Immunoarchitectural characterization of a human skin model reconstructed in vitro

CONTEXT AND OBJECTIVE: Over the last few years, different models for human skin equivalent reconstructed in vitro (HSERIV) have been reported for clinical usage and applications in research for the pharmaceutical industry. Before release for routine use as human skin replacements, HSERIV models need to be tested regarding their similarity with in vivo skin, using morphological (architectural) and immunohistochemical (functional) analyses. A model for HSERIV has been developed in our hospital, and our aim here was to further characterize its immunoarchitectural features by comparing them with human skin, before it can be tested for clinical use, e.g. for severe burns or wounds, whenever ancillary methods are not indicated. DESIGN AND SETTING: Experimental laboratory study, in the Skin Cell Culture Laboratory, School of Medical Sciences, Universidade Estadual de Campinas. METHODS: Histological sections were stained with hematoxylin-eosin, Masson's trichrome for collagen fibers, periodic acid-Schiff reagent for basement membrane and glycogen, Weigert-Van Gieson for elastic fibers and Fontana-Masson for melanocytes. Immunohistochemistry was used to localize cytokeratins (broad spectrum of molecular weight, AE1/AE3), high molecular weight cytokeratins (34βE12), low molecular weight cytokeratins (35βH11), cytokeratins 7 and 20, vimentin, S-100 protein (for melanocytic and dendritic cells), CD68 (KP1, histiocytes) and CD34 (QBend, endothelium). RESULTS: Histology revealed satisfactory similarity between HSERIV and in vivo skin. Immunohistochemical analysis on HSERIV demonstrated that the marker pattern was similar to what is generally present in human skin in vivo. CONCLUSION: HSERIV is morphologically and functionally compatible with human skin observed in vivo.

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Necroptosis Contributes to Urban Particulate Matter-Induced Airway Epithelial Injury

Cellular Physiology and Biochemistry ◽

10.1159/000488726 ◽

2018 ◽

Vol 46 (2) ◽

pp. 699-712 ◽

Cited By ~ 13

Author(s):

Feng Xu ◽

Man Luo ◽

Lulu He ◽

Yuan Cao ◽

Wen Li ◽

...

Keyword(s):

Particulate Matter ◽

Pulmonary Inflammation ◽

Periodic Acid ◽

Enzyme Linked Immunosorbent Assay ◽

Periodic Acid Schiff ◽

Mucus Production ◽

Gm Csf ◽

Mucin Expression

Background/Aims: Necroptosis, a form of programmed necrosis, is involved in the pathologic process of several kinds of pulmonary diseases. However, the role of necroptosis in particulate matter (PM)–induced pulmonary injury remains unclear. The objective of this study is to investigate the involvement of necroptosis in the pathogenesis of PM-induced toxic effects in pulmonary inflammation and mucus hyperproduction, both in vitro and in vivo. Methods: PM was administered into human bronchial epithelial (HBE) cells or mouse airways, and the inflammatory response and mucus production were assessed. The mRNA expressions of IL6, IL8 and MUC5AC in HBE cells and Cxcl1, Cxcl2, and Gm-csf in the lung tissues were detected by quantitative real-time RT-PCR. The secreted protein levels of IL6 and IL8 in culture supernatants and Cxcl1, Cxcl2, and Gm-csf in bronchoalveolar lavage fluid (BALF) were detected by enzyme-linked immunosorbent assay (ELISA). We used Western blot to measure the protein expressions of necroptosis-related proteins (RIPK1, RIPK3, and Phospho-MLKL), NF-κB (P65 and PP65), AP-1 (P-c-Jun and P-c-Fos) and MUC5AC. Cell necrosis and mitochondrial ROS were detected using flow cytometry. In addition, pathological changes and scoring of lung tissue samples were monitored using hemoxylin and eosin (H&E), periodic acid-schiff (PAS) and immunohistochemistry staining. Results: Our study showed that PM exposure induced RIP and MLKL-dependent necroptosis in HBE cells and in mouse lungs. Managing the necroptosis inhibitor Necrostatin-1 (Nec-1) and GSK’872, specific molecule inhibitors of necroptosis, markedly reduced PM-induced inflammatory cytokines, e.g., IL6 and IL8, and MUC5AC in HBE cells. Similarly, administering Nec-1 significantly reduced airway inflammation and mucus hyperproduction in PM-exposed mice. Mechanistically, we found PM–induced necroptosis was mediated by mitochondrial reactive oxygen species-dependent early growth response gene 1, which ultimately promoted inflammation and mucin expression through nuclear factor κB and activator protein-1 pathways, respectively. Conclusions: Our results demonstrate that necroptosis is involved in the pathogenesis of PM–induced pulmonary inflammation and mucus hyperproduction, and suggests that it may be a novel target for treatment of airway disorders or disease exacerbations with airborne particulate pollution.

