Search

Drug‐mediated or medical condition‐mediated disruption of hERG function accounts for the main cause of acquired long‐QT syndrome (acLQTs), which predisposes affected individuals to ventricular arrhythmias (VA) and sudden death. Many Chinese herbal medicines, especially alkaloids, have risks of arrhythmia in clinical application. The characterized mechanisms behind this adverse effect are frequently associated with inhibition of cardiac hERG channels. The present study aimed to assess the potent effect of Rutaecarpine (Rut) on hERG channels. hERG‐HEK293 cell was applied for evaluating the effect of Rut on hERG channels and the underlying mechanism. hERG current (IhERG) was measured by patch‐clamp technique. Protein levels were analysed by Western blot, and the phosphorylation of Sp1 was determined by immunoprecipitation. Optical mapping and programmed electrical stimulation were used to evaluate cardiac electrophysiological activities, such as APD, QT/QTc, occurrence of arrhythmia, phase singularities (PSs), and dominant frequency (DF). Our results demonstrated that Rut reduced the IhERG by binding to F656 and Y652 amino acid residues of hERG channel instantaneously, subsequently accelerating the channel inactivation, and being trapped in the channel. The level of hERG channels was reduced by incubating with Rut for 24 hours, and Sp1 in nucleus was inhibited simultaneously. Mechanismly, Rut reduced threonine (Thr)/ tyrosine (Tyr) phosphorylation of Sp1 through PI3K/Akt pathway to regulate hERG channels expression. Cell‐based model unables to fully reveal the pathological process of arrhythmia. In vivo study, we found that Rut prolonged QT/QTc intervals and increased induction rate of ventricular fibrillation (VF) in guinea pig heart after being dosed Rut for 2 weeks. The critical reasons led to increased incidence of arrhythmias eventually were prolonged APD90 and APD50 and the increase of DF, numbers of PSs, incidence of early after‐depolarizations (EADs). Collectively, the results of this study suggest that Rut could reduce the IhERG by binding to hERG channels through F656 and Y652 instantaneously. While, the PI3K/Akt/Sp1 axis may play an essential role in the regulation of hERG channels, from the perspective of the long‐term effects of Rut (incubating for 24 hours). Importantly, the changes of electrophysiological properties by Rut were the main cause of VA.

Hanper ewes that were either monotocous or polytocous provided ovarian follicles of diameter 3 mm in the follicular phase and, in the luteal phase, samples of corpora lutea that had developed from follicles of diameter 3 mm. Differentially expressed mRNAs (monotocous versus polytocous) were then identified, and their functions were predicted. Results showed that 1508 mRNAs were differentially expressed in the follicular phase, with 885 being in the luteal tissues. Those which were differentially expressed in the follicular phase were mainly involved in the regulation of the ferroptosis and lysosome signalling pathways, whereas, for the luteal tissue, the differentially expressed mRNAs were mainly involved in the regulation of steroid biosynthesis. Based on the results, it was inferred that these pathways could explain variations in the fecundity of sheep.

The global impact of malaria remains staggering despite extensive efforts to eradicate the disease. With increasing drug resistance and the absence of a clinically available vaccine, there is an urgent need for novel, affordable, and safe drugs for prevention and treatment of malaria. Previously, we described a novel antimalarial acridone chemotype that is potent against both blood-stage and liver-stage malaria parasites. Here, we describe an optimization process that has produced a second-generation acridone series with significant improvements in efficacy, metabolic stability, pharmacokinetics, and safety profiles. These findings highlight the therapeutic potential of dual-stage targeting acridones as novel drug candidates for further preclinical development.

Thioridazine (THIO) is a phenothiazine derivative that is mainly used for the treatment of psychotic disorders. However, cardiac arrhythmias especially QT interval prolongation associated with the application of this compound have received serious attention after its introduction into clinical practice, and the mechanisms underlying the cardiotoxicity induced by THIO have not been well defined. The present study was aimed at exploring the long-term effects of THIO on the hERG and L-type calcium channels, both of which are relevant to the development of QT prolongation. The hERG current (IhERG) and the calcium current (ICa‐L) were measured by patch clamp techniques. Protein levels were analyzed by Western blot, and channel-chaperone interactions were determined by coimmunoprecipitation. Reactive oxygen species (ROS) were determined by flow cytometry and laser scanning confocal microscopy. Our results demonstrated that THIO induced hERG channel deficiency but did not alter channel kinetics. THIO promoted ROS production and stimulated endoplasmic reticulum (ER) stress and the related proteins. The ROS scavenger N-acetyl cysteine (NAC) significantly attenuated hERG reduction induced by THIO and abolished the upregulation of ER stress marker proteins. Meanwhile, THIO increased the degradation of hERG channels via disrupting hERG-Hsp70 interactions. The disordered hERG proteins were degraded in proteasomes after ubiquitin modification. On the other hand, THIO increased ICa‐L density and intracellular Ca2+ ([Ca2+]i) in neonatal rat ventricular cardiomyocytes (NRVMs). The specific CaMKII inhibitor KN-93 attenuated the intracellular Ca2+ overload, indicating that ROS-mediated CaMKII activation promoted calcium channel activation induced by THIO. Optical mapping analysis demonstrated the slowing effects of THIO on cardiac repolarization in mouse hearts. THIO significantly prolonged APD50 and APD90 and increased the incidence of early afterdepolarizations (EADs). In human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), THIO also resulted in APD prolongation. In conclusion, dysfunction of hERG channel proteins and activation of L-type calcium channels via ROS production might be the ionic mechanisms for QT prolongation induced by THIO.

