Inhibitory Effects of Interleukin-4 on Human Renal Cell Carcinoma Cells In Vitro: In Combination with Interferon-α, Tumor Necrosis Factor-α or Interleukin-2

Background: Immune cytokines have been shown to play important roles in regulating the growth of neoplastic cells, as well as the function of immune cells. The present study assessed the effects of interleukin (IL)-4 alone, and in combination with recombinant interferon (IFN)-α2b, or with IL-2, or with tumor necrosis factor (TNF)-α on the in vitro proliferation of human renal cell carcinoma (RCC) cell-lines.
Methods: Growth-inhibitory effects of IL-4 alone, and in combination with other cytokines, on three human RCC cell-lines, Caki-1, CURC-II, and A-498, were measured by the [³H]thymidine incorporation assay.
Results: IL-4 inhibited proliferation of all three human RCC cell-lines (P< 0.001). The maximum growth inhibition of RCC cell-lines by IL-4 alone was observed at the concentration of 1 to 3 ng/mL, depending on the cell-line. Antihuman IL-4 antisera was able to reverse the growth-inhibitory effects of IL-4 on Caki-1 in a dose-dependent manner, proving that the growth inhibition was mediated by IL-4 itself. When other cytokines were added in combination with IL-4, only IFN-α2b resulted in significant additional growth inhibition ( P < 0.005). However, when the proliferation was compared to that of RCC cells that were not treated with any cytokine, all combinations produced marked growth inhibition. Conclusion: Our data suggest that IL-4 alone, or in combination with IFN-α2b, can be used to develop new strategies for treatment of human RCC.     

Influence of Dialysis Membranes on Interleukin-1β and Interleukin-1 Receptor Antagonist Production by Peripheral Blood Mononuclear Cells

Abstract: We investigated the production of interleukin (1L)-1β and IL-1 receptor antagonist (Ra) by peripheral blood mononuclear cell (PBMC) in vitro during hemodialysis of 7 dialysis patients using 4 differential dialysis membranes (regenerated cellulose [RC], polyamide [PA], polysulfone [PSI and AN-69). Blood sampling was performed before dialysis (0 min), 15 min after starting dialysis, and after dialysis (240 min) during the last session of each treatment. The cellular content of fresh cells and the production of IL-1β and IL-1Ra with and without lipopolysaccharide (LPS) stimulation of the cells were evaluated and measured by enzyme-linked immunosorbent assay (ELISA). The level of IL-1β with LPS stimulation using RC, PA, and PS membranes was significantly reduced at 15 min and was not changed at 240 min as compared with the level at 0 min. On the other hand, the level of IL-1β with LPS stimulation using an AN-69 membrane at 15 and 240 min was not significantly different from that at 0 min. Neither initial cellular content nor spontaneous production of IL-1β were detected at 0, 15, or 240 min in any of the membranes. The spontaneous production of IL-1Ra at 15 and 240 min was not significantly different from that at 0 min in any of the membranes. The cellular content of IL-1Ra using the RC membrane was significantly lower at 15 min and did not differ at 240 min from the level at 0 min. The cellular content of IL-1Ra using PA, PS, and AN-69 membranes was not significantly different at 15 and 240 min from that at 0 min. However, the IL-1Ra level with LPS stimulation using RC and PA membranes was significantly reduced from that at 0 min, but the level using PS and AN-69 membranes was not different from that at 0 min. Because IL-1β and IL-1Ra levels 15 min after starting dialysis using bioincompatible dialysis membranes were reduced from the levels at 0 min, the findings suggest that measurement of cytokines during dialysis treatment at an early stage is a useful marker for evaluating the biocompatibility of a dialysis membrane.     

The Effect of Alendronate on Cytokine Production, Adhesion Molecule Expression, and Transendothelial Migration of Human Peripheral Blood Mononuclear Cells

Since both osteoclasts and macrophages belong to the mononuclear phagocytic system it is conceivable that bisphosphonates not only affect bone metabolism but also inflammatory responses. The migration of mononuclear cells into perivascular tissue is a central event in inflammatory reactions. We studied the effects of the aminobisphosphonate alendronate on the transendothelial migration of human peripheral blood mononuclear cells in an in vitro model. Alendronate (at a concentration of 100 μM) significantly increased the percentage of peripheral blood mononuclear cells that migrated through endothelial cell monolayers. Similar results were obtained with another aminobisphosphonate, viz, pamidronate. An overnight treatment of the endothelial cell monolayers with alendronate did not alter the rate of peripheral blood mononuclear cells that subsequently migrated. The overnight cultivation of the peripheral blood mononuclear cells in the presence of alendronate resulted in an increased surface expression of CD54 (intercellular adhesion molecule-1, ICAM-1) in both CD14⁺ and CD3⁺ cells; in CD14⁺ cells also the expression of CD49d (α₄ subunit of late activation antigen-4, VLA-4) increased after alendronate treatment. Alendronate treatment of peripheral blood mononuclear cells also resulted in an increased production of interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and interferon-γ (IFN-γ). We conclude that alendronate has a distinct effect on the transendothelial migration of human peripheral blood mononuclear cells in vitro. Alendronate may either directly or indirectly, e.g., by augmenting the production of proinflammatory cytokines, influence the expression of certain adhesion molecules and thereby facilitate transendothelial migration. These effects could be related to the transient leukopenia reported following intravenous administration of relatively high doses of aminobisphosphonates for the treatment of hypercalcemia of malignancy. Received: 11 September 1997 / Accepted: 20 February 1998     

