A Common Variant in the Adaptor Mal Regulates Interferon Gamma Signaling.

Humans that are heterozygous for the common S180L polymorphism in the Toll-like receptor (TLR) adaptor Mal (encoded by TIRAP) are protected from a number of infectious diseases, including tuberculosis (TB), whereas those homozygous for the allele are at increased risk. The reason for this difference in susceptibility is not clear. We report that Mal has a TLR-independent role in interferon-gamma (IFN-γ) receptor signaling. Mal-dependent IFN-γ receptor (IFNGR) signaling led to mitogen-activated protein kinase (MAPK) p38 phosphorylation and autophagy. IFN-γ signaling via Mal was required for phagosome maturation and killing of intracellular Mycobacterium tuberculosis (Mtb). The S180L polymorphism, and its murine equivalent S200L, reduced the affinity of Mal for the IFNGR, thereby compromising IFNGR signaling in macrophages and impairing responses to TB. Our findings highlight a role for Mal outside the TLR system and imply that genetic variation in TIRAP may be linked to other IFN-γ-related diseases including autoimmunity and cancer.

Pharmacological Inhibition of the Nod-Like Receptor Family Pyrin Domain Containing 3 Inflammasome with MCC950.

Activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome drives release of the proinflammatory cytokines interleukin (IL)-1ß and IL-18 and induces pyroptosis (lytic cell death). These events drive chronic inflammation, and as such, NLRP3 has been implicated in a large number of human diseases. These range from autoimmune conditions, the simplest of which is NLRP3 gain-of-function mutations leading to an orphan disease, cryopyrin-associated period syndrome, to large disease burden indications, such as atherosclerosis, heart failure, stroke, neurodegeneration, asthma, ulcerative colitis, and arthritis. The potential clinical utility of NLRP3 inhibitors is substantiated by an expanding list of indications in which NLRP3 activation has been shown to play a detrimental role. Studies of pharmacological inhibition of NLRP3 in nonclinical models of disease using MCC950 in combination with human genetics, epigenetics, and analyses of the efficacy of biologic inhibitors of IL-1ß, such as anakinra and canakinumab, can help to prioritize clinical trials of NLRP3-directed therapeutics. Although MCC950 shows excellent (nanomolar) potency and high target selectivity, its pharmacokinetic and toxicokinetic properties limited its therapeutic development in the clinic. Several improved, next-generation inhibitors are now in clinical trials. Hence the body of research in a plethora of conditions reviewed herein may inform analysis of the potential translational value of NLRP3 inhibition in diseases with significant unmet medical need. SIGNIFICANCE STATEMENT: The nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is one of the most widely studied and best validated biological targets in innate immunity. Activation of NLRP3 can be inhibited with MCC950, resulting in efficacy in more than 100 nonclinical models of inflammatory diseases. As several next-generation NLRP3 inhibitors are entering proof-of-concept clinical trials in 2020, a review of the pharmacology of MCC950 is timely and significant.

4-Octyl-Itaconate and Dimethyl Fumarate Inhibit COX2 Expression and Prostaglandin Production in Macrophages.

PGs are important proinflammatory lipid mediators, the significance of which is highlighted by the widespread and efficacious use of nonsteroidal anti-inflammatory drugs in the treatment of inflammation. 4-Octyl itaconate (4-OI), a derivative of the Krebs cycle-derived metabolite itaconate, has recently garnered much interest as an anti-inflammatory agent. In this article, we show that 4-OI limits PG production in murine macrophages stimulated with the TLR1/2 ligand Pam3CSK4. This decrease in PG secretion is due to a robust suppression of cyclooxygenase 2 (COX2) expression by 4-OI, with both mRNA and protein levels decreased. Dimethyl fumarate, a fumarate derivative used in the treatment of multiple sclerosis, with properties similar to itaconate, replicated the phenotype observed with 4-OI. We also demonstrate that the decrease in COX2 expression and inhibition of downstream PG production occurs in an NRF2-independent manner. Our findings provide a new insight into the potential of 4-OI as an anti-inflammatory agent and also identifies a novel anti-inflammatory function of dimethyl fumarate.

