Renal expression of trefoil factor 3 mRNA in association with tubulointerstitial fibrosis in IgA nephropathy.

AIM: Trefoil factor 3 (TFF3) is a small peptide that is involved in mucosal protection. TFF3 is widely expressed in multiple tissues including kidney tissue. Previous studies have reported that the levels of urinary TFF3 are significantly increased in patients with chronic kidney disease. The aim of this study is to detect the TFF3 mRNA in kidney and elucidate the relationship between renal TFF3 mRNA and tubulointerstitial fibrosis in IgA nephropathy (IgAN). METHODS: We investigated the renal mRNA expression of TFF3 by real-time PCR analysis in biopsy specimens from patients with IgAN, other glomerulonephritis (OGN) and minor glomerular abnormalities (MGA). We also determined the renal localization of TFF3 and the levels of urinary TFF3 by immunostaining and ELISA, respectively. RESULTS: The renal TFF3 mRNA expression was significantly associated with the urinary TFF3 secretion and the tubulointerstitial fibrosis score in the IgAN group alone. Immunostaining of the renal specimen of IgAN patients revealed that TFF3 is located in the renal tubular epithelial cells. The locations were almost the same as those that showed uromodulin positivity; specifically, the thick ascending limb (TAL) of the loop of Henle and the early portion of the distal tubule. The urinary TFF3 levels were positively correlated with the levels of urinary biomarkers of tubulointerstitial injury in such patients. CONCLUSION: Renal TFF3 mRNA is associated with renal tubulointerstitial fibrosis in IgAN patients. The TFF3 located in the renal tubular epithelial cells may play a role in the progression of tubulointerstitial fibrosis in IgAN patients.

Differences between gastric signet-ring cell carcinoma and poorly differentiated adenocarcinoma: A comparison of histopathologic features determined by mucin core protein and trefoil factor family peptide immunohistochemistry.

We investigated differences between the pathological features of gastric signet-ring cell carcinoma (sig) and poorly differentiated adenocarcinoma (por) by examining the expressions of the trefoil factor family peptides (TFFs) and mucin core proteins (MUCs). Ninety-seven tissues of 97 gastric cancer patients were selected for this study. After gastrectomy, the major histopathologic types were determined to be sig, solid-type poorly differentiated adenocarcinoma (por1), non-solid type poorly differentiated adenocarcinoma (por2), and well-differentiated tubular adenocarcinoma (tub1). We evaluated the prevalence of positive staining for MUCs (MUC5AC and MUC2) and TFFs (TFF1 and TFF3) and assessed the correlation between MUCs and TFFs in each histopathological type. The rate of MUC2 expression significantly differed between sig and por2 (50.0% vs 11.7%, P = 0.011). TFF3 expression in sig significantly differed from TFF3 expression in both por2 (100% vs 17.6%, P < 0.0001) and por1 (100% vs 33.3%, P = 0.0004). MUC5AC and TFF1 expressions were significantly correlated in por1 (r = 0.705, P = 0.002), por2 (r = 0.535, P = 0.0009), and tub1 (r = 0.470, P = 0.0034), while MUC2 and TFF3 expressions were significantly correlated only in sig (r = 0.593, P = 0.040). The expression and correlation patterns of the TFFs and MUCs suggest that the histopathologic features of gastric sig differ from those of por.

Alkali treatment stabilizes fluctuations of urine AQP2 values measured by ELISA.

