Age-related increase of autoantibodies to interleukin 1α in healthy Japanese blood donors

Although autoantibodies to interleukin-1α (IL-1α autoantibodies) are known to be present in sera of apparently healthy humans, their frequency of occurrence and significance are unclear. To determine the prevalence of detectable IL-1α autoantibodies in normal human blood, we screened the plasma of blood donors (6290 subjects : 3977 men and 2313 women, ages 16 to 64 yr) by a radioimmunoassay which we developed using a method that could detect over 5 ng/ml. Moreover, we investigated immunoglobulin class of IL-1α autoantibodies and also their function. IL-1α autoantibodies were detected in 14.6% of the 6290 donors. Their frequency was higher in males than females (16.6% vs.11.2%, p<0.01) and increased with age in both sexes. The proportion of subjects with a high IL-1α autoantibodies titers also increased with age. We showed that IL-1α autoantibodies were of the IgG class and that they had neutralizing function to IL-1α by receptor assay. Neutralizing activity was only shown in plasma with concentration of IL-1α autoantibodies, the level of which was over 1000 ng/ml. The affinity of the IL-1α autoantibodies in plasma was between 2.1 X 10-10and 1.2 X 10-9 M (mean 6.4 X 10-10M). Our results provide a basis for comparison with IL-1α autoantibodies prevalence between healthy states and disease states, and suggest that IL-1α autoantibodies may play a significant role in modulating the effects of excessive IL-1α at local site or in systemic regions

Pathophysiological Investigation of the Gastric Surface Mucous Gel Layer of Patients with Helicobacter pylori Infection by Using Immunoassays for Trefoil Factor Family 2 and Gastric Gland Mucous Cell-Type Mucin in Gastric Juice

Background The trefoil factor family (TFF) 2 protein is produced by gastric gland mucous cells (GMCs), and the secreted TFF2 shares a mucosal barrier function with GMC-type mucin. Recently, we presented an enzyme-linked immunosorbent assay (ELISA) method for measurement of GMC-type mucin in the gastric juice. Aims We aimed to develop an ELISA for TFF2 and to assess pathophysiological changes in the gastric surface mucous gel layer (SMGL) of patients with Helicobacter pylori infection. Methods The distribution of TFF2 and GMC-type mucin in the SMGL was immunohistochemically determined. The ELISA for TFF2 was based on a polyclonal goat antibody. Recombinant TFF2 was employed to prepare the calibrators. TFF2 and GMC-type mucin in the gastric juice in healthy individuals (n = 33) and patients with gastritis (n = 37), gastric ulcer (n = 16), and duodenal ulcer (n = 10) were assayed using ELISA. Results TFF2 and GMC-type mucin were immunohistochemically co-localized in the gastric SMGL and GMCs. The TFF2 levels in the patients were significantly higher than those in the healthy individuals. Further, the TFF2 levels in the H. pylori-positive patients were significantly higher than those in the H. pylori-negative patients, and decreased after the eradication of the infection. GMC-type mucin levels showed a tendency similar to that of TFF2 levels. Conclusions The upregulation of TFF2 and GMC-type mucin secretion may reflect the response of the gastric mucosa to H. pylori-induced injuries. TFF2 and GMC-type mucin secreted into the SMGL may protect the gastric mucosa against H. pylori.ArticleDIGESTIVE DISEASES AND SCIENCES. 56(12):3498-3506 (2011)journal articl

Correlation between salivary secretion and salivary AQP5 levels in health and disease

Saliva samples are useful for noninvasive diagnosis of oral and systemic diseases. The water channel protein aquaporin-5 (AQP5) is released into human saliva. Salivary AQP5 levels show a diurnal variation with the secretion of high levels during the waking hours. An age-related decrease in salivary AQP5 levels parallels a decrease in the volume of saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induces the release of AQP5. Changes in salivary AQP5 levels after cevimeline administration occur simultaneously with changes in saliva flow rate. AQP5 and lipid rafts are released separately from human salivary glands upon M3 mAChR stimulation. In patients with diabetes mellitus or Sjögren’s syndrome, a decrease in salivary secretion occurs concomitantly with low salivary AQP5 levels. Salivary AQP5 levels correlate with salivary secretion in both healthy and disease states, suggesting that changes in salivary AQP5 levels can be used as an indicator of salivary flow rate and the effect of M3 mAChR agonists on human salivary glands

Autoantibodies to IL-1α in sera from rapidly progressive idiopathic pulmonary fibrosis

