Development of PainFace software to simplify, standardize, and scale up mouse grimace analyses.

ABSTRACT: Facial grimacing is used to quantify spontaneous pain in mice and other mammals, but scoring relies on humans with different levels of proficiency. Here, we developed a cloud-based software platform called PainFace (http://painface.net) that uses machine learning to detect 4 facial action units of the mouse grimace scale (orbitals, nose, ears, whiskers) and score facial grimaces of black-coated C57BL/6 male and female mice on a 0 to 8 scale. Platform accuracy was validated in 2 different laboratories, with 3 conditions that evoke grimacing-laparotomy surgery, bilateral hindpaw injection of carrageenan, and intraplantar injection of formalin. PainFace can generate up to 1 grimace score per second from a standard 30 frames/s video, making it possible to quantify facial grimacing over time, and operates at a speed that scales with computing power. By analyzing the frequency distribution of grimace scores, we found that mice spent 7x more time in a "high grimace" state following laparotomy surgery relative to sham surgery controls. Our study shows that PainFace reproducibly quantifies facial grimaces indicative of nonevoked spontaneous pain and enables laboratories to standardize and scale-up facial grimace analyses.

Interleukin-13: A pivotal target against influenza-induced exacerbation of chronic lung diseases.

Non-communicable, chronic respiratory diseases (CRDs) affect millions of individuals worldwide. The course of these CRDs (asthma, chronic obstructive pulmonary disease, and cystic fibrosis) are often punctuated by microbial infections that may result in hospitalization and are associated with increased risk of morbidity and mortality, as well as reduced quality of life. Interleukin-13 (IL-13) is a key protein that regulates airway inflammation and mucus hypersecretion. There has been much interest in IL-13 from the last two decades. This cytokine is believed to play a decisive role in the exacerbation of inflammation during the course of viral infections, especially, in those with pre-existing CRDs. Here, we discuss the common viral infections in CRDs, as well as the potential role that IL-13 plays in the virus-induced disease pathogenesis of CRDs. We also discuss, in detail, the immune-modulation potential of IL-13 that could be translated to in-depth studies to develop IL-13-based therapeutic entities.

Smoking and COVID-19: What we know so far.

The ongoing COVID-19 pandemic has placed a spotlight on infectious diseases and their associations with host factors and underlying conditions. New data on the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) virus are entering the public domain at a rapid rate such that their distillation often lags behind. To minimise weak associations becoming perceived as established paradigms, it is imperative that methodologies and outputs from different studies are appropriately critiqued and compared. In this review, we examine recent data on a potential relationship between smoking and COVID-19. While the causal role of smoking has been firmly demonstrated in regard to lung cancer and chronic obstructive pulmonary disease, such associations have the benefit of decades' worth of multi-centre epidemiological and mechanistic data. From our analysis of the available studies to date, it appears that a relationship is emerging in regard to patients with a smoking history having a higher likelihood of developing more severe symptoms of COVID-19 disease than non-smokers. Data on whether COVID-19 has a greater incidence in smokers than non-smokers is thus far, contradictory and inconclusive. There is therefore a need for some caution to be exercised until further research has been conducted in a wider range of geographical settings with sufficient numbers of patients that have been carefully phenotyped in respect of smoking status and adequate statistical control for confounding factors.

Physicochemical stability of voriconazole in elastomeric devices.

OBJECTIVES: Voriconazole is the drug of choice for invasive aspergillosis (IA), a leading cause of mortality and morbidity in immunocompromised patients. Prolong intravenous administration of voriconazole is often needed in such patients due to high incidence of oral mucositis and unreliable bioavailability of oral dosage form. Administration of voriconazole through elastomeric pump may facilitate early hospital discharge of clinically stable immunocompromised patients needing prolonged intravenous treatment. Therefore, we investigated the physicochemical stability of voriconazole in one of the commonly used elastomeric pumps at three different temperatures for various time points. METHODS: A total of 18 elastomeric pumps were prepared and 6 containing 2 mg/mL of voriconazole (3 in 0.9% sodium chloride and 3 in 5% glucose) were stored at either 4°C for 96 hours, 25°C for 4 hours or at 35°C for 4 hours. An aliquot withdrawn immediately before storage (time 0) and at various time points was analysed for chemical stability using high-performance liquid chromatography and for physical stability using visual, pH and microscopic analyses. RESULTS: Voriconazole was stable for at least 96 hours, 4 hours and 4 hours at 4°C, 25°C and 35°C, respectively, when admixed with either 0.9% sodium chloride or 5% glucose. No evidence of particle formation, colour change or pH change was observed throughout the study period. CONCLUSIONS: These findings would allow early hospital discharge using elastomeric intravenous administration of voriconazole in patients in whom oral route of administration is not available.