The Trypanosome-Derived Metabolite Indole-3-Pyruvate Inhibits Prostaglandin Production in Macrophages by Targeting COX2.

The protozoan parasite Trypanosoma brucei is the causative agent of the neglected tropical disease human African trypanosomiasis, otherwise known as sleeping sickness. Trypanosomes have evolved many immune-evasion mechanisms to facilitate their own survival, as well as prolonging host survival to ensure completion of the parasitic life cycle. A key feature of the bloodstream form of T. brucei is the secretion of aromatic keto acids, which are metabolized from tryptophan. In this study, we describe an immunomodulatory role for one of these keto acids, indole-3-pyruvate (I3P). We demonstrate that I3P inhibits the production of PGs in activated macrophages. We also show that, despite the reduction in downstream PGs, I3P augments the expression of cyclooxygenase (COX2). This increase in COX2 expression is mediated in part via inhibition of PGs relieving a negative-feedback loop on COX2. Activation of the aryl hydrocarbon receptor also participates in this effect. However, the increase in COX2 expression is of little functionality, as we also provide evidence to suggest that I3P targets COX activity. This study therefore details an evasion strategy by which a trypanosome-secreted metabolite potently inhibits macrophage-derived PGs, which might promote host and trypanosome survival.

Concurrent transposon engineering and CRISPR/Cas9 genome editing of primary CLL-1 chimeric antigen receptor-natural killer cells.

BACKGROUND: Natural killer (NK) cell genome editing promises to enhance the innate and alloreactive anti-tumor potential of NK cell adoptive transfer. DNA transposons are versatile non-viral gene vectors now being adapted to primary NK cells, representing important tools for research and clinical product development. AIMS AND METHODS: We set out to generate donor-derived, primary chimeric antigen receptor (CAR)-NK cells by combining the TcBuster transposon system with Epstein-Barr virus-transformed lymphoblastoid feeder cell-mediated activation and expansion. RESULTS: This approach allowed for clinically relevant NK-cell expansion capability and CAR expression, which was further enhanced by immunomagnetic selection based on binding to the CAR target protein.The resulting CAR-NK cells targeting the myeloid associated antigen CLL-1 efficiently targeted CLL-1-positive AML cell lines and primary AML populations, including a population enriched for leukemia stem cells. Subsequently, concurrent delivery of CRISPR/Cas9 cargo was applied to knockout the NK cell cytokine checkpoint cytokine-inducible SH2-containing protein (CIS, product of the CISH gene), resulting in enhanced cytotoxicity and an altered NK cell phenotype. CONCLUSIONS: This report contributes a promising application of transposon engineering to donor-derived NK cells and emphasizes the importance of feeder mediated NK cell activation and expansion to current protocols.

Circadian clock protein BMAL1 regulates IL-1ß in macrophages via NRF2.

A variety of innate immune responses and functions are dependent on time of day, and many inflammatory conditions are associated with dysfunctional molecular clocks within immune cells. However, the functional importance of these innate immune clocks has yet to be fully characterized. NRF2 plays a critical role in the innate immune system, limiting inflammation via reactive oxygen species (ROS) suppression and direct repression of the proinflammatory cytokines, IL-1ß and IL-6. Here we reveal that the core molecular clock protein, BMAL1, controls the mRNA expression of Nrf2 via direct E-box binding to its promoter to regulate its activity. Deletion of Bmal1 decreased the response of NRF2 to LPS challenge, resulting in a blunted antioxidant response and reduced synthesis of glutathione. ROS accumulation was increased in Bmal1-/- macrophages, facilitating accumulation of the hypoxic response protein, HIF-1α. Increased ROS and HIF-1α levels, as well as decreased activity of NRF2 in cells lacking BMAL1, resulted in increased production of the proinflammatory cytokine, IL-1ß. The excessive prooxidant and proinflammatory phenotype of Bmal1-/- macrophages was rescued by genetic and pharmacological activation of NRF2, or through addition of antioxidants. Our findings uncover a clear role for the molecular clock in regulating NRF2 in innate immune cells to control the inflammatory response. These findings provide insights into the pathology of inflammatory conditions, in which the molecular clock, oxidative stress, and IL-1ß are known to play a role.

