Injectable Thermosensitive Nanocomposites Based on Poly(N-vinylcaprolactam) and Silica Particles for Localized Release of Hydrophilic and Hydrophobic Drugs.

The systemic delivery of drugs employed by conventional methods has shown to be less effective than a localized delivery system. Many drugs have the effectiveness reduced by fast clearance, increasing the amount required for an efficient treatment. One way to overcome this drawback is through the use of thermoresponsive polymers that undergo a sol-gel transition at physiological temperature, allowing their injection directly in the desired site. In this work, thermosensitive nanocomposites based on poly(N-vinylcaprolactam) and silica particles with 80 and 330 nm were synthesized to be employed as delivery systems for hydrophobic (naringin) and hydrophilic (doxorubicin hydrochloride) drugs. The insertion of SiO2 increased the rheological properties of the nanocomposite at 37 °C, which helps to prevent its diffusion away from the site of injection. The synthesized materials were also able to control the drug release for a period of 7 days under physiological conditions. Due to its higher hydrophobicity and better interaction with the PNVCL matrix, naringin presented a more controlled release. The Korsmeyer-Peppas model indicated different release mechanisms for each drug. At last, a preliminary in vitro study of DOX-loaded nanocomposites cultured with L929 and MB49 cells showed negligible toxic effects on healthy cells and better efficient inhibition of carcinoma cells.

Analyzing the Effects of Silica Nanospheres on the Sol-Gel Transition Profile of Thermosensitive Hydrogels.

The insertion of nanoparticles into smart hydrogels can diversify their functionalities by a synergistic combination of the components properties within the hydrogels. While these hybrid systems are attractive to the biomaterials field, careful design and control of their properties are required since the new interactions between the polymer and the nanoparticles can result in changes or the loss of hydrogels stimuli response. In order to understand the physicochemical aspects of the thermoresponsive systems, nanocomposites of poly(N-vinylcaprolactam) (PNVCL) and silica nanoparticles with different sizes and concentrations were synthesized. The UV-vis and DLS techniques showed that the PNVCL has a sharp phase transition at 34 °C, while the nanocomposites have a diffuse transition. The nanocomposites showed an initial coil-globule transition before the phase transition takes place. This was identified by the evolution of the hydrodynamic diameter of the nanocomposite globules before the cloud point temperature (Tcp), which remained constant for PNVCL. This new transition profile can be described by two stages in which microscopic volume transitions occur first, followed by the macroscopic transition that forms the hydrogel. These results show that the proposed nanocomposites can be designed to have tunable stimuli response to smaller temperature variations with the formation of intermediate globule states.

Inhibition of the sphingosine-1-phosphate pathway promotes the resolution of neutrophilic inflammation.

Sphingosine-1-phosphate (S1P) is an important sphingolipid derived from plasma membrane and has a known role in productive phase of inflammation, but its role in neutrophil survival and resolution phase of inflammation is unknown. Here, we investigated the effects of inhibition of S1P receptors and the blockade of S1P synthesis in BALB/c mice and human neutrophils. S1P and S1PR1-3 receptors expression were increased in cells from the pleural cavity stimulated with LPS. Using different antagonists of S1PRs and inhibitors of different steps of the metabolic pathway of S1P production, we show that S1P and its receptors are involved in regulating neutrophil survival and resolution of inflammation in the pleural cavity. Given the role of the S1P-S1PR axis in resolution of inflammation, we sought to identify whether blockade at different levels of the sphingosine-1-phosphate synthesis pathway could affect neutrophil survival in vitro. Inhibitors of the S1P pathway were also able to induce human neutrophil apoptosis. In addition, blockade of S1P synthesis or its receptor facilitated the efferocytosis of apoptotic neutrophil. Taken together, our data demonstrate a fundamental role for S1P in regulating the outcome of inflammatory responses, and position S1P-S1PR axis as a potential target for treatment of neutrophilic inflammation.

Molecular basis of carrageenan-induced cytokines production in macrophages.

