Controlled dual release of dihydrotestosterone and flutamide from polycaprolactone electrospun scaffolds accelerate burn wound healing.

Wound healing is a complex process involving multiple independent and overlapping sequential physiological mechanisms. In addition to cutaneous injury, a severe burn stimulates physiological derangements that induce a systemic hypermetabolic response resulting in impaired wound healing. Topical application of the anti-androgen drug, flutamide accelerates cutaneous wound healing, whereas paradoxically systemic dihydrotestosterone (DHT) improves burn wound healing. We developed and characterized a PCL scaffold that is capable of controlled release of androgen (DHT) and anti-androgen (F) individually or together. This study aims to investigate whether local modification of androgen actions has an impact on burn injury wound healing. In a full-thickness burn wound healing, mouse model, DHT/F-scaffold showed a significantly faster wound healing compared with F-scaffold or DHT-scaffold. Histology analysis confirmed that DHT/F-scaffold exhibited higher re-epithelization, cell proliferation, angiogenesis, and collagen deposition. Dual release of DHT and F from PCL scaffolds promoted cell proliferation of human keratinocytes and alters the keratinocyte cell cycle. Lastly, no adverse effects on androgen-dependent organs, spleen and liver were observed. In conclusion, we demonstrated DHT plus F load PCL scaffolds accelerated burn wound healing when loading alone did not. These findings point to a complex role of androgens in burn wound healing and open novel therapeutic avenues for treating severe burn patients.

Prescribing of low-dose rivaroxaban in patients with atherosclerotic cardiovascular disease in the United Kingdom and the Netherlands.

AIMS: Low-dose rivaroxaban has been indicated for the management of atherosclerotic cardiovascular disease (ASCVD) after recent (2019-2020) updates to European guidelines. We aimed to describe prescription trends of low-dose rivaroxaban in ASCVD patients over the period 2015-2022 in two European countries, to compare the trends before and after guideline changes, and to determine the characteristics of users. METHODS: In a cross-sectional interrupted time series analysis, utilization of low-dose rivaroxaban (2.5 mg, twice daily) was measured in Clinical Practice Research Datalink Aurum (United Kingdom [UK]) and the PHARMO Database Network (the Netherlands) from 1 January 2015 to 28 February 2022 in patients with an ASCVD diagnosis. Incidence rates (IRs) and incidence rate ratios (IRRs) of new use (within 182 days) compared to the reference period, 2015-2018, were calculated. Age, sex and comorbidities of users were compared to those of nonusers. RESULTS: In the UK, from 721 271 eligible subjects the IR of new use of low-dose rivaroxaban in the period 2015-2018, before guideline changes, was 12.4 per 100 000 person-years and after guideline changes in 2020-2022 was 124.0 (IRR 10.0, 95% confidence interval [CI] 8.5, 11.8). In the Netherlands from 394 851 subjects, the IR in 2015-2018 was 2.4 per 100 000 person-years and in 2020 was 16.3 (IRR 6.7, 95% CI 4.0, 11.4). Users were younger (UK mean difference [MD] -6.1 years, Netherlands -2.4 years; P < .05) and more likely to be male (UK difference 11.5%, Netherlands 13.4%; P < .001) than nonusers. CONCLUSIONS: There was a statistically significant increase in the use of low-dose rivaroxaban for the management of ASCVD after guideline changes in the UK and the Netherlands. There were international differences, but low-dose rivaroxaban has not been put into widespread practice.

Investigation of Plasma-Derived Lipidome Profiles in Experimental Cerebral Malaria in a Mouse Model Study.

Cerebral malaria (CM), a fatal complication of Plasmodium infection that affects children, especially under the age of five, in sub-Saharan Africa and adults in South-East Asia, results from incompletely understood pathogenetic mechanisms. Increased release of circulating miRNA, proteins, lipids and extracellular vesicles has been found in CM patients and experimental mouse models. We compared lipid profiles derived from the plasma of CBA mice infected with Plasmodium berghei ANKA (PbA), which causes CM, to those from Plasmodium yoelii (Py), which does not. We previously showed that platelet-free plasma (18k fractions enriched from plasma) contains a high number of extracellular vesicles (EVs). Here, we found that this fraction produced at the time of CM differed dramatically from those of non-CM mice, despite identical levels of parasitaemia. Using high-resolution liquid chromatography-mass spectrometry (LCMS), we identified over 300 lipid species within 12 lipid classes. We identified 45 and 75 lipid species, mostly including glycerolipids and phospholipids, with significantly altered concentrations in PbA-infected mice compared to Py-infected and uninfected mice, respectively. Total lysophosphatidylethanolamine (LPE) levels were significantly lower in PbA infection compared to Py infection and controls. These results suggest that experimental CM could be characterised by specific changes in the lipid composition of the 18k fraction containing circulating EVs and can be considered an appropriate model to study the role of lipids in the pathophysiology of CM.