Single-Dose Gene-Replacement Therapy for Spinal Muscular Atrophy.

BACKGROUND: Spinal muscular atrophy type 1 (SMA1) is a progressive, monogenic motor neuron disease with an onset during infancy that results in failure to achieve motor milestones and in death or the need for mechanical ventilation by 2 years of age. We studied functional replacement of the mutated gene encoding survival motor neuron 1 (SMN1) in this disease. METHODS: Fifteen patients with SMA1 received a single dose of intravenous adeno-associated virus serotype 9 carrying SMN complementary DNA encoding the missing SMN protein. Three of the patients received a low dose (6.7×1013 vg per kilogram of body weight), and 12 received a high dose (2.0×1014 vg per kilogram). The primary outcome was safety. The secondary outcome was the time until death or the need for permanent ventilatory assistance. In exploratory analyses, we compared scores on the CHOP INTEND (Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders) scale of motor function (ranging from 0 to 64, with higher scores indicating better function) in the two cohorts and motor milestones in the high-dose cohort with scores in studies of the natural history of the disease (historical cohorts). RESULTS: As of the data cutoff on August 7, 2017, all 15 patients were alive and event-free at 20 months of age, as compared with a rate of survival of 8% in a historical cohort. In the high-dose cohort, a rapid increase from baseline in the score on the CHOP INTEND scale followed gene delivery, with an increase of 9.8 points at 1 month and 15.4 points at 3 months, as compared with a decline in this score in a historical cohort. Of the 12 patients who had received the high dose, 11 sat unassisted, 9 rolled over, 11 fed orally and could speak, and 2 walked independently. Elevated serum aminotransferase levels occurred in 4 patients and were attenuated by prednisolone. CONCLUSIONS: In patients with SMA1, a single intravenous infusion of adeno-associated viral vector containing DNA coding for SMN resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts. Further studies are necessary to confirm the safety and efficacy of this gene therapy. (Funded by AveXis and others; ClinicalTrials.gov number, NCT02122952 .).

Gene Therapy Corrects Brain and Behavioral Pathologies in CLN6-Batten Disease.

CLN6-Batten disease, a form of neuronal ceroid lipofuscinosis is a rare lysosomal storage disorder presenting with gradual declines in motor, visual, and cognitive abilities and early death by 12-15 years of age. We developed a self-complementary adeno-associated virus serotype 9 (scAAV9) vector expressing the human CLN6 gene under the control of a chicken ß-actin (CB) hybrid promoter. Intrathecal delivery of scAAV9.CB.hCLN6 into the cerebrospinal fluid (CSF) of the lumbar spinal cord of 4-year-old non-human primates was safe, well tolerated, and led to efficient targeting throughout the brain and spinal cord. A single intracerebroventricular (i.c.v.) injection at post-natal day 1 in Cln6 mutant mice delivered scAAV9.CB.CLN6 directly into the CSF, and it prevented or drastically reduced all of the pathological hallmarks of Batten disease. Moreover, there were significant improvements in motor performance, learning and memory deficits, and survival in treated Cln6 mutant mice, extending survival from 15 months of age (untreated) to beyond 21 months of age (treated). Additionally, many parameters were similar to wild-type counterparts throughout the lifespan of the treated mice.

The Induction of Pro-IL-1ß by Lipopolysaccharide Requires Endogenous Prostaglandin E2 Production.

PGE2 has been shown to increase the transcription of pro-IL-1ß. However, recently it has been demonstrated that PGE2 can block the maturation of IL-1ß by inhibiting the NLRP3 inflammasome in macrophages. These apparently conflicting results have led us to reexamine the effect of PGE2 on IL-1ß production. We have found that in murine bone marrow-derived macrophages, PGE2 via the cAMP/protein kinase A pathway is potently inducing IL-1ß transcription, as well as boosting the ability of LPS to induce IL-1ß mRNA and pro-IL-1ß while inhibiting the production of TNF-α. This results in an increase in mature IL-1ß production in macrophages treated with ATP. We also examined the effect of endogenously produced PGE2 on IL-1ß production. By blocking PGE2 production with indomethacin, we made a striking finding that endogenous PGE2 is essential for LPS-induced pro-IL-1ß production, suggesting a positive feedback loop. The effect of endogenous PGE2 was mediated by EP2 receptor. In primary human monocytes, where LPS alone is sufficient to induce mature IL-1ß, PGE2 boosted LPS-induced IL-1ß production. PGE2 did not inhibit ATP-induced mature IL-1ß production in monocytes. Because PGE2 mediates the pyrogenic effect of IL-1ß, these effects might be especially relevant for the role of monocytes in the induction of fever. A positive feedback loop from IL-1ß and back to PGE2, which itself is induced by IL-1ß, is likely to be operating. Furthermore, fever might therefore occur in the absence of a septic shock response because of the inhibiting effect of PGE2 on TNF-α production.