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Mitochondria-Targeted Antioxidant Mito-Tempo Protects Against Aldosterone-Induced Renal Injury In Vivo

Cellular Physiology and Biochemistry ◽

10.1159/000485287 ◽

2017 ◽

Vol 44 (2) ◽

pp. 741-750 ◽

Cited By ~ 14

Author(s):

Wei Ding ◽

Tingyan Liu ◽

Xiao Bi ◽

Zhiling Zhang

Keyword(s):

Electron Microscopy ◽

Renal Function ◽

Nlrp3 Inflammasome ◽

Renal Injury ◽

Periodic Acid ◽

Inflammasome Activation ◽

Periodic Acid Schiff ◽

Nlrp3 Inflammasome Activation

Background/Aims: Growing evidence suggests mitochondrial dysfunction (MtD) and the Nlrp3 inflammasome play critical roles in chronic kidney disease (CKD) progression. We previously reported that Aldosterone (Aldo)-induced renal injury in vitro is directly caused by mitochondrial reactive oxygen species (mtROS)-mediated activation of the Nlrp3 inflammasome. Here we aimed to determine whether a mitochondria-targeted antioxidant (Mito-Tempo) could prevent Aldo-induced kidney damage in vivo. Methods: C57BL/6J mice were treated with Aldo and/or Mito-Tempo (or ethanol as a control) for 4 weeks. Renal injury was evaluated by Periodic Acid-Schiff reagent or Masson’s trichrome staining and electron microscopy. ROS were measured by DCFDA fluorescence and ELISA. MtD was determined by real-time PCR and electron microscopy. Activation of the Nlrp3 inflammasome and endoplasmic reticulum stress (ERS) was detected via western blot. Results: Compared with control mice, Aldo-infused mice showed impaired renal function, increased mtROS production and MtD, Nlrp3 inflammasome activation, and elevated ERS. We showed administration of Mito-Tempo significantly improved renal function and MtD, and reduced Nlrp3 inflammasome activation and ERS in vivo. Conclusion: Mitochondria-targeted antioxidants may attenuate Aldo-infused renal injury by inhibiting MtD, the Nlrp3 inflammasome, and ERS in vivo. Therefore, targeting mtROS might be an effective strategy for preventing CKD.

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Gastrin Attenuates Renal Ischemia/Reperfusion Injury by a PI3K/Akt/Bad-Mediated Anti-apoptosis Signaling

Frontiers in Pharmacology ◽

10.3389/fphar.2020.540479 ◽

2020 ◽

Vol 11 ◽

Author(s):

Chao Liu ◽

Ken Chen ◽

Huaixiang Wang ◽

Ye Zhang ◽

Xudong Duan ◽

...