BACKGROUND: Low molecular weight carrageenan (Cg) is a seaweed-derived sulfated polysaccharide widely used as inflammatory stimulus in preclinical studies. However, the molecular mechanisms of Cg-induced inflammation are not fully elucidated. The present study aimed to investigate the molecular basis involved in Cg-induced macrophages activation and cytokines production. METHODS: Primary culture of mouse peritoneal macrophages were stimulated with Kappa Cg. The supernatant and cell lysate were used for ELISA, western blotting, immunofluorescence. Cg-induced mouse colitis was also developed. RESULTS: Here we show that Cg activates peritoneal macrophages to produce pro-inflammatory cytokines such as TNF and IL-1ß. While Cg-induced TNF production/secretion depends on TLR4/MyD88 signaling, the production of pro-IL-1ß relies on TLR4/TRIF/SYK/reactive oxygen species (ROS) signaling pathway. The maturation of pro-IL1ß into IL-1ß is dependent on canonical NLRP3 inflammasome activation via Pannexin-1/P2X7/K+ efflux signaling. In vivo, Cg-induced colitis was reduced in mice in the absence of NLRP3 inflammasome components. CONCLUSIONS: In conclusion, we unravel a critical role of the NLRP3 inflammasome in Cg-induced pro-inflammatory cytokines production and colitis, which is an important discovery on the pro-inflammatory properties of this sulfated polysaccharide for pre-clinical studies. Video abstract Carrageenan (Cg) is one the most used flogistic stimulus in preclinical studies. Nevertheless, the molecular basis of Cg-induced inflammation is not totally elucidated. Herein, Lopes et al. unraveled the molecular basis for Cg-induced macrophages production of biological active IL-1ß. The Cg-stimulated macrophages produces pro-IL-1ß depends on TLR4/TRIF/Syk/ROS, whereas its processing into mature IL-1ß is dependent on the canonical NLRP3 inflammasome.

Subversion of early innate antiviral responses during antibody-dependent enhancement of Dengue virus infection induces severe disease in immunocompetent mice.

Dengue is a mosquito-borne disease caused by one of four serotypes of Dengue virus (DENV-1-4). Epidemiologic and observational studies demonstrate that the majority of severe dengue cases, dengue hemorrhagic fever and dengue shock syndrome (DHF/DSS), occurs predominantly in either individuals with cross-reactive immunity following a secondary heterologous infection or in infants with primary DENV infections born from dengue-immune mothers, suggesting that B-cell-mediated and antibody responses impact on disease evolution. We demonstrate here that B cells play a pivotal role in host responses against primary DENV infection in mice. After infection, µMT(-/-) mice showed increased viral loads followed by severe disease manifestation characterized by intense thrombocytopenia, hemoconcentration, cytokine production and massive liver damage that culminated in death. In addition, we show that poly and monoclonal anti-DENV-specific antibodies can sufficiently increase viral replication through a suppression of early innate antiviral responses and enhance disease manifestation, so that a mostly non-lethal illness becomes a fatal disease resembling human DHF/DSS. Finally, treatment with intravenous immunoglobulin containing anti-DENV antibodies confirmed the potential enhancing capacity of subneutralizing antibodies to mediate virus infection and replication and induce severe disease manifestation of DENV-infected mice. Thus, our results show that humoral responses unleashed during DENV infections can exert protective or pathological outcomes and provide insight into the pathogenesis of this important human pathogen.

Measuring NLR Oligomerization II: Detection of ASC Speck Formation by Confocal Microscopy and Immunofluorescence.

Inflammasomes are crucial sentinels of the innate immune system that sense clues of infection, cellular stress, or metabolic imbalances. Upon activation, the inflammasome sensor (e.g., NLRP3) recruits the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC). ASC rapidly oligomerizes to form a micron-sized structure termed "ASC speck." These are crucial for the activation of caspase-1 and downstream inflammatory signals released following a specific form of lytic cell death called pyroptosis. Hence, due to their considerably large size, ASC specks can be easily visualized by microscopy as a simple upstream readout for inflammasome activation. Here, we provide three detailed protocols for imaging ASC specks: (1) live-cell imaging of macrophage cell lines expressing a fluorescent protein fusion form of ASC, (2) imaging of human primary cells using immunofluorescence staining of endogenous ASC, and (3) visualization and quantification of specks on a single-cell level using imaging flow cytometry.