To clarify the clinical significance of autoantibodies to interleukin-1α (IL-1α autoantibodies) in rapidly progressive idiopathic pulmonary fibrosis ( IPF ), we measured the level of IL-1α autoantibodies in serum of 11 patients on the first hospital day, when patients were admitted due to severe symptoms, and on the 21st hospital day. IL-1α autoantibodies in serum were measured using radioimmunoassay, and the limitation of this assay for IL-1α autoantibodies was 5 ng/ml. These antibodies were detected in 5 of 11 patients on the first hospital day. On the 21st hospital day, these antibodies were detected in all patients, and its level was increased compared with that on the first hospital day. IL-1α autoantibodies that appeared in patients corresponded to that of IgG. The half life of exogeneous autoantibodies was investigated following administration of autoantibody rich plasma obtained from healthy blood donors to 6 control patients (CP) and 6 progressive IPF patients. These autoantibody levels in their serum were less than 5 ng/ml before administration. Serum was obtained at the indicated time after administration of IL-1α autoantibodies and the level of these autoantibodies in serum was measured, then the half life was calculated. Half life of exogeneous IL-1α autoantibodies in progressive IPF patients was significantly shorter than that in CP (71.3±31.8 hr vs 352.0±98.3 hr, p<0.01).These findings suggested that IL-1α autoantibodies were generated in response to the inflammatory process of rapidly progressive IPF and may act as a regulatory factor for IL-1α

Pathophysiological Investigation of the Gastric Surface Mucous Gel Layer of Patients with Helicobacter pylori Infection by Using Immunoassays for Trefoil Factor Family 2 and Gastric Gland Mucous Cell-Type Mucin in Gastric Juice

Background The trefoil factor family (TFF) 2 protein is produced by gastric gland mucous cells (GMCs), and the secreted TFF2 shares a mucosal barrier function with GMC-type mucin. Recently, we presented an enzyme-linked immunosorbent assay (ELISA) method for measurement of GMC-type mucin in the gastric juice. Aims We aimed to develop an ELISA for TFF2 and to assess pathophysiological changes in the gastric surface mucous gel layer (SMGL) of patients with Helicobacter pylori infection. Methods The distribution of TFF2 and GMC-type mucin in the SMGL was immunohistochemically determined. The ELISA for TFF2 was based on a polyclonal goat antibody. Recombinant TFF2 was employed to prepare the calibrators. TFF2 and GMC-type mucin in the gastric juice in healthy individuals (n = 33) and patients with gastritis (n = 37), gastric ulcer (n = 16), and duodenal ulcer (n = 10) were assayed using ELISA. Results TFF2 and GMC-type mucin were immunohistochemically co-localized in the gastric SMGL and GMCs. The TFF2 levels in the patients were significantly higher than those in the healthy individuals. Further, the TFF2 levels in the H. pylori-positive patients were significantly higher than those in the H. pylori-negative patients, and decreased after the eradication of the infection. GMC-type mucin levels showed a tendency similar to that of TFF2 levels. Conclusions The upregulation of TFF2 and GMC-type mucin secretion may reflect the response of the gastric mucosa to H. pylori-induced injuries. TFF2 and GMC-type mucin secreted into the SMGL may protect the gastric mucosa against H. pylori.ArticleDIGESTIVE DISEASES AND SCIENCES. 56(12):3498-3506 (2011)journal articl

Association of mast cell-derived VEGF and proteases in dengue shock syndrome

Background: Recent in-vitro studies have suggested that mast cells are involved in Dengue virus infection. To clarify the role of mast cells in the development of clinical Dengue fever, we compared the plasma levels of several mast cell-derived mediators (vascular endothelial cell growth factor [VEGF], soluble VEGF receptors [sVEGFRs], tryptase, and chymase) and -related cytokines (IL-4, -9, and -17) between patients with differing severity of Dengue fever and healthy controls. Methodology/Principal Findings: The study was performed at Children\u27s Hospital No. 2, Ho Chi Minh City, and Vinh Long Province Hospital, Vietnam from 2002 to 2005. Study patients included 103 with Dengue fever (DF), Dengue hemorrhagic fever (DHF), and Dengue shock syndrome (DSS), as diagnosed by the World Health Organization criteria. There were 189 healthy subjects, and 19 febrile illness patients of the same Kinh ethnicity. The levels of mast cell-derived mediators and -related cytokines in plasma were measured by ELISA. VEGF and sVEGFR-1 levels were significantly increased in DHF and DSS compared with those of DF and controls, whereas sVEGFR-2 levels were significantly decreased in DHF and DSS. Significant increases in tryptase and chymase levels, which were accompanied by high IL-9 and -17 concentrations, were detected in DHF and DSS patients. By day 4 of admission, VEGF, sVEGFRs, and proteases levels had returned to similar levels as DF and controls. In-vitro VEGF production by mast cells was examined in KU812 and HMC-1 cells, and was found to be highest when the cells were inoculated with Dengue virus and human Dengue virus-immune serum in the presence of IL-9. Conclusions: As mast cells are an important source of VEGF, tryptase, and chymase, our findings suggest that mast cell activation and mast cell-derived mediators participate in the development of DHF. The two proteases, particularly chymase, might serve as good predictive markers of Dengue disease severity

