流式细胞术的培训实践及技术平台建设探讨

流式细胞分析技术(flow cytometry,FCM),又称流式细胞术,是一种在功能水平上对单细胞或其他生物粒子进行定量分析和分选的技术,具有广泛的科研应用价值.采用理论与实践并举,结合讨论的教学方式,在教师队伍开设针对性的培训课程,对深刻理解并在科研工作中有效利用该技术具有及其重要意义.同时以教学促科研,以科研促技...     

Degradation of a chromogenic substrate by α2-macroglobulin from plasma of patients with rheumatoid arthritis

We have shown previously that serum from patients with rheumatoid arthritis (RA) contains a polyclonal B cell activator that is associated with α₂-macroglobulin (α₂M). Some biologic effects of this activator appear to be due to a trypsin-like protease attached to α₂M. Therefore, in the present study, we used an anti-α₂M antibody solid-phase assay, with Chromozym-Try as a substrate, to determine the level of α₂M-protease complexes in plasma α₂M. We found higher levels of these complexes in RA patients than in 2 control groups. Since α₂M-protease complexes have been shown to induce RA-like inflammation in experimental animals and to be produced by lymphoid cells, we speculate that they may be involved in the pathogenesis of RA. However, the role of the other cells or enzyme systems in the formation of these complexes has not yet been ruled out. Results of these investigations could lead to another link between activation of the immune system and joint inflammation.     

How to Regulate Nonbiological Complex Drugs (NBCD) and Their Follow-on Versions: Points to Consider

The aim of this critical review is to reach a global consensus regarding the introduction of follow-on versions of nonbiological complex drugs (NBCD). A nonbiological complex drug is a medicinal product, not being a biological medicine, where the active substance is not a homo-molecular structure, but consists of different (closely related and often nanoparticulate) structures that cannot be isolated and fully quantitated, characterized and/or described by state of the art physicochemical analytical means and where the clinical meaning of the differences is not known. The composition, quality and in vivo performance of NBCD are highly dependent on manufacturing processes of both the active ingredient as well as in most cases the formulation. The challenges posed by the development of follow-on versions of NBCD are illustrated in this paper by discussing the ‘families’ of liposomes, iron–carbohydrate (‘iron–sugar’) drugs and glatiramoids. It is proposed that the same principles for the marketing authorization of copies of NBCD as for biosimilars be used: the need for animal and/or clinical data and the need to show similarity in quality, safety and efficacy. The regulatory approach of NBCD will have to take into consideration the specific characteristics of the drugs, their formulation and manufacturing process and the resulting critical attributes to achieve their desired quality, safety and efficacy. As with the biosimilars, for the NBCD product, family-specific methods should be evaluated and applied where scientifically proven, including sophisticated quality methods, pharmacodynamic markers and animal models. Concerning substitution and interchangeability of NBCD, it is also advisable to take biosimilars as an example, i.e. (1) substitution without the involvement of a healthcare professional should be discouraged to ensure traceability of the treatment of individual patients, (2) keep an individual patient on a specific treatment if the patient is doing well and only switch if unavoidable and (3) monitor the safety and efficacy of the new product if switching occurs.     

Minocycline is a useful adjuvant therapy for pemphigus

Pemphigus is an autoimmune blistering disease with high mortality if untreated. The cases of 10 patients who had minocycline 100 mg daily added as adjuvant therapy are reported. Prior to the use of minocycline, all patients had active disease, nine were on prednisolone (10–40 mg) and five were on azathioprine (100–200 mg). The response was assessed on clinical improvement and reduction of immunosuppressive (IS) drugs. It was graded into four categories: major, minor, equivocal and no significant response. A major response was seen in four patients, minor in two, equivocal in one and no improvement in three patients. The prednisolone dose in the six responders was reduced to 0–6 mg (0 mg in three patients), with an average decrease of 21 mg. The average time to respond was 8 months. Of the six responders, three were on azathioprine, which was ceased in two patients and reduced by two-thirds in the other patient. No patient ceased minocycline because of side effects. In conclusion, minocycline 100 mg daily is a simple, safe and well tolerated treatment that should be tried in patients with pemphigus to reduce disease activity and/or the dose of potent IS agents.     

Podocytes are new cellular targets of haemoglobin‐mediated renal damage

Recurrent and massive intravascular haemolysis induces proteinuria, glomerulosclerosis, and progressive impairment of renal function, suggesting podocyte injury. However, the effects of haemoglobin (Hb) on podocytes remain unexplored. Our results show that cultured human podocytes or podocytes isolated from murine glomeruli bound and endocytosed Hb through the megalin–cubilin receptor system, thus resulting in increased intracellular Hb catabolism, oxidative stress, activation of the intrinsic apoptosis pathway, and altered podocyte morphology, with decreased expression of the slit diaphragm proteins nephrin and synaptopodin. Hb uptake activated nuclear factor erythroid‐2‐related factor 2 (Nrf2) and induced expression of the Nrf2‐related antioxidant proteins haem oxygenase‐1 (HO‐1) and ferritin. Nrf2 activation and Hb staining was observed in podocytes of mice with intravascular haemolysis. These mice developed proteinuria and showed podocyte injury, characterized by foot process effacement, decreased synaptopodin and nephrin expression, and podocyte apoptosis. These pathological effects were enhanced in Nrf2‐deficient mice, whereas Nrf2 activation with sulphoraphane protected podocytes against Hb toxicity both in vivo and in vitro. Supporting the translational significance of our findings, we observed podocyte damage and podocytes stained for Hb, HO‐1, ferritin and phosphorylated Nrf2 in renal sections and urinary sediments of patients with massive intravascular haemolysis, such as atypical haemolytic uraemic syndrome and paroxysmal nocturnal haemoglobinuria. In conclusion, podocytes take up Hb both in vitro and during intravascular haemolysis, promoting oxidative stress, podocyte dysfunction, and apoptosis. Nrf2 may be a potential therapeutic target to prevent loss of renal function in patients with intravascular haemolysis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.     

Thrombospondin mediates the cytoadherence of Plasmodium falciparum- infected red cells to vascular endothelium in shear flow conditions

Cerebral malaria is thought to involve specific attachment of Plasmodium falciparum-infected knobby red cells to venular endothelium. The nature of surface ligands on host endothelial cells that may mediate cytoadherence is poorly understood. We have investigated the effects of soluble thrombospondin, rabbit antiserum raised against thrombospondin, and human immune serum on cytoadherence of parasitized erythrocytes in ex vivo mesocecum vasculature. Preincubation of infected red cells with soluble thrombospondin or human immune serum inhibits binding of infected red cells to rat venular endothelium. Infusion of the microcirculatory preparation with rabbit antithrombospondin antibodies before perfusion of parasitized erythrocytes also resulted in decreased cytoadherence. In addition, incubation of infected cells with human immune sera obtained from malaria patients significantly inhibited the observed cytoadherence. Our results indicate that thrombospondin mediates binding of infected red cells to venular endothelium and may thus be involved in the pathogenesis of cerebral malaria.