Keyword(s):

Protective Effect ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Periodic Acid ◽

Drug Candidate ◽

Gastrointestinal Hormone ◽

Akt Inhibitor ◽

Periodic Acid Schiff

Ischemic/reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI). Gastrin, a gastrointestinal hormone, is involved in the regulation of kidney function of sodium excretion. However, whether gastrin has an effect on kidney I/R injury is unknown. Here we show that cholecystokinin B receptor (CCKBR), the gastrin receptor, was significantly up-regulated in I/R-injured mouse kidneys. While pre-administration of gastrin ameliorated I/R-induced renal pathological damage, as reflected by the levels of serum creatinine and blood urea nitrogen, hematoxylin and eosin staining and periodic acid-Schiff staining. The protective effect could be ascribed to the reduced apoptosis for gastrin reduced tubular cell apoptosis both in vivo and in vitro. In vitro studies also showed gastrin preserved the viability of hypoxia/reoxygenation (H/R)-treated human kidney 2 (HK-2) cells and reduced the lactate dehydrogenase release, which were blocked by CI-988, a specific CCKBR antagonist. Mechanistically, the PI3K/Akt/Bad pathway participates in the pathological process, because gastrin treatment increased phosphorylation of PI3K, Akt and Bad. While in the presence of wortmannin (1 μM), a PI3K inhibitor, the gastrin-induced phosphorylation of Akt after H/R treatment was blocked. Additionally, wortmannin and Akt inhibitor VIII blocked the protective effect of gastrin on viability of HK-2 cells subjected to H/R treatment. These studies reveals that gastrin attenuates kidney I/R injury via a PI3K/Akt/Bad-mediated anti-apoptosis signaling. Thus, gastrin can be considered as a promising drug candidate to prevent AKI.

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Evaluation of co-cultured spermatogonial stem cells encapsulated in alginate hydrogel with Sertoli cells and their transplantation into azoospermic mice

Zygote ◽

10.1017/s0967199421000733 ◽

2021 ◽

pp. 1-8

Author(s):

Mohammad Veisi ◽

Kamran Mansouri ◽

Vahideh Assadollahi ◽

Cyrus Jalili ◽

Afshin Pirnia ◽

...

Keyword(s):

Sertoli Cells ◽

Periodic Acid ◽

Three Dimensional ◽

3D Culture ◽

Alginate Hydrogel ◽

Periodic Acid Schiff ◽

Expression Levels ◽

Pas Staining

Summary An in vitro spermatogonial stem cell (SSC) culture can serve as an effective technique to study spermatogenesis and treatment for male infertility. In this research, we compared the effect of a three-dimensional alginate hydrogel with Sertoli cells in a 3D culture and co-cultured Sertoli cells. After harvest of SSCs from neonatal mice testes, the SSCs were divided into two groups: SSCs on a 3D alginate hydrogel with Sertoli cells and a co-culture of SSCs with Sertoli cells for 1 month. The samples were evaluated by quantitative reverse transcription polymerase chain reaction (qRT-PCR) assays and bromodeoxyuridine (BrdU) tracing, haematoxylin and eosin (H&E) and periodic acid–Schiff (PAS) staining after transplantation into an azoospermic testis mouse. The 3D group showed rapid cell proliferation and numerous colonies compared with the co-culture group. Molecular assessment showed significantly increased integrin alpha-6, integrin beta-1, Nanog, Plzf, Thy-1, Oct4 and Bcl2 expression levels in the 3D group and decreased expression levels of P53, Fas, and Bax. BrdU tracing, and H&E and PAS staining results indicated that the hydrogel alginate improved spermatogenesis after transplantation in vivo. This finding suggested that cultivation of SSCs on alginate hydrogel with Sertoli cells in a 3D culture can lead to efficient proliferation and maintenance of SSC stemness and enhance the efficiency of SSC transplantation.

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3D-printed chitosan-based scaffolds: An in vitro study of human skin cell growth and an in-vivo wound healing evaluation in experimental diabetes in rats

Carbohydrate Polymers ◽

10.1016/j.carbpol.2018.07.057 ◽

2018 ◽

Vol 199 ◽

pp. 593-602 ◽

Cited By ~ 36

Author(s):

Claudio Intini ◽

Lisa Elviri ◽

Jaydee Cabral ◽

Sonya Mros ◽

Carlo Bergonzi ◽

...

