Single-cell analysis is of increasing importance in many fields, but is challenging due to the ultra-small volumes (picoliters) of single cells. Indeed, analysis of a specific analyte might require ...the analysis of a single molecule or several molecules. Analytical processes usually include sampling, chemical processing, and detection. Although several papers have reported chemical processing and detection methods for single cells, a sampling method compatible with maintaining the viability of a single cell during sampling has yet to be developed. Here, we propose a femtoliter sampling method from a living single cell using micro/nanofluidic device technology. The sampling of 39 fL of cytoplasm from a single human aortic endothelial cell was demonstrated and its viability after sampling was confirmed.
Summary
Background
LL‐37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis ...(SSc).
Objectives
To elucidate the potential role of LL‐37 in SSc.
Methods
The expression of target molecules was evaluated by immunostaining and quantitative reverse‐transcription real‐time polymerase chain reaction in human and murine skin. The mechanisms regulating LL‐37 expression in endothelial cells were examined by gene silencing and chromatin immunoprecipitation. Serum LL‐37 levels were determined by enzyme‐linked immunosorbent assay.
Results
In SSc lesional skin, LL‐37 expression was increased in dermal fibroblasts, perivascular inflammatory cells, keratinocytes and, particularly, dermal small vessels. Expression positively correlated with interferon‐α expression, possibly reflecting LL‐37‐dependent induction of interferon‐α. In SSc animal models, bleomycin‐treated skin exhibited the expression pattern of CRAMP, a murine homologue of LL‐37, similar to that of LL‐37 in SSc lesional skin. Furthermore, Fli1+/− mice showed upregulated expression of CRAMP in dermal small vessels. Fli1 binding to the CAMP (LL‐37 gene) promoter and Fli1 deficiency‐dependent induction of LL‐37 were also confirmed in human dermal microvascular endothelial cells. In the analysis of sera, patients with SSc had serum LL‐37 levels significantly higher than in healthy controls. Furthermore, serum LL‐37 levels positively correlated with skin score and the activity of alveolitis and were significantly elevated in patients with digital ulcers compared with those without.
Conclusions
LL‐37 upregulation, induced by Fli1 deficiency at least in endothelial cells, potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in SSc.
What's already known about this topic?
LL‐37, an antimicrobial peptide, has been shown to be upregulated in systemic sclerosis dermal fibroblasts.
LL‐37 potentially contributes to dermal fibrosis through its antiapoptotic effect on those cells.
What does this study add?
LL‐37 potentially contributes to the development of skin sclerosis, interstitial lung disease and digital ulcers in systemic sclerosis.
This further supports the critical role of antimicrobial peptides in the development of autoimmune and inflammatory diseases.
What is the translational message?
LL‐37 induction due to Fli1 deficiency in endothelial cells supports the notion that Fli1 deficiency is a key predisposing factor in the pathogenesis of systemic sclerosis.
This has recently been demonstrated by the establishment of a new systemic sclerosis animal model, with mice double heterozygous for the Klf5 and Fli1 genes.
Genital necrosis with cutaneous thrombosis after COVID‐19 mRNA vaccination Kuzumi, A.; Yoshizaki, A.; Chiba, K. ...
JEADV. Journal of the European Academy of Dermatology and Venereology/Journal of the European Academy of Dermatology and Venereology,
March 2022, Letnik:
36, Številka:
3
Journal Article
Summary
Background
Endothelial protein C receptor (EPCR), expressed predominantly on endothelial cells, plays a critical role in the regulation of the coagulation system and also mediates various ...cytoprotective effects by binding and activating protein C. So far, the role of EPCR has not been studied in systemic sclerosis (SSc).
Objectives
To investigate the potential contribution of EPCR to the development of SSc.
Methods
EPCR expression was examined in skin samples and cultivated dermal microvascular endothelial cells by immunostaining, immunoblotting and/or quantitative reverse‐transcription polymerase chain reaction. Fli1, binding to the PROCR promoter, was assessed by chromatin immunoprecipitation. Serum EPCR levels were determined by enzyme‐linked immunosorbent assay in 65 patients with SSc and 20 healthy subjects.
Results
EPCR expression was decreased in dermal small vessels of SSc lesional skin compared with those of healthy control skin. Transcription factor Fli1, deficiency of which is implicated in SSc vasculopathy, occupied the PROCR promoter, and EPCR expression was suppressed in Fli1 small interfering RNA‐treated endothelial cells and dermal small vessels of Fli1+/− mice. In patients with SSc, decreased serum EPCR levels were associated with diffuse skin involvement, interstitial lung disease and digital ulcers. Furthermore, serum EPCR levels inversely correlated with plasma levels of plasmin–α2‐plasmin inhibitor complex (PIC). Importantly, bosentan significantly reversed circulating EPCR and PIC levels in patients with SSc, and the expression of Fli1 and EPCR in dermal small vessels was elevated in patients treated with bosentan compared with untreated patients.
Conclusions
Endothelial EPCR downregulation due to Fli1 deficiency may contribute to hypercoagulation status leading to tissue fibrosis and impaired peripheral circulation in SSc.
What's already known about this topic?
Endothelial protein C receptor (EPCR) plays a critical role in the coagulation system and mediates various cytoprotective effects by binding and activating protein C.
The role of EPCR has not been studied in the impaired coagulation/fibrinolysis system of systemic sclerosis.
What does this study add?
EPCR downregulation potentially contributes to the development of digital ulcers and tissue fibrosis in systemic sclerosis.
This further supports the critical role of an impaired coagulation/fibrinolysis system in this disease.
Linked Comment: Jinnin, Br J Dermatol 2016; 174: 263.
