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Both academia and dermatological and cosmetic industries have acknowledged that healthy skin microbiota contribute to overall skin integrity and well-being. This implies that formulations developed for personal care (skin, scalp, hair etc) or (medical and cosmetic) treatment need to be compatible with microbiota conservation or possibly even improvement. The various chemical and biological components and mixtures thereof intended for direct application to the skin should not extensively affect the qualitative and quantitative composition of the skin microbiota. A compound should promote beneficial microbes and inhibit pathogens. Compounds but also final products could be considered at least theoretically “microbiome friendly” while in some cases changes to the microbiota may even be considered beneficial. An important hurdle lies in the practical and methodological approaches to be used for defining microbiota inertia of compounds and formulations. Clear guidelines for assessing microbiome friendliness are lacking. We propose three testing concepts that may help to define microbiome friendliness based on the assessment of minimal microbiota perturbation and possibly elimination of potential pathogens. Methods to prove microbiome friendliness should ultimately be based upon (metagenomic rather than amplicon-based) next generation sequencing of naive versus compound- or final product-exposed skin microbiota in vivo, but preferably also including in vitro and ex vivo pre-screening methodologies to build an understanding of their consequences. As in many domains of microbiome research, the development of experimental process controls and internal standards, which are essentially lacking to date, should be taken as a future prerequisite. There is also a requirement from regulatory agencies to define and harmonize acceptance criteria.

Ischemia–reperfusion injury (IRI) is a complex inflammatory process that detrimentally affects the function of transplanted organs. Neutrophils are important contributors to the pathogenesis of renal IRI. Signaling by G-CSF, a regulator of neutrophil development, trafficking, and function, plays a key role in several neutrophil-associated inflammatory disease models. In this study, we investigated whether targeting neutrophils with a neutralizing mAb to G-CSFR would reduce inflammation and protect against injury in a mouse model of warm renal IRI. Mice were treated with anti–G-CSFR 24 h prior to 22-min unilateral renal ischemia. Renal function and histology, complement activation, and expression of kidney injury markers, and inflammatory mediators were assessed 24 h after reperfusion. Treatment with anti–G-CSFR protected against renal IRI in a dose-dependent manner, significantly reducing serum creatinine and urea, tubular injury, neutrophil and macrophage infiltration, and complement activation (plasma C5a) and deposition (tissue C9). Renal expression of several proinflammatory genes (CXCL1/KC, CXCL2/MIP-2, MCP-1/CCL2, CXCR2, IL-6, ICAM-1, P-selectin, and C5aR) was suppressed by anti–G-CSFR, as was the level of circulating P-selectin and ICAM-1. Neutrophils in anti–G-CSFR–treated mice displayed lower levels of the chemokine receptor CXCR2, consistent with a reduced ability to traffic to inflammatory sites. Furthermore, whole transcriptome analysis using RNA sequencing showed that gene expression changes in IRI kidneys after anti–G-CSFR treatment were indistinguishable from sham-operated kidneys without IRI. Hence, anti–G-CSFR treatment prevented the development of IRI in the kidneys. Our results suggest G-CSFR blockade as a promising therapeutic approach to attenuate renal IRI.

In present study transdermal drug delivery of Physostigmine was developed to overcome the first pass metabolism and to reduce frequency of dosing compared to oral route. Matrix type of transdermal patches was developed by using polymers Eudragit-L100, HPMCk4M and HPMCk15M.Transdermal patches were prepared by employing solvent casting method. Propylene glycol and Tween80 were selected as permeation enhancer and plasticizer. Among all the formulations P6 formulation was found to be best and shown 96.5% drug release in 12 hours. For P6 formulation release kinetics were applied and it was observed that the formulation was following peppas mechanism of drug release.

