TNFAIP3 Reduction-of-Function Drives Female Infertility and CNS Inflammation.

Women with autoimmune and inflammatory aetiologies can exhibit reduced fecundity. TNFAIP3 is a master negative regulator of inflammation, and has been linked to many inflammatory conditions by genome wide associations studies, however its role in fertility remains unknown. Here we show that mice harbouring a mild Tnfaip3 reduction-of-function coding variant (Tnfaip3I325N) that reduces the threshold for inflammatory NF-κB activation, exhibit reduced fecundity. Sub-fertility in Tnfaip3I325N mice is associated with irregular estrous cycling, low numbers of ovarian secondary follicles, impaired mammary gland development and insulin resistance. These pathological features are associated with infertility in human subjects. Transplantation of Tnfaip3I325N ovaries, mammary glands or pancreatic islets into wild-type recipients rescued estrous cycling, mammary branching and hyperinsulinemia respectively, pointing towards a cell-extrinsic hormonal mechanism. Examination of hypothalamic brain sections revealed increased levels of microglial activation with reduced levels of luteinizing hormone. TNFAIP3 coding variants may offer one contributing mechanism for the cause of sub-fertility observed across otherwise healthy populations as well as for the wide variety of auto-inflammatory conditions to which TNFAIP3 is associated. Further, TNFAIP3 represents a molecular mechanism that links heightened immunity with neuronal inflammatory homeostasis. These data also highlight that tuning-up immunity with TNFAIP3 comes with the potentially evolutionary significant trade-off of reduced fertility.

MIC-1/GDF15 Overexpression Is Associated with Increased Functional Recovery in Traumatic Spinal Cord Injury.

Spinal cord injury (SCI) has devastating consequences, with limited therapeutic options; therefore, improving its functional outcome is a major goal. The outcome of SCI is contributed to by neuroinflammation, which may be a target for improved recovery and quality of life after injury. Macrophage inhibitory cytokine-1/growth differentiation factor 15 (MIC-1/GDF15) has been identified as a potential novel therapy for central nervous system (CNS) injury because it is an immune regulatory cytokine with neurotrophic properties. Here we used MIC-1/GDF15 knockout (KO) and overexpressing/transgenic (Tg) and wild type (WT) animals to explore its putative therapeutic benefits in a mouse model of contusive SCI. MIC-1/GDF15 Tg mice had superior locomotor recovery and reduced secondary tissue loss at 28 days compared with their KO and WT counterparts. Overexpression of MIC-1/GDF15 coincided with increased expression of monocyte chemoattractant protein-1 (MCP-1)/C-C Motif Chemokine Ligand 2 (CCL2) at the lesion site (28 days post-SCI) and enhanced recruitment of inflammatory cells to the injured spinal cord. This inflammatory cellular infiltrate included an increased frequency of macrophages and dendritic cells (DCs) that mostly preceded recruitment of cluster of differentiation (CD)4+ and CD8+ T cells. Collectively, our findings suggest hat MIC-1/GDF15 is associated with beneficial changes in the clinical course of SCI that are characterized by altered post-injury inflammation and improved functional outcome. Further investigation of MIC-1/GDF15 as a novel therapeutic target for traumatic SCI appears warranted.

Boron-Pleuromutilins as Anti- Wolbachia Agents with Potential for Treatment of Onchocerciasis and Lymphatic Filariasis.

A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.

Validation of ultrasound bioimaging to predict worm burden and treatment efficacy in preclinical filariasis drug screening models.

Filariasis is a global health problem targeted for elimination. Curative drugs (macrofilaricides) are required to accelerate elimination. Candidate macrofilaricides require testing in preclinical models of filariasis. The incidence of infection failures and high intra-group variation means that large group sizes are required for drug testing. Further, a lack of accurate, quantitative adult biomarkers results in protracted timeframes or multiple groups for endpoint analyses. Here we evaluate intra-vital ultrasonography (USG) to identify B. malayi in the peritonea of gerbils and CB.17 SCID mice and assess prognostic value in determining drug efficacy. USG operators, blinded to infection status, could detect intra-peritoneal filarial dance sign (ipFDS) with 100% specificity and sensitivity, when >5 B. malayi worms were present in SCID mice. USG ipFDS was predictive of macrofilaricidal activity in randomized, blinded studies comparing flubendazole, albendazole and vehicle-treated SCID mice. Semi-quantification of ipFDS could predict worm burden >10 with 87-100% accuracy in SCID mice or gerbils. We estimate that pre-assessment of worm burden by USG could reduce intra-group variation, obviate the need for surgical implantations in gerbils, and reduce total SCID mouse use by 40%. Thus, implementation of USG may reduce animal use, refine endpoints and negate invasive techniques for assessing anti-filarial drug efficacy.

