Skin-resident dendritic cells mediate postoperative pain via CCR4 on sensory neurons.

Inflammatory pain, such as hypersensitivity resulting from surgical tissue injury, occurs as a result of interactions between the immune and nervous systems with the orchestrated recruitment and activation of tissue-resident and circulating immune cells to the site of injury. Our previous studies identified a central role for Ly6Clow myeloid cells in the pathogenesis of postoperative pain. We now show that the chemokines CCL17 and CCL22, with their cognate receptor CCR4, are key mediators of this response. Both chemokines are up-regulated early after tissue injury by skin-resident dendritic and Langerhans cells to act on peripheral sensory neurons that express CCR4. CCL22, and to a lesser extent CCL17, elicit acute mechanical and thermal hypersensitivity when administered subcutaneously; this response abrogated by pharmacological blockade or genetic silencing of CCR4. Electrophysiological assessment of dissociated sensory neurons from naïve and postoperative mice showed that CCL22 was able to directly activate neurons and enhance their excitability after injury. These responses were blocked using C 021 and small interfering RNA (siRNA)-targeting CCR4. Finally, our data show that acute postoperative pain is significantly reduced in mice lacking CCR4, wild-type animals treated with CCR4 antagonist/siRNA, as well as transgenic mice depleted of dendritic cells. Together, these results suggest an essential role for the peripheral CCL17/22:CCR4 axis in the genesis of inflammatory pain via direct communication between skin-resident dendritic cells and sensory neurons, opening therapeutic avenues for its control.

γδ T Cells Modulate Myeloid Cell Recruitment but Not Pain During Peripheral Inflammation.

Circulating immune cells, which are recruited to the site of injury/disease, secrete various inflammatory mediators that are critical to nociception and pain. The role of tissue-resident immune cells, however, remains poorly characterized. One of the first cells to be activated in peripheral tissues following injury are γδT cells, which serve important roles in infection, disease, and wound healing. Using a mouse line lacking these cells, we sought to identify their contribution to inflammatory pain. Three distinct models of peripheral inflammatory pain were used: intraplantar injection of formalin (spontaneous inflammatory pain), incisional wound (acute inflammatory pain), and intraplantar injection of complete Freund's adjuvant (chronic inflammatory pain). Our results show that absence of γδT cells does not alter baseline sensitivity, nor does it result in changes to mechanical or thermal hypersensitivity after tissue injury. Myeloid cell recruitment did show differential changes between models of acute and chronic inflammatory pain. These results were consistent in both male and female mice, suggesting that there are no sex differences in these outcomes. This comprehensive characterization suggests that γδT cells do not contribute to basal sensitivity or the development and maintenance of inflammatory pain.

Skin-Resident γδ T Cells Exhibit Site-Specific Morphology and Activation States.

Skin-resident γδ T cells play an important role in maintaining the immune barrier at the epithelial surface. Their roles in wound healing, regulation of immune response to injury, and reepithelialization have been characterized extensively in the mouse, though their function in human skin remains largely unknown. Human skin-resident γδ T cells sparsely populate the skin and are often small and rounded in appearance. Those in the mouse ear and back, which line the dermal barrier, are highly arborized cells with many processes extending from the cell body. To date, these cells have been studied primarily in the mouse ear and back; however, it is important to further identify and characterize γδ T cells in other body sites to better understand their function and study their contribution to injury and disease. We developed a novel method to visualize these cells in the skin (whole-mount and cryosections) that when combined with flow cytometry allowed us to assess differences in skin-resident γδ T cell numbers, morphology, and activation state in the ear, back, and footpad (chosen for their importance in immunological and pain research). In comparing cell length, number of dendritic processes, and expression of the activation marker CD69, we found that γδ T cell morphology and activation states vary significantly among the three tissue environments. Specifically, γδ T cells in the footpad are smaller, have fewer processes, and show the highest levels of activation compared to back- and ear-resident cells. Our observations suggest that our understanding of skin-resident γδ T cell functionality, drawn from the experiments performed in the ear and back tissue, may not be applicable to all skin environments. The footpad-resident cells also more closely resemble γδ T cells in human skin, suggesting that cells in this tissue environment may serve as a better translational model when studying γδ T cell function/activity.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses.

