Interactions between skin-resident dendritic and Langerhans cells and pain-sensing neurons.

Various immune cells in the skin contribute to its function as a first line of defense against infection and disease, and the skin's dense innervation by pain-sensing sensory neurons protects the host against injury or damage signals. Dendritic cells (DCs) are a heterogeneous population of cells that link the innate immune response to the adaptive response by capturing, processing, and presenting antigens to promote T-cell differentiation and activation. DCs are abundant across peripheral tissues, including the skin, where they are found in the dermis and epidermis. Langerhans cells (LCs) are a DC subset located only in the epidermis; both populations of cells can migrate to lymph nodes to contribute to broad immune responses. Dermal DCs and LCs are found in close apposition with sensory nerve fibers in the skin and express neurotransmitter receptors, allowing them to communicate directly with the peripheral nervous system. Thus, neuroimmune signaling between DCs and/or LCs and sensory neurons can modulate physiologic and pathophysiologic pathways, including immune cell regulation, host defense, allergic response, homeostasis, and wound repair. Here, we summarize the latest discoveries on DC- and LC-neuron interaction with neurons while providing an overview of gaps and areas not previously explored. Understanding the interactions between these 2 defence systems may provide key insight into developing therapeutic targets for treating diseases such as psoriasis, neuropathic pain, and lupus.

Advanced Dynamic Weight Bearing as an Observer-independent Measure of Hyperacute Hypersensitivity in Mice.

Background: Standard methods assessing pain in rodents are often observer dependent, potentially resulting in biased outcomes. Advanced dynamic weight bearing (ADWB) offers an observer-independent approach that can provide objective, reliable data in preclinical pain research. Aims: The aim of this study was to characterize the use of ADWB in assessing murine responses to allyl isothiocyanate (AITC)-induced hyperacute hypersensitivity and identify best practices for use of the device. Methods: Male C57BL/6J mice received intraplantar injections of saline or 0.1% AITC solution and were assessed using the ADWB system; simultaneous observer-dependent durations of paw licking and biting were measured. ADWB data were analyzed using the proprietary software from Bioseb and correlated to observer-dependent results, with parameters assessed to optimize data collected. Results: ADWB detected pain-directed changes in weight and surface area distribution in AITC-treated mice, with paw weight and surface area placement correlating to paw licking and biting. Optimization of adjustable threshold parameters allowed for reduced coefficients of variability and increased duration of validated data. Conclusions: The ADWB assay provides an efficient and unbiased measure of chemical-induced hyperacute hypersensitivity in mice. ADWB detection parameters influence amount of validated data and variability, a consideration for data analysis in future studies.

Contexte: Les méthodes standard d'évaluation de la douleur chez les rongeurs dépendent souvent de l'observateur, ce qui peut fausser les résultats. La mise en charge dynamique avancée offre une approche indépendante de l'observateur qui peut fournir des données objectives et fiables dans la recherche préclinique sur la douleur.Objectifs: L'objectif de cette étude était de caractériser l'utilisation de la mise en charge dynamique avancée dans l'évaluation des réponses murines à l'hypersensibilité hyperaiguë induite par l'isothiocyanate d'allyle et de répertorier les meilleures pratiques d'utilisation de l'appareil.Méthodes: Des souris C57BL/6J mâles ont reçu des injections intraplantaires de solution saline ou de solution d'isothiocyanate d'allyle à 0,1 % et ont été évaluées à l'aide du système de mise en charge dynamique avancée; les durées simultanées de léchage et de morsure des pattes, dépendantes de l'observateur, ont été mesurées. Les données obtenues par la mise en charge dynamique avancée ont été analysées à l'aide du logiciel propriétaire de Bioseb et corrélées aux résultats dépendants de l'observateur, avec des paramètres évalués pour optimiser les données collectées.Résultats: L'essai réalisé à l'aide de la mise en charge dynamique avancée a détecté des changements de poids et de distribution de surface liés à la douleur chez les souris traitées à l'isothiocyanate d'allyle, le poids des pattes et le placement de la surface étant corrélés au léchage et à la morsure des pattes. L'optimisation des paramètres de seuil ajustables a permis de réduire les coefficients de variabilité et d'augmenter la durée des données validées.Conclusion: L'essai réalisé à l'aide de la mise en charge dynamique avancée fournit une mesure efficace et impartiale de l'hypersensibilité hyperaiguë induite par les produits chimiques chez la souris. Les paramètres de détection du système de mise en charge dynamique avancée influencent la quantité de données validées et la variabilité, ce qui doit être pris en compte pour l'analyse des données dans les études futures.