Corrigendum: Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

This corrects the article DOI: 10.1038/srep23458.

Author Correction: Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

A correction to this article has been published and is linked from the HTML version of this paper. The error has been fixed in the paper.

Albendazole and antibiotics synergize to deliver short-course anti-Wolbachia curative treatments in preclinical models of filariasis.

Elimination of filariasis requires a macrofilaricide treatment that can be delivered within a 7-day period. Here we have identified a synergy between the anthelmintic albendazole (ABZ) and drugs depleting the filarial endosymbiont Wolbachia, a proven macrofilaricide target, which reduces treatment from several weeks to 7 days in preclinical models. ABZ had negligible effects on Wolbachia but synergized with minocycline or rifampicin (RIF) to deplete symbionts, block embryogenesis, and stop microfilariae production. Greater than 99% Wolbachia depletion following 7-day combination of RIF+ABZ also led to accelerated macrofilaricidal activity. Thus, we provide preclinical proof-of-concept of treatment shortening using antibiotic+ABZ combinations to deliver anti-Wolbachia sterilizing and macrofilaricidal effects. Our data are of immediate public health importance as RIF+ABZ are registered drugs and thus immediately implementable to deliver a 1-wk macrofilaricide. They also suggest that novel, more potent anti-Wolbachia drugs under development may be capable of delivering further treatment shortening, to days rather than weeks, if combined with benzimidazoles.

Short-Course, High-Dose Rifampicin Achieves Wolbachia Depletion Predictive of Curative Outcomes in Preclinical Models of Lymphatic Filariasis and Onchocerciasis.

Lymphatic filariasis (LF) and onchocerciasis are priority neglected tropical diseases targeted for elimination. The only safe drug treatment with substantial curative activity against the filarial nematodes responsible for LF (Brugia malayi, Wuchereria bancrofti) or onchocerciasis (Onchocerca volvulus) is doxycycline. The target of doxycycline is the essential endosymbiont, Wolbachia. Four to six weeks doxycycline therapy achieves >90% depletion of Wolbachia in worm tissues leading to blockade of embryogenesis, adult sterility and premature death 18-24 months post-treatment. Long treatment length and contraindications in children and pregnancy are obstacles to implementing doxycycline as a public health strategy. Here we determine, via preclinical infection models of Brugia malayi or Onchocerca ochengi that elevated exposures of orally-administered rifampicin can lead to Wolbachia depletions from filariae more rapidly than those achieved by doxycycline. Dose escalation of rifampicin achieves >90% Wolbachia depletion in time periods of 7 days in B. malayi and 14 days in O. ochengi. Using pharmacokinetic-pharmacodynamic modelling and mouse-human bridging analysis, we conclude that clinically relevant dose elevations of rifampicin, which have recently been determined as safe in humans, could be administered as short courses to filariasis target populations with potential to reduce anti-Wolbachia curative therapy times to between one and two weeks.

Minocycline as a re-purposed anti-Wolbachia macrofilaricide: superiority compared with doxycycline regimens in a murine infection model of human lymphatic filariasis.