The influence of ultrasound-assisted immersion freezing (UF), immersion freezing (IF), and air freezing (AF) on the protein oxidation, structure, and thermal stability of chicken breast during frozen storage was evaluated in this study. Compared to IF and AF samples, the UF samples had a lower carbonyl content, dityrosine content, and surface hydrophobicity of myofibrillar protein (MP) (P 0.05), as well as a higher free amino group content and total and reactive sulfhydryl content (P 0.05). Moreover, UF significantly delayed the deterioration of protein secondary and tertiary structures and the decrease in protein thermal stability during frozen storage (P 0.05). Additionally, the UF samples at 180 days had similar protein structures and quality characteristics to the IF samples at 90 days or the AF samples at 60 days. Overall, UF treatment can effectively retard protein oxidation, protein structure deterioration, and protein thermal stability loss caused by frozen storage.

AIMS: Small molecule compound tyrphostin A9 (A9), an inhibitor of platelet-derived growth factor (PDGF) receptor, was previously reported by our group to stimulate extracellular signal-regulated kinase 1 (ERK1) and 2 (ERK2) in neuronal cells in a PDGF receptor-irrelevant manner. The study aimed to investigate whether A9 could protect axons in experimental autoimmune encephalomyelitis through activation of ERKs. MAIN METHODS: A9 treatment on the protection on neurite outgrowth in SH-SY5Y neuroblastoma cells and primary substantia nigra neuron cultures from the neurotoxin MPP+ were analyzed. Then, clinical symptoms as well as ERK1/2 activation, axonal protection induction, and the abundance increases of the regeneration biomarker GAP-43 in the CNS in the relapsing-remitting experimental autoimmune encephalomyelitis (EAE) model were verified. KEY FINDINGS: A9 treatment could stimulate neurite outgrowth in SH-SY5Y neuroblastoma cells and protect primary substantia nigra neuron cultures from the neurotoxin MPP(+). In the relapsing-remitting EAE model, oral administration of A9 successfully ameliorated clinical symptoms, activated ERK1/2, induced axonal protection, and increased the abundance of the regeneration biomarker GAP-43 in the CNS. Interestingly, gene deficiency of ERK1 or ERK2 disrupted the beneficial effects of A9 in MOG-35–55-induced EAE. SIGNIFICANCE: These results demonstrated that small molecule compounds that stimulate persistent ERK activation in vitro and in vivo may be useful in protective or restorative treatment for neurodegenerative diseases.

Robenidine is a veterinary drug used in the poultry industry to treat coccidiosis caused by parasites in the

Litter size is an important trait that determines the production efficiency of sheep bred for meat. Its detailed investigation can reveal the molecular mechanisms that control the fecundity of sheep and possibly accelerate the breeding process of new varieties of sheep that have high prolificacy. Long non-coding RNAs (lncRNAs) have proven to be an important factor in the regulation of follicular development. However, the mechanisms by which lncRNAs regulate litter size in sheep remain unclear. In the present study, ovarian tissues from the follicular (F) or luteal phase (L) of Hanper sheep that were either monotocous (M) or polytocous (P; FM, FP, LM and LP groups) were collected and sequenced to identify differentially expressed lncRNAs and predict their function. The results indicate that the number of up- and down-regulated lncRNAs in the follicular phase (FM vs. FP) was 95 and 111 and 109 and 49, respectively, in the luteal phase (LM vs. LP). The functional enrichment of the different lncRNAs coexpressed with mRNA was analysed. The results demonstrated that the KISS1-GnRH-LH/FSH-E2 and EGF-EGFR-RAS-PI3K signalling pathways promoted the initiation of the primordial period, follicular development and ovulation in the follicular phase (FM vs. FP). During the luteal phase (LM vs. LP), the production and development of the corpus luteum in ewes was influenced by the KITLG-KIT/FGF-FGFR/HGF-MET-RAS-ERK signalling pathway. STEM clustering functional enrichment analysis of the differentially expressed lncRNAs indicated that profile11 was principally enriched in the Cytokine-Jak-STAT, PDGF-PDGFR-PI3K and KITLG-KIT-RAS-ERK signalling pathways. By analysis of the differential expression of the lncRNAs and their expression in each group, lncRNAs Xist (loc101112291) and Gtl2 (loc101123329) were found to be highly expressed, suggesting that regulation of follicular development was mediated through methylation processes.

ELQ-300 is a preclinical antimalarial drug candidate that is active against liver, blood, and transmission stages of Plasmodium falciparum. While ELQ-300 is highly effective when administered in a low multi-dose regimen, poor aqueous solubility and high crystallinity have hindered its clinical development. To overcome its challenging physiochemical properties, a number of bioreversible alkoxycarbonate ester prodrugs of ELQ-300 were synthesized. These bioreversible prodrugs are converted to ELQ-300 by host and parasite esterase action in the liver and bloodstream of the host. One such alkoxycarbonate prodrug, ELQ-331, is curative against Plasmodium yoelii with a single low dose of 3 mg/kg in a murine model of patent malaria infection. ELQ-331 is at least as fully protective as ELQ-300 in a murine malaria prophylaxis model when delivered 24 hours before sporozoite inoculation at an oral dose of 1 mg/kg. Here, we show that ELQ-331 is a promising prodrug of ELQ-300 with improved physiochemical and metabolic properties and excellent potential for clinical formulation.