Efficient Treatment of Experimental Cerebral Malaria by an Artemisone-SMEDDS System: Impact of Application Route and Dosing Frequency.

Artemisone (ART) has been successfully tested in vitro and in animal models against several diseases. However, its poor aqueous solubility and limited chemical stability are serious challenges. We developed a self-microemulsifying drug delivery system (SMEDDS) that overcomes these limitations. Here, we demonstrate the efficacy of this formulation against experimental cerebral malaria in mice and the impact of its administration using different routes (gavage, intranasal delivery, and parenteral injections) and frequency on the efficacy of the treatment. The minimal effective daily oral dose was 20 mg/kg. We found that splitting a dose of 20 mg/kg ART given every 24 h, by administering two doses of 10 mg/kg each every 12 h, was highly effective and gave far superior results compared to 20 mg/kg once daily. We obtained the best results with nasal treatment; oral treatment was ranked second, and the least effective route of administration was intraperitoneal injection. A complete cure of experimental cerebral malaria could be achieved through choosing the optimal route of application, dose, and dosing interval. Altogether, the developed formulation combines easy manufacturing with high stability and could be a successful and very versatile carrier for the delivery of ART in the treatment of human severe malaria.

Safety of Intravenous Olanzapine Administration at a Tertiary Academic Medical Center.

BACKGROUND: Although approved by the Food and Drug Administration for intramuscular administration only, analyses have described the administration of intravenous push (IVP) olanzapine, particularly for acute agitation. The safety and efficacy of IVP olanzapine has mostly been limited to emergency department patients. OBJECTIVE: To evaluate the safety of IVP olanzapine administration in the inpatient setting. METHODS: This single-center, retrospective analysis was conducted between July 1, 2018, and December 31, 2019, at Brigham and Women's Hospital in Boston, Massachusetts. Adult patients who received at least 1 dose of IVP olanzapine were included in the analysis. Safety endpoints analyzed included the following adverse drug events (ADEs): hypotension, bradycardia, cardiac arrhythmias, extrapyramidal adverse effects, and respiratory depressive events. Data on IV site reactions, including phlebitis and infiltration, were also collected. RESULTS: A total of 1,247 IVP administrations of olanzapine were identified across 252 patients. Two or more doses were received by 159 patients (63.1%). Most administrations (66%) took place in intensive care units, with 33% administered on general medicine wards. Overall, 104 administrations (8.3%) were associated with at least 1 ADE. Hypotension and bradycardia occurred in 62 (5.2%) and 16 (1.3%) administrations, respectively. Phlebitis occurred with 8 administrations (1.4%). All other adverse events were rare (<1%). CONCLUSION AND RELEVANCE: Hypotension, the most commonly noted ADE, occurred more frequently than in previous studies. IVP olanzapine appears to be a safe route of administration in hospitalized patients, including those receiving multiple doses. Further studies are required to evaluate the effect of IV olanzapine on hemodynamics.

Safety of Intravenous Push Lacosamide Compared With Intravenous Piggyback at a Tertiary Academic Medical Center.