Acute inflammatory response via neutrophil activation protects against the development of chronic pain.

The transition from acute to chronic pain is critically important but not well understood. Here, we investigated the pathophysiological mechanisms underlying the transition from acute to chronic low back pain (LBP) and performed transcriptome-wide analysis in peripheral immune cells of 98 participants with acute LBP, followed for 3 months. Transcriptomic changes were compared between patients whose LBP was resolved at 3 months with those whose LBP persisted. We found thousands of dynamic transcriptional changes over 3 months in LBP participants with resolved pain but none in those with persistent pain. Transient neutrophil-driven up-regulation of inflammatory responses was protective against the transition to chronic pain. In mouse pain assays, early treatment with a steroid or nonsteroidal anti-inflammatory drug (NSAID) also led to prolonged pain despite being analgesic in the short term; such a prolongation was not observed with other analgesics. Depletion of neutrophils delayed resolution of pain in mice, whereas peripheral injection of neutrophils themselves, or S100A8/A9 proteins normally released by neutrophils, prevented the development of long-lasting pain induced by an anti-inflammatory drug. Analysis of pain trajectories of human subjects reporting acute back pain in the UK Biobank identified elevated risk of pain persistence for subjects taking NSAIDs. Thus, despite analgesic efficacy at early time points, the management of acute inflammation may be counterproductive for long-term outcomes of LBP sufferers.

Spinal cord injury in mice affects central and peripheral pathology in a severity-dependent manner.

ABSTRACT: Chronic pain is a common medical complication experienced by those living with spinal cord injury (SCI) and leads to worsened quality of life. The pathophysiology of SCI pain is poorly understood, hampering the development of safe and efficacious therapeutics. We therefore sought to develop a clinically relevant model of SCI with a strong pain phenotype and characterize the central and peripheral pathology after injury. A contusion (50 kdyn) injury, with and without sustained compression (60 seconds) of the spinal cord, was performed on female C57BL/6J mice. Mice with compression of the spinal cord exhibited significantly greater heat and mechanical hypersensitivity starting at 7 days postinjury, concomitant with reduced locomotor function, compared with those without compression. Immunohistochemical analysis of spinal cord tissue revealed significantly less myelin sparing and increased macrophage activation in mice with compression compared with those without. As measured by flow cytometry, immune cell infiltration and activation were significantly greater in the spinal cord (phagocytic myeloid cells and microglia) and dorsal root ganglia (Ly6C+ monocytes) after compression injury. We also decided to investigate the gastrointestinal microbiome, as it has been shown to be altered in patients with SCI and has recently been shown to play a role in immune system maturation and pain. We found increased dysbiosis of the gastrointestinal microbiome in an injury severity-dependent manner. The use of this contusion-compression model of SCI may help advance the preclinical assessment of acute and chronic SCI pain and lead to a better understanding of mechanisms contributing to this pain.

Weight bearing as a measure of disease progression in experimental autoimmune encephalomyelitis.

Motor disability in multiple sclerosis is often modeled using experimental autoimmune encephalomyelitis (EAE) and assessed using the clinical score (CS), an observer-dependent tool that can lead to potential bias. The Advanced Dynamic Weight Bearing (ADWB) system was evaluated as an observer-independent measurement of EAE symptoms. ADWB detected weight shifts onto the front paws as mice develop hindlimb motor disability. CS and ADWB were strongly correlated, indicated that these measures are comparable and suggesting that ADWB may be an appropriate observer-independent tool for the assessment of EAE progression.

Oral delivery of fish oil in oil-in-water nanoemulsion: development, colloidal stability and modulatory effect on in vivo inflammatory induction in mice.

