Functional characterization of two ecto-nucleoside triphosphate diphosphohydrolase 2 genes in Japanese flounder (Paralichthys olivaceus) head kidney macrophages.

Ecto-nucleoside triphosphate diphosphohydrolases (ENTPDases) are pivotal regulators of extracellular ATP-mediated purinergic immune signaling. ENTPDase2 is a member of the cell surface-bound ecto-nucleoside triphosphate diphosphohydrolase (ENTPDase) protein family that hydrolyzes extracellular nucleoside 5'-triphosphates and nucleoside 5'-diphosphates. However, the immune relevance of ENTPDase2 in fish has not been elucidated. In the present study, from a comparative immunological perspective, we functionally characterized two ENTPDase2 transcript variants (namely ENTPDase2 and ENTPDase2a) from Japanese flounder (Paralichthys olivaceus). Sequence analysis indicates that the deduced Japanese flounder ENTPDase2 and ENTPDase2a proteins possess two conserved transmembrane domains and five apyrase conserved regions that are present in ENTPDase family proteins. However, these proteins only share 54% amino acid sequence identity. Tissue expression analysis revealed that both ENTPDase2 and ENTPDase2a mRNA transcripts are ubiquitously expressed in all examined Japanese flounder tissues, whereas ENTPDase2 is dominantly expressed in blood and ENTPDase2a is abundantly expressed in muscle. Immune challenge experiments showed that ENTPDase2 and ENTPDase2a were significantly upregulated by both inflammatory stimulation and Edwardsiella tarda infection. In addition, the expression of ENTPDase2 and ENTPDase2a was modulated by extracellular ATP (eATP) stimulation in a dose-dependent manner. Furthermore, immunolocalization and functional studies demonstrated that both ENTPDase2 and ENTPDase2a are functional glycosylated plasma membrane proteins. However, ENTPDase2a exhibits greater activity in the hydrolysis of eATP than ENTPDase2 and ENTPDase1 proteins. Finally, knockdown of the ENTPDase2 gene by small interfering RNA significantly upregulated the expression of eATP-induced proinflammatory cytokines IL-1beta, TNF-alpha and G-CSF in Japanese flounder head kidney macrophages, while knockdown of ENTPDase2a only upregulated eATP-induced IL-1beta expression. Taken together, our findings suggest that the two functional Japanese flounder ENTPDase2 isoforms play an essential role in the downregulation of eATP-induced proinflammatory cytokine expression in fish by degrading the available ATP levels in the extracellular milieu.

Microfluidic Immunoaffinity Basophil Activation Test for Point-of-Care Allergy Diagnosis.

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.

Updates on the surface antigens of basophils: CD16 on basophils of patients with respiratory or insect venom allergy and the rejection of CD203c and CD63 externalization decoupling by bisindolylmaleimides.

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 CD16dim . Using CD16dim 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.

Generation of a potential koi herpesvirus live vaccine by simultaneous deletion of the viral thymidine kinase and dUTPase genes.

Koi herpesvirus (KHV, Cyprinidherpesvirus 3) causes a fatal disease of koi and common carp. To obtain safe and efficacious live vaccines, we generated deletion mutants of KHV lacking the nonessential genes encoding two enzymes of nucleotide metabolism, thymidine kinase (TK, ORF55) and deoxyuridine-triphosphatase (DUT, ORF123). Since single-deletion mutants based on a KHV isolate from Israel (KHV-I) only exhibited partial attenuation (Fuchs W, Fichtner D, Bergmann SM, Mettenleiter TC. Arch Virol 2011;156 : 1059-1063), a corresponding double mutant was generated and tested in vivo, and shown to be almost avirulent but still protective. To overcome the low in vitro virus titres of KHV-I (≤105 p.f.u. ml-1), single and double TK and DUT deletions were also introduced into a cell culture-adapted KHV strain from Taiwan (KHV-T). The deletions did not affect in vitro virus replication, and all KHV-T mutants exhibited wild-type-like plaque sizes and titres exceeding 107 p.f.u. ml-1, as a prerequisite for economic vaccine production. Compared to wild-type and revertant viruses, the single-deletion mutants of KHV-T were significantly attenuated in vivo, and immersion of juvenile carp in water containing high doses of the double mutant caused almost no fatalities. Nevertheless, the deletion mutants induced similar levels of KHV-specific serum antibodies to the parental wild-type virus, and conferred solid protection against disease after challenge with wild-type KHV. For the convenient differentiation of DNA samples prepared from gill swabs of carp infected with wild-type and TK-deleted KHV we developed a triplex real-time PCR. Thus, KHV-TΔDUT/TK might be suitable as a genetic DIVA vaccine in the field.

Phase I Trials of Anti-ENPP3 Antibody-Drug Conjugates in Advanced Refractory Renal Cell Carcinomas.

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.

Basophil biomarkers as useful predictors for sublingual immunotherapy in allergic rhinitis.

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 (T0) and after 12 months (T12) 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.

Role of Atorvastatin in Treatment of Chronic Spontaneous Urticaria Patients: A Controlled Clinical Trial.

