79 cytological specimens (35 follicular carcinomas and 44 follicular adenomas) were studied. The positive rates of galectin-3 and CD44v6 were 89 and 74% in follicular carcinoma, respectively, and 25 and 30% in follicular adenoma, respectively. There were no significant correlations between the expression of galectin-3 or CD44v6 in follicular carcinoma and characteristics such as capsular invasion, vascular invasion, metastasis, or tumor size. Positive staining of either galectin-3 or CD44v6 resulted in a diagnostic sensitivity of 97% and a specificity of 52% for follicular carcinoma among follicular tumors. Immunostaining of galectin-3 or CD44v6 using cytological specimens can provide independent information on conventional morphological findings of cytology to distinguish follicular carcinoma from adenoma.     

甲状腺过氧化物酶抗体在甲亢、甲状腺占位性病变诊治中的临床意义

目的观察不同临床治疗阶段甲亢、甲状腺占位性病变患者血清甲状腺过氧化物酶抗体(TPOAb)浓度的变化,探讨其临床指导价值。方法采用固相化学发光酶免疫分析法观察血清TPOAb浓度变化与甲状腺功能减退(化验室检查结果)的关系。结果89例患者中,有50例患者治疗前TPOAb正常或仅轻度增高,治疗后呈现阳性者31例,其中19例发生了甲减或亚临床型;39例治疗前TPOAb阳性,治疗后转阴2例,26例浓度降低,发生甲减3例,29例患者服用不同剂量的甲状腺片而未出现甲减或亚临床型。结论甲亢和甲状腺占位性病变患者在治疗过程可伴有TPOAb浓度的变化,TPOAb的明显增高,预示出现甲减的可能性大,TPOAb阳性的患者应被关注,高滴度TPOAb的甲亢患者治疗方案的选择要慎重。     

Suppressive effect of hyperbaric oxygenation on immune responses of normal and autoimmune mice.

We studied the effect of hyperbaric oxygenation (HBO) on immune responses in normal and autoimmune mice. Mice were exposed to HBO in an animal chamber at a pressure of 252.5 kPa for 1 h and once a day for 5 days. The immunization of C3H/He mice with sheep erythrocytes induced marked anti-sheep erythrocyte antibody response on day 7. However, this response was markedly suppressed in HBO-treated mice. The suppression is dependent on the duration of HBO and it works on the early and the late stage of antibody responses. HBO suppresses the development of both sheep erythrocyte-specific B cells and helper T cells after the immunization. Then, we tried to expose autoimmune mice to HBO. Spontaneous immunoglobulin production of NZB and MRL/lpr spleen cells was also significantly suppressed by HBO. Furthermore, long term HBO exposure results in the suppression of the development of autoimmune symptoms such as proteinuria, facial erythema and lymphadenopathy in MRL/lpr mice. All these results suggest that HBO is applicable for the treatment of autoimmune diseases.     

SLE患者IgG类抗-Sm/抗-U_1RNP自身抗体进入存活Hep-2细胞内的观察

本文采用生物素-亲合素-碱性磷酸酶法(ABC-AKP)检测10例正常人血清和7例系统性红斑狼疮(SLE)病人血清中IgG类抗-Sm/抗-U_1RNP抗体对Hep-2细胞的进入作用.结果表明:自身抗体能够进入存活的Hep-2细胞,患者组阳性者4例(57.14%),对照组皆为阴性(P<0.05).讨论了自身抗体进入活的靶细胞在自身免疫性疾病发病中的意义.     

Effect of potassium iodide and perchlorate on the secretion of thyroid hormones

The effect of potassium iodide and perchlorate was studied on parameters characterizing the secretion of thyroid hormones: the cyclic AMP level in the thyroid gland tissue and the number of intracellular colloid drops during stimulation with thyroid hormones. Excess of iodide was shown to depress these indices whereas perchlorate prevented the inhibitory effect of iodide. The results support the view that the adenylate-cyclase system of the thyroid gland is sensitive to the organic iodine concentration. Excess of iodide evidently reduces the ability of perchlorate to affect its concentration in the gland, so that the process of organification of iodine and the secretion of thyroid hormones are maintained at the optimal level.Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 86, No. 12, pp. 653–655, December, 1978.     

不同年龄组儿童甲状腺测量值及其变化的研究

调查了90具童尸用状腺的基本形态,可分四型。其中以甲状腺由两侧叶及峡部组成者为最多,占50.22%。测量了甲状腺各部的长、宽、厚。分年龄组进行了数据的统计学处理,并计算出儿童与成人甲状腺各部相应值的百分比。结果显示:小儿甲状腺侧叶的长度和宽度在幼儿期(1—3岁)就已发育近成人的一半。随着年龄的增长,甲状腺各部的均值逐渐增加。除各部的宽度以及锥体叶的长度外,各相邻两组间同项均数的比较,经双侧T检验发现差异具有高度显著性(P<0.01)。     

Liver-infiltrating T helper cells in autoimmune chronic active hepatitis stimulate the production of autoantibodies against the human asialoglycoprotein receptor in vitro.

