Up-regulation of oxytocin receptors on peripheral sensory neurons mediates analgesia in chemotherapy-induced neuropathic pain.

BACKGROUND AND PURPOSE: Chemotherapy-induced neuropathic pain (CINP) currently has limited effective treatment. Although the roles of oxytocin (OXT) and the oxytocin receptor (OXTR) in central analgesia have been well documented, the expression and function of OXTR in the peripheral nervous system remain unclear. Here, we evaluated the peripheral antinociceptive profiles of OXTR in CINP. EXPERIMENTAL APPROACH: Paclitaxel (PTX) was used to establish CINP. Quantitative real-time polymerase chain reaction (qRT-PCR), in situ hybridization, and immunohistochemistry were used to observe OXTR expression in dorsal root ganglia (DRG). The antinociceptive effects of OXT were assessed by hot-plate and von Frey tests. Whole-cell patch clamp was performed to record sodium currents, excitability of DRG neurons, and excitatory synapse transmission. KEY RESULTS: Expression of OXTR in DRG neurons was enhanced significantly after PTX treatment. Activation of OXTR exhibited antinociceptive effects, by decreasing the hyperexcitability of DRG neurons in PTX-treated mice. Additionally, OXTR activation up-regulated the phosphorylation of protein kinase C (pPKC) and, in turn, impaired voltage-gated sodium currents, particularly the voltage-gated sodium channel 1.7 (NaV 1.7) current, that plays an indispensable role in PTX-induced neuropathic pain. OXT suppressed excitatory transmission in the spinal dorsal horn as well as excitatory inputs from primary afferents in PTX-treated mice. CONCLUSION AND IMPLICATIONS: The OXTR in small-sized DRG neurons is up-regulated in CINP and its activation relieved CINP by inhibiting the neural excitability by impairment of NaV 1.7 currents via pPKC. Our results suggest that OXTR on peripheral sensory neurons is a potential therapeutic target to relieve CINP.

Diagnostic Value of Serum Chitinase-3-Like Protein 1 for Liver Fibrosis: A Meta-analysis.

Background: Serum chitinase-3-like protein 1 (CHI3L1) is a promising marker for diagnosing liver fibrosis. This meta-analysis was carried out to assess the diagnostic performance of serum CHI3L1 for the estimation of liver fibrosis. Methods: Systematic searches were performed on PubMed, Embase, Web of Science, Scopus, the Cochrane Library, Google Scholar, Sinomed, the China National Knowledge Infrastructure (CNKI), the Chinese Medical Journal Database, and the Wanfang databases for available studies. The primary studies were screened strictly according to inclusion and exclusion criteria, and sensitivity, specificity, and other measures of accuracy of serum CHI3L1 for evaluating liver fibrosis were pooled with 95% confidence intervals. I 2 was calculated to assess heterogeneity, and sources of heterogeneity were explored by subgroup analysis. Deeks' test was used to assess for publication bias, and likelihood ratio was used to determine posttest probability. Results: Our research integrated 11 articles, accounting for 1897 patients older than 18 years old. The pooled sensitivity and specificity for significant fibrosis, advanced fibrosis, and cirrhosis were 0.79 and 0.82 with an area under the receiver operating characteristic curve (AUC) of 0.85, 0.81 and 0.83 with an AUC of 0.91, and 0.72 and 0.74 with an AUC of 0.85, respectively. Random-effects models were used to assess for significant heterogeneity, and subgroup analysis showed that age and aetiology of included patients were likely sources of heterogeneity. No potential publication bias was found for serum CHI3L1 in the diagnosis of significant fibrosis, advanced fibrosis, or cirrhosis, and posttest probability was moderate. Conclusion: Measurement of serum CHI3L1 is a feasible diagnostic tool for liver fibrosis.

Ribonuclease T2 from Aspergillus fumigatus promotes T helper type 2 responses through M2 polarization of macrophages.