Modified Titanium Dioxide as a Potential Visible-Light-Activated Photosensitizer for Bladder Cancer Treatment.

Low oxygen concentration inside the tumor microenvironment represents a major barrier for photodynamic therapy of many malignant tumors, especially urothelial bladder cancer. In this context, titanium dioxide, which has a low cost and can generate high ROS levels regardless of local O2 concentrations, could be a potential type of photosensitizer for treating this type of cancer. However, the use of UV can be a major disadvantage, since it promotes breakage of the chemical bonds of the DNA molecule on normal tissues. In the present study, we focused on the cytotoxic activities of a new material (Ti(OH)4) capable of absorbing visible light and producing high amounts of ROS. We used the malignant bladder cell line MB49 to evaluate the effects of multiple concentrations of Ti(OH)4 on the cytotoxicity, proliferation, migration, and production of ROS. In addition, the mechanisms of cell death were investigated using FACS, accumulation of lysosomal acid vacuoles, caspase-3 activity, and mitochondrial electrical potential assays. The results showed that exposure of Ti(OH)4 to visible light stimulates the production of ROS and causes dose-dependent necrosis in tumor cells. Also, Ti(OH)4 was capable of inhibiting the proliferation and migration of MB49 in low concentrations. An increase in the mitochondrial membrane potential associated with the accumulation of acid lysosomes and low caspase-3 activity suggests that type II cell death could be initiated by autophagic dysfunction mechanisms associated with high ROS production. In conclusion, the characteristics of Ti(OH)4 make it a potential photosensitizer against bladder cancer.

Prevention of self-grooming in rats: Evidence for a rebound effect.

Rodent self-grooming is a stereotyped behavior that can rise due to stressors such as novelty. In the present study, the occurrence of a rebound effect was investigated by means of manipulation of contextual novelty and of the possibility of self-grooming (with an Elizabethan collar which blocked head-body contact). Fourty-six male rats were submitted to an experiment latter replicated with 43 females. Half of the animals were submitted to habituation sessions (30 min) to the test box in three days. The other half was similarly handled but habituated to small cages. On the fourth day, rats from both conditions were assigned to Elizabethan or sham collar groups. Each animal was observed for 15 min with its respective collar and for other 15 min with no collars. Increased locomotion was observed in the rats not habituated to the test box. Such habituation did not affect any grooming parameter. On the other hand, the animals wearing Elizabethan collars during the first half of the test, as compared to those which wore sham collars, showed increased self-grooming during the second half of the test (i.e., with no collars). Females showed pretty similar results. Present results, thus, evidence a rebound effect in self-grooming.

Nanobodies dismantle post-pyroptotic ASC specks and counteract inflammation in vivo.

Inflammasomes sense intracellular clues of infection, damage, or metabolic imbalances. Activated inflammasome sensors polymerize the adaptor ASC into micron-sized "specks" to maximize caspase-1 activation and the maturation of IL-1 cytokines. Caspase-1 also drives pyroptosis, a lytic cell death characterized by leakage of intracellular content to the extracellular space. ASC specks are released among cytosolic content, and accumulate in tissues of patients with chronic inflammation. However, if extracellular ASC specks contribute to disease, or are merely inert remnants of cell death remains unknown. Here, we show that camelid-derived nanobodies against ASC (VHHASC ) target and disassemble post-pyroptotic inflammasomes, neutralizing their prionoid, and inflammatory functions. Notably, pyroptosis-driven membrane perforation and exposure of ASC specks to the extracellular environment allowed VHHASC to target inflammasomes while preserving pre-pyroptotic IL-1ß release, essential to host defense. Systemically administrated mouse-specific VHHASC attenuated inflammation and clinical gout, and antigen-induced arthritis disease. Hence, VHHASC neutralized post-pyroptotic inflammasomes revealing a previously unappreciated role for these complexes in disease. VHHASC are the first biologicals that disassemble pre-formed inflammasomes while preserving their functions in host defense.