Orally Administered Enoxaparin Ameliorates Acute Colitis by Reducing Macrophage-Associated Inflammatory Responses.

Inflammatory bowel diseases, such as ulcerative colitis, cause significant morbidity and decreased quality of life. The currently available treatments are not effective in all patients, can be expensive and have potential to cause severe side effects. This prompts the need for new treatment modalities. Enoxaparin, a widely used antithrombotic agent, is reported to possess anti-inflammatory properties and therefore we evaluated its therapeutic potential in a mouse model of colitis. Acute colitis was induced in male C57BL/6 mice by administration of dextran sulfate sodium (DSS). Mice were treated once daily with enoxaparin via oral or intraperitoneal administration and monitored for colitis activities. On termination (day 8), colons were collected for macroscopic evaluation and cytokine measurement, and processed for histology and immunohistochemistry. Oral but not intraperitoneal administration of enoxaparin significantly ameliorated DSS-induced colitis. Oral enoxaparin-treated mice retained their body weight and displayed less diarrhea and fecal blood loss compared to the untreated colitis group. Colon weight in enoxaparin-treated mice was significantly lower, indicating reduced inflammation and edema. Histological examination of untreated colitis mice showed a massive loss of crypt architecture and goblet cells, infiltration of immune cells and the presence of edema, while all aspects of this pathology were alleviated by oral enoxaparin. Reduced number of macrophages in the colon of oral enoxaparin-treated mice was accompanied by decreased levels of pro-inflammatory cytokines. Oral enoxaparin significantly reduces the inflammatory pathology associated with DSS-induced colitis in mice and could therefore represent a novel therapeutic option for the management of ulcerative colitis.

Fucoidan Extracts Ameliorate Acute Colitis.

Inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease, are an important cause of morbidity and impact significantly on quality of life. Overall, current treatments do not sustain a long-term clinical remission and are associated with adverse effects, which highlight the need for new treatment options. Fucoidans are complex sulphated, fucose-rich polysaccharides, found in edible brown algae and are described as having multiple bioactivities including potent anti-inflammatory effects. Therefore, the therapeutic potential of two different fucoidan preparations, fucoidan-polyphenol complex (Maritech Synergy) and depyrogenated fucoidan (DPF) was evaluated in the dextran sulphate sodium (DSS) mouse model of acute colitis. Mice were treated once daily over 7 days with fucoidans via oral (Synergy or DPF) or intraperitoneal administration (DPF). Signs and severity of colitis were monitored daily before colons and spleens were collected for macroscopic evaluation, cytokine measurements and histology. Orally administered Synergy and DPF, but not intraperitoneal DPF treatment, significantly ameliorated symptoms of colitis based on retention of body weight, as well as reduced diarrhoea and faecal blood loss, compared to the untreated colitis group. Colon and spleen weight in mice treated with oral fucoidan was also significantly lower, indicating reduced inflammation and oedema. Histological examination of untreated colitis mice confirmed a massive loss of crypt architecture and goblet cells, infiltration of immune cells and oedema, while all aspects of this pathology were alleviated by oral fucoidan. Importantly, in this model, the macroscopic changes induced by oral fucoidan correlated significantly with substantially decreased production of at least 15 pro-inflammatory cytokines by the colon tissue. Overall, oral fucoidan preparations significantly reduce the inflammatory pathology associated with DSS-induced colitis and could therefore represent a novel nutraceutical option for the management of IBD.

