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Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD 600nm  = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Cyclic nucleotides cAMP and cGMP are important regulators of immune cell functions. Phosphodiesterases (PDEs) hydrolyze cAMP and/or cGMP and, thus, play crucial roles in cyclic nucleotide homeostasis. Abnormal alterations of PDE expression have been implicated in several diseases. To understand the function of PDEs in macrophages, we screened for all PDE genes in both peritoneal and alveolar macrophages from C57BL/6J mice and found that PDE4B and PDE10A are highly induced by LPS. A number of PDE4 inhibitors have been used clinically for the treatment of inflammatory lung diseases. However, the role of PDE10A in inflammation is still poorly understood. We therefore investigated the role of PDE10A in macrophage inflammatory response in vitro and acute lung inflammation in vivo. We found that LPS induces a sustained PDE10A expression in macrophages, which is different from a transient induction by PDE4B. PDE10A inhibition blocked LPS-induced MCP-1 expression, but not TNF-α, whereas PDE4B inhibition blocked LPS-induced TNF-α expression, but not MCP-1. In addition, PDE10A inhibition or deficiency decreased LPS-induced HIF-1α protein expression and subsequently suppressed MCP-1 expression. In vivo, PDE10A expression was also elevated in lung tissue after LPS exposure. Global PDE10A knockout or systemic administration of the PDE10A inhibitor TP-10 in mice significantly suppressed inflammatory molecule levels in the lung tissue and bronchoalveolar lavage fluid as well as inflammatory cell infiltration. These findings show that PDE10A plays a critical role in lung inflammation by promoting the activation of resident macrophages and infiltration of neutrophils., (Copyright © 2021 by The American Association of Immunologists, Inc.)

Protection against Staphylococcus aureus is determined by the polarization of the anti-bacterial immune effector mechanisms. Virulence factors of S. aureus can modulate these and induce differently polarized immune responses in a single individual. We proposed that this may be due to intrinsic properties of the bacterial proteins. To test this idea, we selected two virulence factors, the serine protease-like protein B (SplB) and the glycerophosphoryl diester phosphodiesterase (GlpQ). In humans naturally exposed to S. aureus , SplB induces a type 2-biased adaptive immune response, whereas GlpQ elicits type 1/type 3 immunity. We injected the recombinant bacterial antigens into the peritoneum of S. aureus -naïve C57BL/6N mice and analyzed the immune response. This was skewed by SplB toward a Th2 profile including specific IgE, whereas GlpQ was weakly immunogenic. To elucidate the influence of adjuvants on the proteins' polarization potential, we studied Montanide ISA 71 VG and Imject™Alum, which promote a Th1 and Th2 response, respectively. Alum strongly increased antibody production to the Th2-polarizing protein SplB, but did not affect the response to GlpQ. Montanide enhanced the antibody production to both S. aureus virulence factors. Montanide also augmented the inflammation in general, whereas Alum had little effect on the cellular immune response. The adjuvants did not override the polarization potential of the S. aureus proteins on the adaptive immune response., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Mrochen, Trübe, Jorde, Domanska, Brandt and Bröker.)

Bites evoked by Brown spiders (Loxosceles genus) are associated with skin injuries (cutaneous rash, itching, swelling, erythema and dermonecrosis) and systemic manifestations. Transcriptome analyses of Loxosceles venom glands showed that the venom has a complex composition containing toxins such as phospholipases-D, metalloproteases and hyaluronidases. Here, by screening the RNA from L. intermedia venom glands, we cloned a novel allergen toxin, and named LALLT (LoxoscelesAllergen-Like Toxin). Sequence analysis showed that LALLT is closely related to allergens from other spiders and RNA screening indicated the presence of LALLT orthologues in the venom of other Loxosceles spiders. Recombinant LALLT was expressed (~45 kDa) in baculovirus-infected insect cells and purified by affinity chromatography. Antibodies against different Loxosceles venoms cross-reacted with LALLT and antibodies against LALLT recognized three Loxosceles venoms, revealing epitopes identity. LALLT triggered paw edema in mice and erythema, edema and leukocyte infiltration into the dermis of rabbit skin. Also, LALLT induced vascular permeability in mice, degranulation of rat mesentery mast cells, as well as prompted degranulation and increased calcium influx in RBL-2H3 cells. Data reported suggest for the first time the existence of allergens in Loxosceles venoms and make LALLT available for clinical studies about allergenic events arisen by Loxosceles envenoming., Competing Interests: Declaration of competing interest None., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Loxoscelism pose a health issue in the South America. The treatment for these accidents is based on the administration of antivenom produced in animals immunized with Loxosceles venom. In this work, a previously produced non-toxic multiepitopic chimeric protein (rMEPlox), composed of epitopes derived from the main toxins families (sphyngomielinase-D, metalloproteases, and hyaluronidases) of Loxosceles spider venoms, was used as antigen to produce monoclonal antibodies (mAbs). A selected anti-rMEPlox mAb (Lox-mAb3) reacted with metalloprotease from L. intermedia venom and showed cross-reactivity with metalloproteses from Brazilian and Peruvian Loxosceles laeta and Loxosceles gaucho venoms in immunoassays. The sequence recognized by Lox-mAb3 ( 184 ENNTRTIGPFDYDSIMLYGAY 205 ) corresponds to the C-terminal region of Astacin-like metalloprotease 1 and the amino acid sequence IGPFDYDSI, conserved among the homologs metalloproteases sequences, is important for antibody recognition. Lox-mAb3 neutralizes the fibrinogenolytic activity caused by metalloprotease from L. intermedia spider venom in vitro, which may lead to a decrease in hemorrhagic disturbances caused by Loxosceles envenomation. Our results show, for the first time, the use of a non-toxic multiepitopic protein for the production of a neutralizing monoclonal antibody against a metalloprotease of medically important Loxosceles venoms. These results contribute for the production improvement of therapeutic antivenom against loxoscelism., Competing Interests: Declaration of competing interest The authors state no conflict of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

The Hemiscorpius lepturus scorpion and brown spider Loxosceles intermedia represent a public health problem in Asia and America, respectively. Although distinct, these organisms contain similar toxins responsible for the principal clinical signs of envenomation. To better understand the properties of these toxins, we designed a study to compare recombinant Heminecrolysin (rHNC) and rLiD1, the major phospholipase D toxins of scorpion and spider venom, respectively. Using a competitive ELISA and a hemolytic inhibition test, we come to spot a cross reaction between scorpion and spider venoms along with an epitopic similarity between rHNC and rLiD1 associated with neutralizing antibodies. Results show that the ability of the rHNC to hydrolyze lysophosphatidylcholine (LPC) is equivalent to that of rLiD1 to hydrolyze sphingomyelin and vice-versa. rHNC exclusively catalyze transphosphatidylation of LPC producing cyclic phosphatidic acid (cPA). The in-silico analysis of hydrogen bonds between LPC and toxins provides a possible explanation for the higher transphosphatidylase activity of rHNC. Interestingly, for the first time, we reveal that lysophosphatidic acid (LPA) can be a substrate for both enzymes using cellular and enzymatic assays. The finding of the usage of LPA as a substrate as well as the formation of cPA as an end product could shed more light on the molecular basis of Hemiscorpius lepturus envenomation as well as on loxoscelism.