The complement system is a potent mediator of ischemia–reperfusion injury (IRI), which detrimentally affects the function and survival of transplanted kidneys. Human complement receptor 1 (HuCR1) is an integral membrane protein that inhibits complement activation by blocking the convertases that activate C3 and C5. We have previously reported that CSL040, a truncated form of recombinant soluble HuCR1 (sHuCR1), has enhanced complement inhibitory activity and improved pharmacokinetic properties compared to the parent molecule. Here, we compared the capacity of CSL040 and full-length sHuCR1 to suppress complement-mediated organ damage in a mouse model of warm renal IRI. Mice were treated with two doses of CSL040 or sHuCR1, given 1 h prior to 22 min unilateral renal ischemia and again 3 h later. 24 h after reperfusion, mice treated with CSL040 were protected against warm renal IRI in a dose-dependent manner, with the highest dose of 60 mg/kg significantly reducing renal dysfunction, tubular injury, complement activation, endothelial damage, and leukocyte infiltration. In contrast, treatment with sHuCR1 at a molar equivalent dose to 60 mg/kg CSL040 did not confer significant protection. Our results identify CSL040 as a promising therapeutic candidate to attenuate renal IRI and demonstrate its superior efficacy over full-length sHuCR1 in vivo.

Deficiency in the membrane-bound complement regulators CD55 and CD59 exacerbates renal ischemia-reperfusion injury (IRI) in mouse models, but the effect of increasing CD55 and CD59 activity has not been examined. In this study, we investigated the impact of overexpression of human (h) CD55 ± hCD59 or treatment with soluble rhCD55 in a mouse model of renal IRI. Unilaterally nephrectomised mice were subjected to 18 (mild IRI) or 22 min (moderate IRI) warm renal ischemia, and analyzed 24 h after reperfusion for renal function (serum creatinine and urea), complement deposition (C3b/c and C9), and infiltration of neutrophils and macrophages. Transgenic mice expressing hCD55 alone were protected against mild renal IRI, with reduced creatinine and urea levels compared with wild type littermates. However, the renal function of the hCD55 mice was not preserved in the moderate IRI model, despite a reduction in C3b/c and C9 deposition and innate cell infiltration. Mice expressing both hCD55 and hCD59, on the other hand, were protected in the moderate IRI model, with significant reductions in all parameters measured. Wild type mice treated with rhCD55 immediately after reperfusion were also protected in the moderate IRI model. Thus, manipulation of CD55 activity to increase inhibition of the C3 and C5 convertases is protective against renal IRI, and the additional expression of hCD59, which regulates the terminal complement pathway, provides further protection. Therefore, anti-complement therapy using complement regulatory proteins may provide a potential clinical option for preventing tissue and organ damage in renal IRI.

Background Complement activation plays an important role in the pathogenesis of renal ischemia-reperfusion (IR) injury (IRI), but whether this involves damage to the vasculoprotective endothelial glycocalyx is not clear. We investigated the impact of complement activation on glycocalyx integrity and renal dysfunction in a mouse model of renal IRI. Methods Right nephrectomized male C57BL/6 mice were subjected to 22 minutes left renal ischemia and sacrificed 24 hours after reperfusion to analyze renal function, complement activation, glycocalyx damage, endothelial cell activation, inflammation, and infiltration of neutrophils and macrophages. Results Ischemia-reperfusion induced severe renal injury, manifested by significantly increased serum creatinine and urea, complement activation and deposition, loss of glycocalyx, endothelial activation, inflammation, and innate cell infiltration. Treatment with the anti-C5 antibody BB5.1 protected against IRI as indicated by significantly lower serum creatinine (P = 0.04) and urea (P = 0.003), tissue C3b/c and C9 deposition (both P = 0.004), plasma C3b (P = 0.001) and C5a (P = 0.006), endothelial vascular cell adhesion molecule-1 expression (P = 0.003), glycocalyx shedding (tissue heparan sulfate [P = 0.001], plasma syndecan-1 [P = 0.007], and hyaluronan [P = 0.02]), inflammation (high mobility group box-1 [P = 0.0003]), and tissue neutrophil (P = 0.0009) and macrophage (P = 0.004) infiltration. Conclusions Together, our data confirm that the terminal pathway of complement activation plays a key role in renal IRI and demonstrate that the mechanism of injury involves shedding of the glycocalyx.

