足细胞病与相关循环因子的表达

肾小球足细胞的损伤不仅是遗传性肾小球病的发病基础,还在很多后天的肾小球疾病中发挥重要作用。常见的足细胞病以微小病变性肾病(Minimal Change Disease,MCD)和局灶阶段性肾小管硬化(Focal Segmental glomerulosclerosis,FSGS)为主,有实验证明,足细胞损伤同时参与了各种不同类型的肾小球疾病,比如糖尿病肾病,HIV相关肾病,膜性肾病及其他获得性肾小球病等,因此,了解足细胞病损伤的机制尤为重要。临床观察提出局灶节段行肾小球硬化患者体内可能存在一种与大量蛋白尿发生有关的循环因子,相关的动物实验和体外观察同时也证实了循环因子的存在。越来越多的研究支持T细胞功能不全,细胞因子异常释放可能是循环因子的来源之一。如果能对患者循环因子进行检测,借助它来指导治疗方案的选择(免疫抑制剂、血浆置换),可能成为局灶节段行肾小球硬化诊断和治疗中的一个突破。本文以国内外研究文献为基础,对文献资料进行分析、归纳和总结,综述了足细胞病中相关循环因子的表达,探讨足细胞病发生及复发的机理,为足细胞病的定向治疗提供帮助。     

Sorting of Clathrin‐Independent Cargo Proteins Depends on Rab35 Delivered by Clathrin‐Mediated Endocytosis

Clathrin‐mediated endocytosis (CME) and clathrin‐independent endocytosis (CIE) co‐exist in most cells but little is known about their communication and coordination. Here we show that when CME was inhibited, endocytosis by CIE continued but endosomal trafficking of CIE cargo proteins was altered. CIE cargo proteins that normally traffic directly into Arf6‐associated tubules after internalization and avoid degradation (CD44, CD98 and CD147) now trafficked to lysosomes and were degraded. The endosomal tubules were also absent and Arf6‐GTP levels were elevated. The altered trafficking, loss of the tubular endosomal network and elevated Arf6‐GTP levels caused by inhibition of CME were rescued by expression of Rab35, a Rab associated with clathrin‐coated vesicles, or its effector ACAPs, Arf6 GTPase activating proteins (GAP) that inactivate Arf6. Furthermore, siRNA knockdown of Rab35 recreated the phenotype of CME ablation on CIE cargo trafficking without altering endocytosis of transferrin. These observations suggest that Rab35 serves as a CME detector and that loss of CME, or Rab35 input, leads to elevated Arf6‐GTP and shifts the sorting of CIE cargo proteins to lysosomes and degradation.      

CD14, TLR4 and TRAM Show Different Trafficking Dynamics During LPS Stimulation

Toll‐like receptor 4 (TLR4) is responsible for the immediate response to Gram‐negative bacteria and signals via two main pathways by recruitment of distinct pairs of adaptor proteins. Mal‐MyD88 [Mal (MyD88‐adaptor‐like) ‐ MYD88 (Myeloid differentiation primary response gene (88))] is recruited to the plasma membrane to initiate the signaling cascade leading to production of pro‐inflammatory cytokines while TRAM‐TRIF [TRAM (TRIF‐related adaptor molecule)‐TRIF (TIR‐domain‐containing adapter‐inducing interferon‐β)] is recruited to early endosomes to initiate the subsequent production of type I interferons. We have investigated the dynamics of TLR4 and TRAM during lipopolysaccharide (LPS) stimulation. We found that LPS induced a CD14‐dependent immobile fraction of TLR4 in the plasma membrane. Total internal reflection fluorescence microscopy (TIRF) revealed that LPS stimulation induced clustering of TLR4 into small punctate structures in the plasma membrane containing CD14/LPS and clathrin, both in HEK293 cells and the macrophage model cell line U373‐CD14. These results suggest that laterally immobilized TLR4 receptor complexes are being formed and prepared for endocytosis. RAB11A was found to be involved in localizing TRAM to the endocytic recycling compartment (ERC) and to early sorting endosomes. Moreover, CD14/LPS but not TRAM was immobilized on RAB11A‐positive endosomes, which indicates that TRAM and CD14/LPS can independently be recruited to endosomes.      

Lymphocyte senescence in COPD is associated with loss of glucocorticoid receptor expression by pro-inflammatory/cytotoxic lymphocytes

Background

Glucocorticoid (GC) resistance is a major barrier in COPD treatment. We have shown increased expression of the drug efflux pump, Pgp1 in cytotoxic/pro-inflammatory lymphocytes in COPD. Loss of lymphocyte co-stimulatory molecule CD28 (lymphocyte senescence) was associated with a further increase in their pro-inflammatory/cytotoxic potential and resistance to GC. We hypothesized that lymphocyte senescence and increased Pgp1 are also associated with down-regulation of the GC receptor (GCR).