The Basophil Activation Test (BAT) is a valuable allergy diagnostic tool but is time-consuming and requires skilled personnel and cumbersome processing, which has limited its clinical use. We therefore investigated if a microfluidic immunoaffinity BAT (miBAT) technique can be a reliable diagnostic method. Blood was collected from allergic patients and healthy controls. Basophils were challenged with negative control, positive control (anti-FcεRI), and two concentrations of a relevant and non-relevant allergen. CD203c and CD63 expression was detected by fluorescent microscopy and flow cytometry. In basophils from allergic patients the CD63% was significantly higher after allergen activation as compared to the negative control (p<.0001-p=.0004). Activation with non-relevant allergen showed equivalent CD63% expression as the negative control. Further, the miBAT data were comparable to flow cytometry. Our results demonstrate the capacity of the miBAT technology to measure different degrees of basophil allergen activation by quantifying the CD63% expression on captured basophils., (Copyright © 2019 Elsevier Inc. All rights reserved.)

LiTCTP is a toxin from the Translationally Controlled Tumor Protein (TCTP) family identified in Loxosceles brown spider venoms. These proteins are known as histamine-releasing factors (HRF). TCTPs participate in allergic and anaphylactic reactions, which suggest their potential role as therapeutic targets. The histaminergic effect of TCTP is related to its pro-inflammatory functions. An initial characterization of LiTCTP in animal models showed that this toxin can increase the microvascular permeability of skin vessels and induce paw edema in a dose-dependent manner. We evaluated the role of LiTCTP in vitro and in vivo in the inflammatory and allergic aspects that undergo the biological responses observed in Loxoscelism, the clinical condition after an accident with Loxosceles spiders. Our results showed LiTCTP recombinant toxin (LiRecTCTP) as an essential synergistic factor for the dermonecrotic toxin actions (LiRecDT1, known as the main toxin in the pathophysiology of Loxoscelism), revealing its contribution to the exacerbated inflammatory response clinically observed in envenomated patients.

Autotaxin (ATX) is a secreted glycoprotein, widely present in biological fluids including blood. ATX catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a growth factor-like, signaling phospholipid. LPA exerts pleiotropic effects mediated by its G-protein-coupled receptors that are widely expressed and exhibit overlapping specificities. Although ATX also possesses matricellular properties, the majority of ATX reported functions in adulthood are thought to be mediated through the extracellular production of LPA. ATX-mediated LPA synthesis is likely localized at the cell surface through the possible interaction of ATX with integrins or other molecules, while LPA levels are further controlled by a group of membrane-associated lipid-phosphate phosphatases. ATX expression was shown to be necessary for embryonic development, and ATX deficient embryos exhibit defective vascular homeostasis and aberrant neuronal system development. In adult life, ATX is highly expressed in the adipose tissue and has been implicated in diet-induced obesity and glucose homeostasis with multiple implications in metabolic disorders. Additionally, LPA has been shown to affect multiple cell types, including stromal and immune cells in various ways. Therefore, LPA participates in many processes that are intricately involved in the pathogenesis of different chronic inflammatory diseases such as vascular homeostasis, skeletal and stromal remodeling, lymphocyte trafficking and immune regulation. Accordingly, increased ATX and LPA levels have been detected, locally and/or systemically, in patients with chronic inflammatory diseases, most notably idiopathic pulmonary fibrosis (IPF), chronic liver diseases, and rheumatoid arthritis. Genetic and pharmacological studies in mice have confirmed a pathogenetic role for ATX expression and LPA signaling in chronic inflammatory diseases, and provided the proof of principle for therapeutic interventions, as exemplified by the ongoing clinical trials for IPF., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Background: The flow cytometry-based basophil activation test (BAT) is used for the diagnosis of allergic response. However, flow cytometry is time-consuming, requiring skilled personnel and cumbersome processing, which has limited its use in the clinic. Here, we introduce a novel microfluidic-based immunoaffinity BAT (miBAT) method., Methods: The microfluidic device, coated with anti-CD203c, was designed to capture basophils directly from whole blood. The captured basophils are activated by anti-FcεRI antibody followed by optical detection of CD63 expression (degranulation marker). The device was first characterized using a basophil cell line followed by whole blood experiments. We evaluated the device with ex vivo stimulation of basophils in whole blood from healthy controls and patients with allergies and compared it with flow cytometry., Results: The microfluidic device was capable of capturing basophils directly from whole blood followed by in vitro activation and quantification of CD63 expression. CD63 expression was significantly higher ( P = 0.0002) in on-chip activated basophils compared with nonactivated cells. The difference in CD63 expression on anti-FcεRI-activated captured basophils in microfluidic chip was significantly higher ( P = 0.03) in patients with allergies compared with healthy controls, and the results were comparable with flow cytometry analysis ( P = 0.04). Furthermore, there was no significant difference of CD63% expression in anti-FcεRI-activated captured basophils in microfluidic chip compared with flow cytometry., Conclusions: We report on the miBAT. This device is capable of isolating basophils directly from whole blood for on-chip activation and detection. The new miBAT method awaits validation in larger patient populations to assess performance in diagnosis and monitoring of patients with allergies at the point of care., (© 2019 American Association for Clinical Chemistry.)

Objective: To describe a novel antibody biomarker of neurologic paraneoplastic autoimmunity specific for phosphodiesterase 10A (PDE10A), a striatum-enriched phosphodiesterase, and to characterize the clinical phenotype of patients with PDE10A immunoglobulin G (IgG)., Methods: We describe 7 patients with autoantibodies specific for PDE10A identified in the Mayo Clinic Neuroimmunology Laboratory. Patient specimens (sera, 7; CSF, 4) produced identical basal ganglia-predominant synaptic staining of murine brain tissue by indirect immunofluorescence. The autoantigen was identified by immunoprecipitation and mass spectrometry as PDE10A, and confirmed by antigen-specific recombinant Western blot and cell-based assays, and immune absorption experiments., Results: The median patient age was 70 years (range 66-76); 4 were men. Four patients with clinical information available had movement disorders (hyperkinetic in 3 [chorea, ballismus, dystonia] and parkinsonism in 1). All patients but one had cancer (lung [adenocarcinoma 1, squamous cell carcinoma 1, poorly differentiated mesenchymal carcinoma 1], renal adenocarcinoma 2, and pancreatic adenocarcinoma 1). Two of the 7 patients developed hyperkinetic movement disorders during treatment with immune checkpoint inhibitors (nivolumab and pembrolizumab), though none of 26 cancer control patients treated with immune checkpoint inhibitors harbored PDE10A IgG in their serum. MRIs from those 2 patients with hyperkinetic movement disorders demonstrated fluid-attenuated inversion recovery/T2 basal ganglia hyperintensities, and their CSF harbored unique oligoclonal bands. One of those 2 patients had substantial improvement after corticosteroids. One patient's renal adenocarcinoma expressed PDE10A by immunohistochemistry., Conclusions: PDE10A IgG defines a novel rare neurologic autoimmune syndrome and expands the spectrum of diagnosable paraneoplastic CNS disorders. The intracellular location of PDE10A suggests a T-cell-mediated pathology targeting cells displaying MHC1-bound PDE10A peptides., (Copyright © 2019 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.)