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.

Purinergic system displays an anti-inflammatory profile in serum of silver catfish experimentally infected with Streptococcus agalactiae: An attempt to ameliorate the inflammatory response.

The purinergic system is recognized to modulate extracellular adenosine triphosphate (ATP) and related nucleotides through the activities of triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and adenosine deaminase (ADA), thereby playing an essential role in the immunoregulation of inflammatory and immune responses. Thus, the aim of this study was to evaluate whether the purinergic system can improve the inflammatory response in fish experimentally infected with Streptococcus agalactiae through the modulation of seric NTPDase, 5'-nucleotidase and ADA activities. Seric NTPDase (ATP as substrate) and 5'-nucleotidase activities increased in silver catfish experimentally infected with S. agalactiae compared with the uninfected control group, while seric ADA activity decreased. Based on this evidence, our findings suggest that regulation of adenine nucleotide hydrolysis occurs in an attempt to restrict the inflammatory process and improve the immune system by hydrolyzing excess extracellular ATP. On the other hand, downregulation of seric ADA activity may be an attempt to augment extracellular adenosine (a molecule with anti-inflammatory effects) levels. In summary, the purinergic system is capable of modulating the immune and inflammatory responses during fish streptococcosis.

Blocking Allergic Reaction through Targeting Surface-Bound IgE with Low-Affinity Anti-IgE Antibodies.

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.

Myalgic encephalomyelitis/chronic fatigue syndrome and gulf war illness patients exhibit increased humoral responses to the herpesviruses-encoded dUTPase: Implications in disease pathophysiology.

Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) and Gulf War Illness (GWI) are debilitating diseases with overlapping symptomology and there are currently no validated tests for definitive diagnosis of either syndrome. While there is evidence supporting the premise that some herpesviruses may act as possible triggers of ME/CFS, the involvement of herpesviruses in the pathophysiology of GWI has not been studied in spite of a higher prevalence of ME/CFS in these patients. We have previously demonstrated that the deoxyuridine triphosphate nucleotidohydrolases (dUTPase) encoded by Epstein-Barr virus (EBV), human herpesvirus-6 (HHV-6), and varicella-zoster virus (VZV) possess novel functions in innate and adaptive immunity. The results of this study demonstrate that a significant percentage of patients with ME/CFS (30.91-52.7%) and GWI (29.34%) are simultaneously producing antibodies against multiple human herpesviruses-encoded dUTPases and/or the human dUTPase when compared to controls (17.21%). GWI patients exhibited significantly higher levels of antibodies to the HHV-6 and human dUTPases than controls (P = 0.0053 and P = 0.0036, respectively), while the ME/CFS cohort had higher anti-EBV-dUTPase antibodies than in both GWI patients (P = 0.0008) and controls (P < 0.0001) as well as significantly higher anti-human dUTPase antibodies than in controls (P = 0.0241). These results suggest that screening of patients' sera for the presence of various combinations of anti-dUTPase antibodies could be used as potential biomarkers to help identify/distinguish patients with these syndromes and better direct treatment.

Does Epoetin Beta Still Have a Place in Peginterferon Alpha-2a Plus Ribavirin Treatment Strategies for Chronic Hepatitis C?

To investigate the impact of epoetin beta (EPO) on sustained virological response (SVR) in hepatitis C virus (HCV)-infected patients treated with peginterferon-ribavirin (RBV). Controlled, randomized, pragmatic multicenter study to assess 2 strategies, ie, the use (EPO group) or nonuse (control group) of EPO in terms of achieving SVR in treatment-naive, genotype non-2/non-3 HCV-infected patients receiving a 48-week treatment regimen of pegylated interferon α-2a (peg-IFN) plus RBV (randomization 2:1). The single-nucleotide polymorphisms of interferon lambda 3 (IFNL3) (rs12979860 and rs8099917), interferon lambda 4 (IFNL4) (ss469415590), and inosine triphosphatase (ITPA) (rs1127354 and rs7270101) were determined retrospectively. Two hundred twenty-seven patients were included in the study. In the global population (n = 227), the overall SVR rate was 52% (118/227). Nonresponse and relapse occurred in respectively 46/227 (20.3%) and 42/227 (18.5%) patients. In the intention-to-treat analysis, 55.5% of patients with anemia (n = 164) had a SVR, specifically 57.4% in the EPO group versus 52.4% in the control group, but the difference was not statistically significant. In the anemic population, independent factors associated with SVR were IFNL3 and IFNL4 polymorphisms, pretreatment HCV RNA level, iron level, and aspartate aminotransferase/alanine aminotransferase (AST/ALT) ratio. EPO has little impact on SVR in patients treated with peg-IFN+RBV and should be recommended only for patients with severe anemia.

[Preparation and identification of rabbit polyclonal antibodies against vacuolar proton pyrophosphatase type I of Toxoplasma gondii].

