Effect of Platelet-Riched Plasma on Corneal Epithelium Regeneration during Its Chronic Erosion (Experimental Study)

Relevance. In a number of pathological conditions accompanied by defects of the basal membrane, such as chemical or thermal burn, mechanical trauma, Schegren’s syndrome, herpetic keratitis processes of corneal epithelization are slowed down, and sometimes epithelization does not occur at all. Slow epithelization or its complete absence creates conditions for infection, thinning, and sometimes perforation of the cornea. That is why the problem of chronic corneal erosions is very relevant. The most perspective method of treatment is the use of autologous platelet-riched plasma (PRP). Presence of platelet growth factors, adhesive molecules and cytokines in PRP allows to use it for acceleration of regeneration of corneal defects, and the presence of live platelets allows to refer this procedure to autologous cell transplantations.Objective: To evaluate the clinical and morphological features of corneal epithelium regeneration in response to the application of PRP in conditions of experimental chronic corneal erosion (ECCE).Materials and methods. Researches were carried out on 12 rabbits of Chinchilla breed (24 eyes). At first stage the model of ECCE was reproduced for all animals: local ultraviolet (UV) irradiation with the exposure time of 45 min. on the preliminary de-epithelized corneal surface. At the second stage all animals under study were divided into three groups (4 rabbits in each group). I-st main group (MG) was instillated with PRP at a rate of 1 drop / 1 min within 10 minutes (total of 10 drops) once, in the II MG treatment was carried out according to the above method, every day for 5 days. In the control group (CG) PRP treatment was not applied.Results. 45-min. UV irradiation causes persistent ECCE up to 30 days of the experiment and reverts only after surface vessels are ingrowed in the affected area. Single use of PRP has insufficient therapeutic effect. Instillation within 5 days accelerates the cornea reparative regeneration in ECCE conditions. The absence of epithelial defect, newly formed vessels and corneal clouding was registered on the 9th day. Histologically, on the 30th day a normal multilayer non-squamous epithelium was found, while the stroma had an organized structure with no signs of inflammation.Conclusions. Therapeutic effect of PRP in ECCE conditions is based on reforming of the normal «adhesion complex» between epithelium, olfactory membrane and multifactor stimulation of reparative regeneration due to growth factors located in it, adhesive molecules and cytokines, as a result of which the typical corneal epithelium is restored.

Neutrophil activation and circulating neutrophil extracellular traps are increased in venous thromboembolism patients for at least one year after the clinical event

Neutrophil activation and neutrophil extracellular traps (NETs) have been associated with the pathogenesis of venous thromboembolism (VTE). Considering VTE-associated chronic sequelae, which suggest that some pathological mechanisms remain after the acute episode, we investigated whether neutrophil activation is increased in patients with a prior VTE at least one year before this investigation. Thirty-seven patients with prior VTE and 37 individuals with no history of VTE were included. Neutrophil activity was evaluated by the expression of the adhesive molecule activation-specific epitopes LFA-1 (CD11a) and MAC-1 (CD11b), chemotaxis, reactive oxygen species (ROS) and by MPO-DNA complexes as markers of NETs. The adhesive molecules sICAM-1 and sVCAM-1, involved in the cross talk between neutrophil and endothelial cells, were also evaluated. Patient neutrophils presented increased CD11a expression before and after TNF-α stimulus, whereas increased CD11b expression was observed only after TNF-α stimulus, as compared to controls. Neutrophil chemotaxis on both, basal state and after IL-8 stimulus, on circulating levels of sICAM-1 and sVCAM-1, and on MPO-DNA complexes were also increased in VTE patients. ROS release was similar between patients and controls. This is, to our knowledge, the first study to investigate neutrophil inflammatory activity in VTE patients a long period after an acute event (approximately 2 years). The results showed altered neutrophil activation patterns in these patients. While activated neutrophils can cause endothelial activation and injury, the activated endothelium can induce the release of NETs with consequent endothelial cytotoxicity, creating a vicious cycle of activation between neutrophils and endothelium that can lead to thrombosis. Graphical abstract VTE patients (approximately 2 years after the clinical event) present an altered neutrophil activation state evidenced by increased activity of the LFA-1 and Mac-1 adhesive molecules, as well as increased chemotaxis and circulating levels of NETs remnants. Circulating levels of ICAM-1 and VCAM-1, which are endothelial adhesive molecules, are also increased in VTE patients, suggesting not only an exacerbated endothelial activation and dysfunction, but also an interaction of the neutrophil adhesive molecules with their endothelial ligands, favoring the migration process of neutrophil.