Keyword(s):

Wound Healing ◽

Cell Growth ◽

Human Skin ◽

Experimental Diabetes ◽

In Vitro Study ◽

Vitro Study ◽

Skin Cell ◽

3D Printed

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Thermosensitive hydrogels as a controlled release system for alkannin to improve localized treatment of Candida vaginitis after external beam radiotherapy in vitro and in vivo

Pathogens and Disease ◽

10.1093/femspd/ftz041 ◽

2019 ◽

Vol 77 (4) ◽

Author(s):

Jin Su ◽

Jia Ma ◽

Fei Mo ◽

Xianwei Yang ◽

Peipei Zhang ◽

...

Keyword(s):

External Beam Radiotherapy ◽

Periodic Acid ◽

High Dose ◽

External Beam ◽

Topical Formulation ◽

Vaginal Tissue ◽

Thermosensitive Hydrogel ◽

Periodic Acid Schiff

ABSTRACT External beam radiotherapy increases the risk of Candida vaginitis in cervical cancer patients, which brings a lot of insufferable influence to their life. Here, we explored the efficacy of alkannin in the treatment of Candida vaginitis after external beam radiotherapy. We exploit thermosensitive hydrogel-mediated alkannin as the topical formulation in a rat model established in our work. Periodic acid-Schiff of vaginas indicated little Candida albicans adhered to the vaginal tissue in treatment group. Additionally, hematoxylin and eosin stain revealed that inflammatory response of high dose alkannin was reduced. Above all, the animal model was first established in our work for the clinical desire. Our results suggested the promising application of alkannin for the disease with satisfying fungicidal activity and anti-inflammatory activity.

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Investigation of the Mechanism Underlying Calcium Dobesilate-Mediated Improvement of Endothelial Dysfunction and Inflammation Caused by High Glucose

Mediators of Inflammation ◽

10.1155/2019/9893682 ◽

2019 ◽

Vol 2019 ◽

pp. 1-12

Author(s):

Yijun Zhou ◽

Chaojun Qi ◽

Shu Li ◽

Xinghua Shao ◽

Zhaohui Ni

Keyword(s):

Endothelial Dysfunction ◽

Gene Silencing ◽

High Glucose ◽

Umbilical Vein ◽

Periodic Acid ◽

Protective Effects ◽

Calcium Dobesilate ◽

Periodic Acid Schiff

Background/Aims. Diabetic kidney disease (DKD) is a leading cause of end-stage renal disease. Calcium dobesilate (CaD) is widely used to treat diabetic retinopathy. Recent studies have demonstrated that CaD exerts protective effects against diabetic nephropathy. The aim of this study was to elucidate the molecular and cellular mechanisms underlying the protective effects of CaD. Methods. Human umbilical vein endothelial cells (HUVECs) were cultured with different D-glucose concentrations to determine the effects of high glucose on HUVEC gene expression. HUVECs were also incubated with CaD (25 μM, 50 μM, and 100 μM) for 3 days to determine the effects of CaD on HUVEC viability. db/db mice were treated with CaD. 2-[(Aminocarbonyl)amino]-5-(4-fluorophenyl)-3-thiophenecarboxamide (TPCA-1) blocked the nuclear factor-κB (NF-κB) pathway in HUVECs. A pentraxin 3 (PTX3) small interfering RNA (siRNA) intervention experiment was performed in the cells. An adenovirus-encapsulated PTX3 siRNA intervention experiment was performed in db/db mice. Western blot and real-time PCR analyses were used to detect PTX3, p-IKBa/IKBa (I-kappa-B-alpha), and p-eNOS/eNOS (endothelial nitric oxide synthase) expression in mice and HUVECs. Hematoxylin-eosin (HE) staining and periodic acid-Schiff (PAS) staining were used to observe renal tissue damage in mice. PTX3 expression was observed by immunohistochemical staining. Results. CaD downregulated the expression of PTX3 and p-IKBa/IKBa and upregulated the expression of p-eNOS/eNOS in vitro. When TPCA-1 was used, high glucose induced high PTX3 expression, and the expression of p-eNOS/eNOS increased. After PTX3 gene silencing, the expression of p-eNOS/eNOS also increased. In vivo, CaD reduced the expression of PTX3 and p-IKBa/IKBa in the kidneys of db/db mice and increased the expression of p-eNOS/eNOS. After PTX3 gene silencing, the urine protein and renal function of db/db mice were ameliorated, the glomerular extracellular matrix was decreased, and the expression of p-eNOS/eNOS was increased. Conclusions. Our results suggested that CaD may inhibit the expression of PTX3 by altering the IKK/IKB/NF-κB pathway, thereby improving endothelial dysfunction in HUVECs. PTX3 may be a potential therapeutic target for DKD.

