Cathepsin B and D activity in stimulated peritoneal macrophages

Summary Highly sensitive and specific synthetic substrates were used to quantitate cathepsin B and D activity in peritoneal macrophages in response to stimulation in vivo with mineral oil and thioglycollate. After intraperitoneal instillation of mineral oil the activity of cathepsin B increased significantly (to 15 300 units/mg protein versus 7 340 in saline controls), reaching values approaching those found in alveolar macrophages (18 400 units/mg protein). Significantly greater stimulation of enzyme activity was obtained after intraperitoneal instillation of thioglycollate (23 600 units/mg protein). Cathepsin D activity also increased significantly after both mineral oil and thioglycollate. However, the increase was moderate (from 806 to about 1 200 units/mg protein), remaining still more than six times lower-than in alveolar macrophages. The data are the first to demonstrate that cathepsin B activity can be stimulated in vivo in peritoneal macrophages by instillation of agents that induce acute inflammation. They also point to a differential control of expression of cathepsin B and D activity in both peritoneal and alveolar macrophages in spite of the common lysosomal origin of the two enzymes.Abbreviations Cbz -N-benxyloxycarbonyl - 2NA 2-naphthylamine - EDTA ethylenediamine tetraacetate - DMSO dimethylsulfoxide - PBS phosphate-buffered saline - PM peritoneal macrophage - AM alveolar macrophage     

Electrokinetic study of the reactions of peritoneal macrophages and eosinophils with IgG immune complexes

Electrophoretic light scattering (laser Doppler electrophoresis) has been employed to study the effects of guinea pig IgG immune complexes on the electrophoretic mobility distributions of guinea pig resident peritoneal cells. The resident population of cells is composed of macrophages (approximately 75%) and eosinophils (approximately 25%). These cells were separated according to the well-established method of Boyum. Populations of resident macrophages, eosinophils, and the unfractionated samples were incubated with soluble immune complexes, antigen alone, or antibody alone. The mean mobility of the resident macrophages decreased approximately 60% when incubated in the presence of immune complexes, although no effect could be discerned in the presence of antigen or antibody alone. The width of the resulting macrophage mobility distribution was larger than that of the control distributions, with a broad shoulder on the high-mobility side, indicating a heterogeneous response of the macrophages to the immune complexes. Eosinophils react in two distinct fashions. One population of eosinophils is present near the control experiments. The second population reacts in a manner very similar to that of macrophages. This suggest that at least two populations of eosinophils are present in the unstimulated guinea pig peritoneal cavity. Results that are intermediate between these two cases are found when unfractionated samples are studied.     

Macrophage function in tumor-bearing mice: dissociation of phagocytic and chemotactic responsiveness

Infusion of CBA mice with lymphoid cells from the H-2 compatible but Mls-antigen incompatible C3H × CBA hybrid results in a specifically reduced capacity of the recipients lymphocytes to react in the MLC against C3H-cells. Although this reduction is immunologically specific the results of this investigation have shown that such mice exhibit a strongly reduced capacity to produce humoral antibodies against heterologous erythrocytes and a T-cell independent antigen (PVP).     

The role of WNT5A and Ror2 in peritoneal membrane injury

Patients on peritoneal dialysis are at risk of developing peritoneal fibrosis and angiogenesis, which can lead to dysfunction of the peritoneal membrane. Recent evidence has identified cross-talk between transforming growth factor beta (TGFB) and the WNT/β-catenin pathway to induce fibrosis and angiogenesis. Limited evidence exists describing the role of non-canonical WNT signalling in peritoneal membrane injury. Non-canonical WNT5A is suggested to have different effects depending on the receptor environment. WNT5A has been implicated in antagonizing canonical WNT/β-catenin signalling in the presence of receptor tyrosine kinase-like orphan receptor (Ror2). We co-expressed TGFB and WNT5A using adenovirus and examined its role in the development of peritoneal fibrosis and angiogenesis. Treatment of mouse peritoneum with AdWNT5A decreased the submesothelial thickening and angiogenesis induced by AdTGFB. WNT5A appeared to block WNT/β-catenin signalling by inhibiting phosphorylation of glycogen synthase kinase 3 beta (GSK3B) and reducing levels of total β-catenin and target proteins. To examine the function of Ror2, we silenced Ror2 in a human mesothelial cell line. We treated cells with AdWNT5A and observed a significant increase in fibronectin compared with AdWNT5A alone. We also analysed fibronectin and vascular endothelial growth factor (VEGF) in a TGFB model of mesothelial cell injury. Both fibronectin and VEGF were significantly increased in response to Ror2 silencing when cells were exposed to TGFB. Our results suggest that WNT5A inhibits peritoneal injury and this is associated with a decrease in WNT/β-catenin signalling. In human mesothelial cells, Ror2 is involved in regulating levels of fibronectin and VEGF.     