The zinc finger transcription factor, KLF2, protects against COVID-19 associated endothelial dysfunction

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its patho-mechanism being incompletely understood. Emerging evidence has demonstrated that endothelial dysfunction precipitates COVID-19 and its accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. The objective of the present study is to evaluate whether kruppel-like factor 2 (KLF2), a master regulator of vascular homeostasis, represents a therapeutic target for COVID-19-induced endothelial dysfunction. Here, we demonstrate that the expression of KLF2 was reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1β and TNF-α, two cytokines elevated in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Pharmacologic (atorvastatin and tannic acid) and genetic (adenoviral overexpression) approaches to augment KLF2 levels attenuated COVID-19-serum-induced increase in endothelial inflammation and monocyte adhesion. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis, and reduced angiogenesis). Finally, knockdown of KLF2 partially reversed the ameliorative effect of atorvastatin on COVID-19-serum-induced endothelial inflammation and monocyte adhesion. Collectively, the present study implicates loss of KLF2 as an important molecular event in the development of COVID-19-induced vascular disease and suggests that efforts to augment KLF2 levels may be therapeutically beneficial.

The CNS/PNS Extracellular Matrix Provides Instructive Guidance Cues to Neural Cells and Neuroregulatory Proteins in Neural Development and Repair

Background. The extracellular matrix of the PNS/CNS is unusual in that it is dominated by glycosaminoglycans, especially hyaluronan, whose space filling and hydrating properties make essential contributions to the functional properties of this tissue. Hyaluronan has a relatively simple structure but its space-filling properties ensure micro-compartments are maintained in the brain ultrastructure, ensuring ionic niches and gradients are maintained for optimal cellular function. Hyaluronan has cell-instructive, anti-inflammatory properties and forms macro-molecular aggregates with the lectican CS-proteoglycans, forming dense protective perineuronal net structures that provide neural and synaptic plasticity and support cognitive learning. Aims. To highlight the central nervous system/peripheral nervous system (CNS/PNS) and its diverse extracellular and cell-associated proteoglycans that have cell-instructive properties regulating neural repair processes and functional recovery through interactions with cell adhesive molecules, receptors and neuroregulatory proteins. Despite a general lack of stabilising fibrillar collagenous and elastic structures in the CNS/PNS, a sophisticated dynamic extracellular matrix is nevertheless important in tissue form and function. Conclusions. This review provides examples of the sophistication of the CNS/PNS extracellular matrix, showing how it maintains homeostasis and regulates neural repair and regeneration.

Bacillus subtilis revives conventional antibiotics against Staphylococcus aureus osteomyelitis

As treatment of Staphylococcus aureus (S. aureus) osteomyelitis is often hindered by the development of antibiotic tolerance, novel antibacterial therapeutics are required. Here we found that the cell-free supernatant of Bacillus subtilis (B. subtilis CFS) killed planktonic and biofilm S. aureus, and increased S. aureus susceptibility to penicillin and gentamicin as well. Further study showed that B. subtilis CFS suppressed the expression of the genes involved in adhesive molecules (Cna and ClfA), virulence factor Hla, quorum sensing (argA, argB and RNAIII) and biofilm formation (Ica and sarA) in S. aureus. Additionally, our data showed that B. subtilis CFS changed the membrane components and increased membrane permeabilization of S. aureus. Finally, we demonstrated that B. subtilis CFS increased considerably the susceptibility of S. aureus to penicillin and effectively reduced S. aureus burdens in a mouse model of implant-associated osteomyelitis. These findings support that B. subtilis CFS may be a potential resistance-modifying agent for β-lactam antibiotics against S. aureus.