Multiple sclerosis (MS) is a chronic demyelinating disease of the central nervous system (CNS) characterized by heterogeneous clinical symptoms including gradual muscle weakness, fatigue, and cognitive impairment. The disease course of MS can be classified into a relapsing-remitting (RR) phase defined by periods of neurological disabilities, and a progressive phase where neurological decline is persistent. Pathologically, MS is defined by a destructive immunological and neuro-degenerative interplay. Current treatments largely target the inflammatory processes and slow disease progression at best. Therefore, there is an urgent need to develop next-generation therapeutic strategies that target both neuroinflammatory and degenerative processes. It has been shown that elevating second messengers (cAMP and cGMP) is important for controlling inflammatory damage and inducing CNS repair. Phosphodiesterases (PDEs) have been studied extensively in a wide range of disorders as they breakdown these second messengers, rendering them crucial regulators. In this review, we provide an overview of the role of PDE inhibition in limiting pathological inflammation and stimulating regenerative processes in MS.

Persistent T cell antigen receptor (TCR) signaling by CD8 T cells is a feature of cancer and chronic infections and results in the sustained expression of, and signaling by, inhibitory receptors, which ultimately impair cytotoxic activity via poorly characterized mechanisms. We have previously determined that the LPA 5 GPCR expressed by CD8 T cells, upon engaging the lysophosphatidic acid (LPA) bioactive serum lipid, functions as an inhibitory receptor able to negatively regulate TCR signaling. Notably, the levels of LPA and autotaxin (ATX), the phospholipase D enzyme that produces LPA, are often increased in chronic inflammatory disorders such as chronic infections, autoimmune diseases, obesity, and cancer. In this report, we demonstrate that LPA engagement selectively by LPA 5 on human and mouse CD8 T cells leads to the inhibition of several early TCR signaling events including intracellular calcium mobilization and ERK activation. We further show that, as a consequence of LPA 5 suppression of TCR signaling, the exocytosis of perforin-containing granules is significantly impaired and reflected by repressed in vitro and in vivo CD8 T cell cytolytic activity. Thus, these data not only document LPA 5 as a novel inhibitory receptor but also determine the molecular and biochemical mechanisms by which a naturally occurring serum lipid that is elevated under settings of chronic inflammation signals to suppress CD8 T cell killing activity in both human and murine cells. As diverse tumors have repeatedly been shown to aberrantly produce LPA that acts in an autocrine manner to promote tumorigenesis, our findings further implicate LPA in activating a novel inhibitory receptor whose signaling may be therapeutically silenced to promote CD8 T cell immunity.

Recent accumulating evidence indicates the biological actions of Autotaxin-Lysophosphatidic acid (ATX-LPA) signaling axis in malignant tumors. However, the role of Autotaxin-Lysophosphatidic acid signaling axis in breast cancer has not been reported. The present study aims to examine the alterations of serum autotaxin in breast cancer and discuss whether serum autotaxin could be useful as a novel parameter of breast cancer.Serum autotaxin antigen was measured in 112 patients with breast cancer and 50 healthy volunteers by ELISA. The association of serum autotaxin antigen levels with clinicopathological parameters and outcomes of breast cancer was analyzed.Serum autotaxin antigen was significantly higher in breast cancer patients than healthy volunteers (291.32 ± 38.02 ng/ml vs 254.04 ± 21.03 ng/ml, respectively; P < .0001). Serum autotaxin measurement successfully discriminated breast cancer patients from normal and healthy controls (AUC = 0.798, 95% CI: 0.732-0.864) with an optimal cut-off value of 267.34 ng/ml (sensitivity = 0.741, specificity = 0.800). Increased serum autotaxin was associated with breast cancer nodal status (P = .007), Tumor-Node- Metastasis (TNM) stage (P = .009) and Ki-67 index (P = .004). Univariate and multivariate Cox regression analysis revealed that elevated serum autotaxin showed an independent prognostic value for poor Disease-free survival.Our present study confirmed the elevation, potential diagnostic, and independent prognostic value of serum autotaxin for breast cancer. Serum autotaxin could serve as a reliable novel biomarker for breast cancer.

Background: CD16 was previously suggested to be a new marker of basophils that is subject to downregulation by FcεRI crosslinking. Certain compounds, including supraoptimal concentrations of the PKC inhibitors, bisindolylmaleimides, decouple the release of granules containing CD203c, CD63 and histamine, and may thus help to identify the mechanisms related to the CD16 externalization., Objective: We hypothesized that CD16 is differentially expressed on the surface of basophils in patients with birch pollen or insect venom allergy and is subject to a regulation in response to allergens. We also employed CD203c and CD63 externalization decoupling by bisindolylmaleimides., Methods: We performed a basophil activation test coupled with CD16 and histamine detection using cells isolated from patients with allergy to birch pollen or insect venom and negative controls. We employed two PKC inhibitors, bisindolylmaleimide II and Ro 31-8220 at their supraoptimal concentrations and, after difficulties reproducing previously published data, we analyzed the fluorescence of these inhibitors alone. We identified the CD16 isoforms by sequencing nested RT-PCR amplicons from flow cytometry sorted basophils and by cleaving the CD16b GPI anchor using a phospholipase C., Results: We provide the first evidence that CD16a is expressed as a surface antigen on a small subpopulation of human basophils in patients with respiratory and insect venom allergy, and this antigen shows increased surface expression following allergen challenge or FcεRI crosslinking. We rejected the apparent decoupling of the surface expression of basophil activation markers following the administration of bisindolylmaleimides., Conclusions & Clinical Relevance: The inclusion of αCD16 in negative selection cocktails selects against a subset of basophils that are CD16 + or CD16 dim . Using CD16 dim basophils and unstained leucocytes, we show that previous studies with supraoptimal concentrations of bisindolylmaleimides are likely flawed and are not associated with the differential expression of CD203c and CD63., (© 2018 John Wiley & Sons Ltd.)

Objectives: Borrelia miyamotoi disease (BMD) is an emerging tick-borne disease in the Northern hemisphere. Serodiagnosis by measuring antibodies against glycerophosphodiester-phosphodiesterase (GlpQ) has been performed experimentally but has not been extensively clinically validated. Because we had previously shown the differential expression of antigenic variable major proteins (Vmps) in B. miyamotoi, our aim was to study antibody responses against GlpQ and Vmps in PCR-proven BMD patients and controls., Methods: We assessed seroreactivity against GlpQ and four Vmps in a well-described, longitudinal cohort of sera from BMD patients (n=182), healthy blood donors (n=136) and controls (n=68). All samples were tested by ELISA and positive sera were tested by western blot, and antibody dynamics and diagnostic value were assessed., Results: IgM antibodies against GlpQ and Vmps peaked between 11 and 20 days, and IgG between 21 and 50 days, after disease onset. Various combinations of GlpQ and Vmps increased sensitivity and/or specificity compared to single antigens. Notably, the GlpQ or variable large protein (Vlp)-15/16 combination yielded a sensitivity of 94.7% (95% CI: 75.4-99.7) 11-20 days after disease onset and a specificity of 96.6% (92.7-98.4) for IgM. A specificity of 100% (97.8-100) for IgM, and 98.3% for IgG (95.2-100), was found when positivity was defined as reactivity to GlpQ and any Vmp, with maximum sensitivities of 79% (56.7-91.5) for IgM and 86.7% (62.1-97.6) for IgG., Conclusions: We clearly demonstrate here the diagnostic potential of these seromarkers. Our findings will facilitate future epidemiological and clinical studies on BMD and lead to the development of a serologic test to be used in clinical practice., (Copyright © 2018 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