Purpose: The novel coronavirus SARS-CoV-2 (COVID-19) and the resultant nationwide lockdown and travel restrictions led to difficulty in providing timely and regular treatment to patients with childhood cancers such as retinoblastoma. This study is aimed at assessing the demography, clinical presentation, treatment strategies, and outcome of treatment defaulters due to the lockdown. Methods: Cross-sectional, observational study of retinoblastoma patients at a tertiary care ocular oncology center during the first wave of COVID-19 and the resulting nationwide lockdown. Results: Of the 476 eyes of 326 patients undergoing active management with a median age of 57 months (range: 4–214 months), 205 (63%) patients returned for follow-up after a mean delay of 45.8 ± 24.3 weeks (range: 8–80 weeks) and 121 (37%) were defaulters according to the data analyzed till June 30, 2021. Distance of residence was ≥1000 km for 148 patients (46%). In terms of need for active treatment, the number of emergent cases was 2 (

Purpose Contemporary business is rather global. Food imports and exports are expanding beyond borders to meet increasing domestic and international food and consumer demands. Genetically modified (GM) food emerged as a potential sustainable solution which aims at meeting consumer demands and to mitigate urgent global food security problem. Because of its nature of existence, GM food is a controversial topic in several countries and has varied acceptance rates by consumers. Both government and consumers are antagonistic towards GM foods in most European countries. In contrast, most Asian consumers are neutral and so do not oppose GM foods. The purpose of this viewpoint paper is to examine the factors determining the difference in acceptance of GM foods between cultures. Such information can facilitate policy implications for governments in global agri-food trade and for producers in segregating markets. Design/methodology/approach Extensive literature review is done to base the discussions of this viewpoint paper. Findings This paper presents five factors that describe the difference in acceptance of GM foods between Europeans and Asians: knowledge and trust over the institutions performing research, uncertainty avoidance and health, gender differences, risk perception and material benefits and food for survival. Originality/value There is no systematic study that compares factors determining acceptance of GM foods across cultures.

BACKGROUND Transgenic expression of human thrombomodulin (hTBM), which has the potential to solve the problem of coagulation dysregulation in pig-to-primate xenotransplantation, may have additional benefits by neutralizing the proinflammatory cytokine high-mobility group box 1 (HMGB1). The aim of this study was to investigate HMGB1-mediated effects on porcine aortic endothelial cells (PAEC) from wild-type (WT) and hTBM transgenic pigs. METHODS Porcine aortic endothelial cells were treated with HMGB1, human (h)TNFα or lipopolysaccharide (LPS). Procoagulant and proinflammatory responses were assessed by measuring expression of cell surface markers (adhesion molecules, fibrinogen-like protein 2, plasminogen activator inhibitor (PAI)-1), secretion of porcine cytokines and chemokines (HMGB1, TNFα, IL-8, monocyte chemotactic protein-1), and formation of PAI-1/tissue plasminogen activator complexes. Thrombin-mediated degradation of HMGB1 in the presence of PAEC was examined by Western blot and functional assay. RESULTS High-mobility group box 1 potently activated WT PAEC, increasing the expression of E-selectin, vascular cell adhesion molecule-1, intercellular adhesion molecule-1, fibrinogen-like protein 2, and PAI-1, the secretion of TNFα, IL-8, and monocyte chemotactic protein-1 and the formation of PAI-1/tissue plasminogen activator complexes. Human TNFα- or LPS-induced activation of WT PAEC was inhibited by treatment with rabbit anti-HMGB1 antibody. Transgenic expression of hTBM significantly reduced the activation of PAEC by HMGB1 or hTNFα, and significantly enhanced thrombin-induced HMGB1 cleavage. Chemically induced shedding of the lectin-like domain of TBM resulted in significantly increased HMGB1-induced PAEC activation. CONCLUSIONS High-mobility group box 1 exerts powerful proinflammatory and procoagulant effects on WT PAEC, and appears to be an important downstream mediator for the actions of hTNFα and LPS. Human thrombomodulin transgenic PAECs are less sensitive to activation by either HMGB1 or hTNFα, an effect that appears to be dependent on the lectin-like domain of TBM.