BACKGROUND: Aquaporin-2 (AQP2) in urine is now measured in many water-balance disorders and regarded as a useful biomarker for diagnosis and prognosis. An enzyme-linked immunosorbent assay (ELISA) method has been developed for measurement of large numbers of clinical samples. However, fluctuations in the measured values were sometimes observed depending on storage conditions. Urine AQP2 is present in exosome membranes and we speculated that this structural organization causes the fluctuations. METHODS: Human urine samples from healthy subjects were measured by ELISA. Effects of maneuvers to disrupt the exosome membrane mechanically (freezing and thawing at different temperatures) and chemically (treating with alkali and detergents) prior to ELISA were examined. RESULTS: Urine samples stored at 4 or -80 °C did not show significant AQP2 values, whereas those stored at -25 °C for more that 2 weeks provided the values. Urine samples treated with 0.4 N NaOH and 0.5 % Triton X-305 showed the consistent and comparable values to those stored at -25 °C. CONCLUSION: Pretreatment with alkali (0.4 N NaOH) to disrupt exosome membranes allowed consistent ELISA measurements of urinary AQP2. This simple method is applicable to ELISA of other membrane proteins included in exosomes.

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.

Urine Levels of Defensin α1 Reflect Kidney Injury in Leptospirosis Patients.

Leptospirosis is a zoonotic disease whose severe forms are often accompanied by kidney dysfunction. In the present study, urinary markers were studied for potential prediction of disease severity. Urine samples from 135 patients with or without leptospirosis at San Lazaro Hospital, the Philippines, were analyzed. Urine levels of defensin α1 (uDA1) were compared with those of neutrophil gelatinase-associated lipocalin (uNGAL) and N-acetyl-ß-d-glucosidase (uNAG). Serum creatinine (Cr) was used as a marker of kidney injury. The levels of uDA1/Cr, uNGAL/Cr, and uNAG/Cr were positive in 46%, 90%, and 80% of leptospirosis patients, and 69%, 70%, and 70% of non-leptospirosis patients, respectively. In leptospirosis patients, the correlation of uDA1/Cr, uNGAL/Cr and uNAG/Cr levels with serum Cr were r = 0.3 (p < 0.01), r = 0.29 (p < 0.01), and r = 0.02 (p = 0.81), respectively. uDA1/Cr levels were correlated with uNGAL/Cr levels (r = 0.49, p < 0.01) and uNAG/Cr levels (r = 0.47, p < 0.0001) in leptospirosis patients. These findings suggest that uDA1, uNGAL, and uNAG were elevated in leptospirosis patients and reflected various types of kidney damage. uDA1 and uNGAL can be used to track kidney injury in leptospirosis patients because of their correlation with the serum Cr level.

Exploring urinary biomarkers in autosomal dominant polycystic kidney disease.

BACKGROUND: Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited kidney disease, is a progressive disease characterized by a bilateral proliferation and enlargement of renal cysts. Recent reports have shown that tolvaptan, a vasopressin V2 receptor antagonist, has been effective in inhibiting renal cyst proliferation and enlargement in ADPKD patients, although no biomarker has identified to predict the effects of tolvaptan. We explored the effective urinary biomarkers in ADPKD in human and in an animal model. METHODS: We measured 28 biomarkers in urine taken from ADPKD patients to compare with that of healthy subjects. Next, a gene expression analysis of the kidney from DBA/2FG-pcy mice (ADPKD model animals) was performed to identify prospective biomarkers. Additionally, we investigated the DBA/2FG-pcy mouse urine samples to determine the biomarkers' efficacy. RESULTS: There were statistically significant differences in 12 of the 28 prospective urinary biomarkers between urine from ADPKD patients and that from healthy subjects. Six of these matched with highly expressed gene products of DBA/sFG-pcy mouse kidneys. Among those 6 biomarkers, NGAL, M-CSF, and MCP-1 showed significantly higher values in the urine of DBA/2FG-pcy mice than that of wild type. CONCLUSIONS: This study suggests that NGAL, M-CSF, MCP-1 are potential candidates of urinary biomarkers in ADPKD.

Influence of Helicobacter pylori status and eradication on the serum levels of trefoil factors and pepsinogen test: serum trefoil factor 3 is a stable biomarker.