Methods

Blood was collected from 10 COPD and 10 healthy aged-matched controls. Flow cytometry was applied to assess intracellular pro-inflammatory cytokines, CD28, Pgp1, GCR, steroid binding and relative cytoplasm/nuclear GCR by CD28+ and CD28null T, NKT-like cells. GCR localization was confirmed by fluorescent microscopy.

Results

COPD was associated with increased numbers of CD28nullCD8+ T and NKT-like cells. Loss of CD28 was associated with an increased percentage of T and NKT-like cells producing IFNγ or TNFα and associated with a loss of GCR and Dex-Fluor staining but unchanged Pgp1. There was a significant loss of GCR in CD8 + CD28null compared with CD8 + CD28+ T and NKT-like cells from both COPD and controls (eg, mean ± SEM 8 ± 3% GCR + CD8 + CD28null T-cells vs 49 ± 5% GCR + CD8 + CD28+ T-cells in COPD). There was a significant negative correlation between GCR expression and IFNγ and TNFα production by T and NKT-like cells(eg, COPD: T-cell IFNγ R = −.615; ) and with FEV1 in COPD (R = −.777).

Conclusions

COPD is associated with loss of GCR in senescent CD28null and NKT-like cells suggesting alternative treatment options to GC are required to inhibit these pro-inflammatory/cytotoxic cells.     

Treatment with a sphingosine analog after the inception of house dust mite-induced airway inflammation alleviates key features of experimental asthma

Background

In vivo phosphorylation of sphingosine analogs with their ensuing binding and activation of their cell-surface sphingosine-1-phosphate receptors is regarded as the main immunomodulatory mechanism of this new class of drugs. Prophylactic treatment with sphingosine analogs interferes with experimental asthma by impeding the migration of dendritic cells to draining lymph nodes. However, whether these drugs can also alleviate allergic airway inflammation after its onset remains to be determined. Herein, we investigated to which extent and by which mechanisms the sphingosine analog AAL-R interferes with key features of asthma in a murine model during ongoing allergic inflammation induced by Dermatophagoides pteronyssinus.

Methods

BALB/c mice were exposed to either D. pteronyssinus or saline, intranasally, once-daily for 10 consecutive days. Mice were treated intratracheally with either AAL-R, its pre-phosphorylated form AFD-R, or the vehicle before every allergen challenge over the last four days, i.e. after the onset of allergic airway inflammation. On day 11, airway responsiveness to methacholine was measured; inflammatory cells and cytokines were quantified in the airways; and the numbers and/or viability of T cells, B cells and dendritic cells were assessed in the lungs and draining lymph nodes.

Results

AAL-R decreased airway hyperresponsiveness induced by D. pteronyssinus by nearly 70%. This was associated with a strong reduction of IL-5 and IL-13 levels in the airways and with a decreased eosinophilic response. Notably, the lung CD4⁺ T cells were almost entirely eliminated by AAL-R, which concurred with enhanced apoptosis/necrosis in that cell population. This inhibition occurred in the absence of dendritic cell number modulation in draining lymph nodes. On the other hand, the pre-phosphorylated form AFD-R, which preferentially acts on cell-surface sphingosine-1-phosphate receptors, was relatively impotent at enhancing cell death, which led to a less efficient control of T cell and eosinophil responses in the lungs.

Conclusion

Airway delivery of the non-phosphorylated sphingosine analog, but not its pre-phosphorylated counterpart, is highly efficient at controlling the local T cell response after the onset of allergic airway inflammation. The mechanism appears to involve local induction of lymphocyte apoptosis/necrosis, while mildly affecting dendritic cell and T cell accumulation in draining lymph nodes.     

Cardiac telocytes are double positive for CD34/PDGFR‐α

Telocytes (TCs) are a distinct type of interstitial cells, which are featured with a small cellular body and long and thin elongations called telopodes (Tps). TCs have been widely identified in lots of tissues and organs including heart. Double staining for CD34/PDGFR‐β (Platelet‐derived growth factor receptor β) or CD34/Vimentin is considered to be critical for TC phenotyping. It has recently been proposed that CD34/PDGFR‐α (Platelet‐derived growth factor receptor α) is actually a specific marker for TCs including cardiac TCs although the direct evidence is still lacking. Here, we showed that cardiac TCs were double positive for CD34/PDGFR‐α in primary culture. CD34/PDGFR‐α positive cells (putative cardiac TCs) also existed in mice ventricle and human cardiac valves including mitral valve, tricuspid valve and aortic valve. Over 87% of cells in a TC‐enriched culture of rat cardiac interstitial cells were positive for PDGFR‐α, while CD34/PDGFR‐α double positive cells accounted for 30.25% of the whole cell population. We show that cardiac TCs are double positive for CD34/PDGFR‐α. Better understanding of the immunocytochemical phenotypes of cardiac TCs might help using cardiac TCs as a novel source in cardiac repair.     