Shear Rheology and Molecular Properties of Biobased Adhesives Through Molecular Dynamics Simulation

The study of shear rheology and molecular architecture through molecular dynamics simulation of starch-based and gelatin-based adhesives formulated from Eleusine coracana, and cow hide has been successfully achieved. This research has revealed that esterification of natural dextrins with 20 % polyvinyl acetate (PVAc) will yield adhesives product with improved gel properties suitable for applications. Method of extraction, production of the adhesives by varying the quantity of fatty acid ester and quality assessment was performed and 20% PVAc incorporation found to be suitable for application. The physical features of the macromolecular complexes formed by the synergistic interaction of starch/gelatin and PVAc in the presence of tetraethylamine (TEA) has been studied from the rheological point of view. On examining the impact of the molecular structure and electronic properties of the adhesive molecules on the adhesive efficiency, quantum chemical calculations were carried out. Rheological analysis shows the adhesives are consistent and the computed free energy obtained from molecular dynamics simulation reveals that the adhesive molecules are spontaneous, hence efficient.

The Immuno-Inflammatory Effect of IL-27 in Preterm Labor

Objective To investigate the effect of IL-27 on Th1 cells infiltration in human fetal membranes (FMs) and the underlying mechanisms in preterm labor. Methods The expression of IL-27 receptor a subunit (IL-27R𝛼) and Th1 cells were studied in human FMs from pregnant women with preterm labor (PL) and term labor (TL). In vitro, we investigated the effects of IL-27 in human amniotic cell lineage on the Th1 related chemokines and adhesive molecules and the underlying intracellular signaling molecules. Results The expression of IL-27R𝛼 was higher in human fetal membranes from PL group compared with TL group. Compared with TL group, the PL group had more Th1 cells infiltration in human FMs. Meanwhile, it was confirmed the expression of CXCL9, CXCL10, CXCL11 and ICAM-1 were higher in FMs from PL group to TL group. Moreover, IL-27 stimulation can up-regulate the expression of those chemokines in human amniotic cell lineage via JAK2/STAT1/STAT3 signaling pathway. Conclusions The expression of IL-27R𝛼 and number of Th1 cells infiltration were higher at FMs of PL group than TL group. IL-27 could promote Th1 related chemokines and adhesive molecules in human amniotic cell lineage mainly through JAK2/STAT1/STAT3 signaling pathway.

Nanomechanical mechanisms of Lyme disease spirochete motility enhancement in extracellular matrix

As opposed to pathogens passively circulating in the body fluids of their host, pathogenic species within the Spirochetes phylum are able to actively coordinate their movement in the host to cause systemic infections. Based on the unique morphology and high motility of spirochetes, we hypothesized that their surface adhesive molecules might be suitably adapted to aid in their dissemination strategies. Designing a system that mimics natural environmental signals, which many spirochetes face during their infectious cycle, we observed that a subset of their surface proteins, particularly Decorin binding protein (Dbp) A/B, can strongly enhance the motility of spirochetes in the extracellular matrix of the host. Using single-molecule force spectroscopy, we disentangled the mechanistic details of DbpA/B and decorin/laminin interactions. Our results show that spirochetes are able to leverage a wide variety of adhesion strategies through force-tuning transient molecular binding to extracellular matrix components, which concertedly enhance spirochetal dissemination through the host.