BACKGROUND: There are limited data regarding the incidence of adverse events associated with administering lacosamide by intravenous push (IVP) compared with IV piggyback (IVPB). OBJECTIVE: The objective of this analysis was to compare the safety profile, including cardiovascular effects, sedative effects, and IV site reactions of IVP and IVPB lacosamide administration. METHODS: A retrospective pre/post cohort analysis comparing patients who received lacosamide via IVP and IVPB was conducted. Safety end points included hypotension, bradycardia, medication-related sedation, and IV site reactions. The relationship between patient characteristics and the incidence of safety end points was analyzed using the Student t-test and χ2 test as appropriate. RESULTS: Bradycardia occurred after 0.19% of IVP administrations and 1.09% of IVPB administrations assessed (P = 0.07). Hypotension was observed in 3.16% of IVP administrations compared to 1.59% in the IVPB cohort (P = 0.12). Post lacosamide-related sedation was noted in 11.32% and 11.68% of the IVP and IVPB cohorts, respectively (P = 0.87). Infusion site reaction rates of 1.80% and 0.84% were documented in the IVP and IVPB cohorts, respectively (P = 0.33). Of note, only 1 adverse event required clinical intervention. One 200-mg dose in the IVP cohort required a fluid bolus postadministration. CONCLUSION AND RELEVANCE: IVP lacosamide was associated with a similar incidence of cardiovascular, neurological, and infusion site-related adverse events compared with IVPB, in which nearly every adverse event was deemed clinically insignificant. Lacosamide administered via IVP may be considered a safe alternative method of administration in the acute care setting.

A systematic review of how missing data are handled and reported in multi-database pharmacoepidemiologic studies.

PURPOSE: Pharmacoepidemiologic multi-database studies (MDBS) provide opportunities to better evaluate the safety and effectiveness of medicines. However, the issue of missing data is often exacerbated in MDBS, potentially resulting in bias and precision loss. We sought to measure how missing data are being recorded and addressed in pharmacoepidemiologic MDBS. METHODS: We conducted a systematic literature search in PubMed for pharmacoepidemiologic MDBS published between 1st January 2018 and 31st December 2019. Included studies were those that used ≥2 distinct databases to assess the same safety/effectiveness outcome associated with a drug exposure. Outcome variables extracted from the studies included strategies to execute a MDBS, reporting of missing data (type, bias evaluation) and the methods used to account for missing data. RESULTS: Two thousand seven hundred and twenty-six articles were identified, and 62 studies were included: using data from either North America (56%), Europe (31%), multiple regions (11%) or East-Asia (2%). Thirty-five (56%) articles reported missing data: 11 of these studies reported that this could have introduced bias and 19 studies reported a method to address missing data. Thirteen (68%) carried out a complete case analysis, 2 (11%) applied multiple imputation, 2 (11%) used both methods, 1 (5%) used mean imputation and 1 (5%) substituted information from a similar variable. CONCLUSIONS: Just over half of the recent pharmacoepidemiologic MDBS reported missing data and two-thirds of these studies reported how they accounted for it. We should increase our vigilance for database completeness in MDBS by reporting and addressing the missing data that could introduce bias.

Manipulating fenestrations in young and old liver sinusoidal endothelial cells.

Fenestrations are pores within liver sinusoidal endothelial cells (LSECs) that enable the transfer of substrates (particularly insulin and lipoproteins) between blood and hepatocytes. With increasing age, there are marked reductions in fenestrations, referred to as pseudocapillarization. Currently, fenestrations are thought to be regulated by vascular endothelial growth factor and nitric oxide (NO) pathways promoting remodeling of the actin cytoskeleton and cell membrane lipid rafts. We investigated the effects of drugs that act on these pathways on fenestrations in old (18-24 mo) and young mice (3-4 mo). Isolated LSECs were incubated with either cytochalasin 7-ketocholesterol, sildenafil, amlodipine, simvastatin, 2, 5-dimethoxy-4-iodoamphetamine (DOI), bosentan, TNF-related apoptosis-inducing ligand (TRAIL) or nicotinamide mononucleotide (NMN). LSECs were visualized under scanning electron microscopy to quantify fenestration porosity, diameter, and frequency, as well as direct stochastic optical reconstruction microscopy to examine actin and NO synthase. In young and old LSECs, fenestration porosity, diameter and frequency were increased by 7-ketocholesterol, while porosity and/or frequency were increased with NMN, sildenafil, amlodipine, TRAIL, and cytochalasin D. In old mice only, bosentan and DOI increased fenestration porosity and/or frequency. Modification of the actin cytoskeleton was observed with all agents that increased fenestrations, while NO synthase was only increased by sildenafil, amlodipine, and TRAIL. In conclusion, agents that target NO, actin, or lipid rafts promote changes in fenestrations in mice LSECs. Regulation of fenestrations occurs via both NO-dependent and independent pathways. This work indicates that age-related defenestration can be reversed pharmacologically, which has potential translational relevance for dyslipidemia and insulin resistance. NEW & NOTEWORTHY We demonstrate the effects of multiple nitric oxide-dependent and -independent pharmaceutical agents on fenestrations of the liver sinusoidal endothelium. Fenestrations are reorganized in response to nicotinamide mononucleotide, sildenafil, amlodipine, and TNF-related apoptosis-inducing ligand. This work indicates that age-related defenestration can be reversed pharmacologically, which has potential translational relevance for dyslipidemia and insulin resistance in old age.