Aims Although surfactant protein-D (SP-D) is a pneumoprotein that is predominantly synthesized by type II epithelial cells in the lung, individuals with increased circulating levels of SP-D are at an elevated risk of mortality from ischemic heart disease. Whether SP-D contributes directly to atherosclerosis is unknown. We determined the effects of SP-D gene deletion in a mouse model of atherosclerosis. Methods and results SP-D knockout (KO) mice were crossed with hyperlipidemic and atherosclerosis-prone apolipoprotein E (ApoE) KO mice to generate SP-D/ApoE double knockout (DKO) mice. Mice were placed on a high-fat diet for 12 weeks beginning at 8 weeks of age. Compared with ApoE KO mice, SP-D/ApoE DKO mice had significantly less atherosclerosis with reduced macrophage accumulation, decreased local macrophage proliferation, and increased smooth muscle cell coverage in plaques. Interestingly, SP-D deficiency worsened hypercholesterolemia and induced obesity and insulin resistance but suppressed plasma interleukin-6 (IL-6) levels. SP-D deficiency also reduced blood monocytes and neutrophils counts in ApoE KO mice. Conclusion SP-D deficiency reduces atherosclerosis in part by decreasing the accumulation and proliferation of macrophages and by reducing IL-6 levels systemically. SP-D is a promising therapeutic target for cachectic COPD patients with elevated circulating SP-D levels who are at increased risk of cardiovascular morbidity and mortality.

BackgroundThe potential benefits of long-acting injectable chemoprotection (LAI-C) against malaria have been recently recognized, prompting a call for suitable candidate drugs to help meet this need. On the basis of its known pharmacodynamic and pharmacokinetic profiles after oral dosing, ELQ-331, a prodrug of the parasite mitochondrial electron transport inhibitor ELQ-300, was selected for study of pharmacokinetics and efficacy as LAI-C in mice.MethodsFour trials were conducted in which mice were injected with a single intramuscular dose of ELQ-331 or other ELQ-300 prodrugs in sesame oil with 1.2% benzyl alcohol; the ELQ-300 content of the doses ranged from 2.5 to 30 mg/kg. Initial blood stage challenges with Plasmodium yoelii were used to establish the model, but the definitive study measure of efficacy was outcome after sporozoite challenge with a luciferase-expressing P.yoelii, assessed by whole-body live animal imaging. Snapshot determinations of plasma ELQ-300 concentration ([ELQ-300]) were made after all prodrug injections; after the highest dose of ELQ-331 (equivalent to 30 mg/kg ELQ-300), both [ELQ-331] and [ELQ-300] were measured at a series of timepoints from 6 hours to 5 ½ months after injection.ResultsA single intramuscular injection of ELQ-331 outperformed four other ELQ-300 prodrugs and, at a dose equivalent to 30 mg/kg ELQ-300, protected mice against challenge with P. yoelii sporozoites for at least 4 ½ months. Pharmacokinetic evaluation revealed rapid and essentially complete conversion of ELQ-331 to ELQ-300, a rapidly achieved (< 6 hours) and sustained (4-5 months) effective plasma ELQ-300 concentration, maximum ELQ-300 concentrations far below the estimated threshold for toxicity, and a distinctive ELQ-300 concentration vs. time profile. Pharmacokinetic modeling indicates a high-capacity, slow-exchange tissue compartment which serves to accumulate and then slowly redistribute ELQ-300 into blood, and this property facilitates an extremely long period during which ELQ-300 concentration is sustained above a minimum fully-protective threshold (60-80 nM).ConclusionsExtrapolation of these results to humans clearly predicts that ELQ-331 should be capable of meeting and far-exceeding currently published duration-of-effect goals for antimalarial LAI-C. Allometric scaling from mice to humans would predict a several-fold enhancement in the relationship between duration-of-effect and dose, and available drug engineering and formulation technologies would be expected to offer significant improvement over the simple powder in sesame oil used here. Furthermore, the distinctive pharmacokinetic profile of ELQ-300 after treatment with ELQ-331 may facilitate durable protection using a variety of delivery and formulation options, and may enable protection for far longer than 3 months. Particularly in light of the favorable pharmacodynamic profile of ELQ-300, ELQ-331 warrants consideration as a leading prototype for LAI-C.

Malaria remains one of the deadliest diseases in the world today. Novel chemoprophylactic and chemotherapeutic antimalarials are needed to support the renewed eradication agenda. We have discovered a novel antimalarial acridone chemotype with dual-stage activity against both liver-stage and blood-stage malaria. Several lead compounds generated from structural optimization of a large library of novel acridones exhibit efficacy in the following systems: (1) picomolar inhibition of in vitro Plasmodium falciparum blood-stage growth against multidrug-resistant parasites; (2) curative efficacy after oral administration in an erythrocytic Plasmodium yoelii murine malaria model; (3) prevention of in vitro Plasmodium berghei sporozoite-induced development in human hepatocytes; and (4) protection of in vivo P. berghei sporozoite-induced infection in mice. This study offers the first account of liver-stage antimalarial activity in an acridone chemotype. Details of the design, chemistry, structure-activity relationships, safety, metabolic/pharmacokinetic studies, and mechanistic investigation are presented herein.

Porcine reproductive and respiratory syndrome (PRRS) caused by PRRS virus (PRRSV) is a severe infectious disease in the swine industry. PRRSV infection is mediated by porcine CD163 (pCD163). Scavenger receptor cysteine-rich domain 5 coded by exon 7 of pCD163 is essential for PRRSV infection. In this study, we generated CD163 exon 7 deleted (CD163E7D) pigs using CRISPR/Cas9 mediated homologous recombination and somatic cell nuclear transfer (SCNT). The deletion of exon 7 had no adverse effects on CD163-associated functions. Pigs were further challenged with a highly pathogenic PRRSV (HP-PRRSV) strain. The CD163E7D pigs exhibited mild clinical symptoms and had decreased viral loads in blood. All CD163E7D pigs survived the viral challenge, while all the WT pigs displayed severe symptoms, and 2 out of 6 WT pigs died during the challenge. Our results demonstrated that CD163 exon 7 deletion confers resistance to HP-PRRSV infection without impairing the biological functions of CD163.