胸腔肿瘤患者围术期外周血NK活力血浆SIL－2R表达水平的动态监测

对２１例胸腔恶性肿瘤和８例胸腔良性肿瘤二组患者围术期作ＮＫ细胞活力、血浆ＳＩＬ－２Ｒ表达水平的动态监测结果表明：胸腔恶性肿瘤患者ＮＫ细胞活力明显低于良性肿瘤组（Ｐ＜０．０５），血浆ＳＩＬ－２Ｒ表达水平明显高于良性肿瘤组（Ｐ＜０．００１）；二组患者在术始２～３小时ＮＫ活性均明显升高，术后３～５天则显著回落，而ＳＩＬ－２Ｒ水平则表现为一反向变化。结果提示胸腔恶性肿瘤患者抗肿瘤免疫功能严重受损；术中ＮＫ细胞活性一过性升高可能对手术操作引起的肿瘤细胞播散有遏制作用，但术后免疫功能的抑制对防止肿瘤的复发和转移是不利的，应引起重视     

Effect of Hemodialysis on T Lymphocyte Subsets, Con-A-Activated Suppressor Cell Activity, and Interleukin-2 Receptor Expression on Lymphocytes in Chronic Uremic Patients

Effect of hemodialysis (HD) on some indices of immune response was studied in nine chronic uremics. Total lymphocyte, OKT4+, and OKT8+ cell numbers significantly decreased during the first 20 min of HD, and they were decreased till the third hour of the procedure, whereas the OKT4+/OKT8+ cell number ratio did not change significantly. Before HD, Con-A--activated suppressor cells exerted a stimulatory action on autologous responder cells measured in two-step culture. During HD, Con-A-activated suppressor cell activity transiently appeared, with its peak at 60 min after the start of HD. It was accompanied by a transient rise in lymphocyte count with spontaneous interleukin-2 (IL-2) receptor expression, whereas the number of cells expressing IL-2 receptor following phytohemaglutinin (PHA) stimulation was progressively decreased during HD. A significant correlation was found between the increment of Con-A-activated suppressor cell activity and the increment of spontaneous IL-2 receptor expression on lymphocytes during one single blood flow through the dialyzer. The results supply further evidence that HD may impose additional disturbances on immune regulation in chronic uremics.     

2型糖尿病肾病患者外周血CD28/CTLA4共刺激分子的表达及其意义

目的:探讨2型糖尿病(T2DM)肾病患者外周血T细胞亚群、共刺激分子CD 28及细胞毒T淋巴细胞抗原(CTLA)4的表达及其临床意义。方法:选取2015年07月~2018年06月至我科室和内分泌科治疗的患者,根据尿微量清蛋白排泄率(UAER)将其分为DM组(T2DM无肾病,n=42)和DN组(T2DM合并肾病,n=40),同时选取健康人群40例作为对照(NC组)。采用流式细胞技术测定两组患者外周血T淋巴细胞上CD4、CD8的表达,并用流式细胞技术测定CD4^+、CD8^+T淋巴细胞表面CD28、CTLA4分子的表达。结果:三组外周血CD4^+和CD4^+/CD8^+按NC组-DM组-DN组顺序均呈显著递增趋势(P<0.01),CD3^+、CD8^+按照顺序呈显著递减趋势(P<0.01),且各组间差异明显(P<0.01);三组外周血CD4^+CD28^+、CD8^+CD28^+ T细胞按NC组-DM组-DN组顺序均呈显著递增趋势(P<0.01),CD4^+CTLA4+及CD8^+CTLA4+T细胞按照顺序呈显著递减趋势(P<0.01),且各组间差异明显(P<0.01)。结论:T2DM患者T细胞亚群失衡,其中合并肾病患者表达失衡更为严重,提示糖尿病肾病患者存在自身免疫调节异常。同时T2DM合并肾病患者外周血CD28和CTLA4表达也显著异于正常对照,提示共刺激分子可能参与了T2DM合并肾病的免疫功能紊乱。     