泌尿器科癌患者末梢血におけるInterleukin-1α, Interleukin-1β産生能の検討

対象は60例の泌尿器科癌患者.その内訳は膀胱癌30例, 腎癌12例, 前立腺癌18例であった.また, 対照群は健常者16例.測定方法はヘパリン採血した全血0.1mlずつを, 6段階(0, 0.1, 0.3, 1, 3, 5μg/ml)の濃度のリポポリサッカライド添加無血清培地にて24時間培養し, その上清をIL-1α, IL-1βに対するモノクローナル抗体を用いたELISA法にて測定した.リポポリサッカライドを含まない条件での全血1mlあたりのIL-1α, IL-1β産生量は, 膀胱癌患者, 腎癌患者, 前立腺癌患者では健常者と比較して有意差を認めなかった.次に5段階濃度のリポポリサッカライドを用いた場合, 膀胱癌患者, 腎癌患者, 前立腺癌患者, 健常者におけるIL-1α, IL-1β産生量はdose responseを示した.また, IL-1βの産生量はIL-1αより高値であった.しかし, 膀胱癌患者, 腎癌患者, 前立腺癌患者と対照群の間でのIL-1α, IL-1β産生量は有意差を認めなかったThe activities of interleukin-1 alpha (IL-1 alpha) and interleukin-1 beta) were investigated in peripheral whole blood from 30 patients with bladder cancer, 12 patients with renal cell carcinoma, 18 patients with prostatic cancer and 16 healthy subjects. Heparinized blood was cultured in the absence and presence of various concentrations of bacterial lipopolysaccharide (LPS). The culture supernatants were obtained and activities of IL-1 alpha and IL-1 beta were determined by enzyme-linked immunosorbent assay (ELISA). In the absence of LPS stimulation, neither IL-1 alpha nor IL-1 beta was spontaneously produced in blood cultures from patients with bladder cancer, renal cell carcinoma or prostatic cancer compared with control subjects. After stimulation with various concentrations of LPS, blood cultures from patients with bladder cancer, renal cell carcinoma, prostatic cancer, those from control subjects produced IL-1 alpha and IL-1 beta in a dose-dependent manner, and IL-1 beta was predominant in all supernatants. The activities of IL-1 alpha and IL-1 beta showed no significant differences between the patients with bladder cancer, renal cell carcinoma or prostatic cancer and control subjects. This study suggested that the patients with bladder cancer renal cell carcinoma and prostatic cancer did not spontaneously produce IL-1 alpha or IL-1 beta, but that the ability to produce IL-1 alpha and IL-1 beta in response to LPS stimulation was not significantly impaired

Disruption of Membranes of Extracellular Vesicles Is Necessary for ELISA Determination of Urine AQP2: Proof of Disruption and Epitopes of AQP2 Antibodies

Aquaporin-2 (AQP2) is present in urine extracellular vesicles (EVs) and is a useful biomarker for water balance disorders. We previously found that pre-treatment of urine with alkali/detergent or storage at −25 °C is required for enzyme-linked immunosorbent assay (ELISA) measurement. We speculated that disruptions of EVs membranes are necessary to allow for the direct contact of antibodies with their epitopes. Human urine EVs were prepared using an ultracentrifugation method. Urine EV samples were stored at different temperatures for a week. Electron microscopy showed abundant EVs with diameters of 20–100 nm, consistent with those of exosomes, in normal urine, whereas samples from alkali/detergent pre-treated urine showed fewer EVs with large swollen shapes and frequent membrane disruptions. The abundance and structures of EVs were maintained during storage at −80 °C, but were severely damaged at −25 °C. Binding and competitive inhibition assays showed that epitopes of monoclonal antibody and polyclonal antibody were the hydrophilic Loop D and C-terminus of AQP2, respectively, both of which are present on the inner surface of EVs. Thus, urine storage at −25 °C or pre-treatment with alkali/detergent disrupt EVs membranes and allow AQP2 antibodies to bind to their epitopes located inside EVs