Club (Clara) cell protein 16 (CC-16) is a protein that is synthesized predominantly in the lungs and is detectable in serum. Its expression decreases with lung injury and smoking, and is thus a marker of bronchial cell dysfunction.To evaluate the possibility of using serum CC-16 as a biomarker for disease progression in chronic obstructive pulmonary disease (COPD).We measured serum CC-16 levels from 4,724 subjects with mild-to-moderate airflow limitation in the Lung Health Study. Using a linear regression model, we determined the relationship of serum CC-16 concentrations to decline in lung function over 9 years. In addition, to determine whether CC-16 plays a major role in the pathogenesis of mild COPD, we exposed CC-16-deficient (-/-) mice to 6 months of cigarette smoke.Reduced serum concentrations of CC-16 were associated with accelerated decline in FEV1 over 9 years (P0.0001), and this association persisted after adjustments for age, sex, race, smoking status, airway reactivity, body mass index, and baseline FEV1 (P = 0.0002). However, CC-16(-/-) mice did not demonstrate an enhanced risk of emphysema or small airway remodeling in response to cigarette smoke.Serum CC-16 is associated with disease progression, and may assist in the identification of "rapid progressors." However, the absence of CC-16 does not appear to modify the risk of cigarette-related COPD in mice.

Antimalarial combination therapies play a crucial role in preventing the emergence of drug-resistant Plasmodium parasites. Although artemisinin-based combination therapies (ACTs) comprise the majority of these formulations, inhibitors of the mitochondrial cytochrome bc 1 complex (cyt bc 1 ) are among the few compounds that are effective for both acute antimalarial treatment and prophylaxis. There are two known sites for inhibition within cyt bc 1 : atovaquone (ATV) blocks the quinol oxidase (Q o ) site of cyt bc 1 , while some members of the endochin-like quinolone (ELQ) family, including preclinical candidate ELQ-300, inhibit the quinone reductase (Q i ) site and retain full potency against ATV-resistant Plasmodium falciparum strains with Q o site mutations. Here, we provide the first in vivo comparison of ATV, ELQ-300, and combination therapy consisting of ATV plus ELQ-300 (ATV:ELQ-300), using P. yoelii murine models of malaria. In our monotherapy assessments, we found that ATV functioned as a single-dose curative compound in suppressive tests whereas ELQ-300 demonstrated a unique cumulative dosing effect that successfully blocked recrudescence even in a high-parasitemia acute infection model. ATV:ELQ-300 therapy was highly synergistic, and the combination was curative with a single combined dose of 1 mg/kg of body weight. Compared to the ATV:proguanil (Malarone) formulation, ATV:ELQ-300 was more efficacious in multiday, acute infection models and was equally effective at blocking the emergence of ATV-resistant parasites. Ultimately, our data suggest that dual-site inhibition of cyt bc 1 is a valuable strategy for antimalarial combination therapy and that Q i site inhibitors such as ELQ-300 represent valuable partner drugs for the clinically successful Q o site inhibitor ATV.

Studies of Leishmania donovani have shown that both ornithine decarboxylase and spermidine synthase, two enzymes of the polyamine biosynthetic pathway, are critical for promastigote proliferation and required for maximum infection in mice. However, the importance of arginase (ARG), the first enzyme of the polyamine pathway in Leishmania , has not been analyzed in L. donovani . To test ARG function in intact parasites, we generated Δ arg null mutants in L. donovani and evaluated their ability to proliferate in vitro and trigger infections in mice. The Δ arg knockout was incapable of growth in the absence of polyamine supplementation, but the auxotrophic phenotype could be bypassed by addition of either millimolar concentrations of ornithine or micromolar concentrations of putrescine or by complementation with either glycosomal or cytosolic versions of ARG. Spermidine supplementation of the medium did not circumvent the polyamine auxotrophy of the Δ arg line. Although ARG was found to be essential for ornithine and polyamine synthesis, ornithine decarboxylase appeared to be the rate-limiting enzyme for polyamine production. Mouse infectivity studies revealed that the Δ arg lesion reduced parasite burdens in livers by an order of magnitude but had little impact on the numbers of parasites recovered from spleens. Thus, ARG is essential for proliferation of promastigotes but not intracellular amastigotes. Coupled with previous studies, these data support a model in which L. donovani amastigotes readily salvage ornithine and have some access to host spermidine pools, while host putrescine appears to be unavailable for salvage by the parasite.

Lung infections are associated with acute lung injury (ALI), systemic inflammation, and vascular events. Clinical studies suggest that statins improve health outcomes of patients with pneumonia and ALI. The mechanisms by which this occurs are unknown. The aim of this study was to determine whether statins attenuate systemic inflammation and cardiovascular dysfunction related to ALI in mice. Simvastatin (SS; 20 mg/kg) or vehicle solution was instilled intraperitoneally into mice 24 h before and again just prior to intratracheal LPS instillation (1 μg/g). These mice were then anesthetized with 2.0% isoflurane and underwent a short surgical procedure to instill LPS. Four hours later, IL-6 levels in bronchoalveolar lavage fluid and in arterial and venous serum were measured. Cardiac function was evaluated using 2-D echocardiography, and endothelial function was determined using wire myography on aortic sections. LPS induced a significant increase in serum IL-6 levels. SS reduced venous ( P = 0.040) but not arterial concentrations of IL-6 ( P = 0.112). SS improved the maximal vasodilatory response of the aorta to ACh ( P = 0.004) but not to sodium nitroprusside ( P = 1.000). SS also improved cardiac output ( P = 0.023). Vasodilatory response to ACh was impaired when aorta from untreated mice was incubated with ex vivo IL-6 ( P = 0.004), whereas in the aorta from mice pretreated with SS, the vasodilatory response did not change with IL-6 incubation ( P = 0.387). SS significantly improved LPS-induced cardiovascular dysfunction possibly by reducing systemic expression of IL-6 and its downstream signaling pathways. These findings may explain how statins improve health outcomes in patients with ALI.