BACKGROUND AND AIM: Emerging data indicate that serum trefoil factors (TFFs), especially TFF3, could be potential biomarkers for gastric cancer risk. We aimed to evaluate the influence of Helicobacter pylori (H. pylori) status and eradication on serum TFFs and the pepsinogen test. METHODS: Healthy individuals who underwent a thorough medical checkup were enrolled in study 1, and gastric ulcer patients who undertook H. pylori eradication therapy were enrolled in studies 2 and 3. Serum levels of the TFFs (TFF1, TFF2 and TFF3), H. pylori antibody and pepsinogen test were examined in all studies. In study 3, TFF expressions in biopsy samples of the gastric mucosa were additionally examined before and 2 months after eradication. RESULTS: In 1,260 healthy individuals enrolled in study 1, serum TFF1 and TFF2 levels were markedly different between H. pylori antibody-positive and -negative participants (P < 0.0001). Differences in serum TFF3 levels between H. pylori antibody-positive (5.85 ± 3.93 ng/ml) and -negative subjects (5.27 ± 2.38 ng/ml) were statistically significant (P = 0.002) but small in absolute value. In 178 gastric ulcer patients enrolled in study 2, serum TFF1, TFF2 and positive rates of the pepsinogen test significantly decreased 2 months after H. pylori eradication therapy (P < 0.001). In contrast, serum TFF3 levels and positive rates of high TFF3 levels (≥7 ng/ml) did not significantly change with H. pylori-eradication until 5 years after eradication. In 18 gastric ulcer patients (study 3), TFF1 and TFF2 were mainly expressed in the foveolar epithelium, and TFF2 was additionally expressed in the pyloric glands. These expressions significantly decreased with H. pylori eradication. TFF3s were scarcely expressed in the gastric mucosa except in goblet cells of intestinal metaplasia, which did not change with H. pylori eradication. CONCLUSION: In serum TFFs and pepsinogen tests, only serum TFF3s were not significantly affected by H. pylori eradication, suggesting that serum TFF3 could be a stable biomarker of gastric cancer risk even after H.pylori eradication in contrast with the pepsinogen test.

Diagnostic utility of trefoil factor families for the early detection of lung cancer and their correlation with tissue expression.

Trefoil factors (TFFs) are upregulated in numerous types of cancer, including those of the breast, the colon, the lung and the pancreas, suggesting their potential utility as biomarkers for screening. In the present study, the clinical relevance of serum or urinary TFFs as biomarkers were comprehensively evaluated and the correlation with TFF expression levels in lung cancer tissue was examined. Serum and urine were collected from 199 patients with lung cancer and 198 healthy individuals. Concentrations of serum and urinary TFF1, TFF2 and TFF3 were measured using ELISA and the potential of TFF levels to discriminate between cancer and non-cancer samples was evaluated. In 100 of the cancer cases, expression of TFF1-3 was analyzed using immunohistochemical staining of paraffin sections. Furthermore, the relationship between TFF levels and clinicopathological factors among these cancer cases was analyzed using immunohistochemistry of tissue specimens, quantified and statistically analyzed. While serum levels of all TFFs measured using ELISA were significantly higher in patients with lung cancer compared with those in healthy individuals, urinary TFFs were lower. Areas under the curve (AUC) of the receiver operating characteristic curves for serum/urinary TFF1, TFF2 and TFF3 were 0.709/0.594, 0.722/0.501 and 0.663/0.665, respectively. Furthermore, the combination of serum TFF1, TFF2, TFF3 and urinary TFF1 and TFF3 demonstrated the highest AUC (0.826). In the clinicopathological analysis, serum TFF1 was higher in the early pathological T-stage (pTis/1/2) compared with the later stage (pT3/4) and TFF2 was higher in the pN0/1 than the pN2 group. With regards to the histological types, urinary TFF1 was higher in squamous cell carcinoma than adenocarcinoma (AC), but TFF2 tended to be higher in AC. Using immunohistochemical analysis, although TFF1 and TFF3 expression showed positive correlation with serum concentrations, TFF2 was inversely correlated. In conclusion, serum and urinary TFF levels are promising predictive biomarkers, and their measurements provide a useful in vivo and non-invasive diagnostic screening tool. In particular, TFF1 and TFF3 could be surrogate markers of clinicopathological profiles of human lung cancer.