Non-Anticoagulant Fractions of Enoxaparin Suppress Inflammatory Cytokine Release from Peripheral Blood Mononuclear Cells of Allergic Asthmatic Individuals.

BACKGROUND: Enoxaparin, a low-molecular-weight heparin, is known to possess anti-inflammatory properties. However, its clinical exploitation as an anti-inflammatory agent is hampered by its anticoagulant effect and the associated risk of bleeding. OBJECTIVE: The aim of the current study was to examine the ability of non-anticoagulant fractions of enoxaparin to inhibit the release of key inflammatory cytokines in primed peripheral blood mononuclear cells derived from allergic mild asthmatics. METHODS: Peripheral blood mononuclear cells from allergic asthmatics were activated with phytohaemag glutinin (PHA), concanavalin-A (ConA) or phorbol 12-myristate 13-acetate (PMA) in the presence or absence of enoxaparin fractions before cytokine levels were quantified using specific cytokine bead arrays. Together with nuclear magnetic resonance analysis,time-dependent and target-specific effects of enoxaparin fractions were used to elucidate structural determinants for their anti-inflammatory effect and gain mechanistic insights into their anti-inflammatory activity. RESULTS: Two non-anticoagulant fractions of enoxaparin were identified that significantly inhibited T-cell activation. A disaccharide fraction of enoxaparin inhibited the release of IL-4, IL-5, IL-13 and TNF-α by more than 57% while a tetrasaccharide fraction was found to inhibit the release of tested cytokines by more than 68%. Our data suggest that the observed response is likely to be due to an interaction of 6-O-sulfated tetrasaccharide with cellular receptor(s). CONCLUSION AND CLINICAL RELEVANCE: The two identified anti-inflammatory fractions lacked anticoagulant activity and are therefore not associated with risk of bleeding. The findings highlight the potential therapeutic use of enoxaparin-derived fractions, in particular tetrasaccharide, in patients with chronic inflammatory disorders.

In-vitro suppression of IL-6 and IL-8 release from human pulmonary epithelial cells by non-anticoagulant fraction of enoxaparin.

BACKGROUND: Enoxaparin, a mixture of anticoagulant and non-anticoagulant fractions, is widely used as an anticoagulant agent. However, it is also reported to possess anti-inflammatory properties. Our study indicated that enoxaparin inhibits the release of IL-6 and IL-8 from A549 pulmonary epithelial cells. Their release causes extensive lung tissue damage. The use of enoxaparin as an anti-inflammatory agent is hampered due to the risk of bleeding associated with its anticoagulant fractions. Therefore, we aimed to identify the fraction responsible for the observed anti-inflammatory effect of enoxaparin and to determine the relationship between its structure and biological activities. METHODS: A549 pulmonary epithelial cells were pre-treated in the presence of enoxaparin and its fractions. The levels of IL-6 and IL-8 released from the trypsin-stimulated cells were measured by ELISA. The anticoagulant activity of the fraction responsible for the effect of enoxaparin was determined using an anti-factor-Xa assay. The fraction was structurally characterised using nuclear magnetic resonance. The fraction was 2-O, 6-O or N-desulfated to determine the position of sulfate groups required for the inhibition of interleukins. High-performance size-exclusion chromatography was performed to rule out that the observed effect was due to the interaction between the fraction and trypsin or interleukins. RESULTS: Enoxaparin (60 µg/mL) inhibited the release of IL-6 and IL-8 by >30%. The fraction responsible for this effect of enoxaparin was found to be a disaccharide composed of α-L-iduronic-acid and α-D-glucosamine-6-sulfate. It (15 µg/mL) inhibited the release of interleukins by >70%. The 6-O sulphate groups were responsible for its anti-inflammatory effect. The fraction did not bind to trypsin or interleukins, suggesting the effect was not due to an artefact of the experimental model. CONCLUSION: The identified disaccharide has no anticoagulant activity and therefore eliminates the risk of bleeding associated with enoxaparin. Future in-vivo studies should be designed to validate findings of the current study.