The biological mechanisms responsible for an association between elevated concentrations of ambient particulate matter (PM) and increased cardiovascular morbidity and mortality remain unclear. Our laboratory showed that exposure to PM induces systemic inflammation that contributes to vascular dysfunction. This study was designed to determine whether the lung is a major source of systemic inflammatory mediators, using IL-6 as a surrogate marker. We also sought to determine the impact on vascular dysfunction after exposure to PM of less than 10 μm in diameter (PM(10)). C57BL/6 mice were intratracheally exposed to a single instillation of PM(10) (10 or 200 μg) or saline. Four hours or 24 hours after exposure, venous and arterial blood samples were simultaneously collected from the right atrium and descending aorta. Concentrations of IL-6 were measured in bronchoalveolar lavage fluid (BALF) and serum samples. Vascular functional responses to acetylcholine (ACh) and phenylephrine were measured in the abdominal aorta. Concentrations of IL-6 in BALF samples were increased at 4 and 24 hours after exposure to PM(10). At baseline, concentrations of IL-6 in venous blood were higher than those in arterial blood. Exposure to PM(10) reversed this arteriovenous gradient, 4 hours after exposure. The relaxation responses of the abdominal aorta to ACh decreased 4 hours after exposure to 200 μg PM(10). In IL-6 knockout mice, the instillation of recombinant IL-6 increased IL-6 concentrations in the blood, and exposure to PM(10) did not cause vascular dysfunction. These results support our hypothesis that exposure to PM(10) increases pulmonary inflammatory mediators that translocate to the circulation, contributing to systemic inflammation, with downstream effects such as vascular dysfunction.

Ovarian hormones, including progesterone, are known to have immunomodulatory and neuroprotective effects which may alter the disease course of experimental autoimmune encephalomyelitis (EAE). In the current study, we examined the treatment potential of progesterone beginning at the onset of EAE symptoms. Progesterone treated animals showed reduced peak disease scores and cumulative disease indices, and decreased inflammatory cytokine secretion (IL-2 and IL-17). In addition, increased production of IL-10 was accompanied by increased numbers of CD19+ cells and an increase in CD8+ cells. Decreased chemokine and chemokine receptor expression in the spinal cord also contributed to decreased lesions in the spinal cord.

The regulatory role of programmed death 1 (PD-1) was investigated in the development of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Typical EAE could be induced by immunization without pertussis toxin (PTX) in PD-1-null but not in wild-type (WT) mice. However, both strains developed a similar EAE phenotype when immunized with PTX or by adoptive transfer of pathogenic T cells. In WT mice that did not develop EAE after immunization without PTX, the frequency of CD4(+)FoxP3(+) Treg cells was boosted in the periphery but not in the thymus. This increase in Treg frequency was abrogated by PD-1 deficiency or inclusion of PTX. In addition, PD-1 expression was critical to in vitro conversion of naïve myelin-specific CD4 T cells into Treg cells and was directly related to Treg suppressive activity. Finally, PD-1 was markedly down-modulated in the periphery of WT mice after administration of PTX. Therefore, down-modulation of PD-1 in Treg cells may abrogate Treg-mediated immune suppression, permitting the activation of myelin-reactive T cells and induction of EAE.

It is widely postulated that systemic inflammation related to lung infections is largely caused by cytokine translocation from the lungs into the systemic circulation but there is a paucity of animal models to evaluate this hypothesis. In this proof-of-concept study, we developed a murine model to determine whether interleukin (IL)-6, a primary inflammatory cytokine, translocates following airway exposure to endotoxin. We collected central venous blood from the right atrium and arterial blood from the aorta simultaneously at 4 h and 24 h following intratracheal exposure to endotoxin (25 mg) and measured IL-6 in the serum and broncho-alveolar lavage (BAL) fluid (n = 33 mice). We repeated the experiment following 3 d of treatment with dexamethasone (n = 31 mice). Without stimulation, there was no significant arteriovenous gradient (3 pg/ml with interquartile range [IQR] of 3—5 pg/ml in arterial versus 18 pg/ml with IQR of 8—24 pg/ml in venous serum; P = 0.86). A significant arteriovenous difference was observed by 4 h post-exposure to endotoxin (2813 pg/ml with IQR of 1578—4316 pg/ml in arterial versus 1282 pg/ml with IQR of 778—2699 pg/ml in venous serum; P50.0001). The rise in the BAL IL-6 levels correlated with the increases in the arterial serum levels (P50.0001). Administration of intraperitoneal dexamethasone for 3 d attenuated the increased arteriovenous gradient. This murine model facilitates the estimation of cytokine translocation across the lungs and evaluation of compounds to modulate this gradient.