Background: In pig-to-human xenotransplantation, interactions between human natural killer (NK) cells and porcine endothelial cells (pEC) are characterized by recruitment and cytotoxicity. Protection from xenogeneic NK cytotoxicity can be achieved in vitro by the expression of the non-classical human leukocyte antigen-E (HLA-E) on pEC. Thus, the aim of this study was to analyze NK cell responses to vascularized xenografts using an ex vivo perfusion system of pig limbs with human blood. Methods: Six pig forelimbs per group, respectively, stemming from either wild-type (wt) or HLA-E/hCD46 double-transgenic (tg) animals, were perfused ex vivo with heparinized human blood for 12hours. Blood samples were collected at defined time intervals, cell numbers counted, and peripheral blood mononuclear cells analyzed for phenotype by flow cytometry. Muscle biopsies were analyzed for NK cell infiltration. In vitro NK cytotoxicity assays were performed using pEC derived from wt and tg animals as target cells. Results: Ex vivo, a strong reduction in circulating human CD45 leukocytes was observed after 60minutes of xenoperfusion in both wt and tg limb groups. NK cell numbers dropped significantly. Within the first 10minutes, the decrease in NK cells was more significant in the wt limb perfusions as compared to tg limbs. Immunohistology of biopsies taken after 12hours showed less NK cell tissue infiltration in the tg limbs. In vitro, NK cytotoxicity against hCD46 single tg pEC and wt pEC was similar, while lysis of double tg HLA-E/hCD46 pEC was significantly reduced. Finally, circulating cells of pig origin were observed during the ex vivo xenoperfusions. These cells expressed phenotypes mainly of monocytes, B and T lymphocytes, NK cells, as well as some activated endothelial cells. Conclusion: sEx vivo perfusion of pig forelimbs using whole human blood represents a powerful tool to study humoral and early cell-mediated rejection mechanisms of vascularized pig-to-human xenotransplantation, although there are several limitations of the model. Here, we show that (i) transgenic expression of HLA-E/hCD46 in pig limbs provides partial protection from human NK cell-mediated xeno responses and (ii) the emergence of a pig cell population during xenoperfusions with implications for the immunogenicity of xenografts.

Diastereomeric norbornapeptides represent globular scaffolds with geometries determined by the chirality of amino acid residues and sharing structural features of β-turns and α-helices., Double cyclization of short linear peptides obtained by solid phase peptide synthesis was used to prepare bridged bicyclic peptides (BBPs) corresponding to the topology of bridged bicyclic alkanes such as norbornane. Diastereomeric norbornapeptides were investigated by 1H-NMR, X-ray crystallography and CD spectroscopy and found to represent rigid globular scaffolds stabilized by intramolecular backbone hydrogen bonds with scaffold geometries determined by the chirality of amino acid residues and sharing structural features of β-turns and α-helices. Proteome profiling by capture compound mass spectrometry (CCMS) led to the discovery of the norbornapeptide 27c binding selectively to calmodulin as an example of a BBP protein binder. This and other BBPs showed high stability towards proteolytic degradation in serum.

BACKGROUND:Asialoglycoprotein receptor 1 (ASGR1) mediates capture and phagocytosis of platelets in pig to primate liver xenotransplantation. However thrombocytopenia is also observed in xenotransplantation or xenoperfusion of other porcine organs than liver. We therefore assessed ASGR1 expression as well as ASGR1 mediated xenogeneic platelet phagocytosis in vitro and ex vivo on porcine aortic femoral arterial and liver sinusoidal endothelial cells (PAEC/PFAEC/PLSEC). METHODS:Porcine forelimbs were perfused with whole heparinized human or autologous pig blood. Platelets were counted at regular intervals. Pig limb muscle and liver as well as PAEC/PFAEC/PLSEC were characterized for ASGR1 expression. In vitro PAEC cultured on microcarrier beads and incubated with non anticoagulated human blood were used to study binding of human platelets and platelet white blood cell aggregation. Carboxyfluorescein diacetate succinimidyl ester labeled human platelets were exposed to PAEC/PFAEC/PLSEC and analyzed for ASGR1 mediated phagocytosis. RESULTS:Human platelet numbers decreased from 102 {\textpm} 33 at beginning to 13 {\textpm} 6 {\texttimes} 10/µL (P < 0.0001) after 10 minutes of perfusion whereas no significant decrease of platelets was seen during autologous perfusions (171 {\textpm} 26 to 122 {\textpm} 95 {\texttimes} 10/µL). The PAEC PFAEC and PLSEC all showed similar ASGR1 expression. In vitro no correlation was found between reduction in platelet count and platelet white blood cell aggregation. Phagocytosis of human carboxyfluorescein diacetate succinimidyl ester labeled platelets by PAEC/PFAEC/PLSEC peaked at 15 minutes and was inhibited (P < 0.05 to P < 0.0001) by rabbit anti ASGR1 antibody and asialofetuin. CONCLUSIONS:The ASGR1 expressed on aortic and limb arterial pig vascular endothelium plays a role in binding and phagocytosis of human platelets. Therefore ASGR1 may represent a novel therapeutic target to overcome thrombocytopenia associated with vascularized pig to primate xenotransplantation.