Trypanosoma brucei metabolite indolepyruvate decreases HIF-1α and glycolysis in macrophages as a mechanism of innate immune evasion.

The parasite Trypanasoma brucei causes African trypanosomiasis, known as sleeping sickness in humans and nagana in domestic animals. These diseases are a major burden in the 36 sub-Saharan African countries where the tsetse fly vector is endemic. Untreated trypanosomiasis is fatal and the current treatments are stage-dependent and can be problematic during the meningoencephalitic stage, where no new therapies have been developed in recent years and the current drugs have a low therapeutic index. There is a need for more effective treatments and a better understanding of how these parasites evade the host immune response will help in this regard. The bloodstream form of T. brucei excretes significant amounts of aromatic ketoacids, including indolepyruvate, a transamination product of tryptophan. This study demonstrates that this process is essential in bloodstream forms, is mediated by a specialized isoform of cytoplasmic aminotransferase and, importantly, reveals an immunomodulatory role for indolepyruvate. Indolepyruvate prevents the LPS-induced glycolytic shift in macrophages. This effect is the result of an increase in the hydroxylation and degradation of the transcription factor hypoxia-inducible factor-1α (HIF-1α). The reduction in HIF-1α levels by indolepyruvate, following LPS or trypanosome activation, results in a decrease in production of the proinflammatory cytokine IL-1ß. These data demonstrate an important role for indolepyruvate in immune evasion by T. brucei.

MiR-155 deletion reduces ischemia-induced paralysis in an aortic aneurysm repair mouse model: Utility of immunohistochemistry and histopathology in understanding etiology of spinal cord paralysis.

Spinal cord paralysis is relatively common after surgical repair of thoraco-abdominal aortic aneurysm (TAAA) and its etiology is unknown. The present study was designed to examine the histopathology of the disease and investigate whether miR-155 ablation would reduce spinal cord ischemic damage and delayed hindlimb paralysis induced by aortic cross-clamping (ACC) in our mouse model. The loss of locomotor function in ACC-paralyzed mice correlated with the presence of extensive gray matter damage and central cord edema, with minimal white matter histopathology. qRTPCR and Western blotting showed that the spinal cords of wild-type ACC mice that escaped paralysis showed lower miR-155 expression and higher levels of transcripts encoding Mfsd2a, which is implicated in the maintenance of blood-brain barrier integrity. In situ based testing demonstrated that increased miR-155 detection in neurons was highly correlated with the gray matter damage and the loss of one of its targets, Mfsd2a, could serve as a good biomarker of the endothelial cell damage. In vitro, we demonstrated that miR-155 targeted Mfsd2a in endothelial cells and motoneurons and increased endothelial cell permeability. Finally, miR-155 ablation slowed the progression of central cord edema, and reduced the incidence of paralysis by 40%. In sum, the surgical pathology findings clearly indicated that the epicenter of the ischemic-induced paralysis was the gray matter and that endothelial cell damage correlated to Mfsd2a loss is a good biomarker of the disease. MiR-155 targeting therefore offers new therapeutic opportunity for edema caused by traumatic spinal cord injury and diagnostic pathologists, by using immunohistochemistry, can clarify if this mechanism also is important in other ischemic diseases of the CNS, including stroke.

The Incidence of Intradiscal, Intrathecal, and Intravascular Flow During the Performance of Retrodiscal (Infraneural) Approach for Lumbar Transforaminal Epidural Steroid Injections.