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Gastroprotective Effects of Periplaneta americana L. Extract Against Ethanol-Induced Gastric Ulcer in Mice by Suppressing Apoptosis-Related Pathways

Frontiers in Pharmacology ◽

10.3389/fphar.2021.798421 ◽

2021 ◽

Vol 12 ◽

Author(s):

Shu Fu ◽

Jiamei Chen ◽

Chen Zhang ◽

Jinfeng Shi ◽

Xin Nie ◽

...

Keyword(s):

Cell Death ◽

Gastric Ulcer ◽

Programmed Cell Death ◽

Periplaneta Americana ◽

Periodic Acid ◽

Protective Effects ◽

Periodic Acid Schiff ◽

Wide Range

Although Periplaneta americana L. and its modern preparation, Kangfuxin liquid, have been extensively applied for ulcerative diseases in gastrointestinal tract (e.g., gastric ulcer (GU) and ulcerative colitis, the effective components and potential mechanisms) remain unclear. In accordance with the accumulating research evidences, the relieving/exacerbating of GU is noticeably correlated to focal tissue programmed cell death. Herein, gastro-protective effects of the effective Periplaneta americana L. extract (PAE) fraction were assessed in vitro and in vivo, involving in programmed cell death-related signaling channels. To screen the effective PAE fraction exerting gastroprotective effects, several PAE fractions were gained based on a wide range of ethanol solution concentration, and they were assessed on ethanol-induced ulcer mice. Based on HPLC investigation with the use of nucleosides, the chemical composition of screened effective PAE, extracted by 20% ethanol, was analyzed in terms of quality control. Based on CCK-8 assay, the protective effects on GES-1 cells, impaired by ethanol, of PAE were assessed. After 3 days pre-treatment with PAE (200, 400, 800 mg/kg), the gastric lesions were assessed by tissue morphology, and periodic acid-schiff (PAS) staining, as well as hematoxylin and eosin (H&E) based histopathology-related investigation. The levels for inflammation cytokines (IL1-β, TNF-α, IL-18, PGE2, and IL-6), antioxidant indices (SOD and MDA) were examined via ELISA. In the meantime, based on Western Blotting assay, the expression levels of some programmed cell death-related protein targets (NLRP3, caspase-1, NF-κB p65, MyD88, and TLR4) were analyzed. As revealed from the results, PAE is capable of alleviating gastric mucosa impairment, suppressing the inflammatory cytokines, and down-regulating the MyD88/NF-κB channels. Accordingly, 20% ethanol extract of Periplaneta americana L. would contribute its gastroprotective effects, thereby providing the evidence that its anti-GU mechanisms correlated with inhibiting programmed cell death channel.

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LPS Exposure in Early Life Protects Against Mucus Hypersecretion in Ovalbumin-Induced Asthma by Down-Regulation of the IL-13 and JAK-STAT6 Pathways

Cellular Physiology and Biochemistry ◽

10.1159/000489109 ◽

2018 ◽

Vol 46 (3) ◽

pp. 1263-1274 ◽

Cited By ~ 5

Author(s):

Fengxia Ding ◽

Bo Liu ◽

Wenjing Zou ◽

Daiyin Tian ◽

Qubei Li ◽

...