Tests for serum levels of trefoil factor family proteins can improve gastric cancer screening.

BACKGROUND & AIMS: Improving methods for early detection of gastric cancer could reduce mortality. Measurements of serum pepsinogen levels have been used for screening in Japan without satisfactory levels of sensitivity or specificity. Trefoil factor family (TFF) proteins (TFF1, TFF2, and TFF3) are small and stable molecules secreted by the mammalian gastrointestinal tract. Foveolar hyperplasia, spasmolytic polypeptide (TFF2)-expressing metaplasia, and intestinal metaplasia are histologic changes observed in patients with atrophic gastritis; they express TFF1, TFF2, and TFF3, respectively. We investigated whether serum levels of TFF can be used as markers for gastric cancer screening. METHODS: Serum was collected from 183 patients with gastric cancer and 280 healthy individuals without cancer. Serum levels of anti-Helicobacter pylori immunoglobulin G, pepsinogen I, pepsinogen II, TFF1, TFF2, and TFF3 were measured by enzyme-linked immunosorbent assay and associated with gastric cancer. RESULTS: Using a cutoff of 3.6 ng/mL, the level of TFF3 was significantly increased in serum samples from patients with cancer (odds ratio, 18.1; 95% confidence interval, 11.2-29.2); using this test, patients with cancer were identified with 80.9% sensitivity and 81.0% specificity. The test for TFF3 had a significantly higher odds ratio than that for pepsinogen. A test for the combination of TFF3 and pepsinogen had better results than the test for only pepsinogen. CONCLUSIONS: Serum levels of TFF3 are a better marker of gastric cancer than pepsinogen; a test for the combined levels of serum pepsinogen and TFF3 could improve gastric cancer screening.

Daily variance of urinary excretion of AQP2 determined by sandwich ELISA method.

BACKGROUND: Urinary excretion of aquaporin 2 (AQP2) is a useful marker of kidney collecting duct function. A specific and convenient method to measure AQP2 in human urine would help to treat water balance disorders. It is unknown whether urinary excretion of AQP2 shows any daily variance. METHODS: A sandwich enzyme-linked immunosorbent assay (ELISA) method for AQP2 was established using two different kinds of antibodies, and its sensitivity and specificity were examined. Daily variance of urinary excretion of AQP2 and responses to acute water load were examined. RESULTS: The established ELISA specifically detected urine AQP2 with high sensitivity (detected as low as 0.34 pmol/mL). Urinary excretion of AQP2 did not show daily variance between 9 a.m. and 9 p.m. in healthy subjects. CONCLUSIONS: The developed ELISA method using two different antibodies is convenient and highly sensitive, and could be useful in clinical practice. Urinary excretion of AQP2 is relatively constant from morning to night, and spot urine sampling at any time during this time period does not affect the results.

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.

TFF-1 Functions to Suppress Multiple Phenotypes Associated with Lung Cancer Progression.