We show that an innate defense-regulator peptide (IDR-1) was protective in mouse models of infection with important Gram-positive and Gram-negative pathogens, including methicillin-resistant Staphylococcus aureus, vancomycin-resistant Enterococcus and Salmonella enterica serovar Typhimurium. When given from 48 h before to 6 h after infection, the peptide was effective by both local and systemic administration. Because protection by IDR-1 was prevented by in vivo depletion of monocytes and macrophages, but not neutrophils or B- and T-lymphocytes, we conclude that monocytes and macrophages are key effector cells. IDR-1 was not directly antimicrobial: gene and protein expression analysis in human and mouse monocytes and macrophages indicated that IDR-1, acting through mitogen-activated protein kinase and other signaling pathways, enhanced the levels of monocyte chemokines while reducing pro-inflammatory cytokine responses. To our knowledge, an innate defense regulator that counters infection by selective modulation of innate immunity without obvious toxicities has not been reported previously.

Single-dose therapies for malaria have been proposed as a way to reduce the cost and increase the effectiveness of antimalarial treatment. However, no compound to date has shown single-dose activity against both the blood-stage Plasmodium parasites that cause disease and the liver-stage parasites that initiate malaria infection. Here, we describe a subset of cytochrome bc1 (cyt bc1) inhibitors, including the novel 4(1H)-quinolone ELQ-400, with single-dose activity against liver, blood, and transmission-stage parasites in mouse models of malaria. Although cyt bc1 inhibitors are generally classified as slow-onset antimalarials, we found that a single dose of ELQ-400 rapidly induced stasis in blood-stage parasites, which was associated with a rapid reduction in parasitemia in vivo. ELQ-400 also exhibited a low propensity for drug resistance and was active against atovaquone-resistant P. falciparum strains with point mutations in cyt bc1. Ultimately, ELQ-400 shows that cyt bc1 inhibitors can function as single-dose, blood-stage antimalarials and is the first compound to provide combined treatment, prophylaxis, and transmission blocking activity for malaria after a single oral administration. This remarkable multi-stage efficacy suggests that metabolic therapies, including cyt bc1 inhibitors, may be valuable additions to the collection of single-dose antimalarials in current development.

ELQ-300 is a preclinical candidate that targets the liver and blood stages of Plasmodium falciparum , as well as the forms that are crucial to transmission of disease: gametocytes, zygotes, and ookinetes. A significant obstacle to the clinical development of ELQ-300 is related to its physicochemical properties. Its relatively poor aqueous solubility and high crystallinity limit absorption to the degree that only low blood concentrations can be achieved following oral dosing. While these low blood concentrations are sufficient for therapy, the levels are too low to establish an acceptable safety margin required by regulatory agencies for clinical development. One way to address the challenging physicochemical properties of ELQ-300 is through the development of prodrugs. Here, we profile ELQ-337, a bioreversible O-linked carbonate ester prodrug of the parent molecule. At the molar equivalent dose of 3 mg/kg of body weight, the delivery of ELQ-300 from ELQ-337 is enhanced by 3- to 4-fold, reaching a maximum concentration of drug in serum ( C max ) of 5.9 μM by 6 h after oral administration, and unlike ELQ-300 at any dose, ELQ-337 provides single-dose cures of patent malaria infections in mice at low-single-digit milligram per kilogram doses. Our findings show that the prodrug strategy represents a viable approach to overcome the physicochemical limitations of ELQ-300 to deliver the active drug to the bloodstream at concentrations sufficient for safety and toxicology studies, as well as achieving single-dose cures.

The historical antimalarial compound endochin served as a structural lead for optimization. Endochin-like quinolones (ELQ) were prepared by a novel chemical route and assessed for in vitro activity against multidrug resistant strains of Plasmodium falciparum and against malaria infections in mice. Here we describe the pathway to discovery of a potent class of orally active antimalarial 4(1H)-quinolone-3-diarylethers. The initial prototype, ELQ-233, exhibited low nanomolar IC50 values against all tested strains including clinical isolates harboring resistance to atovaquone. ELQ-271 represented the next critical step in the iterative optimization process, as it was stable to metabolism and highly effective in vivo. Continued analoging revealed that the substitution pattern on the benzenoid ring of the quinolone core significantly influenced reactivity with the host enzyme. This finding led to the rational design of highly selective ELQs with outstanding oral efficacy against murine malaria that is superior to established antimalarials chloroquine and atovaquone.

The goal for developing new antimalarial drugs is to find a molecule that can target multiple stages of the parasite's life cycle, thus impacting prevention, treatment, and transmission of the disease. The 4(1H)-quinolone-3-diarylethers are selective potent inhibitors of the parasite's mitochondrial cytochrome bc1 complex. These compounds are highly active against the human malaria parasites Plasmodium falciparum and Plasmodium vivax. They target both the liver and blood stages of the parasite as well as the forms that are crucial for disease transmission, that is, the gametocytes, the zygote, the ookinete, and the oocyst. Selected as a preclinical candidate, ELQ-300 has good oral bioavailability at efficacious doses in mice, is metabolically stable, and is highly active in blocking transmission in rodent models of malaria. Given its predicted low dose in patients and its predicted long half-life, ELQ-300 has potential as a new drug for the treatment, prevention, and, ultimately, eradication of human malaria.

Sontochin was the original chloroquine replacement drug, arising from research by Hans Andersag 2 years after chloroquine (known as “resochin” at the time) had been shelved due to the mistaken perception that it was too toxic for human use. We were surprised to find that sontochin, i.e., 3-methyl-chloroquine, retains significant activity against chloroquine-resistant strains of Plasmodium falciparum in vitro . We prepared derivatives of sontochin, “pharmachins,” with alkyl or aryl substituents at the 3 position and with alterations to the 4-position side chain to enhance activity against drug-resistant strains. Modified with an aryl substituent in the 3 position of the 7-chloro-quinoline ring, Pharmachin 203 (PH-203) exhibits low-nanomolar 50% inhibitory concentrations (IC 50 s) against drug-sensitive and multidrug-resistant strains and in vivo efficacy against patent infections of Plasmodium yoelii in mice that is superior to chloroquine. Our findings suggest that novel 3-position aryl pharmachin derivatives have the potential for use in treating drug resistant malaria.