To improve the oral absorption of fish oil and test its anti-inflammatory effect, a fish oil nanoemulsion was developed using cis-4,7,10,13,16,19-docosahexaenoic fatty acid as a biomarker for oral administration. The colloidal stability tests of the fish oil nanoemulsion showed an average size of 155.44 nm ±â€¯6.46 (4 °C); 163.04 nm ±â€¯9.97 (25 °C) and polydispersity index 0.22 ±â€¯0.02 (4 °C), 0.21 ±â€¯0.02 (25 °C), indicating systems with low polydispersity and stable droplets. The fish oil nanoemulsion did not alter the cell viability of the RAW 264.7 macrophages and, at a concentration of 0.024 mg/mL, was kinetically incorporated into the cells after 18 h of contact. The nanoemulsion was maintained in the gastrointestinal region for a significantly shorter period of time (p ≤ 0.05) compared to the intake of fish oil in free form. Inflammatory tests demonstrated that nanoemulsion and fish oil showed less (p ≤ 0.05) neutrophil infiltration after 24h of sepsis induction and there was a significant reduction (p ≤ 0.05) in the volume of paw edema in female adult Balb/c mice who received the nanoemulsion diet compared to the other experimental groups (control, formalin, fish oil and sunflower oil). These results indicate that the fish oil nanoemulsion was significantly effective in the dietary conditions tested here, presenting satisfactory responses in the modulation of inflammatory disorders, demonstrating interesting and beneficial nutraceutical effects.

Chronic mechanical hypersensitivity in experimental autoimmune encephalomyelitis is regulated by disease severity and neuroinflammation.

Chronic pain severely affects quality of life in more than half of people living with multiple sclerosis (MS). A commonly-used model of MS, experimental autoimmune encephalomyelitis (EAE), typically presents with hindlimb paralysis, neuroinflammation and neurodegeneration. However, this paralysis may hinder the use of pain behavior tests, with no apparent hypersensitivity observed post-peak disease. We sought to adapt the classic actively-induced EAE model to optimize its pain phenotype. EAE was induced with MOG35-55/CFA and 100-600 ng pertussis toxin (PTX), and mice were assessed for mechanical, cold and thermal sensitivity over a 28-day period. Spinal cord tissue was collected at 14 and 28 days post-injection to assess demyelination and neuroinflammation. Only mice treated with 100 ng PTX exhibited mechanical hypersensitivity. Hallmarks of disease pathology, including demyelination, immune cell recruitment, cytokine expression, glial activation, and neuronal damage were higher in EAE mice induced with moderate (200 ng) doses of pertussis toxin, compared to those treated with low (100 ng) levels. Immunostaining demonstrated activated astrocytes and myeloid/microglial cells in both EAE groups. These results indicate that a lower severity of EAE disease may allow for the study of pain behaviors while still presenting with disease pathology. By using this modified model, researchers may better study the mechanisms underlying pain.

A new brilliantly blue-emitting luciferin-luciferase system from Orfelia fultoni and Keroplatinae (Diptera).

Larvae of O. fultoni (Keroplatidae: Keroplatinae), which occur along river banks in the Appalachian Mountains in Eastern United States, produce the bluest bioluminescence among insects from translucent areas associated to black bodies, which are  located mainly in the anterior and posterior parts of the body. Although closely related to Arachnocampa spp (Keroplatidae: Arachnocampininae), O.fultoni has a morphologically and biochemically distinct bioluminescent system which evolved independently, requiring a luciferase enzyme, a luciferin, a substrate binding fraction (SBF) that releases luciferin in the presence of mild reducing agents, molecular oxygen, and no additional cofactors. Similarly, the closely related Neoceroplatus spp, shares the same kind of luciferin-luciferase system of Orfelia fultoni. However, the molecular properties, identities and functions of luciferases, SBF and luciferin of Orfelia fultoni and other  luminescent members of the Keroplatinae subfamily still remain to be fully elucidated. Using O. fultoni as a source of luciferase, and the recently discovered non-luminescent cave worm Neoditomiya sp as the main source of luciferin and SBF, we isolated and initially characterized these compounds. The luciferase of O. fultoni is a stable enzyme active as an apparent trimer (220 kDa) composed of ~70 kDa monomers, with an optimum pH of 7.8. The SBF, which is found in the black bodies in Orfelia fultoni and in smaller dark granules in Neoditomiya sp, consists of a high molecular weight complex of luciferin and proteins, apparently associated to mitochondria. The luciferin, partially purified from hot extracts by a combination of anion exchange chromatography and TLC, is a very polar and weakly fluorescent compound, whereas its oxidized product displays blue fluorescence with an emission spectrum matching the bioluminescence spectrum (~460 nm), indicating that it is oxyluciferin. The widespread occurrence of luciferin and SBF in both luminescent and non-luminescent Keroplatinae larvae indicate an additional important biological function for the substrate, and therefore the name keroplatin.