Purpose – Domestic preparation practices influence the sensory properties and nutritional composition of food products. Information on the variability in actual domestic preparation practices is needed to assess the influence of applied conditions on the sensory and nutritional quality of food. The collection of such information requires a reliable, valid and practical research method. The paper aims to discuss these issues. Design/methodology/approach – Direct in-home observations, observations in a model-kitchen using cameras, and a self-reporting questionnaire were evaluated for reliability and validity, to study domestic food preparation practices by consumers. Broccoli preparation practices by Dutch consumers were checked by these three methods in this research paper. Findings – All three research methods were found to be test-retest, inter-observer, parallel-form reliable; and face, content and concurrent valid. However, the self-reporting questionnaire is the most practical research method that can be administered on a large number of respondents in a short time to capture the wide variations in preparation practices. Originality/value – Consumers can be assisted on domestic food preparation practices that reach their sensory preferences (e.g. texture, colour) as well as have health benefits on consumption.

Acute vascular rejection (AVR), in particular microvascular thrombosis, is an important barrier to successful pig-to-primate xenotransplantation. Here, we report the generation of pigs with decreased tissue factor (TF) levels induced by small interfering (si)RNA-mediated gene silencing. Porcine fibroblasts were transfected with TF-targeting small hairpin (sh)RNA and used for somatic cell nuclear transfer. Offspring were analyzed for siRNA, TF mRNA and TF protein level. Functionality of TF downregulation was investigated by a whole blood clotting test and a flow chamber assay. TF siRNA was expressed in all twelve liveborn piglets. TF mRNA expression was reduced by 94.1 ± 4.7% in TF knockdown (TFkd) fibroblasts compared to wild-type (WT). TF protein expression in PAEC stimulated with 50 ng/mL TNF-α was significantly lower in TFkd pigs (mean fluorescence intensity TFkd: 7136 ± 136 vs. WT: 13 038 ± 1672). TF downregulation significantly increased clotting time (TFkd: 73.3 ± 8.8 min, WT: 45.8 ± 7.7 min, p

In vivo, endothelial cells are crucial for the natural anticoagulation of circulating blood. Consequently, endothelial cell activation leads to blood coagulation. This phenomenon is observed in many clinical situations, like organ transplantation in the presence of pre-formed anti-donor antibodies, including xenotransplantation, as well as in ischemia/reperfusion injury. In order to reduce animal experimentation according to the 3R standards (reduction, replacement and refinement), in vitro models to study the effect of endothelial cell activation on blood coagulation would be highly desirable. However, common flatbed systems of endothelial cell culture provide a surface-to-volume ratio of 1 - 5 cm(2) of endothelium per mL of blood, which is not sufficient for natural, endothelial-mediated anticoagulation. Culturing endothelial cells on microcarrier beads may increase the surface-to-volume ratio to 40 - 160 cm(2)/mL. This increased ratio is sufficient to ensure the "natural" anticoagulation of whole blood, so that the use of anticoagulants can be avoided. Here an in vitro microcarrier-based system is described to study the effects of genetic modification of porcine endothelial cells on coagulation of whole, non-anticoagulated human blood. In the described assay, primary porcine aortic endothelial cells, either wild type (WT) or transgenic for human CD46 and thrombomodulin, were grown on microcarrier beads and then exposed to freshly drawn non-anticoagulated human blood. This model allows for the measurement and quantification of cytokine release as well as activation markers of complement and coagulation in the blood plasma. In addition, imaging of activated endothelial cell and deposition of immunoglobulins, complement- and coagulation proteins on the endothelialized beads were performed by confocal microscopy. This assay can also be used to test drugs which are supposed to prevent endothelial cell activation and, thus, coagulation. On top of its potential to reduce the number of animals used for such investigations, the described assay is easy to perform and consistently reproducible.