Accidents with venomous animals pose a health issue in Brazil, and those involving brown spiders (Loxosceles sp.) figure between the most frequent ones. The accidental envenomation by brown spiders causes a strong local dermonecrotic effect, which can be followed by systemic manifestations that in some cases lead to death. The production of antivenoms for the treatments of such accidents relies on a variety of animal experiments, from the spider venom extraction to the production of antivenom in horses. In the present work, there is an attempt to reduce and optimize animal experiments with the construction and production of a chimeric protein, named Lil, containing immunodominant epitopes previously mapped from the main proteins of the Loxosceles venom, the Sphingomyelinases D. The Lil protein contains epitopes from Sphinomyelinases D of the three-main species found in Brazil and this chimeric protein was found capable of inducing antibodies with the potential to partially neutralize the toxic effects of Loxosceles intermedia venom in an animal model. Therefore, in order to reduce spider usage and to improve the lifespan of the horses used for immunization we suggest the Lil protein as a potential candidate to replace the venom usage in the antivenom production protocols., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Since 2011, Borrelia miyamotoi disease (BMD) has been reported in five countries in the northern hemisphere. The causative agent of BMD is transmitted by Ixodes ticks, which are also vectors of Lyme disease borreliae. In this study, we examined 459 cases of clinically suspected Lyme disease (LD group), and found twelve cases that were seropositive for the glycerophosphodiester phosphodiesterase (GlpQ) antigen derived from B. miyamotoi. The retrospective surveillance revealed that the seroprevalence of anti-GlpQ in the LD group was significantly higher than in a healthy cohort. Seropositive cases were observed from spring through autumn when ticks are active, and the cases were geographically widespread, being found in Hokkaido-Tohoku, Kanto, Chubu, Kinki, and Kyushu-Okinawa regions. Seropositive cases for GlpQ were most frequent in the Chubu region (6.3%) where B. miyamotoi has been found in Ixodes ticks. Out of the twelve cases that were found in the LD group, three cases exhibited concomitant seropositivity to Lyme disease borreliae by western blot assay. This is the first report of serological surveillance for BMD in Japan, and we conclude that BMD occurs nationwide., (Copyright © 2018. Published by Elsevier Ltd.)

Purpose: To determine the safety, pharmacokinetics, and recommended phase II dose of an antibody-drug conjugate (ADC) targeting ectonucleotide phosphodiesterases-pyrophosphatase 3 (ENPP3) conjugated to monomethyl auristatin F (MMAF) in subjects with advanced metastatic renal cell carcinoma (mRCC). Patients and Methods: Two phase I studies were conducted sequentially with 2 ADCs considered equivalent, hybridoma-derived AGS-16M8F and Chinese hamster ovary-derived AGS-16C3F. AGS-16M8F was administered intravenously every 3 weeks at 5 dose levels ranging from 0.6 to 4.8 mg/kg until unacceptable toxicity or progression. The study was terminated before reaching the MTD. A second study with AGS-16C3F started with the AGS-16M8F bridging dose of 4.8 mg/kg given every 3 weeks. Results: The AGS-16M8F study ( n = 26) closed before reaching the MTD. The median duration of treatment was 12 weeks (1.7-83 weeks). One subject had durable partial response (PR; 83 weeks) and 1 subject had prolonged stable disease (48 weeks). In the AGS-16C3F study ( n = 34), the protocol-defined MTD was 3.6 mg/kg, but this was not tolerated in multiple doses. Reversible keratopathy was dose limiting and required multiple dose deescalations. The 1.8 mg/kg dose was determined to be safe and was associated with clinically relevant signs of antitumor response. Three of 13 subjects at 1.8 mg/kg had durable PRs (range, 100-143 weeks). Eight subjects at 2.7 mg/kg and 1.8 mg/kg had disease control >37 weeks (37.5-141 weeks). Conclusions: AGS-16C3F was tolerated and had durable antitumor activity at 1.8 mg/kg every 3 weeks. Clin Cancer Res; 24(18); 4399-406. ©2018 AACR ., (©2018 American Association for Cancer Research.)

Prevalence of allergic diseases is increasing worldwide. Allergen-specific immunotherapy (ASIT) is potentially the only curative treatment for allergy, but there is a lack of reliable methods to monitor the immune responses to ASIT and to predict clinical efficacy. Recently, the definition of allergen sensitivity threshold (CD-Sens) by Basophil Activation Tests has been suggested as potential method in this context. The aim of this study was to compare trends of CD-Sens, measured by the markers CD63 and CD203c, and clinical symptoms in subjects with allergic rhinitis receiving Sublingual Immunotherapy (SLIT). 26 rhinitis patients allergic to Parietaria were selected and matched into two groups; a SLIT treated group (SG) and a reference group (RG) treated by traditional anti-allergic medications. Visual Analogue Scale (VAS) score for the four cardinal symptoms of rhinitis and peripheral blood was collected before the first dose of SLIT (T 0 ) and after 12 months (T 12 ) to define the severity of the symptoms and the sensitivity of basophils to Parietaria. The comparison between T0 and T12 in SG patients showed a significant decrease of symptom severity (VAS score) and an increased tolerability of basophils to Parietaria (CD-Sens) both by CD63 and CD203c. But, only CD203c seems to be correlated with the clinical symptoms. These data corroborate the hypothesis that SLIT could change the immunological course of allergic sensitization already in the first year, and that an immunological parameter as CD-Sens measured by CD63 and CD203c expression on stimulated basophils could be useful to monitor the changes in the immune system., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Chronic spontaneous urticaria (CSU) is a popular disease, affects patients' life. Its etiologic agents are not well known so; treatment of the patients is difficult. CD203c is a marker that is only present on basophils. Statins are drugs used to lower cholesterol. Nowadays, it is well known that they have immunomodulatory effects. This study evaluated the efficacy of a statin, atorvastatin, in combination with antihistamines in treating CSU patients. Forty CSU patients were divided equally into two groups. The first group was treated with antihistamines and atorvastatin, while the second group was treated with antihistamines and placebo. Both groups received the treatment for three months. The effect of treatment on total severity score (TSS), autologous serum skin test (ASST), basophil histamine release (BHR) assay, in vivo basophil CD203c expression (%) and basophil activation test (BAT-CD203c) was assessed. We found statistically significant reduction in TSS, BHR assay, in vivo basophil CD203c expression (%) (P= 0.000 each), diameter of ASST and BAT-CD203c (P= 0.002, 0.017, respectively), in the patients that received the atorvastatin and antihistamines. In conclusion, atorvastatin is effective in treating CSU patients., (Copyright© by the Egyptian Association of Immunologists.)