Telocytes are reduced during fibrotic remodelling of the colonic wall in ulcerative colitis

Ulcerative colitis (UC) is characterized by chronic relapsing intestinal inflammation finally leading to extensive tissue fibrosis and resulting in a stiff colon unable to carry out peristalsis or to resorb fluids. Telocytes, a peculiar type of stromal cells, have been recently identified in the human gastrointestinal tract. Several roles have been proposed for telocytes, including mechanical support, intercellular signalling and modulation of intestinal motility. The aim of the present work was to investigate the presence and distribution of telocytes in colonic specimens from UC patients compared with controls. Archival paraffin‐embedded samples of the left colon from UC patients who underwent elective bowel resection and controls were collected. Tissue sections were stained with Masson's trichrome to detect fibrosis. Telocytes were identified by CD34 immunohistochemistry. In early fibrotic UC cases, fibrosis affected the muscularis mucosae and submucosa, while the muscularis propria was spared. In advanced fibrotic UC cases, fibrosis extended to affect the muscle layers and the myenteric plexus. Few telocytes were found in the muscularis mucosae and submucosa of both early and advanced fibrotic UC colonic wall. In the muscle layers and myenteric plexus of early fibrotic UC, telocytes were preserved in their distribution. In the muscularis propria of advanced fibrotic UC, the network of telocytes was reduced or even completely absent around smooth muscle bundles and myenteric plexus ganglia, paralleling the loss of the network of interstitial cells of Cajal. In UC, a loss of telocytes accompanies the fibrotic remodelling of the colonic wall and might contribute to colonic dysmotility.     

The CD4+AT2R+ T cell subpopulation improves post‐infarction remodelling and restores cardiac function

Myocardial infarction (MI) is a major condition causing heart failure (HF). After MI, the renin angiotensin system (RAS) and its signalling octapeptide angiotensin II (Ang II) interferes with cardiac injury/repair via the AT1 and AT2 receptors (AT1R, AT2R). Our study aimed at deciphering the mechanisms underlying the link between RAS and cellular components of the immune response relying on a rodent model of HF as well as HF patients. Flow cytometric analyses showed an increase in the expression of CD4⁺ AT2R⁺ cells in the rat heart and spleen post‐infarction, but a reduction in the peripheral blood. The latter was also observed in HF patients. The frequency of rat CD4⁺ AT2R⁺ T cells in circulating blood, post‐infarcted heart and spleen represented 3.8 ± 0.4%, 23.2 ± 2.7% and 22.6 ± 2.6% of the CD4⁺ cells. CD4⁺ AT2R⁺ T cells within blood CD4⁺ T cells were reduced from 2.6 ± 0.2% in healthy controls to 1.7 ± 0.4% in patients. Moreover, we characterized CD4⁺ AT2R⁺ T cells which expressed regulatory FoxP3, secreted interleukin‐10 and other inflammatory‐related cytokines. Furthermore, intramyocardial injection of MI‐induced splenic CD4⁺ AT2R⁺ T cells into recipient rats with MI led to reduced infarct size and improved cardiac performance. We defined CD4⁺ AT2R⁺ cells as a T cell subset improving heart function post‐MI corresponding with reduced infarction size in a rat MI‐model. Our results indicate CD4⁺ AT2R⁺ cells as a promising population for regenerative therapy, via myocardial transplantation, pharmacological AT2R activation or a combination thereof.     

羟基化修饰对大鼠胶原热稳定性的影响研究

采用差示扫描量热仪(DSC)研究羟脯氨酸(Hyp)含量对胶原热稳定性的影响。以不同周龄BN大鼠皮肤为原料制备了胶原,分析制备胶原中Hyp的含量;采用DSC测定不同Hyp含量胶原的临界变性温度及焓变;采用圆二色光谱(CD)检测提取胶原的二级结构。结果表明,提取胶原在41.3℃发生三螺旋解聚,CD光谱分析结果表明,当样品经临界变性温度处理后,部分三螺旋结构转化为无规则线圈结构。胶原变性过程中所需热量与羟脯氨酸含量呈正相关,实验建立了胶原热变性过程中焓变与Hyp含量的关系。该研究表明胶原中脯氨酸羟基化修饰是影响胶原热稳定性的关键因素。