A monoclonal antibody (RH1-38) which inhibits multiple systems of cell-mediated cytotoxicity--I. Identification of a novel epitope on a killer cell surface bimolecular complex important in the cytotoxic response

This paper presents the initial characterization of a mouse monoclonal antibody (RH1-38) which blocks, in the absence of complement, three different systems of cell-mediated cytotoxicity. This monoclonal antibody markedly inhibits cytotoxicity mediated by human natural killer cells, a monocyte-like cell [phorbol myristate acetate (PMA) stimulated HL-60], and cytotoxic T-lymphocytes generated in a mixed leukocyte reaction. RH1-38 is not nonspecifically toxic to cells since antibody-dependent cellular cytotoxicity was not inhibited and viability as assessed by trypan blue exclusion was not affected. Inhibition is specific since control hybridoma culture supernatants, parent (NS-1) ascites supernatant, monoclonal anti-HLA and normal mouse IgG were not significantly inhibitory. In the NK system, the inhibitory effect appears to be due to binding of monoclonal antibody to effector cell surface since exposure of targets to antibody followed by washing yielded no inhibition of killing. Inhibition requires the antigen-binding portion of the antibody molecule and thus appears to be related to steric hindrance of an effector cell surface molecule which is important in the expression of cell-mediated cytotoxicity. Immunoprecipitation of surface-radioiodinated membranes from PMA-stimulated HL-60 cells and analysis on sodium dodecyl sulfate-polyacrylamide gels revealed a bimolecular complex (195,000 and 125,000 daltons) without significant change under reducing conditions. Control immunoprecipitates yielded no peaks of activity. This monoclonal antibody should serve as a useful probe of the function and biochemistry of a killer cell surface antigen important in the expression of cell-mediated cytotoxicity. Since RH1-38 inhibits cytotoxicity mediated by at least three apparently unrelated effector cells, the relevant antigen may be part of a common mechanistic step. As the companion paper demonstrates, this monoclonal antibody does not affect the conjugation step, but appears to block a late step in the NK cytolytic mechanism. Thus, RH1-38 recognizes either an epitope district from previously-described anti-LFA-1 antibodies or alternatively recognizes a distinct functional killer cell surface molecule.     

Effect of NMDA on Production of Reactive Oxygen Species by Human Lymphocytes

Low concentrations (<20 M) of N-methyl-D-aspartate (NMDA), an agonist of specific receptors of brain glutamatergic systems, promote the formation of reactive oxygen species (ROS) both in the whole blood and in lymphocyte fraction. Further increase in NMDA concentrations led to progressive increase in ROS content in the whole blood, but to its decrease in lymphocyte suspension. The activating effect of NMDA is abolished by antioxidant N-acetylcysteine (5 mM) and NMDA-type glutamate receptor antagonist MK-801 (5 M). Phorbol myristate acetate (PMA, 1 M) also increased ROS content in the examined structures. This effect was antagonized by N-acetylcysteine, but not MK-801.     

Lucigenin-dependent chemiluminescence as a new assay for NAD(P)H-oxidase activity in particulate fractions of human polymorphonuclear leukocytes

The enzyme responsible for the respiratory burst in human neutrophils is an oxidase that catalyzes the reduction of oxygen to superoxide anion (O-2). Superoxide anion production may be measured by chemiluminescence (CL) in the presence of lucigenin (10,10'-dimethyl-9,9'- biacridinium dinitrate). We established an assay of the oxidase, by measuring the CL of particulate fractions of PMN in the presence of lucigenin . This CL required the addition of NAD(P)H and was very low in fractions of resting cells. In particulate fractions of PMNs stimulated with PMA selectively, the NADPH-dependent CL was found to be increased. CL was linear with protein concentrations up to 100 micrograms and was shown to be at least 10 times more sensitive for the detection of O-2 than the assay based on the spectrophotometric determination of superoxide mediated cytochrome c reduction. CL was abolished by inactivating the enzyme at 56 degrees C.     

Distribution of acid alpha-naphthyl acetate esterase among human T lymphocyte subsets

Human T lymphocyte subsets, identified by means of OKT3, 4 and 8 monoclonal antibodies, were isolated by a fluorescence activated cell sorter (FACS IV) and analyzed for distribution of alpha-naphthyl acetate esterase (ANAE) activity. As compared to OKT8⁺ lymphocytes a higher proportion of OKT4⁺ lymphocytes was ANAE-positive exibiting a spot or dot-like pattern in the cytoplasm. OKT8 and 4 positive subsets showed a similar ANAE distribution in diffuse granular form. Although OKT4 and OKT8 populations presented a different ANAE dot-like reactivity, this marker did not allow as clear a distinction between them as that reported for T_G and T_M lymphocytes.     

Growth,regression and cell death in rat liver as related to tissue levels of the hepatomitogen cyproterone acetate