BACKGROUND: Lumbar transforaminal epidural steroid injections (TFESIs) are often used in the treatment of radicular pain. In light of safety concerns, many practitioners have proposed adopting the retrodiscal (infraneural) approach with the needle tip positioned into Kambin's triangle. With this technique, the needle may inadvertently be directed too far ventrally and enter the intervertebral disc. In addition, the risk of subarachnoid or subdural extra-arachnoid injection may be higher with this technique as well. OBJECTIVE: To determine the incidence of inadvertent intradiscal, intrathecal, and vascular injections during the performance of retrodiscal TFESI. STUDY DESIGN: Retrospective review METHODS: Retrospective review of all retrodiscal approach TFESIs performed from July 2012 to August 2014 by two of the authors (DL and SH). RESULTS: A total of 257 retrodiscal transforaminal injections were performed. There were no neurologic complications. There were no cases of discitis. Inadvertent intradiscal injections occurred in 12/257 injections, 4.7% (95% CI 2.1-7.3%). Intrathecal injections occurred in 8/257 injections, 3.1% (95% CI 0.99- 5.23%). Three were subarachnoid (SA), four were subdural extra-arachnoid (SDXA), and one was both SA and SDXA. Vascular injections occurred in 17/257, 6.6% (95% CI 3.6-9.6%). CONCLUSION: This retrospective review demonstrates that a relatively high rate of inadvertent intradiscal injections occurs in the performance of the retrodiscal approach for TFESI. This has significant implications in terms of the potential risk of disc injury induced by the needle puncture. The high incidence of intrathecal injections may also be of great concern depending upon the injectate delivered.

Changes in vector species composition and current vector biology and behaviour will favour malaria elimination in Santa Isabel Province, Solomon Islands.

BACKGROUND: In 2009, Santa Isabel Province in the Solomon Islands embarked on a malaria elimination programme. However, very little is known in the Province about the anopheline fauna, which species are vectors, their bionomics and how they may respond to intensified intervention measures. The purpose of this study was to provide baseline data on the malaria vectors and to ascertain the possibility of successfully eliminating malaria using the existing conventional vector control measures, such as indoor residual spraying (IRS) and long-lasting insecticidal nets (LLIN). METHODS: Entomological surveys were undertaken during October 2009. To determine species composition and distribution larval surveys were conducted across on the whole island. For malaria transmission studies, adult anophelines were sampled using human landing catches from two villages - one coastal and one inland. RESULTS: Five Anopheles species were found on Santa Isabel: Anopheles farauti, Anopheles hinesorum, Anopheles lungae, Anopheles solomonis, and Anopheles nataliae. Anopheles hinesorum was the most widespread species. Anopheles farauti was abundant, but found only on the coast. Anopheles punctulatus and Anopheles koliensis were not found. Anopheles farauti was the only species found biting in the coastal village, it was incriminated as a vector in this study; it fed early in the night but equally so indoors and outdoors, and had a low survival rate. Anopheles solomonis was the main species biting humans in the inland village, it was extremely exophagic, with low survival rates, and readily fed on pigs. CONCLUSION: The disappearance of the two major vectors, An. punctulatus and An. koliensis, from Santa Isabel and the predominance of An. hinesorum, a non-vector species may facilitate malaria elimination measures. Anopheles farauti was identified as the main coastal vector with An. solomonis as a possible inland vector. The behaviour of An. solomonis is novel as it has not been previously found biting humans in any numbers. Both species appear to be short-lived, a characteristic that will limit their transmission potential. The early night feeding behaviour and a degree of outdoor biting seen in An. farauti and particularly in An. solomonis will require that their response to IRS and LLIN be closely monitored. In coastal villages, where large, favourable breeding sites allow for high numbers of An. farauti may require the addition of larval control to achieve elimination.

Impact of early life stress on the reinforcing and behavioral-stimulant effects of psychostimulants in rhesus monkeys.