Assessment of polyurethane spheres as surrogates for military ballistic head injury.

SYNBONE® spheres were impacted with 7.62 × 39 mm mild steel core ammunition at a mean impact velocity of 654 m/s, SD 7 m/s, to simulate engagement distances of around 50-100 m. The wounds and fracture patterns were assessed by two forensic pathologists familiar with military cranial injury. The overall fracture pattern was assessed as being too comminuted when compared with actual injury. This suggests the SYNBONE® spheres have less utility for simulating military injury than other purposes described in the literature.

Ballistic impacts on an anatomically correct synthetic skull with a surrogate skin/soft tissue layer.

The aim of this work was to further develop a synthetic model of ballistic head injury by the addition of skin and soft tissue layers to an anatomically correct polyurethane skull filled with gelatine 10% by mass. Six head models were impacted with 7.62 x 39 mm full metal jacket mild steel core (FMJ MSC) bullets with a mean velocity of 652 m/s. The impact events were filmed with high-speed cameras. The models were imaged pre- and post-impact using computed tomography. The models were assessed post impact by two experienced Home Office pathologists and the images assessed by an experienced military radiologist. The findings were scored against real injuries. The entry wounds, exit wounds and fracture patterns were scored positively, but the synthetic skin and soft tissue layer was felt to be too extendable. Further work is ongoing to address this.

Visualizing value for money in public health interventions.

Background: The Socio-Technical Allocation of Resources (STAR) has been developed for value for money analysis of health services through stakeholder workshops. This article reports on its application for prioritization of interventions within public health programmes. Methods: The STAR tool was used by identifying costs and service activity for interventions within commissioned public health programmes, with benefits estimated from the literature on economic evaluations in terms of costs per Quality-Adjusted Life Years (QALYs); consensus on how these QALY values applied to local services was obtained with local commissioners. Results: Local cost-effectiveness estimates could be made for some interventions. Methodological issues arose from gaps in the evidence base for other interventions, inability to closely match some performance monitoring data with interventions, and disparate time horizons of published QALY data. Practical adjustment for these issues included using population prevalences and utility states where intervention specific evidence was lacking, and subdivision of large contracts into specific intervention costs using staffing ratios. The STAR approach proved useful in informing commissioning decisions and understanding the relative value of local public health interventions. Conclusions: Further work is needed to improve robustness of the process and develop a visualization tool for use by public health departments.

Blood‒Brain Barrier Pathology and CNS Outcomes in Streptococcus pneumoniae Meningitis.

Streptococcus pneumoniae is a major meningitis-causing pathogen globally, bringing about significant morbidity and mortality, as well as long-term neurological sequelae in almost half of the survivors. Subsequent to nasopharyngeal colonisation and systemic invasion, translocation across the blood‒brain barrier (BBB) by S. pneumoniae is a crucial early step in the pathogenesis of meningitis. The BBB, which normally protects the central nervous system (CNS) from deleterious molecules within the circulation, becomes dysfunctional in S. pneumoniae invasion due to the effects of pneumococcal toxins and a heightened host inflammatory environment of cytokines, chemokines and reactive oxygen species intracranially. The bacteria‒host interplay within the CNS likely determines not only the degree of BBB pathological changes, but also host survival and the extent of neurological damage. This review explores the relationship between S. pneumoniae bacteria and the host inflammatory response, with an emphasis on the BBB and its roles in CNS protection, as well as both the acute and long-term pathogenesis of meningitis.

Changes in orexinergic immunoreactivity of the piglet hypothalamus and pons after exposure to chronic postnatal nicotine and intermittent hypercapnic hypoxia.