Chronic lymphocytic leukemia (CLL) can be immunosuppressive in humans and mice, and CLL cells share multiple phenotypic markers with regulatory B cells that are competent to produce interleukin ...(IL)-10 (B10 cells). To identify functional links between CLL cells and regulatory B10 cells, the phenotypes and abilities of leukemia cells from 93 patients with overt CLL to express IL-10 were assessed. CD5(+) CLL cells purified from 90% of the patients were IL-10-competent and secreted IL-10 following appropriate ex vivo stimulation. Serum IL-10 levels were also significantly elevated in CLL patients. IL-10-competent cell frequencies were higher among CLLs with IgV(H) mutations, and correlated positively with TCL1 expression. In the TCL1-transgenic (TCL1-Tg) mouse model of CLL, IL-10-competent B cells with the cell surface phenotype of B10 cells expanded significantly with age, preceding the development of overt, CLL-like leukemia. Malignant CLL cells in TCL1-Tg mice also shared immunoregulatory functions with mouse and human B10 cells. Serum IL-10 levels varied in TCL1-Tg mice, but in vivo low-dose lipopolysaccharide treatment induced IL-10 expression in CLL cells and high levels of serum IL-10. Thus, malignant IL-10-competent CLL cells exhibit regulatory functions comparable to normal B10 cells that may contribute to the immunosuppression observed in patients and TCL1-Tg mice.
Background
Trappin‐2/pre‐elafin is an endogenous inhibitor of human neutrophil elastase involved in inflammation, innate immunity and vascular remodelling, which consist of the complex pathological ...process of systemic sclerosis (SSc).
Objectives
To clarify the potential role of trappin‐2 in SSc.
Methods
Serum trappin‐2 levels were determined by enzyme‐linked immunosorbent assay in 51 SSc and 18 healthy subjects. Trappin‐2 expression was evaluated in SSc lesional skin and cultured endothelial cells treated with FLI1 siRNA by immunohistochemistry, reverse transcription‐real‐time PCR and/or immunoblotting. Friend leukaemia virus integration 1 (Fli1) binding to the PI3 promoter was assessed by chromatin immunoprecipitation.
Results
Since serum trappin‐2 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction, SSc patients with normal renal function were analysed. Although serum trappin‐2 levels were comparable between diffuse cutaneous SSc, limited cutaneous SSc and control subjects, the prevalence of digital ulcers or elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum trappin‐2 levels than in those with normal levels. Furthermore, serum trappin‐2 levels were significantly increased in SSc patients with digital ulcers or elevated RVSP compared to those without. Moreover, serum trappin‐2 levels positively correlated with RVSP values in SSc patients. Importantly, trappin‐2 expression was enhanced in small vessels of SSc lesional skin. In cultured endothelial cells, trappin‐2 expression was elevated by gene silencing of FLI1 at mRNA and protein levels and Fli1 occupied the PI3 promoter.
Conclusions
Endothelial trappin‐2 up‐regulation partially due to Fli1 deficiency can be associated with the development of SSc vasculopathy.
Summary
Background
Interleukin (IL)‐25 is a member of the IL‐17 family, which can promote and augment T‐helper (Th) type 2 responses. The expression of IL‐25 and its cognate receptor, IL‐25 receptor ...(IL‐25R), is upregulated and correlated with disease activity in Th2‐associated diseases.
Objectives
To examine the expression and function of IL‐25 in cutaneous T‐cell lymphoma (CTCL).
Methods
Expression and location of IL‐25 in lesional skin was investigated with immunohistochemistry. The effect of various cytokines on IL‐25 production from normal human epidermal keratinocytes was assessed by quantitative reverse‐transcription real‐time polymerase chain reaction. Serum IL‐25 levels were measured by enzyme‐linked immunosorbent assay. The direct effect of IL‐25 on tumour cells was also examined using CTCL cell lines and peripheral blood mononuclear cells in patients with Sézary syndrome.
Results
IL‐25 expression was increased in epidermal keratinocytes in lesional skin of CTCL. Th2 cytokines, IL‐4 and IL‐13, and periostin induced IL‐25 expression by normal human epidermal keratinocytes. Serum IL‐25 levels were increased in patients with advanced CTCL and correlated with serum lactate dehydrogenase levels. MyLa cells expressed IL‐25R and its expression was augmented by stimulation with IL‐25. IL‐25 enhanced IL‐13 production from MyLa cells via phosphorylation of signal transducer and activator of transcription 6. Peripheral blood mononuclear cells from one patient with Sézary syndrome expressed IL‐25R and showed increase of IL‐13 production by IL‐25.
Conclusions
Th2 cytokines highly expressed in CTCL lesional skin induce IL‐25 production by epidermal keratinocytes, which may, in turn, lead to formation of a Th2‐dominant microenvironment through the direct induction of IL‐13 by tumour cells.
What's already known about this topic?
Cutaneous T‐cell lymphomas (CTCLs), such as mycosis fungoides and Sézary syndrome, are regarded as T helper 2 (Th2)‐type diseases, and a Th2‐dominant microenvironment is beneficial for tumour cells.
Interleukin (IL)‐25 has the capacity to promote and augment Th2 responses and is associated with several Th2‐type diseases, including atopic dermatitis.
What does this study add?
Th2 cytokines highly expressed in lesional skin of CTCL induce IL‐25 production by epidermal keratinocytes.
IL‐25 directly induces IL‐13 production from CTCL tumour cells through signal transducer and activator of transcription 6 (STAT6) signalling pathways, resulting in the formation of a Th2‐dominant microenvironment.
What is the translational message?
Our results support the notion that activation of STAT6 is a key signalling pathway for the creation of a Th2‐dominant microenvironment in CTCL.
As the destruction of a Th2‐dominant microenvironment is effective for CTCL, IL‐25 and STAT6 can be a therapeutic target for CTCL.
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