Commercial amniotic membrane extract for treatment of corneal ulcers in adult horses.

BACKGROUND: Amniotic membrane extract enhances the rate of epithelialisation after corneal ulceration in several species but has not been studied in the equine cornea. OBJECTIVES: To evaluate the effect of amniotic membrane extract on re-epithelialisation of equine corneal ulcers compared with ulcers treated with antibiotic, antifungal and mydriatic medical therapy alone, and to evaluate equine corneal healing after experimentally induced superficial ulceration. STUDY DESIGN: Masked, randomised, controlled experimental trial. METHODS: Superficial, 8 mm corneal ulcers were created bilaterally in each horse. One eye was treated with amniotic membrane extract and the opposite was control. Both eyes were treated with medical therapy. Treatment eyes received amniotic membrane extract, and control eyes received the amniotic membrane extract vehicle. Ulcers were stained with fluorescein and photographed in 12-hour increments until completely healed. Ulcer surface area was determined by analysing photographs with ImageJ. A mixed linear model was used to compare ulcer surface area and hours until healing between treatment groups. A regression model was also used to calculate corneal re-epithelialisation rate over time. RESULTS: Regardless of therapy, healing occurred in two phases: an initial rapid phase of 0.88 mm2 /hr (95% CI: 0.81-0.94 mm2 /hr) for approximately 48-54 hours followed by a second, slow phase of 0.07 mm2 /hr (95% CI: 0.04-0.09 mm2 /hr). Most eyes healed within 135.5 ± 48.5 hours. Treatment (amniotic membrane extract vs. control) was not significantly associated with size of ulcers over time (P = .984). Discomfort was minimal to absent in all horses. MAIN LIMITATIONS: Results achieved experimental studies may differ from outcomes in the clinical setting. CONCLUSIONS: There was no significant difference in healing rate with addition of amniotic membrane extract to medical therapy for equine superficial corneal ulcers. A biphasic corneal healing process was observed, with an initial rapid phase followed by a slow phase. Further study will be needed to determine whether amniotic membrane extract will be helpful for infected or malacic equine corneal ulcers.

The role and response of primary healthcare services in the delivery of palliative care in epidemics and pandemics: A rapid review to inform practice and service delivery during the COVID-19 pandemic.

BACKGROUND: The increased number of deaths in the community happening as a result of COVID-19 has caused primary healthcare services to change their traditional service delivery in a short timeframe. Services are quickly adapting to new challenges in the practical delivery of end-of-life care to patients in the community including through virtual consultations and in the provision of timely symptom control. AIM: To synthesise existing evidence related to the delivery of palliative and end-of-life care by primary healthcare professionals in epidemics and pandemics. DESIGN: Rapid systematic review using modified systematic review methods, with narrative synthesis of the evidence. DATA SOURCES: Searches were carried out in Medline, Embase, PsychINFO, CINAHL and Web of Science on 7th March 2020. RESULTS: Only five studies met the inclusion criteria, highlighting a striking lack of evidence base for the response of primary healthcare services in palliative care during epidemics and pandemics. All were observational studies. Findings were synthesised using a pandemic response framework according to 'systems' (community providers feeling disadvantaged in terms of receiving timely information and protocols), 'space' (recognised need for more care in the community), 'staff' (training needs and resilience) and 'stuff' (other aspects of managing care in pandemics including personal protective equipment, cleaning care settings and access to investigations). CONCLUSIONS: As the COVID-19 pandemic progresses, there is an urgent need for research to provide increased understanding of the role of primary care and community nursing services in palliative care, alongside hospices and community specialist palliative care providers.

Optimization Techniques for miRNA Expression in Low Frequency Immune Cell Populations.

In this chapter we outline a RNA extraction method for very low immune cell populations isolated from the central nervous system of mice undergoing experimental autoimmune encephalomyelitis. We compare various normalization and quantification techniques to examine miRNA expression. Our data highlight that employing a mean normalization procedure using a number of well-selected housekeeping miRNA genes, followed by absolute quantification with a standard curve generated from a commercial miRNA oligo, gave the most robust and reproducible miRNA expression results.

Simple Methods to Investigate MicroRNA Induction in Response to Toll-Like Receptors.

In this chapter, we describe simple methods to investigate microRNA (miRNA) induction in response to lipopolysaccharide, the ligand for Toll-Like Receptor-4 activation. In brief, we demonstrate how to investigate global miRNA induction and/or repression in bone marrow-derived macrophages using TaqMan MicroRNA Arrays, followed by methods to measure individual miRNAs and target mRNA expression. Moreover, we explain step-by-step instructions on how to modulate endogenous miRNA expression through the use of miRNA inhibitors and mimics as well as highlight how miRNA modulation can be used to confirm mRNA targeting via Luciferase reporter assay. Moreover, these methods can be applied to whichever cell type and cellular function under investigation.

Human invariant NKT cell subsets differentially promote differentiation, antibody production, and T cell stimulation by B cells in vitro.