Loxoscelism is the most important form of araneism in South America. The treatment of these accidents uses heterologous antivenoms obtained from immunization of production animals with crude loxoscelic venom. Due to the scarcity of this immunogen, new alternatives for its substitution in antivenom production are of medical interest. In the present work, three linear epitopes for Loxosceles astacin-like protease 1 (LALP-1) (SLGRGCTDFGTILHE, ENNTRTIGPFDYDSIMLYGAY, and KLYKCPPVNPYPGGIRPYVNV) and two for hyaluronidase (LiHYAL) (NGGIPQLGDLKAHLEKSAVDI and ILDKSATGLRIIDWEAWR) from Loxosceles intermedia spider venom were identified by SPOT-synthesis technique. One formerly characterized linear epitope (DFSGPYLPSLPTLDA) of sphingomyelinase D (SMase D) SMase-I from Loxosceles laeta was also chosen to constitute a new recombinant multiepitopic protein. These epitopes were combined with a previously produced chimeric multiepitopic protein (rCpLi) composed by linear and conformational B-cell epitopes from SMase D from L. intermedia venom, generating a new recombinant multiepitopic protein derived from loxoscelic toxins (rMEPLox). We demonstrated that rMEPLox is non-toxic and antibodies elicited in rabbits against this antigen present reactivity in ELISA and immunoblot assays with Brazilian L. intermedia, L. laeta, L. gaucho , and L. similis spider venoms. In vivo and in vitro neutralization assays showed that anti-rMEPLox antibodies can efficiently neutralize the sphingomyelinase, hyaluronidase, and metalloproteinase activity of L. intermedia venom. This study suggests that this multiepitopic protein can be a suitable candidate for experimental vaccination approaches or for antivenom production against Loxosceles spp. venoms.

Allergic disorders have now become a major worldwide public health issue, but the effective treatment options remain limited. We report a novel approach to block allergic reactivity by targeting the surface-bound IgE of the allergic effector cells via low-affinity anti-human IgE Abs with dissociation constants in the 10 -6 to 10 -8 M range. We demonstrated that these low-affinity anti-IgE mAbs bind to the cell surface-bound IgE without triggering anaphylactic degranulation even at high concentration, albeit they would weakly upregulate CD203c expression on basophils. This is in contrast to the high-affinity anti-IgE mAbs that trigger anaphylactic degranulation at low concentration. Instead, the low-affinity anti-IgE mAbs profoundly block human peanut- and cat-allergic IgE-mediated basophil CD63 induction indicative of anaphylactic degranulation; suppress peanut-, cat-, and dansyl-specific IgE-mediated passive cutaneous anaphylaxis; and attenuate dansyl IgE-mediated systemic anaphylaxis in human FcεRIα transgenic mouse model. Mechanistic studies reveal that the ability of allergic reaction blockade by the low-affinity anti-IgE mAbs was correlated with their capacity to downregulate the surface IgE and FcεRI level on human basophils and the human FcεRIα transgenic mouse bone marrow-derived mast cells via driving internalization of the IgE/FcεRI complex. Our studies demonstrate that targeting surface-bound IgE with low-affinity anti-IgE Abs is capable of suppressing allergic reactivity while displaying an excellent safety profile, indicating that use of low-affinity anti-IgE mAbs holds promise as a novel therapeutic approach for IgE-mediated allergic diseases., (Copyright © 2017 by The American Association of Immunologists, Inc.)

Background: Borrelia miyamotoi causes systemic febrile illness and is transmitted by the same tick species that transmits Borrelia burgdorferi sensu lato and tick-borne encephalitis virus. We describe a serological test using a fragment of glycerophosphodiester phosphodiesterase (GlpQ) as an antigen, and determined its performance in well-defined patient categories., Methods: Serum of patients with PCR-confirmed Borrelia miyamotoi disease (BMD), Lyme borreliosis (LB), tick-borne encephalitis (TBE), and healthy blood donors (HBD) were collected in Udmurt Republic, Russia. Sera of BMD and LB patients were collected at hospital admission, one week, one month and one year after admission., Results: The levels of IgM and IgG anti-GlpQ antibodies, determined as optical density values in Luminex bead-based assays, were significantly higher in the BMD patient group than in LB patients, TBE patients or HBD group (all p<0.05)., Conclusions: By using a strict cut-off value, it was possible to exclude B. miyamotoi infection in LB and TBE patients and to serologically confirm B. miyamotoi infection in 44% to 94% of the PCR-positive BMD patients (95% confidence interval). Thus, sensitive serological assays should not solely rely on rGlpQ, to support the diagnosis of acute BMD., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Ixodes tick-borne borreliosis caused by Borrelia miyamotoi (ITBB-BM) is a previously unknown infectious disease discovered in Russia., Aim: The present study continues the investigation of the clinical features of ITBB-BM in the context of an immune system-pathogen interaction., Subjects and Methods: The study enrolled 117 patients with ITBB-BM and a comparison group of 71 patients with Lyme disease (LD) that is ITBB with erythema migrans. All the patients were treated at the New Hospital, Yekateringburg. More than 100 clinical, epidemiological and laboratory parameters were obtained from each patient's medical history and included in the general database. A subset of patients hospitalized in 2015 and 2016 underwent additional laboratory examinations. Namely, the levels of B. miyamotoi-specific IgM and IgG antibodies were measured by the protein microarray containing GlpQ protein and four variable major proteins (VMPs): Vlp15/16, Vlp18, Vsp1, and Vlp5. The blood concentration of Borrelia was estimated by quantitative real-time PCR., Results: In contrast to LD, first of all (p<0.001) the following clinical features were typical for ITBB-BM: the absence of erythema migrans (in 95% of patients), fever (93%), fatigue (96%), headache (82%), chill (41%), nausea (28%), lymphopenia (56%), thrombocytopenia (46%), the abnormal levels of alanine aminotransferase (54%) and C-reactive protein (98%), proteinuria (61%). Given the set of these indicators, the course of ITBB-BM was more severe in approximately 70% of patients. At admission, only 13% and 38% of patients had antibodies to GlpQ and VMPs, respectively; at discharge, antibodies to GlpQ and VMPs were detected in 88% of patients. There was no statistically significant association of the antibody response with individual clinical manifestations and laboratory parameters of the disease. However, patients with more severe ITBB-BM produced less IgM antibodies to VMPs and GlpQ at the time of discharge., Conclusion: ITBB-BM is a moderate systemic disease accompanied by the production of specific antibodies in virtually all patients.

Loxosceles spiders' venom comprises a complex mixture of biologically active toxins, mostly consisting of low molecular mass components (2-40 kDa). Amongst, isoforms of astacin-like metalloproteases were identified through transcriptome and proteome analyses. Only LALP1 (Loxosceles Astacin-Like protease 1) has been characterized. Herein, we characterized LALP3 as a novel recombinant astacin-like metalloprotease isoform from Loxosceles intermedia venom. LALP3 cDNA was cloned in pET-SUMO vector, and its soluble heterologous expression was performed using a SUMO tag added to LALP3 to achieve solubility in Escherichia coli SHuffle T7 Express LysY cells, which express the disulfide bond isomerase DsbC. Protein purification was conducted by Ni-NTA Agarose resin and assayed for purity by SDS-PAGE under reducing conditions. Immunoblotting analyses were performed with specific antibodies recognizing LALP1 and whole venom. Western blotting showed linear epitopes from recombinant LALP3 that cross-reacted with LALP1, and dot blotting revealed conformational epitopes with native venom astacins. Mass spectrometry analysis revealed that the recombinant expressed protein is an astacin-like metalloprotease from L. intermedia venom. Furthermore, molecular modeling of LALP3 revealed that this isoform contains the zinc binding and Met-turn motifs, forming the active site, as has been observed in astacins. These data confirmed that LALP3, which was successfully obtained by heterologous expression using a prokaryote system, is a new astacin-like metalloprotease isoform present in L. intermedia venom., (Copyright © 2016 Elsevier B.V. and Société Française de Biochimie et Biologie Moléculaire (SFBBM). All rights reserved.)