Profound downregulation of neural transcription factor Npas4 and Nr4a family in fetal mice neurons infected with Zika virus.

Zika virus (ZIKV) infection of neurons leads to neurological complications and congenital malformations of the brain of neonates. To date, ZIKV mechanism of infection and pathogenesis is not entirely understood and different studies on gene regulation of ZIKV-infected cells have identified a dysregulation of inflammatory and stem cell maintenance pathways. MicroRNAs (miRNAs) are post-transcriptional regulators of cellular genes and they contribute to cell development in normal function and disease. Previous reports with integrative analyses of messenger RNAs (mRNAs) and miRNAs during ZIKV infection have not identified neurological pathway defects. We hypothesized that dysregulation of pathways involved in neurological functions will be identified by RNA profiling of ZIKV-infected fetal neurons. We therefore used microarrays to analyze gene expression levels following ZIKV infection of fetal murine neurons. We observed that the expression levels of transcription factors such as neural PAS domain protein 4 (Npas4) and of three members of the orphan nuclear receptor 4 (Nr4a) were severely decreased after viral infection. We confirmed that their downregulation was at both the mRNA level and at the protein level. The dysregulation of these transcription factors has been previously linked to aberrant neural functions and development. We next examined the miRNA expression profile in infected primary murine neurons by microarray and found that various miRNAs were dysregulated upon ZIKV infection. An integrative analysis of the differentially expressed miRNAs and mRNAs indicated that miR-7013-5p targets Nr4a3 gene. Using miRmimics, we corroborated that miR-7013-5p downregulates Nr4a3 mRNA and protein levels. Our data identify a profound dysregulation of neural transcription factors with an overexpression of miR-7013-5p that results in decreased Nr4a3 expression, likely a main contributor to ZIKV-induced neuronal dysfunction.

Influenza A Virus as a Predisposing Factor for Cryptococcosis.

Influenza A virus (IAV) infects millions of people annually and predisposes to secondary bacterial infections. Inhalation of fungi within the Cryptococcus complex causes pulmonary disease with secondary meningo-encephalitis. Underlying pulmonary disease is a strong risk factor for development of C. gattii cryptococcosis though the effect of concurrent infection with IAV has not been studied. We developed an in vivo model of Influenza A H1N1 and C. gattii co-infection. Co-infection resulted in a major increase in morbidity and mortality, with severe lung damage and a high brain fungal burden when mice were infected in the acute phase of influenza multiplication. Furthermore, IAV alters the host response to C. gattii, leading to recruitment of significantly more neutrophils and macrophages into the lungs. Moreover, IAV induced the production of type 1 interferons (IFN-α4/ß) and the levels of IFN-γ were significantly reduced, which can be associated with impairment of the immune response to Cryptococcus during co-infection. Phagocytosis, killing of cryptococci and production of reactive oxygen species (ROS) by IAV-infected macrophages were reduced, independent of previous IFN-γ stimulation, leading to increased proliferation of the fungus within macrophages. In conclusion, IAV infection is a predisposing factor for severe disease and adverse outcomes in mice co-infected with C. gattii.

N-Methyl-d-Aspartate (NMDA) Receptor Blockade Prevents Neuronal Death Induced by Zika Virus Infection.