Lymphatic filariasis and onchocerciasis are parasitic helminth diseases, which cause severe morbidities such as elephantiasis, skin disease and blindness, presenting a major public health burden in endemic communities. The anti-Wolbachia consortium (A·WOL: http://www.a-wol.com/) has identified a number of registered antibiotics that target the endosymbiotic bacterium, Wolbachia, delivering macrofilaricidal activity. Here we use pharmacokinetics/pharmacodynamics (PK/PD) analysis to rationally develop an anti-Wolbachia chemotherapy by linking drug exposure to pharmacological effect. We compare the pharmacokinetics and anti-Wolbachia efficacy in a murine Brugia malayi model of minocycline versus doxycycline. Doxycycline exhibits superior PK in comparison to minocycline resulting in a 3-fold greater exposure in SCID mice. Monte-Carlo simulations confirmed that a bi-daily 25-40 mg/Kg regimen is bioequivalent to a clinically effective 100-200 mg/day dose for these tetracyclines. Pharmacodynamic studies showed that minocycline depletes Wolbachia more effectively than doxycycline (99.51% vs. 90.35%) after 28 day 25 mg/Kg bid regimens with a more potent block in microfilarial production. PK/PD analysis predicts that minocycline would be expected to be 1.7 fold more effective than doxycycline in man despite lower exposure in our infection models. Our findings warrant onward clinical investigations to examine the clinical efficacy of minocycline treatment regimens against lymphatic filariasis and onchocerciasis.

Plasminogen activator inhibitor (PAI)-1 suppresses inhibition of gastric emptying by cholecystokinin (CCK) in mice.

The intestinal hormone cholecystokinin (CCK) delays gastric emptying and inhibits food intake by actions on vagal afferent neurons. Recent studies suggest plasminogen activator inhibitor (PAI)-1 suppresses the effect of CCK on food intake. In this study we asked whether PAI-1 also modulated CCK effects on gastric emptying. Five minute gastric emptying of liquid test meals was studied in conscious wild type mice (C57BL/6) and in transgenic mice over-expressing PAI-1 in gastric parietal cells (PAI-1H/Kß mice), or null for PAI-1. The effects of exogenous PAI-1 and CCK8s on gastric emptying were studied after ip administration. Intragastric peptone delayed gastric emptying in C57BL/6 mice by a mechanism sensitive to the CCK-1 receptor antagonist lorglumide. Peptone did not delay gastric emptying in PAI-1-H/Kß mice. Exogenous CCK delayed gastric emptying of a control test meal in C57BL/6 mice and this was attenuated by administration of PAI-1; exogenous CCK had no effect on emptying in PAI-1-H/Kß mice. Prior administration of gastrin to increase gastric PAI-1 inhibited CCK-dependent effects on gastric emptying in C57BL/6 mice but not in PAI-1 null mice. Thus, both endogenous and exogenous PAI-1 inhibit the effects of CCK (whether exogenous or endogenous) on gastric emptying. The data are compatible with emerging evidence that gastric PAI-1 modulates vagal effects of CCK.

Gastric expression of plasminogen activator inhibitor (PAI)-1 is associated with hyperphagia and obesity in mice.

The adipokine plasminogen activator inhibitor (PAI)-1 is increased in plasma of obese individuals and exhibits increased expression in the stomachs of individuals infected with Helicobacter. To investigate the relevance of gastric PAI-1, we used 1.1 kb of the H(+)/K(+)ß subunit promoter to overexpress PAI-1 specifically in mouse gastric parietal cells (PAI-1-H/Kß mice). We studied the physiological, biochemical, and behavioral characteristics of these and mice null for PAI-1 or a putative receptor, urokinase plasminogen activator receptor (uPAR). PAI-1-H/Kß mice had increased plasma concentrations of PAI-1 and increased body mass, adiposity, and hyperphagia compared with wild-type mice. In the latter, food intake was inhibited by cholecystokinin (CCK)8s, but PAI-1-H/Kß mice were insensitive to the satiating effects of CCK8s. PAI-1-H/Kß mice also had significantly reduced expression of c-fos in the nucleus tractus solitarius in response to CCK8s and refeeding compared with wild-type mice. Exogenous PAI-1 reversed the effects of CCK8s on food intake and c-fos levels in the nucleus tractus solitarius of wild-type mice, but not uPAR-null mice. Infection of C57BL/6 mice with Helicobacter felis increased gastric abundance of PAI-1 and reduced the satiating effects of CCK8s, whereas the response to CCK8s was maintained in infected PAI-1-null mice. In cultured vagal afferent neurons, PAI-1 inhibited stimulation of neuropeptide Y type 2 receptor (Y2R) expression by CCK8s. Thus, gastric expression of PAI-1 is associated with hyperphagia, moderate obesity, and resistance to the satiating effects of CCK indicating a new role in suppressing signals from the upper gut that inhibit food intake.