Invariant NK T (iNKT) cells can provide help for B cell activation and Ab production. Because B cells are also capable of cytokine production, Ag presentation, and T cell activation, we hypothesized that iNKT cells will also influence these activities. Furthermore, subsets of iNKT cells based on CD4 and CD8 expression that have distinct functional activities may differentially affect B cell functions. We investigated the effects of coculturing expanded human CD4(+), CD8α(+), and CD4(-)CD8α(-) double-negative (DN) iNKT cells with autologous peripheral B cells in vitro. All iNKT cell subsets induced IgM, IgA, and IgG release by B cells without needing the iNKT cell agonist ligand α-galactosylceramide. Additionally, CD4(+) iNKT cells induced expansions of cells with phenotypes of regulatory B cells. When cocultured with α-galactosylceramide-pulsed B cells, CD4(+) and DN iNKT cells secreted Th1 and Th2 cytokines but at 10-1000-fold lower levels than when cultured with dendritic cells. CD4(+) iNKT cells reciprocally induced IL-4 and IL-10 production by B cells. DN iNKT cells expressed the cytotoxic degranulation marker CD107a upon exposure to B cells. Remarkably, whereas iNKT cell subsets could induce CD40 and CD86 expression by B cells, iNKT cell-matured B cells were unable to drive proliferation of autologous and alloreactive conventional T cells, as seen with B cells cultured in the absence of iNKT cells. Therefore, human CD4(+), CD8α(+), and DN iNKT cells can differentially promote and regulate the induction of Ab and T cell responses by B cells.

Different reactive oxygen species lead to distinct changes of cellular metal ions in the eukaryotic model organism Saccharomyces cerevisiae.

Elemental uptake and export of the cell are tightly regulated thereby maintaining the ionomic homeostasis. This equilibrium can be disrupted upon exposure to exogenous reactive oxygen species (ROS), leading to reduction or elevation of the intracellular metal ions. In this study, the ionomic composition in the eukaryotic model organism Saccharomyces cerevisiae was profiled using the inductively-coupled plasma optical emission spectrometer (ICP-OES) following the treatment with individual ROS, including hydrogen peroxide, cumen hydroperoxide, linoleic acid hydroperoxide (LAH), the superoxide-generating agent menadione, the thiol-oxidising agent diamide [diazine-dicarboxylic acid-bis(dimethylamide)], dimedone and peroxynitrite. The findings demonstrated that different ROS resulted in distinct changes in cellular metal ions. Aluminium (Al(3+)) level rose up to 50-fold after the diamide treatment. Cellular potassium (K(+)) in LAH-treated cells was 26-fold less compared to the non-treated controls. The diamide-induced Al(3+) accumulation was further validated by the enhanced Al(3+) uptake along the time course and diamide doses. Pre-incubation of yeast with individual elements including iron, copper, manganese and magnesium failed to block diamide-induced Al(3+) uptake, suggesting Al(3+)-specific transporters could be involved in Al(3+) uptake. Furthermore, LAH-induced potassium depletion was validated by a rescue experiment in which addition of potassium increased yeast growth in LAH-containing media by 26% compared to LAH alone. Taken together, the data, for the first time, demonstrated the linkage between ionomic profiles and individual oxidative conditions.

An antioxidant screening assay based on oxidant-induced growth arrest in Saccharomyces cerevisiae.

This report describes a biological screening system to measure the antioxidant capacity of compounds using the oxidant-induced growth arrest response of Saccharomyces cerevisiae. Alternative methods using the nonphysiological free radical compounds such as diphenylpicrylhydrazyl and azinobis ethylbenzothiaziline-6-sulphonate (ABTS) only provide an indication of the ability of a compound to scavenge oxidants. In contrast, this yeast-based method can also measure the ability of a compound to induce cellular resistance to the damaging effects of oxidants. The screening assay was established against a panel of six physiologically relevant oxidants ranging from reactive oxygen species (hydrogen peroxide, cumene peroxide, linoleic acid hydroperoxide), to a superoxide-generating agent (menadione), reactive nitrogen species (peroxynitrite) and a thiol-oxidizing agent (diamide). The antioxidants ascorbate and gallic acid displayed scavenging activity and induced the resistance of cells against a broad range of oxidants using this assay. Lipoic acid, which showed no scavenging activity and thus would not be detected as an antioxidant using a nonphysiological screen was, however, identified in this assay as providing resistance to cells against a range of oxidants. This assay is high throughput, in the format of a 96-well microtitre plate, and will greatly facilitate the search for effective antioxidants.

Zinc starvation induces a stress response in Saccharomyces cerevisiae that is mediated by the Msn2p and Msn4p transcriptional activators.

During the production of wine and beer, the yeast Saccharomyces cerevisiae can encounter an environment that is deficient in zinc, resulting in a 'sluggish' or a 'stuck' ferment. It has been shown that the Zap1p-transcription factor induces the expression of a regulon in response to zinc deficiency; however, it was evident that a separate regulon was also activated during zinc deficiency in a Zap1p-independent manner. This study discovered the Msn2p and Msn4p (Msn2/4p) transcriptional activator proteins to be an additional control mechanism inducing the stress response during zinc deficiency. Promoter sequence analysis identified the stress-response element (STRE) motif, recognized by Msn2/4p, and was significantly enriched in the promoters of genes induced by zinc deficiency. An investigation using genome-wide analyses revealed a distinct regulon consisting of STRE-containing genes whose zinc-responsive expression was abolished in an msn2 msn4 double mutant. An STRE-driven lacZ reporter construct confirmed that expression of the genes within this regulon was perturbed by the deletion of MSN2 and MSN4 and also implicated Hog1p as a contributing factor. This research provides a better understanding of the molecular mechanisms involved in the yeast response to zinc deficiency during fermentation.