Impact of hyperthermic intraperitoneal chemotherapy on Hsp27 protein expression in serum of patients with peritoneal carcinomatosis

Despite the strong rationale for combining cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) in patients with peritoneal carcinomatosis, thermotolerance and chemoresistance might result from heat shock protein overexpression. The aim of the present study was thus to determine whether the heat shock protein 27 (Hsp27), a potential factor in resistance to treatment, could have a higher level in serum from patients under this combined therapy. Patients receiving CRS plus HIPEC for peritoneal carcinomatosis (group 1), patients with cancer or a history of cancer undergoing abdominal surgery (group 2), and patients without malignancies undergoing abdominal surgery (group 3) were included. Hsp27 serum levels were determined before and at different times following CRS and HIPEC using enzyme-linked immunosorbent assay. In group 1 (n = 25), the high Hsp27 levels, observed at the end of surgery compared with before (p < 0.0001), decreased during HIPEC, but remained significantly higher than before surgery (p < 0.0005). In groups 2 (n = 11) and 3 (n = 15), surgery did not significantly increase Hsp27 levels. A targeted molecular strategy, inhibiting Hsp27 expression in tumor tissue, could significantly reduce resistance to the combined CRS plus HIPEC treatment. This approach should be further assessed in a clinical phase I trial.     

Update on the challenging role of biofilms in peritoneal dialysis

Biofilms are commonly associated with an increased risk of patient infection. In peritoneal dialysis (PD), catheter associated infection, especially peritonitis, remains a clinically relevant problem. Although the presence of a biofilm is recognized in relapsing, repeat, and catheter-related peritonitis, it remains poorly characterized. In this review, an update on the role of biofilms in PD infections is presented. The emerging concept that host cells and tissue associated biofilms, in addition to the biofilms on the catheters themselves, contribute to the recalcitrance of infections is discussed. Furthermore, the evidence of biofilms on PD catheters, their developmental stages, and the possible influence of the PD environment are reviewed. The focus is given to ex vivo and in vitro studies that contribute to the elucidation of the interplay between host, microbial, and dialysis factors. The key issues that are still to be answered and the challenges to clinical practice are discussed.     

骨化三醇联合腹膜透析疗法治疗慢性肾功能衰竭的临床疗效及对患者血清ProGRP、Cysc、Chemerin水平的影响

目的:探讨骨化三醇联合腹膜透析疗法治疗慢性肾功能衰竭的临床疗效及对患者血清Pro-Gastrin-Releasing Peptide (ProGRP)、CystatinC(Cysc)、Chemerin水平的影响。方法:选取我院2017年2月至2018年1月收治的98例慢性肾功能衰竭患者,按照随机数表法将其分为观察组(n=51)和对照组(n=47)。对照组采用腹膜透析疗法治疗,观察组采用骨化三醇联合腹膜透析疗法治疗。观察和比较两组治疗前后肾功能指标尿素氮(blood urea nitrogen,BUN)、血清肌酐(Serum creatinine Cr)、24小时尿蛋白(24 h urinary protein,24 h UP),生化指标白蛋白(albumin,Alb)、血红蛋白(hemoglobin,Hb)及红细胞(red blood cell,RBC),胃泌素释放肽前体(ProGRP)、血清胱抑素(Cys C)、趋化素(Chemerin)水平的变化,6个月、1年生存率及不良反应的发生情况。结果:治疗后,观察组BUN、SCr、24hUP水平均显著低于对照组[(13.95±3.06)mmol/L vs.(21.10±3.85)mmol/L,(260.12±40.32)μmol/L vs.(354.93±51.06)μmol/L,(1.75±0.45)g/24 h vs.(2.67±0.80)g/24 h](P0.05);Alb水平显著低于对照组[(27.85±3.58)g/L vs.(33.06±4.27)g/L](P0.05);Hb、RBC显著高于对照组[(91.72±13.46)g/L vs.(82.36±10.15)g/L,(379.47±92.08)×1012/L vs.(315.70±73.24)×1012/L](P0.05);ProGRP、Chemerin水平显著低于对照组[(49.23±4.72)pg/mL vs.(63.87±7.30)pg/mL,(37.02±6.15)μg/L vs.(30.63±4.81)μg/L](P0.05);Cysc水平显著高于对照组[(80.75±16.08)mL/min vs.(98.81±18.07)mL/min](P0.05);6个月、1年生存率均显著高于对照组[96.08%(49/51) vs. 91.49%(43/47),90.20%(46/51) vs. 74.47%(35/47)](P0.05);不良反应总发生率显著低于对照组[17.65%(9/51) vs. 44.68%(21/47)](P0.05)。结论:骨化三醇联合腹膜透析疗法治疗慢性肾功能衰竭的临床效果显著优于单用,其可有效减轻患者的临床症状,纠正电解质紊乱,改善肾功能和预后,可能与降低血清ProGRP、Chemerin水平及提高血清Cysc水平有关。     

Effects of two different nutrition supply methods on improving hybridoma cell production

Hybridoma cells are featured by the effective utilization of both B lymphocytes and immortalized myeloma cells, allowing for the continuous generation of monoclonal antibodies specific to antigens. With regard to conventional hybridoma technology, B lymphocytes must be fused with myeloma cells using various methods to generate hybridoma cells. Nutrition plays an important role in hybridoma cell survival and amplification, which determines the fusion effect and antibody production. Here we compared the growth and survival rates of hybridoma in a commonly used peritoneal macrophage feeder layer (PMFL) nutrition supply system with a commercial hybridoma feeder additive (HFA) nutrition supply system at the post fusion stage and discussed the titer of monoclonal antibodies by enzyme linked immunosorbent assay (ELISA). Our results indicate that commercially available HFA promotes the survival and amplification of hybridoma clones and improves the titer of monoclonal antibodies indirectly.