Allergic bronchopulmonary aspergillosis (ABPA) is an allergic immunological response to Aspergillus fumigatus (Af) exposure, which induces a strong T helper 2 (Th2) response via mechanisms that have yet to be elucidated. The aim of the present study was to investigate the hypothesis that T2 ribonuclease from Af (Af RNASET2) induces M2­type macrophage polarization to produce a T helper 2 (Th2) immune response. Recombinant Af RNASET2 (rAf RNASET2) was expressed and purified in a prokaryotic pET system and BALB/c mice were immunized with rAf RNASET2 for in vivo analyses. Expression levels of M2 polarization factors were evaluated in RAW264.7 macrophages treated with rAf RNASET2 in vitro using flow cytometry, reverse transcription­quantitative PCR, and western blot analysis. The results predicted that the mature Af RNASET2 protein (382 amino acids; GenBank no. MN593022) contained two conserved amino acid sequence (CAS) domains, termed CAS­1 and CAS­2, which are also characteristic of the RNASET2 family proteins. The protein expression levels of the Th2­related cytokines interleukin (IL)­4, IL­10, and IL­13 were upregulated in mice immunized with rAf RNASET2. RAW264.7 macrophages treated with rAf RNASET2 showed increased mRNA expression levels of M2 factors [arginase 1, Il­10, and Il­13]; however, there was no difference in cells treated with rAf RNASET2 that had been inactivated with a ribonuclease inhibitor (RNasin). The protein expression levels of IL­10 in macrophage culture supernatant were also increased following stimulation with rAf RNASET2. In addition, rAf RNASET2 upregulated the expression of phosphorylated mitogen activated protein kinases (MAPKs) in RAW264.7 cells, whereas MAPK inhibitors attenuated rAf RNASET2­induced IL­10 expression in RAW264.7 cells. In conclusion, the present study reveals that high rAf RNASET2 activity is required for rAf RNASET2­induced M2 polarization of macrophages and suggests an important immune regulatory role for Af RNASET2 in ABPA pathogenesis.

Enhanced sensitivity of capture IgE­ELISA based on a recombinant Der f 1/2 fusion protein for the detection of IgE antibodies targeting house dust mite allergens.

The detection of allergen­specific immunoglobulin (Ig)E is an important method for the diagnosis of IgE­mediated allergic diseases. The sensitivity of the indirect IgE­ELISA method against allergen extracts is limited by interference from high IgG titers and low quantities of effectual allergen components in extracts. To overcome these limitations, a novel capture IgE­ELISA based on a recombinant Der f 1/Der f 2 fusion protein (rDer f 1/2) was developed to enhance the sensitivity to IgEs that bind allergens from the house dust mite (HDM) species Dermatophagoides farina. pET28­Der f 1/2 was constructed and expressed in Escherichia coli BL21 (DE3) pLysS. The purified fusion protein was evaluated by IgE western blotting, IgE dot blotting and indirect IgE­ELISA. Capture­ELISA was performed by coating wells with omalizumab and incubating in series with sera, biotinylated Der f 1/2, horseradish peroxidase­conjugated streptavidin and 3,3,5,5­tetramethylbenzidine. The relative sensitivities of indirect­ELISA and capture­ELISA for HDM allergen­specific IgE binding were determined; sera from non­allergic individuals were used as the control group. rDer f 1/2 was expressed in the form of inclusion bodies comprising refolded protein, which were then purified. It exhibited increased IgE­specific binding (24/28, 85.8%) than rDer f 1 (21/28, 75.0%) or rDer f 2 (22/28, 78.6%) with HDM­allergic sera. Furthermore, in a random sample of HDM­allergic sera (n=71), capture­ELISA (71/71, 100%) was more sensitive than indirect­ELISA (68/71, 95.8%) for the detection of HDM­specific IgEs (P<0.01), indicating that this novel method may be useful for the diagnosis of HDM allergy.

Identification of immunodominant IgE epitopes of the major house dust mite allergen Der f 24.