Autoantibodies against the human asialoglycoprotein receptor (ASGPR) occur in the sera of patients with autoimmune liver disorders. Liver-infiltrating T cell clones that specifically recognize the ASGPR have been described in patients with autoimmune chronic active hepatitis (AI-CAH) and primary biliary cirrhosis (PBC). Recently, we have shown that peripheral blood mononuclear cells (PBMC) from patients with AI-CAH or PBC but not chronic viral hepatitis secreted anti-ASGPR antibodies in vitro. In this study we characterized the influence of liver-infiltrating T cells on the secretion of ASGPR-specific autoantibodies by autologous B cells in cell culture supernatants. T cell clones from liver biopsies of three patients with chronic autoimmune liver disorders (one with AI-CAH, two with PBC) were isolated and investigated for their proliferative response to soluble ASGPR and their helper function provided to autoantibody-secreting B lymphocytes. PBMC from these patients secreted autoantibodies spontaneously in their cell culture supernatants and showed a proliferative response to ASGPR. T cell-depleted PBMC, however, lacked spontaneous antibody secretion. Four CD4+CD8- liver-infiltrating T cell clones showed a proliferative response to ASGPR and also induced spontaneous anti-ASGPR antibody production in cell culture supernatants when added to autologous T cell depleted PBMC. Activated supernatants of these T cell clones failed to induce antibody production. None of seven CD4+CD8- and two CD4-CD8+ T cell clones non-responding to ASGPR provided this help for antibody secretion. Anti-ASGPR secretion in vitro could not be inhibited by the addition of MoAbs raised against monomorphic determinants on HLA class II molecules. The addition of purified ASGPR or polyclonal-activating pokeweed mitogen showed no influence on the production of autoantibodies in these cultures. These data show that B lymphocytes require T cell help for the production of ASGPR-specific antibodies. This help can be provided by ASGPR-responsive T helper cells via cellular interactions.     

天然角蛋白自身反应性B细胞亚群及功能的分析

目的:明确天然角蛋白反应性B细胞的亚群及解剖定位,初步分析其分泌天然抗角蛋白自身抗体(anti-keratinautoantibody,AKautoAb)的能力。方法:取SPF级C57BL/6小鼠的脾细胞和腹腔细胞,荧光抗体染色后用流式细胞仪分析角蛋白反应性B细胞亚群。将脾脏和腹腔淋巴细胞体外培养后,用ELISA分析AKautoAb的滴度,用ELISPOT法分析分泌AKautoAb的B细胞数。结果:腹腔中几乎所有结合角蛋白的B细胞均为B-1a细胞,脾脏中结合角蛋白的B细胞以边缘带B细胞为主。腹腔细胞中分泌AKautoAb的细胞数显著多于脾细胞,其培养上清中AKautoAb的滴度显著高于脾细胞。结论:角蛋白反应性B细胞存在于3个成熟B细胞亚群:B-1细胞、滤泡B细胞和边缘带B细胞,其中CD5 的B-1a细胞具有活跃的分泌AKautoAb的能力。     

Adhesion molecule expression in Graves'' thyroid glands; potential relevance of granule membrane protein (GMP-140) and intercellular adhesion molecule-1 (ICAM-1) in the homing and antigen presentation processes.

To assess the potential role of adhesion molecules in the pathogenesis of Graves' disease, we examined the expression of several of these adhesion molecules, including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule (VCAM-1) and granule membrane protein-140 (GMP-140), in sections of Graves' thyroid glands and control thyroids, using immunohistochemical techniques. Up-regulated expression of GMP-140 was frequently observed on endothelial cells (EC) of post-capilliary venules in all Graves' thyroids examined, compared with an occasional weak staining on EC control glands. Some capillary EC around thyroid follicles (perifollicular EC) were strongly positive for GMP-140 in the Graves' thyroids in contrast to a negative staining on the same structures in the control glands. In addition, there was a correlation between the reactivity and frequency of GMP-140 expression on EC and the severity of mononuclear cell (MNC) infiltration in the Graves' thyroids. The expression of ICAM-1 was up-regulated on perifollicular EC and EC of small venules in some thyroids of both Graves' and control groups. Conversely, no significant expression was observed on any type of EC for both endothelial-leucocyte adhesion molecule-1 (ELAM-1) and VCAM-1. However, dendritic-like cells, present within lymphocytic infiltrates, were positive for VCAM-1 in most of the Graves' thyroids examined, especially in those with a severe lymphocytic infiltration. Thyrocytes were constantly negative for the expression of all four adhesion molecules investigated. These data suggest that GMP-140, as well as ICAM-1, could play an important role in the initiation of MNC infiltration in Graves' disease. ELAM-1 and VCAM-1 appear not to be relevant for the migration of MNC from the blood vessels into the target gland, although VCAM-1 expression on dendritic-like cells might play an additively tissue-selective role in autoantigen presentation and subsequent elicitation of autoimmune phenomena.