INTRODUCTION: Trefoil Factor (TFF) is a member of a protein family comprised of three isoforms, of which TFF-1 exhibits antithetical functions; promotion or suppression of cell proliferation, survival and invasion, depending on the cancer type. However, the pathobiological function of TFF-1 in lung carcinoma has been still unclear. METHODS: We examined the expression and secretion of TFF-1 using cultured human lung carcinoma cells by immunoblotting, immunofluorescence, enzyme-linked immunosorbent assay and quantitative real-time PCR analyses. The effects of TFF-1 on various phenotypes were analyzed in two cell lines, including those transfected with cDNA encoding TFF-1. Cell proliferation and death were examined by hemocytometer cell counting and by colorimetric viability/cytotoxicity assay. Cell cycle profile, migration and invasion were also examined by flow cytometry, wound healing assay and Matrigel Transwell assay, respectively. The effect of TFF-1 overexpression was confirmed by additional transfection of TFF-1-specific siRNA. RESULTS: Endogenous TFF-1 protein expression and secretion into the media were observed exclusively in adenocarcinoma-derived cell lines. Forced overexpression of TFF-1 drove cell cycle transition, while the proliferation decreased by 19% to 25% due to increased cell death. This cell death was predominantly caused by apoptosis, as assessed by the activation of caspase 3/7. Cell migration was also suppressed by 71% to 82% in TFF-1-transfected cells. The suppressive effect of TFF-1 on proliferation and migration was restored by transfection of TFF-1 siRNA. Moreover, invasion was also suppressed to 77% to 83% in TFF-1-transfected cells. CONCLUSION: These findings reveal that TFF-1 functions as a suppressor of cancer proliferation by induction of apoptosis, cell migration and invasion and thus may provide a synergistic target for potential treatment strategies for human lung carcinoma.

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva.

BACKGROUND: It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. METHODS: In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). RESULTS: AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. GENERAL SIGNIFICANCE: AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.

Serum evaluation of soluble interferon-alpha/beta receptor and high-sensitivity C-reactive protein for diagnosis of the patients with gastrointestinal and hepatobiliary-pancreatic cancer.

Serum soluble interferon-alpha/beta receptor (sIFN-alpha/betaR) and high-sensitivity C-reactive protein (hs-CRP) levels were evaluated in the patients with gastrointestinal and hepatobiliary-pancreatic cancer. We compared the sensitivity and specificity of serum sIFN-alpha/betaR with that of serum hs-CRP and evaluated the two diagnostic parameters in combination. Serum sIFN-alpha/betaR levels were measured in 92 patients and 25 healthy individuals by enzyme-linked immunosorbent assay. The diagnoses were 37 cases of hepatocellular carcinoma, 17 cases of pancreatic cancer, 15 cases of colon cancer, 13 cases of biliary tract cancer, and 10 cases of gastric cancer. Serum levels of sIFN-alpha/betaR and hs-CRP were significantly higher in the patients than in healthy individuals (p<0.05). The optimal cut-off values of sIFN-alpha/betaR and hs-CRP were 3600pg/ml and 0.5microg/ml, respectively. The sensitivity and specificity for these thresholds were 94.6% and 88.0%, whereas positive predictive and negative predictive values were 96.7% and 81.5%. These results suggest that a combination of serum sIFN-alpha/betaR and hs-CRP thresholds may be more reliable diagnostic parameter for gastrointestinal and hepatobiliary-pancreatic cancer.

Screening Biomarker as an Alternative to Endoscopy for the Detection of Early Gastric Cancer: The Combination of Serum Trefoil Factor Family 3 and Pepsinogen.

OBJECTIVE: The serum pepsinogen test has limitation in its predictive power as a noninvasive biomarker for gastric cancer screening. We aimed to investigate whether the combination of TFF3 and pepsinogen could be an effective biomarker for the detection of gastric cancer even in the early stages. METHODS: In total, 281 patients with early gastric cancer (EGC), who underwent endoscopic submucosal dissection in Korea, and 708 healthy individuals from Japan were enrolled in the derivation cohort. The validation cohort included 30 Korean patients with EGC and 30 Korean healthy control blood donors. Serum TFF3 levels were examined using enzyme-linked immunosorbent assay. RESULTS: Using a cutoff of 6.73 ng/mL in the derivation cohort, the sensitivity of the combination of tests for EGC detection was superior (87.5%) to that of TFF3 (80.4%) or pepsinogen test alone (39.5%). Similarly, in the validation cohort, the sensitivity of TFF3 plus pepsinogen was higher (90.4%) than that of TFF3 (80.0%) or pepsinogen test alone (33.3%). CONCLUSION: The combination of serum TFF3 and pepsinogen is a more effective noninvasive biomarker for gastric cancer detection compared with pepsinogen or TFF3 alone, even in EGC. This trial is registered with NCT03046745.