Loxosceles spider bites often lead to serious envenomings and no definite therapy has yet been established. In such a context, it is of interest to consider an antibody-based targeted therapy. We have previously prepared a murine monoclonal IgG (LiMab7) that binds to 32-35kDa components of Loxosceles intermedia venom and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a recombinant diabody. The protein was produced in bacteria and then it was functionally characterized. It proved to be efficient at neutralizing sphingomyelinase and hemolytic activities of the crude venom despite the slightly altered binding kinetic constants and the limited stability of the dimeric configuration. This is the first report of a specific recombinant antibody for a next-generation of Loxosceles antivenoms., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Background & Aims: T-helper-type 17 (Th17) cells are involved in autoimmune tissue damage. CD39 is an ectonucleotidase that catalyzes extracellular ATP/ADP hydrolysis, culminating in the generation of immunosuppressive adenosine. Functional CD39 expression confers immunosuppressive properties upon immune cells. As the proportion of CD39 lymphocytes is decreased in juvenile autoimmune liver disease (AILD), we have explored whether decreased CD39 expression is present on Th17 cells and whether this phenomenon is associated with heightened effector function and inflammation., Methods: Thirty-eight patients with juvenile AILD (22 autoimmune hepatitis and 16 autoimmune sclerosing cholangitis), 8 disease controls (DC) and 16 healthy subjects (HS) were studied. Peripheral blood cell phenotype was determined by flow cytometry; ability to suppress by inhibition of cell proliferation/effector cytokine production; ectoenzymatic activity by thin layer chromatography; expression of adenosine receptor, adenosine deaminase (ADA) and phosphodiesterases (PDE) by quantitative real-time PCR or by Western Blot., Results: CD39(+) Th17 (Th17(CD39+)) cells from HS appear activated and contain high frequencies of lymphocytes producing regulatory cytokines. In AILD, however, Th17(CD39+) cells are markedly diminished and fail to generate AMP/adenosine, thereby limiting control of both target cell proliferation and IL-17 production. When compared to HS, Th17 cells from AILD patients also show lower A2A adenosine receptor expression while displaying similar levels of PDE4A, PDE4B and ADA. Only rare Th17(CD39+) cells are observed by liver immunohistochemistry., Conclusions: Th17(CD39+) cells in juvenile AILD are both quantitatively decreased and qualitatively deficient. Low levels CD39 and A2A expression may contribute to the perpetuation of Th17 cell effector properties and unfettered inflammation in this disease., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

In the present investigation we used a recombinant LiD1 toxin, named rLiD1his, from Loxosceles intermedia brown spider to elicit specific antibodies in mice carrying different Human Leukocyte Antigens class II (HLAII) {DRB1.0401 (DR4), DQB1.0601 (DQ6) and DQB1.0302 (DQ8)} as well as in BALB/C and C57BL/6 control mice. All mice strains produced high antibody titers against rLiD1his but DR4 mice antibodies (the lower responder mice) were not able to recognize L. intermedia crude venom. The anti-rLiD1his sera, except from DR4 mice, were able to neutralize dermonecrotic, hemorrhagic and edematogenic effects of rLiD1his in naïve rabbits. Overlapping peptides from the amino acid sequence of LiD1 toxin were prepared by SPOT method and differences in LiD1 epitope recognition were observed using different mice anti-rLiD1his sera. The region (160)DKVGHDFSGNDDISDVGK(177) was recognized by transgenic DQ8 and DQ6 mice sera. Other epitopes were recognized by at least two different animals' sera including (10)MGHMVNAIGQIDEFVNLG(27), (37)FDDNANPEYTYHGIP(51), (70)GLRSATTPGNSKYQEKLV(87) and (259)AAYKKKFRVATYDDN(273). Among these epitopes, the epitopes 37-51 and 160-177 have already been shown in previously studies as good candidates to be used alone or combined with other peptides to induce protective immune response against Loxosceles venoms. The results presented here highlight the importance of HLAII in antibody response and recognition of specific B-cell epitopes of rLiD1his spider toxin according to HLAII type and impact in the epitopic vaccine development against this spider., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Bites by spiders from Loxosceles genus often lead to a wide variance in envenomation profile of patients and diagnosis is difficult due to the number of diseases that mimic loxoscelism. In such a context, it is of interest to consider the design of standardized recombinant colorimetric antibodies for diagnosis and specific detection of individual circulating toxins in biological fluids of envenomed patients. We have previously prepared a monoclonal murine IgG (LiMab7) that reacts with Loxosceles intermedia venom components of 32-35kDa and neutralizes the dermonecrotic activity of the venom. Here, we re-engineered LiMab7 into a colorimetric bifunctional protein consisting in the corresponding single-chain antibody fragment (scFv) fused to alkaline phosphatase (AP) of Escherichia coli. The immune tracer was tested in two different types of immunoassays and it proved to be efficient in both. Thus, this recombinant fusion protein (scFv-LiMab7/AP) can be used for rapid and specific immunotitration of L. intermedia venom with a linear range of 39-20000ng/mL and a detection limit of 39ng/mL without any cross-reaction., (Copyright © 2016 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Lysophosphatidic acid (LPA) is a phospholipid that is mainly produced by the hydrolysis of lysophosphatidylcholine (LPC) by lysophospholipase D, which is also called autotaxin (ATX). LPA interacts with specific G-protein coupled receptors and is involved in the regulation of cellular survival, proliferation, differentiation and motility. LPA also has roles in several pathological disorders, such as cancer and pulmonary, dermal and renal fibrosis. The involvement of the ATX-LPA pathway has recently been demonstrated in inflammatory responses and apoptosis of fibroblast-like synoviocytes (FLS) from patients with rheumatoid arthritis and during the development of experimental arthritis. This review summarises the current literature of the ATX-LPA pathway in rheumatoid arthritis., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Background: The first recognized cases of Borrelia miyamotoi disease (BMD) in North America were reported in the northeastern United States in 2013., Objective: To further describe the clinical spectrum and laboratory findings for BMD., Design: Case series., Setting: Patients presenting to primary care offices, emergency departments, or urgent care clinics in 2013 and 2014., Participants: Acutely febrile patients from the northeastern United States in whom the treating health care providers suspected and ordered testing for tick-transmitted infections., Measurements: Whole-blood polymerase chain reaction (PCR) testing was performed for the presence of specific DNA sequences of common tickborne infections (including BMD). Serologic testing for B. miyamotoi was performed using a recombinant glycerophosphodiester phosphodiesterase (rGlpQ) protein. Clinical records were analyzed to identify the major features of acute disease., Results: Among 11,515 patients tested, 97 BMD cases were identified by PCR. Most of the 51 case patients on whom clinical histories were reviewed presented with high fever, chills, marked headache, and myalgia or arthralgia. Twenty-four percent were hospitalized. Elevated liver enzyme levels, neutropenia, and thrombocytopenia were common. At presentation, 16% of patients with BMD were seropositive for IgG and/or IgM antibody to B. miyamotoi rGlpQ. Most (78%) had seropositive convalescent specimens. Symptoms resolved after treatment with doxycycline, and no chronic sequelae or symptoms were observed., Limitation: Findings were based on specimens submitted for testing to a reference laboratory, and medical records of only 51 of the 97 case patients with BMD were reviewed., Conclusion: Patients with BMD presented with nonspecific symptoms, including fever, headache, chills, myalgia, and arthralgia. Laboratory confirmation of BMD was possible by PCR on blood from acutely symptomatic patients who were seronegative at presentation. Borrelia miyamotoi disease may be an emerging tickborne infection in the northeastern United States., Primary Funding Source: IMUGEN.