Zika virus (ZIKV) infection is a global health emergency that causes significant neurodegeneration. Neurodegenerative processes may be exacerbated by N-methyl-d-aspartate receptor (NMDAR)-dependent neuronal excitoxicity. Here, we have exploited the hypothesis that ZIKV-induced neurodegeneration can be rescued by blocking NMDA overstimulation with memantine. Our results show that ZIKV actively replicates in primary neurons and that virus replication is directly associated with massive neuronal cell death. Interestingly, treatment with memantine or other NMDAR blockers, including dizocilpine (MK-801), agmatine sulfate, or ifenprodil, prevents neuronal death without interfering with the ability of ZIKV to replicate in these cells. Moreover, in vivo experiments demonstrate that therapeutic memantine treatment prevents the increase of intraocular pressure (IOP) induced by infection and massively reduces neurodegeneration and microgliosis in the brain of infected mice. Our results indicate that the blockade of NMDARs by memantine provides potent neuroprotective effects against ZIKV-induced neuronal damage, suggesting it could be a viable treatment for patients at risk for ZIKV infection-induced neurodegeneration.IMPORTANCE Zika virus (ZIKV) infection is a global health emergency associated with serious neurological complications, including microcephaly and Guillain-Barré syndrome. Infection of experimental animals with ZIKV causes significant neuronal damage and microgliosis. Treatment with drugs that block NMDARs prevented neuronal damage both in vitro and in vivo These results suggest that overactivation of NMDARs contributes significantly to the neuronal damage induced by ZIKV infection, and this is amenable to inhibition by drug treatment.

Rapid antigen tests for dengue virus serotypes and Zika virus in patient serum.

The recent Zika virus (ZIKV) outbreak demonstrates that cost-effective clinical diagnostics are urgently needed to detect and distinguish viral infections to improve patient care. Unlike dengue virus (DENV), ZIKV infections during pregnancy correlate with severe birth defects, including microcephaly and neurological disorders. Because ZIKV and DENV are related flaviviruses, their homologous proteins and nucleic acids can cause cross-reactions and false-positive results in molecular, antigenic, and serologic diagnostics. We report the characterization of monoclonal antibody pairs that have been translated into rapid immunochromatography tests to specifically detect the viral nonstructural 1 (NS1) protein antigen and distinguish the four DENV serotypes (DENV1-4) and ZIKV without cross-reaction. To complement visual test analysis and remove user subjectivity in reading test results, we used image processing and data analysis for data capture and test result quantification. Using a 30-µl serum sample, the sensitivity and specificity values of the DENV1-4 tests and the pan-DENV test, which detects all four dengue serotypes, ranged from 0.76 to 1.00. Sensitivity/specificity for the ZIKV rapid test was 0.81/0.86, respectively, using a 150-µl serum input. Serum ZIKV NS1 protein concentrations were about 10-fold lower than corresponding DENV NS1 concentrations in infected patients; moreover, ZIKV NS1 protein was not detected in polymerase chain reaction-positive patient urine samples. Our rapid immunochromatography approach and reagents have immediate application in differential clinical diagnosis of acute ZIKV and DENV cases, and the platform can be applied toward developing rapid antigen diagnostics for emerging viruses.

Lipoxin A4 Is Increased in the Plasma of Preeclamptic Women.

BACKGROUND: Excessive inflammation is involved in preeclampsia (PE) pathogenesis. Lipoxin A4 (LXA4) is an eicosanoid that counter-regulates inflammation. The main objective of this study was to determine LXA4 plasma levels in PE women. The correlations among LXA4 levels, ultrasensitive C-reactive protein (us-CRP) levels, and clinical/laboratory parameters of the studied participants were also investigated. METHODS: LXA4 plasma levels were determined by ELISA in 23 nonpregnant, 26 normotensive pregnant, and 27 PE women (early PE (N = 10) and late PE (N = 17)), according to gestational age (GA) at clinical symptoms onset). The clinical/laboratory parameters included in Spearman's correlation analysis were: systolic and diastolic blood pressure (SBP and DBP, respectively), lactate dehydrogenase (LDH) activity, platelet count, proteinuria, and white blood cell count (WBC). RESULTS: LXA4 levels were higher in PE women than in nonpregnant and normotensive pregnant women, and similar between nonpregnant and normotensive pregnant women. LXA4 plasma levels were higher in early PE vs. normotensive pregnancy (GA < 34 weeks) and in late PE vs. normotensive pregnancy (GA ≥ 34 weeks). No significant differences were detected between early and late PE. LXA4 levels were positively correlated with us-CRP levels, SBP, DBP, and WBC. No significant correlation was detected between LXA4 levels and the other laboratory parameters. CONCLUSIONS: Chronic inflammation in PE, in spite of increased levels of LXA4, points to a possible failure in this regulatory pathway. Further studies are necessary to clarify this issue and to evaluate the role of LXA4 and other proresolving mediators of inflammation in the pathogenesis of PE.