S100A9 plays a pivotal role in a mouse model of herpetic neuralgia via TLR4/TNF pathway.

Herpetic neuralgia is a painful condition following herpes zoster disease, which results from Varicella-zoster virus reactivation in the dorsal or trigeminal sensory ganglia. Nevertheless, the pathophysiological mechanisms involved in herpetic neuralgia are not well understood. Recently, we identified, that neuroimmune-glia interactions in the sensory ganglion is a critical mechanism for the development of herpetic neuralgia. Here, we investigate the contribution of S100A9, a well-known pro-inflammatory molecule produced by myeloid cells, for the development of herpetic neuralgia using a murine model of HSV-1 infection. We found that cutaneous HSV-1 infection results in an increase of S100A9 expression in the Dorsal Root Ganglia (DRGs). Infiltrating neutrophils into the DRGs were the main source of S100A9 post HSV-1 infection. Functionally, genetic or pharmacological inhibition of S100A9 impairs the development of HSV-1 infection-induced mechanical pain hypersensitivity. Finally, we found that the pronociceptive role of S100A9 in herpetic neuralgia depends on the TLR4/TNF pathway. These results unraveled previously unknown mechanisms involved in the pathophysiology of herpetic neuralgia and indicate that S100A9 might be an important target for novel therapies aiming acute herpetic neuralgia.

Synergistic Antitumor Efficacy of Magnetohyperthermia and Poly(lactic-co-glycolic acid)-Encapsulated Selol in Ehrlich Breast Adenocarcinoma Treatment in Elderly Swiss Mice.

Nanobiotechnology strategies for cancer treatments are currently being tested with increasing interest, except in elderly groups. It is well established that breast cancer incidence increases with age and that traditional therapies usually generate severe adverse effects, especially for elderly groups. To investigate if the benefits of nanotechnology could be extended to treating cancer in this group, citrate-coated maghemite nanoparticles (NpCit) were used for magnetohyperthermia (MHT) in combination with the administration of PLGA-Selol nanocapsule (NcSel), a formulation with antioxidant and antitumor activity. The combined therapies significantly inhibited breast Ehrlich tumor growth and prevented metastases to the lymph nodes, liver and lungs until 45 days after tumor induction, a better result than the group undergoing conventional drug treatment. The levels of TNF-α, associated with poor prognosis in Ehrlich tumor, were also normalized. Therefore, the results evidenced the potential use of these therapies for future clinical trials in elderly breast cancer patients.

Tumor cell death in orthotopic breast cancer model by NanoALA: a novel perspective on photodynamic therapy in oncology.

Aim: Nano-5-aminolevulic acid (NanoALA)-mediated photodynamic therapy (PDT), an oil-in-water polymeric nanoemulsion of ALA, was evaluated in a murine model of breast cancer. Materials & methods: Analysis of ALA-derived protoporphyrin IX production and acute toxicity test, biocompatibility and treatment efficacy, and long-term effect of NanoALA-PDT on tumor progression were performed. Results: The nanoformulation favored the prodrug uptake by tumor cells in a shorter time (1.5 h). As a result, the adverse effects were negligible and the response rates for primary mammary tumor control were significantly improved. Tumor progression was slower after NanoALA-PDT treatment, providing longer survival. Conclusion: NanoALA is a good proactive drug candidate for PDT against cancer potentially applied as adjuvant/neoadjuvant intervention strategy for breast cancer.

Comparison of the Malpighian tubules and fat body transcriptional profiles of Zophobas morio larvae (Coleoptera: Tenebrionidae).