Background: Autotaxin (ATX) is a secreted enzyme that converts lysophosphatidylcholine to lysophosphatidic acid, a potent bioactive lipid mediator, through its lysophospholipase D activity. Although five alternative splicing isoforms of ATX have been identified as ATXα, ATXβ, ATXγ, ATXδ, and ATXε and the expression patterns of each isoform differ among several tissues, the clinical significance of each isoform remains to be elucidated., Methods: Anti-ATXβ and anti-ATXδ monoclonal antibodies were produced by immunization with recombinant human ATXβ and ATXδ expressed using a baculovirus system, respectively. We then developed enzyme immunoassays to measure the serum concentrations of "classical ATX" (ATXα, ATXβ, and ATXγ) and "novel ATX" (ATXδ and ATXε) antigens and evaluated the usefulness of these assays using human serum samples., Results: The with-run and between-run precision, interference, detection limit, and linearity studies for the present assay were well validated. In healthy subjects, the serum concentrations of classical ATX and novel ATX were significantly (P < 0.01) higher in women than in men, while the ratios of classical ATX or novel ATX to total ATX were not different between women and men. The concentrations of both classical ATX and novel ATX in normal pregnant subjects and patients with chronic liver diseases or follicular lymphoma were significantly higher than those in healthy subjects, while the ratio of both ATX isoforms to total ATX did not vary among these groups., Conclusions: We have developed a new enzyme immunoassay to determine the concentrations of classical ATX and novel ATX in human serum. These assays may be helpful for elucidating the distinct functional roles of each ATX isoform, which are largely unknown at present.

Background: Flowcytometric identification of basophils is a prerequisite for measuring activation of basophils with IgE-dependent or IgE-independent stimuli. Aim of this study was to compare different marker combinations in a simultaneous multicolor flowcytometric measurement., Methods: Ten patients with a grass pollen allergy and three controls were included in the study. Basophilic cells were gated by using anti-CCR3, anti-IgE, anti-CRTH2, anti-CD203c, and anti-CD3. Cells were activated by a monoclonal anti-FcεRI antibody, N-formyl-methionyl-leucyl-phenylalanine (fMLP), and the allergen extract Phleum pratense. The activation marker anti-CD63 was used., Results: The highest relative number of basophils was found with anti-CCR3+ cells, anti-IgE+ and anti-IgE+ /anti-CD203c+ cells, the lowest with CRTH2+/CD203c+/CD3- cells. A very good and good concordance of CCR3+ cells was seen with CCR3+/CD3- cells and CRTH2+/CD203c+/CD3- cells in all experiments. The contamination of the CCR3+ population with CD3+ cells and the contamination of the IgE+-population with CCR3- cells and CD203- cells were the lowest compared to all other marker combinations., Conclusions: As the highest relative number of basophils was identified by anti-CCR3 followed by the anti-IgE and anti-IgE/antiCD203c positive population in most cases, these markers can generally be recommended for identification of basophils. If a basophil population with very high purity is needed, anti-IgE should be chosen., (© 2014 International Clinical Cytometry Society.)

Envenomation caused by spiders Loxosceles induce intense dermonecrosis at the bite site and systemic disease. In this work we described the hyaluronidase and collagenase activities in vitro of the Loxosceles intermedia venom, but no phospholipase A2 activity. In vivo, we evaluated the effect of L. intermedia venom used different strain of mice, C57BL/6, BALB/c and Swiss. All mice developed paw edema after venom injection, persistent for 24 h in BALB/c and C57BL/6 mice. Histopathological analysis of the skin after venom injection revealed vascular congestion in Swiss mice and an inflammatory reaction in BALB/c and C57BL/6 mice. The mobilization of inflammatory cells from bone marrow, spleen and blood was investigated. Typical innate immune response with mobilization of myeloid cells and cytotoxic CD8 T lymphocytes was observed in C57BL/6 mice. In contrast, typical acquired/humoral immune response was observed in BALB/c mice, with preferential involvement of conventional B lymphocytes and CD4 T helper cells. The skin inflammation associated to mobilization of inflammatory cells indicated that mice models are strongly recommended to investigate specific cell types involved with immune response to the envenomation and mechanisms to inhibit skin lesions., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Crosslinking of the immunoglobulin receptor FcεRI activates basophils and mast cells to induce immediate and chronic allergic inflammation. However, it remains unclear how the chronic allergic inflammation is regulated. Here, we showed that ecto-nucleotide pyrophosphatase-phosphodiesterase 3 (E-NPP3), also known as CD203c, rapidly induced by FcεRI crosslinking, negatively regulated chronic allergic inflammation. Basophil and mast cell numbers increased in Enpp3(-/-) mice with augmented serum ATP concentrations. Enpp3(-/-) mice were highly sensitive to chronic allergic pathologies, which was reduced by ATP blockade. FcεRI crosslinking induced ATP secretion from basophils and mast cells, and ATP activated both cells. ATP clearance was impaired in Enpp3(-/-) cells. Enpp3(-/-)P2rx7(-/-) mice showed decreased responses to FcεRI crosslinking. Thus, ATP released by FcεRI crosslinking stimulates basophils and mast cells for further activation causing allergic inflammation. E-NPP3 decreases ATP concentration and suppresses basophil and mast cell activity., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Autotaxin (ATX), through its lysophospholipase D activity controls physiological levels of lysophosphatidic acid (LPA) in blood. ATX is overexpressed in multiple types of cancers, and together with LPA generated during platelet activation promotes skeletal metastasis of breast cancer. However, the pathophysiological sequelae of regulated interactions between circulating LPA, ATX, and platelets remain undefined in cancer. In this study, we show that ATX is stored in α-granules of resting human platelets and released upon tumor cell-induced platelet aggregation, leading to the production of LPA. Our in vitro and in vivo experiments using human breast cancer cells that do not express ATX (MDA-MB-231 and MDA-B02) demonstrate that nontumoral ATX controls the early stage of bone colonization by tumor cells. Moreover, expression of a dominant negative integrin αvβ3-Δ744 or treatment with the anti-human αvβ3 monoclonal antibody LM609, completely abolished binding of ATX to tumor cells, demonstrating the requirement of a fully active integrin αvβ3 in this process. The present results establish a new mechanism for platelet contribution to LPA-dependent metastasis of breast cancer cells, and demonstrate the therapeutic potential of disrupting the binding of nontumor-derived ATX with the tumor cells for the prevention of metastasis., (© 2014 by The American Society of Hematology.)