Early life stress has effects on behavior and stress reactivity, which are linked to enhanced sensitivity to stimulants in rodents. This study investigated whether rhesus monkeys that experienced early life stress would show altered sensitivity to the reinforcing effects of stimulants as compared with controls. Control (n=5) and maternally separated (n=4) monkeys were trained to self-administer cocaine (0.1 mg/kg/injection) under a second-order schedule of intravenous drug delivery. The rate of acquisition and subsequent dose-effect determinations for cocaine (0.01-1.0 mg/kg/injection) and amphetamine (0.003-0.3 mg/kg/injection) provided complementary measures of reinforcing effectiveness. In addition, stimulant-induced increases in home cage activity and dopamine D2 receptor binding potential were quantified with positron emission tomography neuroimaging. Compared with controls, maternally separated monkeys showed lower responding during the acquisition of self-administration and in the dose-response curves for both stimulants, and significantly lower response rates during maintenance of cocaine self-administration. Maternally separated monkeys also failed to exhibit stimulant-induced increases in motor activity. Groups did not differ in dopamine D2 receptor binding potential in the caudate nucleus or the putamen. Taken together, the results of this study do not provide support for early life stress leading to enhanced vulnerability to stimulant use in the nonhuman primate model employed.

The Immunomodulatory Metabolite Itaconate Modifies NLRP3 and Inhibits Inflammasome Activation.

The Krebs cycle-derived metabolite itaconate is highly upregulated in inflammatory macrophages and exerts immunomodulatory effects through cysteine modifications on target proteins. The NLRP3 inflammasome, which cleaves IL-1ß, IL-18, and gasdermin D, must be tightly regulated to avoid excessive inflammation. Here we provide evidence that itaconate modifies NLRP3 and inhibits inflammasome activation. Itaconate and its derivative, 4-octyl itaconate (4-OI), inhibited NLRP3 inflammasome activation, but not AIM2 or NLRC4. Conversely, NLRP3 activation was increased in itaconate-depleted Irg1-/- macrophages. 4-OI inhibited the interaction between NLRP3 and NEK7, a key step in the activation process, and "dicarboxypropylated" C548 on NLRP3. Furthermore, 4-OI inhibited NLRP3-dependent IL-1ß release from PBMCs isolated from cryopyrin-associated periodic syndrome (CAPS) patients, and reduced inflammation in an in vivo model of urate-induced peritonitis. Our results identify itaconate as an endogenous metabolic regulator of the NLRP3 inflammasome and describe a process that may be exploited therapeutically to alleviate inflammation in NLRP3-driven disorders.

Impaired reward responsiveness in schizophrenia.

BACKGROUND: Anhedonia is a core negative symptom of schizophrenia. Schizophrenia patients report largely intact pleasure in consuming rewards, but have impairments in generating motivated behavior to pursue rewards, and show reduced fMRI activation of the reward pathway during presentation of rewarded stimuli. A computer based task measuring the development of a response bias in favor of rewarded stimuli permits assessment of reward-induced motivation. We hypothesized that subjects with schizophrenia would be impaired on this task. METHODS: 58 schizophrenia subjects (SCZ) and 52 healthy controls (CON) were studied with a signal detection task to assess reward responsiveness. In multiple trials over three blocks subjects were asked to correctly identify two stimuli that were paired with unequal chance of monetary reward. The critical outcome variable was response bias, the development of a greater percent correct identification of the stimulus that was rewarded more often. RESULTS: An ANOVA on response bias with Block as a repeated-measures factor and Diagnosis as a between-group factor indicated that SCZ subjects achieved a lower bias to rewarded stimuli than CON subjects (F(1,105)=8.82, p=0.004, η2=0.078). Post hoc tests indicated that SCZ subjects had significantly impaired bias in Block 1 (p=0.002) and Block 2 (p=0.05), indicating that SCZ were slower to achieve normal levels of bias during the session. CONCLUSIONS: SCZ subjects were slower to develop response bias to rewarded stimuli than CON subjects. This finding is consonant with the hypothesis that people with schizophrenia have a blunted capacity to modify behavior in response to reward.

A polymerase chain reaction-based diagnostic to identify larvae and eggs of container mosquito species from the Australian region.