Nicorandil inhibits neutrophil recruitment in carrageenan-induced experimental pleurisy in mice.

Nicorandil is a drug characterized by the coupling of a nitric oxide (NO) donor to nicotinamide. We have previously demonstrated that nicotinamide exhibits activity in different models of pain and inflammation. Now, we investigated the effects induced by per os (p.o.) administration of nicorandil (25, 50 or 100mg/Kg) on neutrophil recruitment in a carrageenan-induced model of pleurisy in mice. Effects induced by nicorandil (100mg/kg) were compared with those induced by equimolar doses of nicotinamide (58mg/kg) and N-(2-hydroxyethyl)-nicotinamide (NHN; 79mg/kg). We also investigated whether effects on the production of inflammatory mediators play a role in the activity of nicorandil. P.o. nicorandil, 0.5h before and 1h after the i.pl. injection of carrageenan, reduced neutrophil recruitment. However, equimolar doses of nicotinamide or NHN failed to induce such effect. Single treatment (previous or late) with nicorandil (100mg/Kg, p.o.) also reduced neutrophils recruitment, although to a lesser extent when compared to the double treatment. Nicorandil reduced the concentrations of interleukin-1ß, CXCL-1 and prostaglandin E2 in the pleural exudate. Concluding, we demonstrated the activity of nicorandil in a model of pleurisy induced by carrageenan. This activity was characterized by reduction of the neutrophil accumulation and inhibition of production of inflammatory mediators. The effects induced by nicorandil on the leukocytes recruitment and production of inflammatory mediators contribute to a better understanding of its clinical benefits and indicate that these benefits may be due to its vasodilating and anti-inflammatory activities.

Activities of 2-phthalimidethanol and 2-phthalimidethyl nitrate, phthalimide analogs devoid of the glutarimide moiety, in experimental models of inflammatory pain and edema.

The reintroduction of thalidomide in the pharmacotherapy greatly stimulated the interest in the synthesis and pharmacological evaluation of phthalimide analogs with new and improved activities and also greater safety. In the present study, we evaluated the activities of two phthalimide analogs devoid of the glutarimide ring, namely 2-phthalimidethanol (PTD-OH) and 2-phthalimidethyl nitrate (PTD-NO), in experimental models of inflammatory pain and edema in male C57BL/6J mice. Intraplantar (i.pl.) injection of carrageenan (300 µg) induced mechanical allodynia and this response was inhibited by previous per os (p.o.) administration of PTD-OH and PTD-NO (750 mg/kg) and also by thalidomide (500 or 750 mg/kg). The edema induced by carrageenan was also inhibited by previous p.o. administration of PTD-OH (500 and 750 mg/kg) and PTD-NO (125, 250, 500 or 750 mg/kg), but not by thalidomide. Carrageenan increased tumor necrosis factor (TNF)-α and CXCL1 concentrations and also the number of neutrophils in the paw tissue. Previous p.o. administration of PTD-NO (500 mg/kg) reduced all the parameters, while PTD-OH (500 mg/kg) reduced only the accumulation of neutrophils. Thalidomide, on the other hand, was devoid of effect on these biochemical parameters. Plasma concentrations of nitrite were increased after p.o. administration of the phthalimide analog coupled to a NO donor, PTD-NO (500 mg/kg), but not after administration of PTD-OH or thalidomide. In conclusion, our results show that small molecules, structurally much simpler than thalidomide or many of its analogs under investigation, exhibit similar activities in experimental models of pain and inflammation. Finally, as there is evidence that the glutarimide moiety contributes to the teratogenic effect of many thalidomide analogs, our results indicate that phthalimide analogs devoid of this functional group could represent a new class of analgesic and anti-inflammatory candidates with potential greater safety.