The Malpighian tubules in insects play an essential role in osmoregulation, through the transport of ions during excretion, whereas the fat body is usually associated with the intermediary metabolism. The tubules also are involved in excretion of organic solutes and xenobiotics. However, with the exception of a preliminary transcriptional survey of the Zophobas morio (Tenebrionidae) larval tubules, there are no detailed transcriptional analysis of this organ in Coleoptera. A luciferase-like enzyme that displays weak luminescence activity in the presence of firefly D-luciferin and ATP was cloned from the tubules of Z. morio larvae. In order to better understand the molecular physiology of Malpighian tubules and fat body in Coleoptera larvae, and to investigate the occurrence and functions of AMP-CoA ligases in these tissues, we performed a comparative transcriptional analysis of these tissues using Z. morio giant-mealworms. As expected, the tubules displayed organic and inorganic transporters, xenobiotic metabolism enzymes, V-ATPases, channels, and pumps. The fat body showed proteins that are synthesized in this tissue and secreted to the hemolymph, as well as enzymes involved in lipid and carbohydrate metabolism. These tissues are also involved in common pathways, such as nitrogen metabolism to degradation/excretion, eye pigments biosynthesis, immunity, and detoxification. The presence of coumarate-CoA ligase-like enzymes in these tissues suggest their involvement in the degradation of coumaric acid derivatives obtained from the diet, or alternatively, in the biosynthesis of compounds structurally related to coumaric acids such as eye pigments. Our results confirm to the physiological versatility of tubules and fat body in larval Coleoptera.

Smoking-induced aggravation of experimental arthritis is dependent of aryl hydrocarbon receptor activation in Th17 cells.

BACKGROUND: Epidemiologic studies have highlighted the association of environmental factors with the development and progression of autoimmune and chronic inflammatory diseases. Among the environmental factors, smoking has been associated with increased susceptibility and poor prognosis in rheumatoid arthritis (RA). However, the immune and molecular mechanism of smoking-induced arthritis aggravation remains unclear. The transcription factor aryl hydrocarbon receptor (AHR) regulates the generation of Th17 cells, CD4 T cells linked the development of autoimmune diseases. AHR is activated by organic compounds including polycyclic aromatic hydrocarbons (PAHs), which are environmental pollutants that are also present in cigarette smoke. In this study, we investigated the role of AHR activation in the aggravation of experiment arthritis induced by exposure to cigarette smoke. METHODS: Mice were exposed to cigarette smoke during the developmental phase of antigen-induced arthritis and collagen-induced arthritis to evaluate the effects of smoking on disease development. Aggravation of articular inflammation was assessed by measuring neutrophil migration to the joints, increase in articular hyperalgesia and changes in the frequencies of Th17 cells. In vitro studies were performed to evaluate the direct effects of cigarette smoke and PAH on Th17 differentiation. We also used mice genetically deficient for AHR (Ahr KO) and IL-17Ra (Il17ra KO) to determine the in vivo mechanism of smoking-induced arthritis aggravation. RESULTS: We found that smoking induces arthritis aggravation and increase in the frequencies of Th17 cells. The absence of IL-17 signaling (Il17ra KO) conferred protection to smoking-induced arthritis aggravation. Moreover, in vitro experiments showed that cigarette smoke can directly increase Th17 differentiation of T cells by inducing AHR activation. Indeed, Ahr KO mice were protected from cigarette smoke-induced arthritis aggravation and did not display increase in TH17 frequencies, suggesting that AHR activation is an important mechanism for cigarette smoke effects on arthritis. Finally, we demonstrate that PAHs are also able to induce arthritis aggravation. CONCLUSIONS: Our data demonstrate that the disease-exacerbating effects of cigarette smoking are AHR dependent and environmental pollutants with AHR agonist activity can induce arthritis aggravation by directly enhancing Th17 cell development.

Neuroimmune-Glia Interactions in the Sensory Ganglia Account for the Development of Acute Herpetic Neuralgia.

