Relaxin-like factor (RLF)/insulin-like peptide 3 (INSL3) is secreted from testicular Leydig cells as a monomeric protein comprising three domains B–C–A with full biological activity in boars

RLF (relaxin-like factor), also known as INSL3 (insulin-like peptide 3), is a novel member of the relaxin/insulin gene family that is expressed in testicular Leydig cells. Despite the implicated role of RLF/INSL3 in testis development, its native conformation remains unknown. In the present paper we demonstrate for the first time that boar testicular RLF/INSL3 is isolated as a monomeric structure with full biological activity. Using a series of chromatography steps, the native RLF/INSL3 was highly purified as a single peak in reverse-phase HPLC. MS/MS (tandem MS) analysis of the trypsinized sample provided 66% sequence coverage and revealed a distinct monomeric structure consisting of the B-, C- and A-domains deduced previously from the RLF/INSL3 cDNA. Moreover, the N-terminal peptide was four amino acid residues longer than predicted previously. MS analysis of the intact molecule and PMF (peptide mass fingerprinting) analysis at 100% sequence coverage confirmed this structure and indicated the existence of three site-specific disulfide bonds. RLF/INSL3 retained full bioactivity in HEK (human embryonic kidney)-293 cells expressing RXFP2 (relaxin/insulin-like family peptide receptor 2), the receptor for RLF/INSL3. Furthermore, RLF/INSL3 was found to be secreted from Leydig cells into testicular venous blood. Collectively, these results indicate that boar RLF/INSL3 is secreted from testicular Leydig cells as a B–C–A monomeric structure with full biological activity

Severity scales of non-IgE-mediated gastrointestinal food allergies in neonates and infants

Background: Non-IgE-mediated gastrointestinal food allergies (non-IgE-GI-FAs) are one type of food allergy found in neonates and infants. Few reports have defined the severity of non-IgE-GI-FAs in these populations. Methods: Grading scales of the severity of non-IgE-GI-FAs according to extra-GI symptoms, such as poor weight gain, as well as systemic symptoms, including fever and shock, were developed and retrospectively applied to patients with non-IgE-GI-FAs. The relationship between the severity of non-IgE-GI-FAs and both clinical and laboratory findings were examined. Results: Elevation of C-reactive protein levels and a decrease in total protein and albumin were observed in accordance with allergy severity. In an endoscopic examination, inflammatory findings were confirmed in large areas of the colonic mucosa in case of higher severity levels, and infiltration of inflammatory cells other than eosinophils was found in the severest grade. Extensively hydrolyzed milk or amino acid-based milk was required for all patients with the severest grade. In addition, the timing of acquiring tolerance tended to be late for this grade. Conclusions: Classification and determination of the severity of non-IgE-GI-FAs in neonates and infants may not only contribute to elucidation of the pathogenesis but may also be useful in the clinical setting. Keywords: Food hypersensitivity, Gastroenteritis, Infant, Milk, Prognosi

Oxidative stress induces MUC5AC expression through mitochondrial damage‐dependent STING signaling in human bronchial epithelial cells

Abstract Oxidative stress increases the production of the predominant mucin MUC5AC in airway epithelial cells and is implicated in the pathogenesis of bronchial asthma and chronic obstructive pulmonary disease. Oxidative stress impairs mitochondria, releasing mitochondrial DNA into the cytoplasm and inducing inflammation through the intracytoplasmic DNA sensor STING (stimulator of interferon genes). However, the role of innate immunity in mucin production remains unknown. We aimed to elucidate the role of innate immunity in mucin production in airway epithelial cells under oxidative stress. Human airway epithelial cell line (NCI‐H292) and normal human bronchial epithelial cells were used to confirm MUC5AC expression levels by real‐time PCR when stimulated with hydrogen peroxide (H2O2). MUC5AC transcriptional activity was increased and mitochondrial DNA was released into the cytosol by H2O2. Mitochondrial antioxidants were used to confirm the effects of mitochondrial oxidative stress where antioxidants inhibited the increase in MUC5AC transcriptional activity. Cyclic GMP‐AMP synthase (cGAS) or STING knockout (KO) cells were generated to investigate their involvement. H2O2‐induced MUC5AC expression was suppressed in STING KO cells, but not in cGAS KO cells. The epidermal growth factor receptor was comparably expressed in STING KO and wild‐type cells. Thus, mitochondria and STING play important roles in mucin production in response to oxidative stress in airway epithelial cells