Previous studies have shown that xenobiotic compounds such as the environmental pollutant -hexa-chlorocyclohexane (-HCH) and the synthetic sex steroid cyproterone acetate (CPA) induce growth of rat liver by hypertrophy and hyperplasia. After withdrawal of the growth stimuli, liver hypertrophy was usually found to be readily reversible. Conflicting observations were made concerning the fate of liver hyperplasia: hepatic hyperplasia persisted when induced by -HCH but was found to be partially reversible when induced by CPA. The present study confirms the reversibility of hepatic hyperplasia induced by CPA in rats: about 30% of liver DNA present at maximal liver enlargement disappeared within 6 days after cessation of CPA treatment. Simultaneously, a dramatic increase in the rate of cell elimination by apoptosis was found. Glutamate-pyruvate transaminase and alkaline phosphatase in serum did not show major increases, suggesting that cell death was not due to lytic membrane damage. Furthermore, if treatment with CPA was continued or resumed, the enhanced DNA content persisted and the number of apoptotic bodies was greatly reduced. These observations suggest that the occurrence of cell death is due to withdrawal of the growth stimulus CPA. It may reflect a regulatory phenomenon serving to maintain homeostasis of cell number.Further studies showed that CPA is rapidly eliminated from rat liver and serum: t 1/2 in the liver is about 11 h. In contrast, -HCH was previously found to be eliminated more slowly: t 1/2 approximately 144 h. The present study revealed that -HCH, CPA and nafenopin lower the number of apoptotic bodies. This suggests that inducers of liver growth can inhibit hepatocellular death by apoptosis. It is concluded that the regression of hyperplasia after CPA withdrawal may be due to its rapid elimination. On the other hand the relatively long persistence of -HCH may result in inhibition of cell death and thereby stabilize hepatic hyperplasia.Abbreviations CPA cyproterone acetate - -HCH -hexachlorocyclohexane - PB phenobarbital - NAF nafenopin - AB apoptotic body - b.w. body weight - p. admin. post-administration - GPT glutamate-pyruvate transaminase - ALP alkaline phosphatase Dedicated to Professor W. Koransky on the occasion of his 65th birthday     

低铅染毒大鼠体内脂质过氧化及抗氧化水平改变

目的：观察低剂量铅染毒后大鼠体内脂质过氧化水平改变,为铅的毒作用机制研究提供依据。方法：以醋酸铅为受试物,雄性Wistar大鼠为受试对象,分别观察其脏器脂质过氧化水平改变。结果：低铅染毒后,大鼠各脏器超氧化物歧化酶（SOD）、谷胱苷肽过氧化物酶（GSH－Px）及过氧化脂质（LPO）均有不同程度改变（P＜0.05,P＜0.01）,而巯基（－SH）含量仅肝脏变化较明显。结论：较低剂量的铅即可引起体内脂质过氧化水平的改变,而这种变化可能是铅的毒作用途径之一。     

腹腔镜联合醋酸亮丙瑞林对子宫内膜异位症合并不育症疗效

目的:探讨腹腔镜联合醋酸亮丙瑞林治疗子宫内膜异位症(EMS)合并不育症对患者妊娠结局的影响。方法:选取2017年3月-2019年3月本院收治的80例EMS合并不育症患者,随机分为对照组(n=40)和观察组(n=40),对照组给予腹腔镜治疗,观察组给予腹腔镜联合醋酸亮丙瑞林治疗,比较两组临床疗效、绝经症状(Kupperman)评分、盆腔疼痛程度(VAS)评分、血清学指标以及1年内复发率和妊娠率。结果:治疗后观察组治疗有效率(95.0%)高于对照组(67.5%),Kupperman(5.80±2.11分)、VAS评分(1.59±0.22分),低于对照组(8.52±3.84分、2.04±0.51分),血清卵泡刺激素、黄体生成素、雌二醇、基质金属蛋白酶-9、基质金属蛋白酶抑制剂-1、人附睾蛋白、糖类抗原125、糖类抗原199水平均低于对照组(均P<0.05),1年复发率(5.0%)低于对照组(25.0%),1年受孕率(45.0%)高于对照组(20.0%)(P<0.05)。结论:腹腔镜联合醋酸亮丙瑞林治疗EMS合并不育症疗效显著,可缓解临床症状和疼痛程度,改善性激素水平,降低复发率,提高受孕率。     

Relationship between a novel human cytotoxin (factor 2) produced by a B cell line (Karpas 160) and phorbol-myristate-acetate-associated cytotoxicity.

We found that 160b cells, a subclone of Karpas 160 human B cell line, spontaneously secreted a novel cytotoxin, factor 2 (F2). F2 was also extracted from the cells by 60% ammonium sulphate, 0.5% CHAPS and 0.28% Triton X-114. We were able to show that phorbol myristate acetate (PMA) greatly enhanced the production of F2, and PMA may also account for part of the putative F2 cytotoxic activity to K562 cells in crude preparations. We compared the cytotoxic effect of F2 with PMA-associated F2-like cytotoxicity to K562 cells as well as the adequacy of our schemes to purified F2 with regard to its separation from PMA. We found that it was possible to separate PMA from F2 preparations by gel filtration and Rotofor preparative isoelectric focusing. The fate of PMA was also monitored with 3H-PMA and chromatographic profiles of 3H-PMA were studied using DE52 and gel filtration chromatography. We were able to establish that less than 2.9% of the cytotoxicity to K562 was due to PMA. We also found that the radioactive peaks and cytotoxicity peaks to K562 were not well correlated, indicating that the cytotoxicity was not mainly due to remaining PMA. Activated charcoal removed virtually all F2 and PMA but not tumour necrosis factor activity. Our results also showed that cytotoxicity to K562 resulting from F2 or PMA-associated proteins had different physicochemical properties, indicating that they are different molecular entities. These findings are consistent with the earlier observation that 160 cells produce F2 spontaneously and that PMA can amplify its production significantly.     

Cyclical use of tamoxifen and high-dose medroxyprogesterone acetate in advanced estrogen receptor positive breast cancer

Summary One-hundred and seventy patients with estrogen receptor positive (10 pmol/g protein) advanced breast cancer have been treated in a prospective randomized study either with continuous tamoxifen 30 mg × 1 daily (TAM), or with TAM 30 mg × 1 daily for 8 weeks alternating with medroxyprogesterone acetate 500 mg × 2 daily for 8 weeks (TAM/HD-MPA). The response rate was 62% in the group treated with cyclic TAM/HD-MPA versus 41% in the TAM alone group (p = 0.02). There was no significant difference in duration of remissions or survival.