The effect of propofol on actin, ERK-1/2 and GABAA receptor content in neurones

AIM: Interaction with the gamma-aminobutyric acid receptor (GABA(A)R) complex is recognized as an important component of the mechanism of many anaesthetic agents, including propofol. The aims of this study were to investigate the effect of propofol on GABA(A)R, to determine whether exposure of neurones to propofol influences the localization of GABA(A)R within the cell and to look for cytoskeletal changes that may be connected with activation, such as the mitogen-activated protein kinase (MAPK) pathway. METHODS: Primary cortical cell cultures from rat, with and without pre-incubation with the GABA(A)R antagonist bicuculline, were exposed to propofol. The cells were lysed and separated into membrane and cytosolic fractions. Immunoblot analyses of filamentous actin (F-actin), the GABA(A)beta(2)-subunit receptor and extracellular signal-regulated kinase-1/2 (ERK-1/2) were performed. RESULTS: Propofol triggers an increase in GABA(A)R, actin content and ERK-1/2 phosphorylation in the cytosolic fraction. In the membrane fraction, there is a decrease in GABA(A)beta(2)-subunit content and an increase in both actin content and ERK-1/2 phosphorylation. The GABA(A)R antagonist bicuculline blocks the propofol-induced changes in F-actin, ERK and GABA(A)beta(2)-subunit content, and ERK-1/2 phosphorylation. CONCLUSION: We believe that propofol triggers a dose-dependent internalization of the GABA(A)beta(2)-subunit. The increase in internal GABA(A)beta(2)-subunit content exhibits a close relationship to actin polymerization and to an increase in ERK-1/2 activation. Actin contributes to the internalization sequestering of the GABA(A)beta(2)-subunit.     

Evidence for abnormal regulation of circulating 1α,25-dihydroxyvitamin D in patients with pulmonary tuberculosis and normal calcium metabolism

Summary Available evidence indicates that hypercalcemia in pulmonary tuberculosis results from increases in circulating 1α,25-dihydroxyvitamin D [1α,25(OH)₂D]. To further characterize vitamin D metabolism in this disorder, the effects of vitamin D, 100,000 units a day for 4 days, were compared in 25 normal subjects and 11 patients with active pulmonary tuberculosis who were normocalcemic and had not had hypercalcemia. Serum calcium, phosphorus, 25-hydroxyvitamin D (25-OHD) and 1α,25(OH)₂D were measured. Whereas vitamin D increased mean serum 25-OHD from 20±2 (±SE) to 40±5 ng/ml (P<0.001) and did not change mean serum 1α,25(OH)₂D in the normals (33±2 vs. 31±2 pg/ml), it increased mean serum 25-OHD from 21±4 to 55±13 ng/ml (P<0.05) and mean serum 1α,25(OH)₂D from 28±2 to 35±3 pg/ml (P<0.05) in the patients. Serum calcium was normal and remained within the normal range in all subjects and patients. The findings indicate that there is a modest but significant abnormality in the regulation of circulating 1α,25(OH)₂D in normocalcemic patients with pulmonary tuberculosis. The results are similar to those previously reported by us in normocalcemic patients with sarcoidosis.     

Regulation of bone turnover and prevention of bone atrophy in ovariectomized beagle dogs by the administration of 24R, 25(OH)2D3

Summary In order to determine whether the administration of 24R,25(OH)₂D₃ had any beneficial effect on the regulation of bone turnover and the prevention of bone atrophy, we examined beagles for 31 months after ovariectomy (OVX). Fourteen beagle dogs (8.54±1.22 kg body wt-b.w.) were divided into four groups. Group 1 (n=3) was the sham, and Group 2 (n=3) served as the OVX control. In Group 3 (n=4) and Group 4 (n=4), 24,25-dihydroxyvitamin D₃(24R,25(OH)₂D₃) was given daily at dose levels of 2 and 10 mcg/kg B.W., respectively. In Group 4, the dose level was increased to 100 mcg/kg by 17 months. During the experiments, urinary hydroxyproline (U-HPr), serum chemistry, serum bone gla-protein (BGP), and vitamin D metabolite levels were monitored. At the end of the experiment, bone mineral content (BMC) in the 6th and 7th lumbar vertebrae and right femur was determined by single photon absorptiometry. The left iliac bone sample was obtained after tetracycline labeling, and undecalcified sections were observed. In Group 2, excretion of U-HPr increased after OVX and had reached a level of approximately twice the baseline values by 10 months; then it gradually came down to the original level. In Group 3, however, U-HPr excretion remained at the same level as the baseline value, as it did in Group 1. In Group 4, it was remarkably reduced down to 50–60% of the baseline values. Serum BGP level was markedly reduced in Group 4. Serum 24,25(OH)₂D levels were markedly increased in Groups 3 and 4. BMC levels of both vertebrae and epi-metaphyseal regions in the femur showed a significant reduction of approximately 25% in Group 2. In Groups 3 and 4, however, they remained at the same level as in Group 1. Histomorphometrical data showed a reduction in the parameters of osteoblast functions in Group 2. In Group 3, both kinetic and static parameters maintained the same level as in Group 1. In Group 4, eroded surface and osteoclast number decreased significantly, but mineral appositional rate and wall thickness maintained the same level as in Group 1. From these findings, it was concluded that, in beagle dogs, the administration of 24R,25(OH)₂D₃ inhibited the increase of bone turnover and prevented the reduction of cancellous bone mass after a long time postovariectomy.     

Treatment of renal osteodystrophy in children with 1αOHD3 and 24, 25(OH)2D3

Three pediatric patients with renal osteodystrophy were treated with 1αOHD₃ and 24, 25(OH)₂D₃. While serum calcium level significantly decreased, no significant effects were found on serum phosphorus, alkaline phosphatase, parathyroid hormone and urinary excretion of calcium. These results suggest that 24, 25(OH)₂D₃ may play some roles in bone and mineral metabolism.