Herpetic neuralgia is the most important symptom of herpes zoster disease, which is caused by Varicella zoster Nevertheless, the pathophysiological mechanisms involved in herpetic neuralgia are not totally elucidated. Here, we examined the neuroimmune interactions at the sensory ganglia that account for the genesis of herpetic neuralgia using a murine model of Herpes Simplex Virus Type-1 (HSV-1) infection. The cutaneous HSV-1 infection of mice results in the development of a zosteriform-like skin lesion followed by a time-dependent increase in pain-like responses (mechanical allodynia). Leukocytes composed mainly of macrophages and neutrophils infiltrate infected DRGs and account for the development of herpetic neuralgia. Infiltrating leukocytes are responsible for driving the production of TNF, which in turn mediates the development of herpetic neuralgia through downregulation of the inwardly rectifying K+ channel Kir4.1 in satellite glial cells. These results revealed that neuroimmune-glia interactions at the sensory ganglia play a critical role in the genesis of herpetic neuralgia. In conclusion, the present study elucidates novel mechanisms involved in the genesis of acute herpetic pain and open new avenues for its control.SIGNIFICANCE STATEMENT Acute herpetic neuralgia is the most important symptom of herpes zoster disease and it is very difficult to treat. Using a model of peripheral infection of mice with HSV-1, we have characterized for the first time the neuroimmune-glia interactions in the sensory ganglia that account for the development of acute herpetic neuralgia. Among these mechanisms, leukocytes composed mainly of macrophages and neutrophils infiltrate infected sensory ganglia and are responsible for driving the production of TNF. TNF, via TNFR1, mediates herpetic neuralgia development through downregulation of the inwardly rectifying K+ channel Kir4.1 in satellite glial cells. This study elucidates novel mechanisms involved in the genesis of acute herpetic neuralgia and open new avenues for its control.

A reduction in CD90 (THY-1) expression results in increased differentiation of mesenchymal stromal cells.

BACKGROUND: Mesenchymal stromal cells (MSCs) are multipotent progenitor cells used in several cell therapies. MSCs are characterized by the expression of CD73, CD90, and CD105 cell markers, and the absence of CD34, CD45, CD11a, CD19, and HLA-DR cell markers. CD90 is a glycoprotein present in the MSC membranes and also in adult cells and cancer stem cells. The role of CD90 in MSCs remains unknown. Here, we sought to analyse the role that CD90 plays in the characteristic properties of in vitro expanded human MSCs. METHODS: We investigated the function of CD90 with regard to morphology, proliferation rate, suppression of T-cell proliferation, and osteogenic/adipogenic differentiation of MSCs by reducing the expression of this marker using CD90-target small hairpin RNA lentiviral vectors. RESULTS: The present study shows that a reduction in CD90 expression enhances the osteogenic and adipogenic differentiation of MSCs in vitro and, unexpectedly, causes a decrease in CD44 and CD166 expression. CONCLUSION: Our study suggests that CD90 controls the differentiation of MSCs by acting as an obstacle in the pathway of differentiation commitment. This may be overcome in the presence of the correct differentiation stimuli, supporting the idea that CD90 level manipulation may lead to more efficient differentiation rates in vitro.

A new orange emitting luciferase from the Southern-Amazon Pyrophorus angustus (Coleoptera: Elateridae) click-beetle: structure and bioluminescence color relationship, evolutional and ecological considerations.

Bioluminescent click-beetles display a wide variation of bioluminescence colors ranging from green to orange, including an unusual intra-specific color variation in the Jamaican Pyrophorus plagiophthalamus. Recently, we collected individuals of the Pyrophorus angustus species from the Southern Amazon forest, in Brazil, which displays an orange light emitting abdominal lantern. This species was also previously described from Central America, but displaying a bioluminescence spectrum from 536 nm (dorsal) to 578 nm (ventral). The biogeographic variation of the bioluminescence color in this species could be an adaptation to environmental reflectance and inter/intraspecific sexual competition. Here, we cloned, sequenced, characterized and performed site-direct mutagenesis of this new orange emitting luciferase. The in vitro luciferase spectrum displayed a peak at 594 nm, KM values for ATP and d-luciferin of 160 µM and 17 µM, respectively, and an optimum pH of approximately 8.5. Comparative multialignment and site-directed mutagenesis using different color emitting click-beetle luciferases from P. angustus, Fulgeochlizus bruchi and Pyrearinus termitilluminans luciferases cloned by our group showed an integral role of residue 247 in bioluminescence color modulation.