Urine Trefoil Factors as Prognostic Biomarkers in Chronic Kidney Disease.

INTRODUCTION: Trefoil factor family (TFF) peptides are increased in serum and urine in patients with chronic kidney disease (CKD). However, whether the levels of TFF predict the progression of CKD remains to be elucidated. METHODS: We determined the TFF levels using peptide-specific ELISA in spot urine samples and performed a prospective cohort study. The association between the levels of urine TFFs and other urine biomarkers as well as the renal prognosis was analyzed in 216 CKD patients (mean age: 53.7 years, 47.7% female, 56.9% with chronic glomerulonephritis, and mean eGFR: 58.5 ml/min/1.73 m2). RESULTS: The urine TFF1 and TFF3 levels significantly increased with the progression of CKD stages, but not the urine TFF2 levels. The TFF1 and TFF3 peptide levels predicted the progression of CKD ≥ stage 3b by ROC analysis (AUC 0.750 and 0.879, resp.); however, TFF3 alone predicted CKD progression in a multivariate logistic regression analysis (odds ratio 3.854, 95% confidence interval 1.316-11.55). The Kaplan-Meier survival curves demonstrated that patients with a higher TFF1 and TFF3 alone, or in combination with macroalbuminuria, had a significantly worse renal prognosis. CONCLUSION: The data suggested that urine TFF peptides are associated with renal progression and the outcomes in patients with CKD.

Serum TFF1 and TFF3 but not TFF2 are higher in women with breast cancer than in women without breast cancer.

Breast cancer remains a common malignancy in women, but the take-up for breast cancer screening programs in Japan is still low, possibly due to its perceived inconvenience. TFF1 and TFF3 are expressed in both breast cancer tissue and normal breast. Serum trefoil proteins were reported as cancer screening markers for gastric, prostate, lung, pancreatic cancer and cholangio carcinoma. The purpose of this study was to examine whether serum trefoil proteins could be screening biomarkers for breast cancer. Serum trefoil proteins in 94 breast cancer patients and 84 health check females were measured by ELISA. Serum TFF1 and TFF3 were significantly higher and serum TFF2 was significantly lower in breast cancer patients. Area under the curve of receiver operating characteristic of TFF1, TFF2, and TFF3 was 0.69, 0.83, and. 0.72, respectively. AUC of the combination of TFF1, TFF2, and TFF3 was 0.96. Immunohistochemically, TFF1 expression was positive in 56.5% and TFF3 was positive in 73.9% of breast cancers, while TFF2 was negative in all tumors. Serum TFF1 had positive correlation with expression of TFF1 in breast cancer tissue. Serum concentrations of TFF1 and TFF3 but not TFF2 are higher in women with breast cancer than in women without breast cancer.

A novel modification of a flow cytometric assay of phosphorylated STAT1 in whole blood lymphocytes for rapid detection of interferon-alpha signal in vivo.

Phosphorylation of signal transducer and activator of transcription factor 1 (STAT1) is a key response in the type I interferon (IFN) signal cascade. We developed a novel flow cytometric assay for phosphotyrosine-STAT1 (p-STAT1) to rapidly monitor in vivo IFN signaling. Mouse blood stimulated with mouse IFN-alpha was hemolyzed with lysis buffer in place of lymphocyte purification, permeabilized with methanol, and stained with an Alexa Fluor 488-conjugated anti-p-STAT1 antibody. The cells were also stained with phycoerythrin (PE)-conjugated anti-CD45 antibody for eliminating debris (CD45-negative) from leukocytes (CD45-positive), and with PE covalently linked to cyanin 5-conjugated anti-Gr-1 antibody for separating lymphocytes (Gr-1-negative) and granulocytes (Gr-1-positive). When whole blood was treated with IFN-alpha, the Alexa Fluor 488 intensity of lymphocytes increased, reaching a peak within 1 h, and this increase was statistically significant at IFN-alpha concentrations of 100 U/mL and higher. When IFN-alpha was administered intravenously to mice, the Alexa Fluor 488 intensity of blood lymphocytes was increased, reaching a peak in 1 h and returning to baseline at 18 h, and this increase was dose-dependent, with statistically significant increases seen at doses of 1,000 U/body and higher. The kinetics and dose-responses of p-STAT1 levels in the spleen, lung, and liver were similar to those in blood lymphocytes. This new flow cytometric assay of p-STAT1 in peripheral blood leukocytes will be useful for examining IFN-alpha signaling and for monitoring tissue response to IFN-alpha in vivo.