KLF2 is a therapeutic target for COVID-19 induced endothelial dysfunction

Coronavirus disease 2019 (COVID-19) is regarded as an endothelial disease (endothelialitis) with its mechanism being incompletely understood. Emerging evidence has demonstrated that the endothelium represents the Achilles' heel in COVID-19 patients and that endothelial dysfunction precipitates COVID-19 and accompanying multi-organ injuries. Thus, pharmacotherapies targeting endothelial dysfunction have potential to ameliorate COVID-19 and its cardiovascular complications. Primary human umbilical vein endothelial cells (HUVECs) and human pulmonary microvascular endothelial cells (HPMECs) were treated with serum from control subjects or COVID-19 patients. Downstream monocyte adhesion and associated gene/protein expression was evaluated in endothelial cells exposed to COVID-19 patient serum in the presence of KLF2 activator (Atorvastatin) or KLF2 overexpression by an adenoviral vector. Here, we demonstrate that the expression of KLF2 was significantly reduced and monocyte adhesion was increased in endothelial cells treated with COVID-19 patient serum due to elevated levels of pro-adhesive molecules, ICAM1 and VCAM1. IL-1β and TNF-α, two cytokines observed in cytokine release syndrome in COVID-19 patients, decreased KLF2 gene expression. Next-generation RNA-sequencing data showed that atorvastatin treatment leads to a cardiovascular protective transcriptome associated with improved endothelial function (vasodilation, anti-inflammation, antioxidant status, anti-thrombosis/-coagulation, anti-fibrosis and reduced angiogenesis). Treatment of HPMECs with atorvastatin or KLF2 adenovirus ameliorate COVID-19 serum-induced increase in endothelial inflammation and monocyte adhesion by increasing KLF2 expression. Altogether, the present study demonstrates that genetic and pharmacological activation of KLF2 represses COVID-19 associated endothelial dysfunction, heralding a potentially new direction to treat endothelialitis accompanying COVID-19.

Morphological features of vascular lesions of the microvasculature in sepsis

Introduction.Sepsis is a public health priority due to its high incidence and mortality. There are no clear criteria for assessing the vessels of the microvasculature in the pathological diagnosis of septic condition.The objective of the work was to characterize changes in the vessels of the microvasculature with different morphological variants of sepsis.Methods and materials. We analyzed 93 cases of death with a clinical diagnosis of sepsis. A study was made of pathomorphological signs of microcirculatory disorders, organ cell damage, and the presence of inflammatory infiltrates.Results. In the course of the study, it was found that the morphological manifestations of sepsis, in addition to purulent metastasis of various degrees of severity, are signs of damage to microvessels, such as the expression of adhesive molecules by the endotheliocytes, their desquamation, leukocyte stasis, marginal standing and output of single leukocytes beyond the vessels, fibrin and hyaline microthrombi as a manifestation of DIC syndrome.Conclusion. Regardless of the severity of the purulent metastatic process, septic lesions are characterized by the same changes in the microvasculature, expressed in increased expression of adhesive molecules by the endotheliocytes (ICAM-1), desquamation of endotheliocytes (51 %), marginal standing of leukocytes (38 %), the output of single leukocytes beyond the vessels (21 %), leukocyte stasis (55 %), the formation of fibrin and hyaline microtrombi (45 %), the sludge phenomenon (91 %), diapedetic hemorrhage in the tissues (72 %), which indicate a leading role in microcirculation disorders and, correspondingly, the formation of multiple organ failure during sepsis of secondary microvascular damage. With septicopyemia, bacterial dissemination leads not only to primary damage of the walls of blood vessels, but also to activation of their secondary lesions, as evidenced by the revealed relationship between an increase in the severity of purulent metastasis and an increase in the number of microtrombiin blood vessels, the frequency of detection of diapedetic hemorrhages in brain tissue, and hyaline membranes in alveoli of the lungs, leukocyte stasis in the vessels of the myocardium and kidneys, necrosis of the vascular loops of the glomeruli of the kidneys.