Dengue outbreaks occur regularly in parts of northern Queensland, Australia, and there is concern that these outbreaks may spread with the introduction and range expansion of the two main vectors Aedes aegypti (L.) and Aedes albopictus (Skuse). Problems encountered in separating larvae of endemic and exotic container mosquito species resulted in the development of a polymerase chain reaction diagnostic procedure that uses a restriction enzyme to cut the internal transcribed spacer region 1 of the ribosomal DNA to separate Ae. aegypti and Ae. albopictus from a number of common local container mosquito species which can be used at any stage of the life cycle, including eggs up to 8 wk of age. Identification was possible using desiccated or alcohol-preserved specimens with a response time of < 24 h after receipt of the specimens.

HIF1α and metabolic reprogramming in inflammation.

HIF1α is a common component of pathways involved in the control of cellular metabolism and has a role in regulating immune cell effector functions. Additionally, HIF1α is critical for the maturation of dendritic cells and for the activation of T cells. HIF1α is induced in LPS-activated macrophages, where it is critically involved in glycolysis and the induction of proinflammatory genes, notably Il1b. The mechanism of LPS-stimulated HIF1α induction involves succinate, which inhibits prolyl hydroxylases (PHDs). Pyruvate kinase M2 (PKM2) is also induced and interacts with and promotes the function of HIF1α. In another critical inflammatory cell type, Th17 cells, HIF1α acts via the retinoic acid-related orphan receptor-γt (RORγt) to drive Th17 differentiation. HIF1α is therefore a key reprogrammer of metabolism in inflammatory cells that promotes inflammatory gene expression.

Area postrema undergoes dynamic postnatal changes in mice and humans.

The postnatal period in mammals represents a developmental epoch of significant change in the autonomic nervous system (ANS). This study focuses on postnatal development of the area postrema, a crucial ANS structure that regulates temperature, breathing, and satiety, among other activities. We find that the human area postrema undergoes significant developmental changes during postnatal development. To characterize these changes further, we used transgenic mouse reagents to delineate neuronal circuitry. We discovered that, although a well-formed ANS scaffold exists early in embryonic development, the area postrema shows a delayed maturation. Specifically, postnatal days 0-7 in mice show no significant change in area postrema volume or synaptic input from PHOX2B-derived neurons. In contrast, postnatal days 7-20 show a significant increase in volume and synaptic input from PHOX2B-derived neurons. We conclude that key ANS structures show unexpected dynamic developmental changes during postnatal development. These data provide a basis for understanding ANS dysfunction and disease predisposition in premature and postnatal humans.

Reducing mechanical activation-induced amorphisation of salbutamol sulphate by co-processing with selected carboxylic acids.

The unintentional generation of amorphous character in crystalline active pharmaceutical ingredients (APIs) is an adverse consequence of mechanical activation during dosage form manufacture. In this study, we assess and compare the ability of low glass transition temperature (Tg) dicarboxylic acids to mitigate amorphisation of a model API, salbutamol sulphate (SS), on both co-milling and co-mixing. SS processed alone, as well as co-milled and co-mixed composites of the API with glutaric acid (GA), adipic acid (AA) and pimelic acid (PA) were characterised by powder X-ray diffraction (pXRD), differential scanning calorimetry (DSC) and dynamic vapour sorption (DVS). Milling and dry mixing of SS both resulted in pXRD amorphous materials. No amorphous content of SS was detected by DVS on co-milling with 50% (w/w) GA, while amorphisation was more than halved, relative to the API milled alone, on co-milling with 50% (w/w) AA and PA, respectively. Co-mixing with each excipient also resulted in a decrease in API amorphicity, although the extent of reduction was considerably less compared to the co-milling experiments. The solubility (Solexcipient) of each excipient in amorphous SS was determined by thermal methods. No further reduction in API amorphisation was achieved on co-mixing with 50% (w/w) excipient, compared to concentrations corresponding to the solubility of each excipient in the amorphous API (SolGA=36%, SolAA=21%, SolPA=22%). PXRD confirmed gradual dissolution over time of GA in amorphous SS on co-mixing. In contrast to co-mixing, co-milling SS at excipient weight fractions above their respective solubilities in the amorphous drug resulted in further reductions in API amorphisation. This is thought to be due to the generation of a molecular dispersion of amorphous API, supersaturated with excipient, thereby leading to a more pronounced composite Tg lowering effect. The results indicate that co-processing with low Tg excipients is an effective strategy at minimising amorphisation of an API on mechanical activation.