INTRODUCTION: Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. METHODS: A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. RESULTS: Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. CONCLUSIONS: Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil.

A Saint Louis encephalitis and Rocio virus serosurvey in Brazilian horses

Introduction Arboviruses are an important public health problem in Brazil, in especially flaviviruses, including the Saint Louis encephalitis virus (SLEV) and the Rocio virus (ROCV), are especially problematic. These viruses are transmitted to humans or other vertebrates through arthropod bites and may cause diseases with clinical manifestations that range from asymptomatic infection, viral hemorrhagic fever to encephalitis. Methods A serological survey of horses from various regions of Brazil using an enzyme-linked immunosorbent assay (ELISA) with recombinant SLEV domain III peptides and ROCV E protein as antigens. Results Overall, 415 (55.1%) of the 753 horses that were screened were seropositive for flavivirus and, among them, monotypic reactions were observed to SLEV in 93 (12.3%) and to ROCV in 46 (6.1%). These results suggested that these viruses, or other closely related viruses, are infecting horses in Brazil. However, none of the studied horses presented central nervous system infection symptoms. Conclusions Our results suggest that SLEV and ROCV previously circulated among horses in northeast, west-central and southeast Brazil. .

Serologic survey of West Nile virus in horses from Central-West, Northeast and Southeast Brazil.

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil.

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Production and diagnostic application of recombinant domain III of West Nile envelope protein in Brazil

INTRODUCTION: West Nile virus (WNV) is a flavivirus with a natural cycle involving mosquitoes and birds. Over the last 11 years, WNV has spread throughout the Americas with the imminent risk of its introduction in Brazil. METHODS: Envelope protein domain III of WNV (rDIII) was bacterially expressed and purified. An enzyme-linked immunosorbent assay with WNV rDIII antigen was standardized against mouse immune fluids (MIAFs) of different flavivirus. RESULTS: WNV rDIII reacted strongly with St. Louis encephalitis virus (SLEV) MIAF but not with other flaviviruses. CONCLUSIONS: This antigen may be a potentially useful tool for serologic diagnosis and may contribute in future epidemiological surveillance of WNV infections in Brazil.

Use of reverse transcriptase polymerase chain reaction (RT-PCR) in molecular screening of Newcastle disease virus in poultry and free-living bird populations.

The aim of this study was to evaluate a simple molecular method of reverse transcriptase polymerase chain reaction (RT-PCR) to differentiate Newcastle disease virus strains according to their pathogenicity, in order to use it in molecular screening of Newcastle disease virus in poultry and free-living bird populations. Specific primers were developed to differentiate LaSota--LS--(vaccine strain) and Sao Joao do Meriti--SJM--strain (highly pathogenic strain). Chickens and pigeons were experimentally vaccinated/infected for an in vivo study to determine virus shedding in feces. Validation of sensitivity and specificity of the primers (SJM and LS) by experimental models used in the present study and results obtained in the molecular analysis of the primers by BLAST made it possible to generalize results. The development of primers that differentiate the level of pathogenicity of NDV stains is very important, mainly in countries where real-time RT-PCR is still not used as a routine test. These primers were able to determine the presence of the agent and to differentiate it according to its pathogenicity.

Domain III peptides from flavivirus envelope protein are useful antigens for serologic diagnosis and targets for immunization.

The Flavivirus genus of the Flaviviridae family includes 70 enveloped single-stranded-RNA positive-sense viruses transmitted by arthropods. Among these viruses, there are a relevant number of human pathogens including the mosquito-borne dengue virus (DENV), yellow fever virus (YFV), Japanese encephalitis virus (JEV) and West Nile virus (WNV), as well as tick-borne viruses such as tick-borne encephalitis virus (TBEV), Langat virus (LGTV) and Omsk hemorrhagic fever (OHFV). The flavivirus envelope (E) protein is a dominant antigen inducing immunologic responses in infected hosts and eliciting virus-neutralizing antibodies. The domain III (DIII) of E protein contains a panel of important epitopes that are recognized by virus-neutralizing monoclonal antibodies. Peptides of the DIII have been used with promising results as antigens for flavivirus serologic diagnosis and as targets for immunization against these viruses. We review here some important aspects of the molecular structure of the DIII as well as its use as antigens for serologic diagnosis and immunization in animal models.