A chimeric protein (rCpLi) was constructed expressing three epitopes of rLiD1, a dermonecrotic toxin from the venom of Loxosceles intermedia spider. We have analyzed the neutralization potential of sera obtained by immunization of horses with rCpLi and rCpLi combined with initial doses of venoms and compared these with antivenom traditionally produced in horses using crude Loxosceles gaucho, Loxosceles laeta and L. intermedia venoms as antigens. We have demonstrated by ELISA that horses immunized with three initial doses of crude venom containing mixtures of L. intermedia, L. gaucho and L. laeta followed by nine doses of rCpLi generate antibodies with the same reactivity as those produced following immunization with traditional antivenom, towards the venoms of the three Loxosceles sp. species. Results from in vivo and in vitro neutralization assays showed that the new horse sera are able to neutralize the dermonecrotic activity of Loxosceles venoms, which are of medical importance in Brazil and some of these sera are capable of meeting the necessary potency requirements that could allow for their therapeutic use in humans. This immunization strategy combining both antigens used approximately 67% less crude Loxosceles venoms compared to traditional immunization protocol and can mean the development of Loxosceles antivenoms with the consequent reduction of devastation of arachnid fauna., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

T cells exhibit high-speed migration within the paracortical T zone of lymph nodes (LNs) as they scan cognate Ags displayed by dendritic cells in the tissue microenvironment supported by the network of stromal cells. Although intranodal T cell migration is controlled in part by chemokines and LFA-1/ICAM-1, the mechanisms underlying their migratory activity independent of these factors remain to be elucidated. In this study, we show that LN stromal cells constitutively express autotaxin (ATX), an ectoenzyme that is important for the generation of lysophosphatidic acid (LPA). Importantly, CCL21(+) stromal cells in the T zone produced and immobilized ATX on their cell surface. Two-photon imaging using LN tissue slices revealed that pharmacological inhibition of ATX or LPA receptors significantly reduced T cell migration, and this was further exacerbated by blockage of Gαi signaling or LFA-1. Therefore, T cell motility mediated by the ATX-LPA axis was independent of Gαi and LFA-1. LPA induced slow intermittent movement of T cells in vitro in a LFA-1-independent manner and enhanced CCL21-induced migration. Moreover, LPA and CCL21 cooperatively augmented RhoA activity in T cells, which was necessary for efficient intranodal T cell migration via the downstream ROCK-myosin II pathway. Taken together, T zone stromal cells control optimal migratory behavior of T cells via multiple signaling cues mediated by chemokines and ATX/LPA., (Copyright © 2014 by The American Association of Immunologists, Inc.)

Systemic loxoscelism is a rare complication after Loxosceles reclusa (brown recluse spider) envenomation. Loxosceles venom contains pro-inflammatory proteins, which have been shown to be elevated in patients with hemophagocytic lymph histiocytosis. We present a case of a 10-year-old male that developed presumed systemic loxoscelism, secondary hemophagocytic lymphohistiocytosis with hepatic dysfunction and renal failure. He was treated with dexamethasone and made a full recovery.

Background: There is need for an accurate diagnostic test in mastocytosis patients with wasp venom allergy (WVA) and monitoring of these patients during immunotherapy (IT). In this study, we aimed to evaluate sensitivity and specificity of the Basophil Activation Test (BAT) as a diagnostic and monitoring test in patients with mastocytosis and WVA., Methods: Seventeen patients with mastocytosis and WVA and six mastocytosis patients without WVA were included. BAT was performed before the start of IT (first visit) and at 6 weeks (second visit) and 1 year (third visit), after reaching the maintenance dose. Of 17 patients included, 11 completed the third visit. In mastocytosis patients with WVA, dose-dependent wasp-venom induced upregulation of CD63 and CD203c expression on basophils was observed compared with mastocytosis patients without WVA. Serum specific IgE, IgG4, and tryptase levels were measured in all patients., Results: BAT had a sensitivity of 87% and specificity of 100% in diagnosing WVA in mastocytosis patients. Basophil allergen threshold sensitivity with respect to CD63 and CD203c was significantly decreased in the second visit compared with the first visit and increased significantly in the third visit compared with the second visit. Specific IgE levels increased significantly in the second visit compared with first and decreased significantly in the third visit compared with the second. Specific IgG4 levels rose significantly in the second visit compared with the first and on the third visit compared with the second. Tryptase levels did not change significantly during the study., Conclusions: BAT represents a diagnostic test with 100% specificity in allergic patients with mastocytosis and these patients are better to be monitored for a longer period during IT., (© 2013 Clinical Cytometry Society.)

We report the production of a neutralizing monoclonal antibody able to recognize the venoms of three major medically important species of Loxosceles spiders in Brazil. The mAb was produced by immunization of mice with a toxic recombinant L. intermedia sphingomyelinase D {SMases D isoform (rLiD1)} [1] and screened by enzyme-linked immunosorbent assay (ELISA) using L. intermedia, L. laeta and L. gaucho venoms as antigens. One clone (LiD1mAb16) out of seventeen anti-rLiD1 hybridomas was cross-reactive with the three whole Loxosceles venoms. 2D Western blot analysis indicated that LiD1mAb16 was capable of interacting with 34 proteins of 29-36kDa in L. intermedia, 33 in L. gaucho and 27 in L. laeta venoms. The results of immunoassays with cellulose-bound peptides revealed that the LiD1mAb16 recognizes a highly conserved linear epitope localized in the catalytic region of SMases D toxins. The selected mAb displayed in vivo protective activity in rabbits after challenge with rLiD1. These results show the potential usefulness of monoclonal antibodies for future therapeutic approaches and also opens up the perspective of utilization of these antibodies for immunodiagnostic assays in loxoscelism., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Lysophosphatidic acid (LPA) is a pleiotropic lipid molecule with potent effects on cell growth and motility. Major progress has been made in recent years in deciphering the mechanisms of LPA generation and how it acts on target cells. Most research has been conducted in other disciplines, but emerging data indicate that LPA has an important role to play in immunity. A key discovery was that autotaxin (ATX), an enzyme previously implicated in cancer cell motility, generates extracellular LPA from the precursor lysophosphatidylcholine. Steady-state ATX is expressed by only a few tissues, including high endothelial venules in lymph nodes, but inflammatory signals can upregulate ATX expression in different tissues. In this article, we review current thinking about the ATX/LPA axis in lymphocyte homing, as well as in models of allergic airway inflammation and asthma. New insights into the role of LPA in regulating immune responses should be forthcoming in the near future.

Increased FcεR1α expression with upregulated CD203c expression on peripheral basophils is seen in patients with chronic urticaria (CU). However, there has been no published report on the association between CD203c expression level and clinical disease activity in CU patients. To investigate whether the increase of basophil activation is associated with the disease activity of CU, we measured basophil CD203c expression using a tricolor flow cytometric method in 82 CU patients and 21 normal controls. The relationship between the percentage of CD203c-expressing basophils and clinical parameters was analyzed. The mean basophil CD203c expression was significantly higher in CU patients than in healthy controls (57.5% vs 11.6%, P < 0.001). The basophil CD203c expression in severe CU patients was significantly higher than in non-severe CU (66.5% ± 23.3% vs 54.0% ± 23.3%, P = 0.033). Multiple logistic regression analysis indicated that both ≥ 72% basophil CD203c expression and urticaria activity score (UAS)≥ 13 were significant predictors of severe CU (P = 0.005 and P = 0.032, respectively). These findings suggest that the quantification of basophil activation with CD203c at baseline may be used as a potential predictor of severe CU requiring another treatment option beyond antihistamines.