Previously, a ubiquinol­cytochrome c reductase binding protein (UQCRB) homolog was identified in the house dust mite (HDM) species Dermatophagoides farinae (Der f) as a major allergen. In the present study, the immunodominant immunoglobulin E (IgE) epitope of the protein Der f 24 was investigated. Analysis of the homologous amino acid (aa) sequences in Der f and human UQCRB was performed. Four different recombinant Der f 24 and hybrid proteins formed by integrating Der f and human UQCRB sequences were expressed in Escherichia coli, purified using Ni­NTA resins, and IgE­binding activity was determined using IgE­western blotting and enzyme­linked immunosorbent assay (ELISA) experiments. IgE epitopes were further identified by IgE­dot blotting and IgE­ELISA with synthetic polypeptides and HDM­allergic sera. Three­dimensional (3D) structural modeling was used to analyze the position of the immunodominant IgE epitope. The amino acid sequence homology between Der f 24 and the human UQCRB protein was determined to be 39.34%. IgE­ELISA and western blot analysis showed that all of the Der f­human UQCRB hybrid proteins generated, except for the one lacking 59 residues of the N­terminal region of Der f 24, were bound by allergic serum IgE. A synthetic polypeptide consisting of 32 residues of the N­terminal reacted with IgEs from HDM­allergic sera and could be used to generate high titer specific IgG or specific IgE antibodies in immunized mice. The 32­aa N­terminal region of Der f 24 was localized to a structural protrusion, which may facilitate specific IgE­binding. These results indicate that the immunodominant IgE epitope of Der f 24 is located mainly in a 32­residue region of the N­terminus. These findings may inform the mechanisms of HDM allergy sensitization and allergy immunotherapy development.

Identification of Der f 23 as a new major allergen of Dermatophagoides farinae.

House dust mites (HDM) are common allergen sources worldwide. At present, 32 of the 37 internationally recognized HDM allergen groups have been identified in Dermatophagoides farinae. The present study study describes the identification of the first known D. farinae Group 23 allergen (Der f 23). Recombinant Der f 23 protein (rDer f 23) was cloned, expressed and purified. The open reading frame of rDer f 23 was 525 base pairs and encoded a 174­amino acid protein (GenBank accession no., KU166910.1). ELISAs indicated that 72/129 HDM allergic serum samples (55.8%) had specific immunoglobulin E (sIgE) binding activity to rDer f 23. Additionally, 3/10 patients with HDM allergies (30%) exhibited positive skin prick test reactions to rDer f 23. IgE western blot analysis data suggested that only 4/11 HDM allergic sera had a positive sIgE binding result. Sequence homology analysis revealed an extra P2 region (Ser56­Thr117) in Der f 23 that was not present in the D. pteronyssinus homolog, which may affect sIgE binding. Der f 23ΔP2 demonstrated binding with HDM allergic sera, whereas the P2 peptide alone did not. The sIgE binding ability of Der f 23 ΔP2 (Der f 23 with a truncated P2 region) was more marked compared with that of Der f 23 in an IgE ELISA. These data indicate that P2 region in Der f 23 attenuates IgE binding ability. In conclusion, the results of the present study indicate that Der f 23 is a major HDM allergen with predominantly conformational sIgE binding epitopes. The allergenic identification of Der f 23 and its inclusion in World Health Organization/International Union of Immunological Societies database contributes to the theoretical basis underlying the diagnosis and treatment of HDM allergic diseases.

Identification of immunodominant IgE binding epitopes of Der p 24, a major allergen of Dermatophagoides pteronyssinus.

BACKGROUND: The identification of house dust mite (HDM) allergens and epitopes is important for allergy diagnosis and treatment. We sought to identify the Dermatophagoides pteronyssinus group 24 allergen (Der p 24) and to identify its immunodominant IgE epitope(s). METHODS: Der p 24 cDNA was cloned and expressed in a pET expression system. The IgE binding activity of purified recombinant (r)Der p 24 was evaluated by western blotting. Truncated Der p 24 proteins and overlapping synthetic polypeptides were subjected to IgE binding assays. Balb/c mice were immunized to investigate IgE epitope induction of IgE production. IgE binding of the 32 N-terminal residues of Der p 24 was compared to other Der p epitopes in enzyme-linked immunosorbent assays and dot blot assays. Human skin prick tests (SPTs) were performed. RESULTS: We cloned and expressed Der p 24 cDNA (GenBank accession no. KP893174.1). HDM allergic sera bound rDer p 24 in vitro and 5/10 HDM allergic patients (50%) had positive SPT reactions to rDer p 24. The immunodominant IgE epitope of Der p 24 was localized to the N-terminal 32-residue region, which produced a high specific IgE antibody titer in vivo and promoted mast cell ß-hexosaminidase release. The IgE binding activity this N-terminal epitope of Der p 24 was stronger than that of Der p 1 or Der p 2 IgE epitopes. CONCLUSIONS: We identified Der p 24 as a major HDM allergen with strong IgE binding activity via an immunodominant IgE epitope in the N-terminal 32-residue region, which triggers IgE production in vivo. The identified Der p 24 epitope may support HDM allergy diagnosis and treatment.