Opposing effects of low molecular weight heparins on the release of inflammatory cytokines from peripheral blood mononuclear cells of asthmatics.

BACKGROUND: T-cell-mediated inflammatory cytokines, such as interleukin (IL)-4, IL-5, IL-13 and tumor necrosis factor-alpha (TNF-α), play an important role in the initiation and progression of inflammatory airways diseases. Low-molecular-weight heparins (LMWHs), widely used anticoagulants, possess anti-inflammatory properties making them potential treatment options for inflammatory diseases, including asthma. In the current study, we investigated the modulating effects of two LMWHs (enoxaparin and dalteparin) on the release of cytokines from stimulated peripheral blood mononuclear cells (PBMCs) of asthmatic subjects to identify the specific components responsible for the effects. METHODS: PBMCs from asthmatic subjects (consist of ~75% of T-cells) were isolated from blood taken from ten asthmatic subjects. The PBMCs were pre-treated in the presence or absence of different concentrations of LMWHs, and were then stimulated by phytohaemagglutinin for the release of IL-4, IL-5, IL-13 and TNF-α. LMWHs were completely or selectively desulfated and their anticoagulant effect, as well as the ability to modulate cytokine release, was determined. LMWHs were chromatographically fractionated and each fraction was tested for molecular weight determination along with an assessment of anticoagulant potency and effect on cytokine release. RESULTS: Enoxaparin inhibited cytokine release by more than 48%, whereas dalteparin increased their release by more than 25%. The observed anti-inflammatory effects of enoxaparin were independent of their anticoagulant activities. Smaller fractions, in particular dp4 (four saccharide units), were responsible for the inhibitory effect of enoxaparin. Whereas, the larger fractions, in particular dp22 (twenty two saccharide units), were associated with the stimulatory effect of dalteparin. CONCLUSION: Enoxaparin and dalteparin demonstrated opposing effects on inflammatory markers. These observed effects could be due to the presence of structurally different components in the two LMWHs arising from different methods of depolymerisation. This study provides a platform for further studies investigating the usefulness of enoxaparin in various inflammatory diseases.

Filtration of crushed tablet suspensions has potential to reduce infection incidence in people who inject drugs.

INTRODUCTION AND AIMS: The medical complications of injecting preparations from crushed tablets can be severe, and most can be attributed to the injection of insoluble particles and micro-organisms. Previously we have shown that most of the particles can be removed by filtration, but it was not known whether bacteria could also be filtered in the presence of a high particle load. This study aims to determine the feasibility of filtration to remove bacteria from injections prepared from tablets. DESIGN AND METHODS: Injections were prepared from crushed slow-release morphine tablets, in mixed bacterial suspensions of Staphylococcus aureus, Streptococcus pyogenes and Pseudomonas aeruginosa. The injection suspensions were passed through syringe filters of porosity 0.45 or 0.20 µm, or combined 0.8 then 0.2 µm, and the bacterial load was counted. RESULTS: Bacterial concentrations in unfiltered injections were 2.5-4.3 × 10(6) colony forming units mL(-1) . Both the 0.20 and 0.45 µm filters blocked unless a prefilter (cigarette filter) was used first. The 0.2 µm filter and the combined 0.8/0.2 µm filter reduced the bacteria to the limit of detection (10 colony forming units mL(-1) ) or below. Filtration through a 0.45 µm filter was slightly less effective. DISCUSSION AND CONCLUSIONS: Use of a 0.2 µm filter, together with other injection hygiene measures, offers the prospect of greatly reducing the medical complications of injecting crushed tablets and should be considered as a highly effective harm reduction method. It is very likely that these benefits would also apply to other illicit drug injections, although validation studies are needed.

Identification of pro- and anti-proliferative oligosaccharides of heparins.