COPD is a chronic inflammatory disorder with high risk of cardiovascular morbidity and mortality. Adiponectin is a hormone that has anti inflammatory, antidiabetic, and anti atherogenic activities. We investigated the relationship of serum adiponectin to health outcomes in COPD.We measured adiponectin levels in serum samples from participants of the Lung Health Study, who were smokers with mild to moderate airflow limitation. We determined the relationship of serum adiponectin to hospitalization and mortality using a Cox proportional hazards model and to baseline lung function measurements and bronchial reactivity using multiple regression methods.Serum adiponectin concentrations were inversely related to hospitalizations and mortality from coronary heart disease (hazard ratio [HR], 0.73; 95% CI, 0.62-0.86) and to cardiovascular disease (HR, 0.83; 95% CI, 0.73-0.94) and positively related to deaths from respiratory causes (HR, 2.09; 95% CI, 1.41-3.11). However, serum adiponectin concentrations were not significantly related to total mortality (HR, 1.10; 95% CI, 0.93-1.29) or cancer-related mortality(HR, 1.11; 95% CI, 0.92-1.34). Serum adiponectin concentrations were significantly related to increased bronchial reactivity and an accelerated decline in lung function (both P , .0001). Smoking status had no material influence on serum adiponectin concentrations.Adiponectin is a complex serum biomarker in COPD that is associated with decreased risk of cardiovascular events but increased risk of respiratory mortality. Because serum adiponectin is not significantly influenced by smoking status, it is a very promising biomarker of cardiovascular outcomes in COPD.

Exposure to tobacco carcinogens is causally associated with head and neck squamous cell carcinoma (HNSCC), but the underlying molecular mechanisms remain unclear. Here, we reported that AKT is activated at a higher frequency in both HNSCC tumors and the adjacent mucosa from HNSCC patients who are smokers than those from HNSCC patients who are non-smokers. Adding physiologically relevant concentrations of 4-(methylnitrosamino)-1-(3-pyridyl)-1-1butanone (NNK), a major tobacco carcinogen, to normal head and neck epithelial cells and HNSCC cell lines, rapidly and constitutively activated AKT through phosphorylation in a dose- and time-dependent manner. AKT phosphorylation was associated with activation of downstream signaling mediators BAD, MDM2, GSK-3β, mTOR. These alterations correlated with increased proliferation and decreased etoposide-induced apoptosis in NNK-exposed cells. Finally, NNK exposure to mouse head and neck epithelia resulted in epithelial hyperproliferation and reduced apoptosis, which is correlated with AKT activation. Our results suggest that AKT activation is an early event and plays a pivotal role in mediating tobacco-induced HNSCC carcinogenesis.

Acute lung injury (ALI) is associated with systemic inflammation and cardiovascular dysfunction. IL-6 is a biomarker of this systemic response and a predictor of cardiovascular events, but its possible causal role is uncertain. Inhaled corticosteroids and long-acting β2 agonists (ICS/LABA) down-regulate the systemic expression of IL-6, but whether they can ameliorate the cardiovascular dysfunction related to ALI is uncertain. We sought to determine whether IL-6 contributes to the cardiovascular dysfunction related to ALI, and whether budesonide/formoterol ameliorates this process. Wild-type mice were pretreated for 3 hours with intratracheal budesonide, formoterol, or both, before LPS was sprayed into their tracheas. IL-6-deficient mice were similarly exposed to LPS. Four hours later, bronchoalveolar lavage fluid (BALF) and serum were collected, and endothelial and cardiac functions were measured, using wire myography of the aortic tissue and echocardiography, respectively. LPS significantly impaired vasodilatory responses to acetylcholine (P < 0.001) and cardiac output (P = 0.002) in wild-type but not IL-6-deficient mice. Intratracheal instillations of exogenous IL-6 into IL-6-deficient mice restored these impairments (vasodilatory responses to acetylcholine, P = 0.005; cardiac output, P = 0.025). Pretreatment with the combination of budesonide and formoterol, but not either alone, ameliorated the vasodilatory responses to acetylcholine (P = 0.018) and cardiac output (P < 0.001). These drugs also attenuated the rise in the systemic expression of IL-6 (P < 0.05) related to LPS. IL-6 contributes to the cardiovascular dysfunction related to LPS, and pretreatment with budesonide/formoterol reduces the systemic expression of IL-6 and improves cardiovascular dysfunction. ICS/LABA may reduce acute cardiovascular events related to ALI.

Background Remission of multiple sclerosis during periods of high ovarian hormone secretion (such as pregnancy) has led to a great deal of interest in the potential for estrogens to treat autoimmune disease. Previous work has established that 17β-estradiol can inhibit onset of experimental autoimmune encephalomyelitis (EAE), while ethinyl estradiol (EE) can reduce the severity of established disease. In the current study, the influence of estrogen receptor-α (ERα) and the G-protein coupled estrogen receptor (GPR30 or GPER) on EE's ability to treat EAE was explored. Results EE reduced disease severity in wild-type and ERα knockout (ERKO) mice, but did not alter disease in the GPR30KO group. Production of anti-inflammatory IL-10 increased in EE-ERKO mice (which showed reduced disease) but not in EE-GPR30KO mice (who did not have improved disease). Conclusions Differential production of IL-10 following EE treatment in ERKO and GPR30KO animals may be responsible for the distinctly different effects on disease severity. Increased IL-10 in ERKO-EE compared to ERKO-Controls is likely to be an important factor in reducing established disease. The inability of EE to reduce disease in GPR30KO mice indicates an important but still undefined role for GPR30 in regulating immune reactivity.