Labeling mesenchymal cells with DMSA-coated gold and iron oxide nanoparticles: assessment of biocompatibility and potential applications.

BACKGROUND: Nanoparticles' unique features have been highly explored in cellular therapies. However, nanoparticles can be cytotoxic. The cytotoxicity can be overcome by coating the nanoparticles with an appropriated surface modification. Nanoparticle coating influences biocompatibility between nanoparticles and cells and may affect some cell properties. Here, we evaluated the biocompatibility of gold and maghemite nanoparticles functionalized with 2,3-dimercaptosuccinic acid (DMSA), Au-DMSA and γ-Fe2O3-DMSA respectively, with human mesenchymal stem cells. Also, we tested these nanoparticles as tracers for mesenchymal stem cells in vivo tracking by computed tomography and as agents for mesenchymal stem cells magnetic targeting. RESULTS: Significant cell death was not observed in MTT, Trypan Blue and light microscopy analyses. However, ultra-structural alterations as swollen and degenerated mitochondria, high amounts of myelin figures and structures similar to apoptotic bodies were detected in some mesenchymal stem cells. Au-DMSA and γ-Fe2O3-DMSA labeling did not affect mesenchymal stem cells adipogenesis and osteogenesis differentiation, proliferation rates or lymphocyte suppression capability. The uptake measurements indicated that both inorganic nanoparticles were well uptaken by mesenchymal stem cells. However, Au-DMSA could not be detected in microtomograph after being incorporated by mesenchymal stem cells. γ-Fe2O3-DMSA labeled cells were magnetically responsive in vitro and after infused in vivo in an experimental model of lung silicosis. CONCLUSION: In terms of biocompatibility, the use of γ-Fe2O3-DMSA and Au-DMSA as tracers for mesenchymal stem cells was assured. However, Au-DMSA shown to be not suitable for visualization and tracking of these cells in vivo by standard computed microtomography. Otherwise, γ-Fe2O3-DMSA shows to be a promising agent for mesenchymal stem cells magnetic targeting.

IL-33 signaling is essential to attenuate viral-induced encephalitis development by downregulating iNOS expression in the central nervous system.

BACKGROUND: Viral encephalitis is a common cause of lethal infections in humans, and several different viruses are documented to be responsible. Rocio virus is a flavivirus that causes a severe lethal encephalitis syndrome in humans and also mice, providing an interesting model to study the CNS compartmentalized immune response. Interleukin 33 (IL-33), a member of the IL-1 family, is an immunomodulatory cytokine that is highly expressed in the CNS. However, the role of IL-33 on viral encephalitis remains unclear. Therefore, we aimed to explore how the IL-33/ST2 axis regulates the local immune response during Rocio virus infection. METHODS: Wild-type (WT), ST2 (ST2(-/-)), and nitric oxide synthase-deficient mice (iNOS(-/-)) and Stat6 (Stat6(-/-))-deficient mice were infected with different concentrations of the Rocio virus by intraperitoneal route, the cytokine mRNA level in CNS was analyzed by qPCR, and cellular immunophenotyping was performed on infected mice by the flow cytometry of isolated CNS mononuclear cells. RESULTS: We have shown that the mRNA expression of IL-33 and ST2 receptors is increased in the CNS of Rocio virus-infected WT mice and that ST2(-/-) mice showed increased susceptibility to infection. ST2 deficiency was correlated with increased tissue pathology, cellular infiltration, and tumor necrosis factor alpha (TNF-α) and interferon-gamma (IFN-γ) mRNA levels and higher viral load in the CNS, compared with wild-type mice. The increased Th1 cytokine levels released in the CNS acted on infiltrating macrophages, as evidenced by flow cytometry characterization of cellular infiltrates, inducing the expression of iNOS, contributing to brain injury. Moreover, iNOS(-/-) mice were more resistant to Rocio virus encephalitis, presenting a lower clinical score and reduced mortality rate, despite the increased tissue pathology. CONCLUSIONS: We provide evidences of a specific role for IL-33 receptor signaling in nitric oxide induction through local IFN-γ modulation, suggesting that nitric oxide overproduction might have an important role in the progression of experimental viral encephalitis.