Myeloid-related protein-8/14 is associated with proinflammatory cytokines in cervical mucus.

Myeloid-related protein-8 (MRP-8), MRP-14, and MRP-8/14 are found in a variety of inflammatory conditions and are involved in the host defense system. The objective of this study was to determine the concentrations of MRP-8, MRP-14, and MRP-8/14 in human cervical mucus and the associations between MRP-8/14 and proinflammatory cytokines. Samples of cervical mucus were obtained using a syringe from sexually active women (n=97) during the preovulatory phase. Samples from seven women were obtained using a swab placed in the cervical canal during the proliferative, preovulatory, and luteal phases. Concentrations of MRP-8, MRP-14, MRP-8/14, IL-1alpha, IL-6, IL-8, and granulocyte elastase were measured using an ELISA. The mean levels of MRP-8, MRP-14, and MRP-8/14 in cervical mucus were 1.87, 0.46, and 23.90microg/ml, respectively. The concentration of MRP-8/14 showed positive correlations with concentrations of IL-1alpha (p<0.0001), IL-8 (p<0.0001), and granulocyte elastase (p<0.0001). However, there were no significant differences in MRP-8/14 levels in the cervical mucus of each patient during the menstrual cycle. MRP-8/14 was mainly detected in human cervical mucus and showed a positive correlation with proinflammatory cytokines. The MRP-8/14 level in cervical mucus may be useful as a marker of inflammation of the uterine cervix.

Adipocyte-derived plasma protein adiponectin acts as a platelet-derived growth factor-BB-binding protein and regulates growth factor-induced common postreceptor signal in vascular smooth muscle cell.

BACKGROUND: Vascular smooth muscle cell proliferation plays an important role in the development of atherosclerosis. We previously reported that adiponectin, an adipocyte-specific plasma protein, accumulated in the human injured artery and suppressed endothelial inflammatory response as well as macrophage-to-foam cell transformation. The present study investigated the effects of adiponectin on proliferation and migration of human aortic smooth muscle cells (HASMCs). Methods and Results- HASMC proliferation was estimated by [(3)H] thymidine uptake and cell number. Cell migration assay was performed using a Boyden chamber. Physiological concentrations of adiponectin significantly suppressed both proliferation and migration of HASMCs stimulated with platelet-derived growth factor (PDGF)-BB. Adiponectin specifically bound to (125)I-PDGF-BB and significantly inhibited the association of (125)I-PDGF-BB with HASMCs, but no effects were observed on the binding of (125)I-PDGF-AA or (125)I-heparin-binding epidermal growth factor (EGF)-like growth factor (HB-EGF) to HASMCs. Adiponectin strongly and dose-dependently suppressed PDGF-BB-induced p42/44 extracellular signal-related kinase (ERK) phosphorylation and PDGF beta-receptor autophosphorylation analyzed by immunoblot. Adiponectin also reduced PDGF-AA-stimulated or HB-EGF-stimulated ERK phosphorylation in a dose-dependent manner without affecting autophosphorylation of PDGF alpha-receptor or EGF receptor. CONCLUSIONS: The adipocyte-derived plasma protein adiponectin strongly suppressed HASMC proliferation and migration through direct binding with PDGF-BB and generally inhibited growth factor-stimulated ERK signal in HASMCs, suggesting that adiponectin acts as a modulator for vascular remodeling.