Heparins, unfractionated heparin (UFH) and low molecular weight heparins (LMWHs), are heterogeneous mixtures of anticoagulant and non-anticoagulant oligosaccharides. In addition to their well-known anticoagulant effect, heparins have shown to mediate a wide range of non-anticoagulant effects, including the modulation of cellular growth. However, contradictory results have been reported with regard to their effects on cellular proliferation, with some studies suggesting anti-proliferative while others indicating pro-proliferative effects. This study investigated the proliferation of human colonic epithelial cancer cells in the presence of UFH and LMWHs (enoxaparin and dalteparin). In our experimental setting, all heparins caused a dose-dependent reduction in cellular growth, which correlated well with the induction of cell cycle arrest in the G1 phase and which was not associated with significant changes in cell viability. The effects on cellular proliferation of 14 different oligosaccharides of enoxaparin obtained through ion-exchange chromatography were also assessed. Surprisingly, only two oligosaccharides showed distinctive anti-proliferative effects while the majority of oligosaccharides actually stimulated proliferation. Interestingly, the smallest oligosaccharide devoid of any anticoagulant activity showed the strongest anti-proliferative effect. Notably, heparins are currently standardised only according to their anticoagulant activity but not based on other non-anticoagulant properties. Our results indicate that slight differences in the composition of heparins' non-anticoagulant oligosaccharides, due to different origins of material and preparation methods, have the potential to cause diverse effects and highlight the need for additional characterisation of non-anticoagulant activities.

Ion exchange chromatographic separation and isolation of oligosaccharides of intact low-molecular-weight heparin for the determination of their anticoagulant and anti-inflammatory properties.

It is well known that enoxaparin, a widely used anticoagulant and low-molecular-weight heparin containing a large number of oligosaccharides, possesses anti-inflammatory activity. Whilst enoxaparin has shown promising results in various inflammatory disorders, some of its oligosaccharides have anti-inflammatory properties and others increase the risk of bleeding due to their anticoagulant effects. The aim of this study was to develop an effective ion exchange chromatographic (IC) technique which allows the separation, isolation and, consequently, the identification of different oligosaccharides of enoxaparin with or without anticoagulant activity. The developed method utilises a semi-preparative CarboPac PA100 (9 × 250 mm) ion exchange column with sodium chloride gradient elution and UV detection at 232 nm. The method successfully resolved enoxaparin into more than 30 different peaks. IC-derived oligosaccharides with high, moderate, low or no anticoagulant activity were identified using an anti-factor Xa assay. The anti-inflammatory activity of selected oligosaccharides was investigated using the Griess assay. Using this technique, the oligosaccharides of enoxaparin with low or no anticoagulant activity, whilst exhibiting significant anti-inflammatory activity, could be fractionated. This technique can provide a platform to identify the oligosaccharides which are devoid of significant anticoagulant activity and are responsible for the therapeutic effects of enoxaparin that have been observed in various inflammatory conditions.

Effects of filtration on the presence of particulate and oxycodone content of injections prepared from crushed OxyContin® tablets.

It is common for injecting drug users (IDU) to prepare injections by crushing tablets which are not designed for parental administration. The injection of insoluble tablet excipients can lead to serious local and systemic medical complications. The aim of the study was to investigate the effectiveness of various types of filters in removing harmful insoluble particles from the injections prepared using crushed oxycodone tablets. Injections were prepared from a sustained-release oxycodone tablet formulation. The filtration of tablet extracts was carried out following procedures used by IDU using makeshift filter and commercially available filters. Particulate contamination and oxycodone content were analysed using light microscopy and spectrophotometer. Unfiltered extracts contained hundreds of thousands of particles of sufficient size to cause harms. Cigarette filters removed large particles but failed to remove small particles. The combination of cigarette filter and syringe filter (0.45 µm or 0.22 µm) reduced the particle count by 90 - 95%. A double membrane syringe filter (0.8/0.2 µm) removed more than 99% of the particles. Recovery of oxycodone was more than 95% with the tested syringe filters. Particulate contamination in injections prepared from crushed tablets can be effectively removed using a combination process of cigarette filter and syringe filters, or a 0.8/0.2 µm syringe filter. Compared to other filters, the 0.8/0.2 µm syringe filter did not block, the filtration was quick and easy to perform, and did not retain oxycodone. The use of a 0.8/0.2 µm syringe filter can provide an important harm reduction measure for IDU.