We recently showed that orexin expression in sudden infant death syndrome (SIDS) infants was reduced by 21% in the hypothalamus and by 40-50% in the pons as compared with controls. Orexin maintains wakefulness/sleeping states, arousal, and rapid eye movement sleep, abnormalities of which have been reported in SIDS. This study examined the effects of two prominent risk factors for SIDS, intermittent hypercapnic hypoxia (IHH) (prone-sleeping) and chronic nicotine exposure (cigarette-smoking), on orexin A (OxA) and orexin B (OxB) expression in piglets. Piglets were randomly assigned to five groups: saline control (n = 7), air control (n = 7), nicotine [2 mg/kg per day (14 days)] (n = 7), IHH (6 min of 7% O2 /8% CO2 alternating with 6-min periods of breathing air, for four cycles) (n = 7), and the combination of nicotine and IHH (N + IHH) (n = 7). OxA/OxB expression was quantified in the central tuberal hypothalamus [dorsal medial hypothalamus (DMH), perifornical area (PeF), and lateral hypothalamus], and the dorsal raphe, locus coeruleus of the pons. Nicotine and N + IHH exposures significantly increased: (i) orexin expression in the hypothalamus and pons; and (ii) the total number of neurons in the DMH and PeF. IHH decreased orexin expression in the hypothalamus and pons without changing neuronal numbers. Linear relationships existed between the percentage of orexin-positive neurons and the area of pontine orexin immunoreactivity of control and exposure piglets. These results demonstrate that postnatal nicotine exposure increases the proportion of orexin-positive neurons in the hypothalamus and fibre expression in the pons, and that IHH exposure does not prevent the nicotine-induced increase. Thus, although both nicotine and IHH are risk factors for SIDS, it appears they have opposing effects on OxA and OxB expression, with the IHH exposure closely mimicking what we recently found in SIDS.

Benzo[b]quinolizinium Derivatives Have a Strong Antimalarial Activity and Inhibit Indoleamine Dioxygenase.

The heme-containing enzymes indoleamine 2,3-dioxygenase-1 (IDO-1) and IDO-2 catalyze the conversion of the essential amino acid tryptophan into kynurenine. Metabolites of the kynurenine pathway and IDO itself are involved in immunity and the pathology of several diseases, having either immunoregulatory or antimicrobial effects. IDO-1 plays a central role in the pathogenesis of cerebral malaria, which is the most severe and often fatal neurological complication of infection with Plasmodium falciparum. Mouse models are usually used to study the underlying pathophysiology. In this study, we screened a natural compound library against mouse IDO-1 and identified 8-aminobenzo[b]quinolizinium (compound 2c) to be an inhibitor of IDO-1 with potency at nanomolar concentrations (50% inhibitory concentration, 164 nM). Twenty-one structurally modified derivatives of compound 2c were synthesized for structure-activity relationship analyses. The compounds were found to be selective for IDO-1 over IDO-2. We therefore compared the roles of prominent amino acids in the catalytic mechanisms of the two isoenzymes via homology modeling, site-directed mutagenesis, and kinetic analyses. Notably, methionine 385 of IDO-2 was identified to interfere with the entrance of l-tryptophan to the active site of the enzyme, which explains the selectivity of the inhibitors. Most interestingly, several benzo[b]quinolizinium derivatives (6 compounds with 50% effective concentration values between 2.1 and 6.7 nM) were found to be highly effective against P. falciparum 3D7 blood stages in cell culture with a mechanism independent of IDO-1 inhibition. We believe that the class of compounds presented here has unique characteristics; it combines the inhibition of mammalian IDO-1 with strong antiparasitic activity, two features that offer potential for drug development.

Production, fate and pathogenicity of plasma microparticles in murine cerebral malaria.

In patients with cerebral malaria (CM), higher levels of cell-specific microparticles (MP) correlate with the presence of neurological symptoms. MP are submicron plasma membrane-derived vesicles that express antigens of their cell of origin and phosphatidylserine (PS) on their surface, facilitating their role in coagulation, inflammation and cell adhesion. In this study, the in vivo production, fate and pathogenicity of cell-specific MP during Plasmodium berghei infection of mice were evaluated. Using annexin V, a PS ligand, and flow cytometry, analysis of platelet-free plasma from infected mice with cerebral involvement showed a peak of MP levels at the time of the neurological onset. Phenotypic analyses showed that MP from infected mice were predominantly of platelet, endothelial and erythrocytic origins. To determine the in vivo fate of MP, we adoptively transferred fluorescently labelled MP from mice with CM into healthy or infected recipient mice. MP were quickly cleared following intravenous injection, but microscopic examination revealed arrested MP lining the endothelium of brain vessels of infected, but not healthy, recipient mice. To determine the pathogenicity of MP, we transferred MP from activated endothelial cells into healthy recipient mice and this induced CM-like brain and lung pathology. This study supports a pathogenic role for MP in the aggravation of the neurological lesion and suggests a causal relationship between MP and the development of CM.