Keyword(s):

Transcription Factor ◽

Periodic Acid ◽

Mucus Secretion ◽

Periodic Acid Schiff ◽

Mucus Hypersecretion ◽

Asthma Model ◽

Pas Staining ◽

Pre Treatment

Background/Aims: Previous studies have shown that lipopolysaccharide (LPS) exposure may have a protective effect on asthma by reducing airway hyper-responsiveness, airway inflammation and serum IgE levels. However, there are few studies investigating the effect of LPS on mucous secretion in asthma. In this study, we evaluate the relationship between LPS pre-treatment in infant mice and airway mucus hypersecretion in an OVA (ovalbumin)-induced asthma model, and further explore the mechanisms behind this effect. Methods: Mice were pre-treated with LPS by intranasal instillation (i.n.) from the 3rd day of life for 10 consecutive days before the OVA-induced asthma model was established. In order to detect mucus secretion, periodic acid-Schiff (PAS) staining was carried out, and the expression of Muc5ac was detected. The IL-13 levels in Bronchoalveolar lavage fluid (BALF) and lung tissue were also detected. In vitro, the expression of Muc5ac mRNA and protein was quantified in IL-13-stimulated 16HBE cells with or without LPS pre-treatment. In addition, proteins in the JAK2/STAT6 pathway, transcription factors (forkhead box transcription factor A2 (FOXA2), activation protein-1(AP-1), NF-κB), and the levels of reactive oxygen species (ROS) were also measured in vivo and in vitro. Results: LPS pre-treatment reduced mucus secretion, as demonstrated by decreased PAS staining and muc5ac expression. Further exploration of the underlying mechanisms of this phenomenon revealed that LPS pre-treatment decreased the production of IL-13, IL-13 induced ROS synthesis was reduced, and the JAK2/STAT6 pathway was inhibited. Decreased stat6 increased transcription factor FOXA2, and the relatively increased FOXA2 further decreased the level of Muc5ac and mucous hypersecretion in OVA-induced asthma. Conclusions: LPS pre-treatment ameliorated mucus hypersecretion in an OVA-induced asthma model by inhibition of IL-13 production and by further inhibiting the JAK2/STAT6 pathway and ROS activity, and up-regulating expression of FOXA2.

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Étude des interactions hôte–Bifidobacterium chez la souris axénique: caractérisation partielle des facteurs bifidigènes du contenu intestinal

Canadian Journal of Microbiology ◽

10.1139/m90-049 ◽

1990 ◽

Vol 36 (4) ◽

pp. 286-291 ◽

Cited By ~ 5

Author(s):

Marie Bénédicte Romond ◽

Monzer Hamze ◽

Charles Romond ◽

Pierre Bourlioux

Keyword(s):

Periodic Acid ◽

Bifidobacterium Bifidum ◽

Periodic Acid Schiff ◽

Intestinal Mucus ◽

Sds Page ◽

Sulfuric Acid Method ◽

Sepharose 4B ◽

Phenol Sulfuric Acid

Growth factors for Bifidobacterium bifidum were detected in faeces of axenic mice strain C3H. Most of these factors were found in the nondialyzable fraction obtained after aqueous extraction and dialysis. SDS–PAGE and filtration chromatography on Sepharose 4B revealed that many glycosylated components harbored a bifidigenic activity. Intestinal colinization of mice by B. bifidum involved the utilization and eventually the disappearance of the intestinal bifidigenic factors. There was no change in the protein concentration in fecal extracts, but the total hexose concentration was lower. Comparison of electrophoretic PAGE profiles after periodic acid Schiff coloration showed that bacteria used up the glycosylated fractions of many glycopeptides, particularly those of mucins and four glycoproteins. There was no correlation between the hexose concentration detected in every active fraction and the degree of in vivo degradation of bifidigenic factors. The attack on active glycopeptides having a molecular mass greater than 670 kDa thus revealed hexose sites that were not detectable previously by the phenol – sulfuric acid method. However, the amount of bifidigenic factors detected in vitro allowed us to measure the importance of the degradation of a component by B. bifidum in vivo. Key words: Bifidobacterium, bifidigenic factors, intestinal mucus, axenic mice. [Journal translation]

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