The mechanism by which oestrogens suppress experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis, is only partially understood. We here demonstrate that treatment with 17beta-oestradiol (E(2)) in C57BL/6 mice boosted the expression of programmed death 1 (PD-1), a negative regulator of immune responses, in the CD4(+) FoxP3(+) regulatory T (Treg) cell compartment in a dose-dependent manner that correlated with the efficiency of EAE protection. Administration of E(2) at pregnancy levels but not lower concentrations also enhanced the frequency of Treg cells. Additionally, E(2) treatment drastically reduced the production of interleukin-17 (IL-17) in the periphery of immunized mice. However, E(2) treatment did not protect against EAE or suppress IL-17 production in PD-1 gene-deficient mice. Finally, E(2) failed to prevent Treg-deficient mice from developing spontaneous EAE. Taken together, our results suggest that E(2)-induced protection against EAE is mediated by upregulation of PD-1 expression within the Treg-cell compartment.

Although estrogens exert a pronounced protective effect on multiple sclerosis and its animal model, experimental autoimmune encephalomyelitis (EAE), their therapeutic application has been limited by undesirable side effects thought to be mediated primarily through estradiol binding to intracellular estrogen receptor α. In this study, we found that signaling through the putative membrane estrogen receptor, G protein-coupled receptor 30 (GPR30), was sufficient to mediate protection against EAE, which was significantly impaired in GPR30 gene-deficient mice. Treatment with G-1, an agonist that selectively activates GPR30 without engagement of the intracellular estrogen receptors, retained the ability of estradiol to protect against clinical and histological EAE without estradiol-associated side effects, deviated cytokine profiles, and enhanced suppressive activity of CD4+Foxp3+ T regulatory cells through a GPR30- and programmed death 1-dependent mechanism. This study is the first to evaluate the protective effect of GPR30 activation on EAE, and provides a strong foundation for the clinical application of GPR30 agonists such as G-1 in multiple sclerosis.

Epidemiologic and animal studies have shown that exposure to particulate matter air pollution (PM) is a risk factor for the development of atherosclerosis. Whether PM-induced lung and systemic inflammation is involved in this process is not clear. We hypothesized that PM exposure causes lung and systemic inflammation, which in turn leads to vascular endothelial dysfunction, a key step in the initiation and progression of atherosclerosis. New Zealand White rabbits were exposed for 5 days (acute, total dose 8 mg) and 4 wk (chronic, total dose 16 mg) to either PM smaller than 10 μm (PM10) or saline intratracheally. Lung inflammation was quantified by morphometry; systemic inflammation was assessed by white blood cell and platelet counts and serum interleukin (IL)-6, nitric oxide, and endothelin levels. Endothelial dysfunction was assessed by vascular response to acetylcholine (ACh) and sodium nitroprusside (SNP). PM10 exposure increased lung macrophages ( P < 0.02), macrophages containing particles ( P < 0.001), and activated macrophages ( P < 0.006). PM10 increased serum IL-6 levels in the first 2 wk of exposure ( P < 0.05) but not in weeks 3 or 4. PM10 exposure reduced ACh-related relaxation of the carotid artery with both acute and chronic exposure, with no effect on SNP-induced vasodilatation. Serum IL-6 levels correlated with macrophages containing particles ( P = 0.043) and ACh-induced vasodilatation ( P = 0.014 at week 1, P = 0.021 at week 2). Exposure to PM10 caused lung and systemic inflammation that were both associated with vascular endothelial dysfunction. This suggests that PM-induced lung and systemic inflammatory responses contribute to the adverse vascular events associated with exposure to air pollution.

Genomic approaches can be exploited to expose the complexities and conservation of biological systems such as the immune network across various mammalian species. In this study, temporal transcriptional expression profiles were analyzed in human and bovine monocytic cells in response to the TLR-4 agonist, LPS, in the presence or absence of their respective host defense peptides. The cathelicidin peptides, human LL-37 and bovine myeloid antimicrobial peptide-27 (BMAP-27), are homologs, yet they have diverged notably in terms of sequence similarity. In spite of their low sequence similarities, both of these cathelicidin peptides demonstrated potent, antiendotoxin activity in monocytic cells at low, physiologically relevant concentrations. Microarray studies indicated that 10 ng/ml LPS led to the up-regulation of 125 genes in human monocytes, 106 of which were suppressed in the presence of 5 μg/ml of the human peptide LL-37. To confirm and extend these data, temporal transcriptional responses to LPS were assessed in the presence or absence of the species-specific host defense peptides by quantitative real-time PCR. The transcriptional trends of 20 LPS-induced genes were analyzed in bovine and human monocytic cells. These studies demonstrated conserved trends of gene responses in that both peptides were able to profoundly suppress many LPS-induced genes. Consistent with this, the human and bovine peptides suppressed LPS-induced translocation of NF-κB subunits p50 and p65 into the nucleus of monocytic cells. However, there were also distinct differences in responses to LPS and the peptides; for example, treatment with 5 μg/ml BMAP-27 alone tended to influence gene expression (RELA, TNF-α-induced protein 2, MAPK phosphatase 1/dual specificity phosphatase 1, IκBκB, NFκBIL1, TNF receptor-associated factor 2) to a greater extent than did the same amount of human LL-37. We hypothesize that the immunomodulatory effects of the species-specific host defense peptides play a critical role in regulating inflammation and represent an evolutionarily conserved mechanism for maintaining homeostasis, although the sequence divergence of these peptides is substantial.