Real-time imaging reveals the dynamics of leukocyte behaviour during experimental cerebral malaria pathogenesis.

During experimental cerebral malaria (ECM) mice develop a lethal neuropathological syndrome associated with microcirculatory dysfunction and intravascular leukocyte sequestration. The precise spatio-temporal context in which the intravascular immune response unfolds is incompletely understood. We developed a 2-photon intravital microscopy (2P-IVM)-based brain-imaging model to monitor the real-time behaviour of leukocytes directly within the brain vasculature during ECM. Ly6C(hi) monocytes, but not neutrophils, started to accumulate in the blood vessels of Plasmodium berghei ANKA (PbA)-infected MacGreen mice, in which myeloid cells express GFP, one to two days prior to the onset of the neurological signs (NS). A decrease in the rolling speed of monocytes, a measure of endothelial cell activation, was associated with progressive worsening of clinical symptoms. Adoptive transfer experiments with defined immune cell subsets in recombinase activating gene (RAG)-1-deficient mice showed that these changes were mediated by Plasmodium-specific CD8(+) T lymphocytes. A critical number of CD8(+) T effectors was required to induce disease and monocyte adherence to the vasculature. Depletion of monocytes at the onset of disease symptoms resulted in decreased lymphocyte accumulation, suggesting reciprocal effects of monocytes and T cells on their recruitment within the brain. Together, our studies define the real-time kinetics of leukocyte behaviour in the central nervous system during ECM, and reveal a significant role for Plasmodium-specific CD8(+) T lymphocytes in regulating vascular pathology in this disease.

Synergistic induction of CXCL10 by interferon-gamma and lymphotoxin-alpha in astrocytes: Possible role in cerebral malaria.

Cerebral malaria (CM) has a high mortality rate and incidence of neurological sequelae in survivors. Hypoxia and cytokine expression in the brain are two mechanisms thought to contribute to the pathogenesis of CM. The cytokines interferon (IFN)-γ and lymphotoxin (LT)-α and the chemokine CXCL10 are essential for the development of CM in a mouse model. Furthermore, serum IFN-γ protein levels are higher in human CM than in controls, and CXCL10 is elevated in both serum and cerebrospinal fluid in Ghanaian paediatric CM cases. Astrocytes actively participate in CNS pathologies, becoming activated in response to various stimuli including cytokines. Astrocyte activation also occurs in murine and human CM. We here determined the responsiveness of mouse and human astrocytes to IFN-γ and LT-α, with the aim of further elucidating the role of astrocytes in CM pathogenesis. Initially we confirmed that Ifn-γ and Cxcl10 are expressed in the brain in murine CM, and that the increased Cxcl10 expression is IFN-γ-dependant. IFN-γ induced CXCL10 production in human and murine astrocytes in vitro. The degree of induction was increased synergistically in the presence of LT-α. IFN-γ induced the expression of receptors for LT-α, while LT-α increased the expression of the receptor for IFN-γ, in the astrocytes. This cross-induction may explain the synergistic effect of the two cytokines on CXCL10 production. Expression of these receptors also was upregulated in the brain in murine CM. The results suggest that astrocytes contribute to CM pathogenesis by producing CXCL10 in response to IFN-γ and LT-α.

The kynurenine pathway of tryptophan degradation is activated during osteoblastogenesis.