OBJECTIVE: To prepare and characterize rabbit polyclonal antibodies against Toxoplasma gondii vacuolar proton pyrophosphatase type I (TgVP1). METHODS AND RESULTS: Two synthesized peptides TgVP1-1 and TgVP1-2 as the haptens were conjugated with KLH to immunize rabbits. Indirect ELISA showed that the titers of rabbit anti-TgVP1-1 polyclonal antibody and rabbit anti-TgVP1-2 polyclonal antibody reached 1:128 000. Western blotting results revealed that both purified polyclonal antibodies could specifically bind to a purified 85 kD T. gondii protein predicted as TgVP1. The protein detected by these two polyclonal antibodies was distributed in the cytoplasm of T. gondii tachyzoite, and this distribution pattern was consistent with that of acidocalcisome. CONCLUSION: The peptide-based method of antibody generation is efficient and the obtained TgVP1 polyclonal antibodies possess a high specificity to facilitate further study of T. gondii acidocalcisome and the diagnosis of toxoplasmosis.

The Activation of Phytophthora Effector Avr3b by Plant Cyclophilin is Required for the Nudix Hydrolase Activity of Avr3b.

Plant pathogens secrete an arsenal of effector proteins to impair host immunity. Some effectors possess enzymatic activities that can modify their host targets. Previously, we demonstrated that a Phytophthora sojae RXLR effector Avr3b acts as a Nudix hydrolase when expressed in planta; and this enzymatic activity is required for full virulence of P. sojae strain P6497 in soybean (Glycine max). Interestingly, recombinant Avr3b produced by E. coli does not have the hydrolase activity unless it was incubated with plant protein extracts. Here, we report the activation of Avr3b by a prolyl-peptidyl isomerase (PPIase), cyclophilin, in plant cells. Avr3b directly interacts with soybean cyclophilin GmCYP1, which activates the hydrolase activity of Avr3b in a PPIase activity-dependent manner. Avr3b contains a putative Glycine-Proline (GP) motif; which is known to confer cyclophilin-binding in other protein substrates. Substitution of the Proline (P132) in the putative GP motif impaired the interaction of Avr3b with GmCYP1; as a result, the mutant Avr3bP132A can no longer be activated by GmCYP1, and is also unable to promote Phytophthora infection. Avr3b elicits hypersensitive response (HR) in soybean cultivars producing the resistance protein Rps3b, but Avr3bP132A lost its ability to trigger HR. Furthermore, silencing of GmCYP1 rendered reduced cell death triggered by Avr3b, suggesting that GmCYP1-mediated Avr3b maturation is also required for Rps3b recognition. Finally, cyclophilins of Nicotiana benthamiana can also interact with Avr3b and activate its enzymatic activity. Overall, our results demonstrate that cyclophilin is a "helper" that activates the enzymatic activity of Avr3b after it is delivered into plant cells; as such, cyclophilin is required for the avirulence and virulence functions of Avr3b.

Generation and Identification of Peptide-Based Monoclonal Antibodies Against Vacuolar Proton Pyrophosphatase of Toxoplasma gondii.

Vacuolar proton pyrophosphatase (V-PPase), an electrogenic proton pump widely distributed in non-mammalian species, is one of the important targets for acidocalcisomes. In this study, a novel method of peptide-based antibody generation was performed to produce monoclonal antibodies (MAbs) against Toxoplasma gondii V-PPase. Three hybridomas were identified and confirmed by ELISA, Western blotting, and immunofluorescence. All of them can react with an 85 kDa band of T. gondii protein in purified acidocalcisomal fraction. The three MAbs were all specific to the synthetic peptide of YTKAADVGADLSGKNEYGMSEDDPRNPAC, corresponding to amino acids at the location of 292aa-320aa of TgVP1 amino acid sequence. These specific MAbs will be valuable tools for further study of T. gondii infection biology, pathogenesis, and host immune response.

Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

Oral vaccination of mice with Trichinella spiralis nudix hydrolase DNA vaccine delivered by attenuated Salmonella elicited protective immunity.

We have previously reported that Trichinella spiralis Nudix hydrolase (TsNd) bound to intestinal epithelial cells (IECs), and the vaccination of mice with recombinant TsNd protein (rTsNd) produced a partial protective immunity against challenge infection in mice. In this study, the full-length cDNA sequence of TsNd gene was cloned into the eukaryotic expression plasmid pcDNA3.1, and the recombinant TsNd DNA was transformed into attenuated Salmonella typhimurium strain ⊿cyaSL1344. Oral immunization of mice with TsNd/S. typhimurium elicited a significant local mucosal IgA response and a systemic Th1/Th2 immune response. Cytokine profiling also showed a significant increase in the Th1 (IFN-γ, IL-2) and Th2 (IL-4, 10) responses in splenocytes of immunized mice upon stimulation with the rTsNd. The oral immunization of mice with TsNd/S. typhimurium displayed a statistically significant 73.32% reduction in adult worm burden and a 49.5% reduction in muscle larvae after challenge with T. spiralis muscle larvae, compared with PBS control group. Our results demonstrated that TsNd DNA delivered by attenuated live S. typhimurium elicited a local IgA response and a mixed Th1/Th2 immune response, and produced a partial protection against T. spiralis infection in mice.