The mechanisms involved in the anabolic effect of interferon gamma (IFNγ) on bone have not been carefully examined. Using microarray expression analysis, we found that IFNγ upregulates a set of genes associated with a tryptophan degradation pathway, known as the kynurenine pathway, in osteogenic differentiating human mesenchymal stem cells (hMSC). We, therefore, hypothesized that activation of the kynurenine pathway plays a role in osteoblastogenesis even in the absence of IFNγ. Initially, we observed a strong increase in tryptophan degradation during osteoblastogenesis with and without IFNγ in the media. We next blocked indoleamine 2,3-dioxygenase-1 (IDO1), the most important enzyme in the kynurenine pathway, using a siRNA and pharmacological approach and observed a strong inhibition of osteoblastogenesis with a concomitant decrease in osteogenic factors. We next examined the bone phenotype of Ido1 knockout (Ido1(-/-)) mice. Compared to their wild-type littermates, Ido1(-/-) mice exhibited osteopenia associated with low osteoblast and high osteoclast numbers. Finally, we tested whether the end products of the kynurenine pathway have an osteogenic effect on hMSC. We identified that picolinic acid had a strong and dose-dependent osteogenic effect in vitro. In summary, we demonstrate that the activation of the kynurenine pathway plays an important role during the commitment of hMSC into the osteoblast lineage in vitro, and that this process can be accelerated by exogenous addition of IFNγ. In addition, we found that mice lacking IDO1 activity are osteopenic. These data therefore support a new role for the kynurenine pathway and picolinic acid as essential regulators of osteoblastogenesis and as potential new targets of bone-forming cells in vivo.

Antibody-induced neutrophil depletion prior to the onset of pneumococcal meningitis influences long-term neurological complications in mice.

During pneumococcal meningitis, clearance of bacteria by recruited neutrophils is crucial for host protection. However, these innate immune mechanisms are often insufficient and treatment with antibiotics is necessary to prevent death. Despite this antibiotic treatment, approximately half of all survivors suffer lifelong neurological problems. There is growing evidence indicating the harmful effects of neutrophils on CNS integrity. Therefore, the present study investigated the roles of neutrophils in the acute inflammatory response and the resulting long-term neuropsychological effects in murine pneumococcal meningitis. Long-term behavioural and cognitive functions in mice were measured using an automated IntelliCage system. Neutrophil depletion with antibody 1A8 as adjunctive therapy was shown to remarkably impair survival in meningitic C57BL/6J mice despite antibiotic (ceftriaxone) treatment. This was accompanied by increased bacterial load in the cerebrospinal fluid (CSF) and an increase in IL-1ß, but decrease in TNF, within the CSF at 20h after bacterial inoculation. In the longer term, the surviving neutrophil-depleted post-meningitic (PM) mice displayed reduced diurnal hypolocomotion compared to PM mice treated with an isotype antibody. However, they showed nocturnal hyperactivity, and greater learning impairment in a patrolling task that is believed to depend upon an intact hippocampus. The data thus demonstrate two important mechanisms: 1. Neutrophil extravasation into the CNS during pneumococcal meningitis influences the pro-inflammatory response and is central to control of the bacterial load, an increase in which may lead to death. 2. Neutrophil-mediated changes in the acute inflammatory response modulate the neuropsychological sequelae in mice that survive pneumococcal meningitis.

IRGM3 contributes to immunopathology and is required for differentiation of antigen-specific effector CD8+ T cells in experimental cerebral malaria.

Gamma interferon (IFN-γ) drives antiparasite responses and immunopathology during infection with Plasmodium species. Immunity-related GTPases (IRGs) are a class of IFN-γ-dependent proteins that are essential for cell autonomous immunity to numerous intracellular pathogens. However, it is currently unknown whether IRGs modulate responses during malaria. We have used the Plasmodium berghei ANKA (PbA) model in which mice develop experimental cerebral malaria (ECM) to study the roles of IRGM1 and IRGM3 in immunopathology. Induction of mRNA for Irgm1 and Irgm3 was found in the brains and spleens of infected mice at times of peak IFN-γ production. Irgm3-/- but not Irgm1-/- mice were completely protected from the development of ECM, and this protection was associated with the decreased induction of inflammatory cytokines, as well as decreased recruitment and activation of CD8+ T cells within the brain. Although antigen-specific proliferation of transferred CD8+ T cells was not diminished compared to that of wild-type recipients following PbA infection, T cells transferred into Irgm3-/- recipients showed a striking impairment of effector differentiation. Decreased induction of several inflammatory cytokines and chemokines (interleukin-6, CCL2, CCL3, and CCL4), as well as enhanced mRNA expression of type-I IFNs, was found in the spleens of Irgm3-/- mice at day 4 postinfection. Together, these data suggest that protection from ECM pathology in Irgm3-/- mice occurs due to impaired generation of CD8+ effector function. This defect is nonintrinsic to CD8+ T cells. Instead, diminished T cell responses most likely result from defective initiation of inflammatory responses in myeloid cells.