Rapid and simple quantification of belimumab in human plasma using ultra-high performance liquid chromatography with tandem mass spectrometry.

OBJECTIVE: Belimumab is a monoclonal antibody against the B-lymphocyte stimulating factor and is approved for the treatment of patients with systemic lupus erythematosus (SLE) not responding adequately to existing therapies. In this study, we established and validated an assay for quantifying belimumab in human plasma. METHODS: From the peptides generated by trypsin digestion of belimumab, in silico analysis was used to search for unique peptides to determine the surrogate peptides. Samples were trypsin digested, pretreated with solid phase extraction, and analyzed by ultra-high performance liquid chromatography with tandem mass spectrometry (UHPLC-MS/MS) to quantify the surrogate peptide in the samples. The assay was validated according to the Food and Drug Administration (FDA) bioanalytical method validation guidance. We used the established assay to quantify plasma belimumab concentrations in two SLE patients treated with belimumab. RESULTS: Among the unique peptides identified by the in silico analysis, the peptide with the best peak shape when measured by UHPLC-MS/MS was selected as the surrogate peptide. The validation results of this assay met the acceptable criteria recommended by the FDA guidance. The lower limit of quantification (LLOQ) for belimumab was 2 µg/mL. Recovery rates and matrix effects when corrected for internal standards were 91.5-114.3 % and 96.9-108.4 %, respectively. Plasma concentrations of belimumab were measured in 12 samples from two belimumab-treated SLE patients. All concentrations were within the calibration range. CONCLUSIONS: We have established and validated a method for measuring plasma belimumab concentrations using UHPLC/MS-MS. By measuring plasma belimumab concentrations in more patients, this method is expected to contribute to appropriate use of belimumab.

Case Report: Solid variant of papillary thyroid carcinoma in a young adult with Turner syndrome with chronic thyroiditis.

Turner syndrome is associated with an increased risk of developing several neoplasms. In particular, a clinical feature of Turner syndrome with chronic thyroiditis implies a relationship with thyroid malignancies. We report a very rare case of a solid variant of papillary thyroid carcinoma that was identified during a follow-up of chronic thyroiditis in a 22-year-old woman with Turner syndrome. The patient had no notable history of radiation exposure. No genetic mutations relating to the occurrence of the solid variant of papillary thyroid carcinoma, including RET/PTC rearrangements and mutations in the BRAF or RAS, were detected by a gene panel test, namely, the Oncomine™ Dx Target test. To the best of our knowledge, this is the first report of a solid variant of papillary thyroid carcinoma in a young adult with Turner syndrome with chronic thyroiditis. Our case suggests that in patients with Turner syndrome, there may be different pathogeneses from those previously reported, including exposure to radiation or known genetic mutations for the development of a solid variant of papillary thyroid carcinoma.

Comprehensive lipidomics of lupus-prone mice using LC-MS/MS identifies the reduction of palmitoylethanolamide that suppresses TLR9-mediated inflammation.

Lipid mediators are known to play crucial roles not only in the onset of the inflammatory response but also in the induction of resolution of inflammation. Here, we report that palmitoylethanolamide (PEA), an endogenous N-acylethanolamine, can suppress the inflammation induced by Toll-like receptor (TLR) signaling both in vitro and in vivo. PEA was found to be significantly reduced in the serum and spleen of lupus-prone MRL/lpr mice analyzed by lipidomics. PEA suppressed pro-inflammatory cytokine production in a mouse macrophage cell line stimulated with TLR ligands such as lipopolysaccharide, peptidoglycan, poly (I:C), imiquimod, and CpG-ODN. PEA also inhibited both mRNA and protein levels of IL-6 in bone marrow-derived dendritic cells (BMDCs) and B cells stimulated with CpG-ODN. Augmentation of cell surface CD86 and CD40 on BMDCs and B cells, IgM production, and cell proliferation of B cells in response to CpG-ODN were attenuated by PEA. Moreover, PEA treatment significantly reduced mortality and serum IL-6 levels in mice injected with CpG-ODN plus D-galactosamine. Taken together, PEA ameliorates inflammation induced by TLR signaling, which could be a novel therapeutic target for inflammatory disorders.

Factors Influencing Plasma Coproporphyrin-I Concentration as Biomarker of OATP1B Activity in Patients With Rheumatoid Arthritis.

Organic anion transporting polypeptides (OATPs) 1B are drug transporters mainly expressed in the sinusoidal membrane. In previous reports, genetic factor, 3-carboxy-4-methyl-5-propyl-2-furanpropanoic acid (CMPF), which is one of the uremic toxins, inflammatory cytokines such as tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) decreased OATP1B1 activity in vitro, but in vivo effects of these factors have not been elucidated. Plasma coproporphyrin-I (CP-I) is spotlighted as a highly accurate endogenous substrate of OATP1B. This study focused on patients with rheumatoid arthritis (RA) and evaluated the influence of several factors comprising gene polymorphisms, uremic toxins, and inflammatory cytokines on OATP1B activity using plasma CP-I concentration. Thirty-seven outpatients with RA who satisfied the selection criteria were analyzed at the time of recruitment (baseline) and at the next visit. OATP1B1*15 carriers tended to have higher CP-I concentration compared with noncarriers. Plasma CP-I correlated positively with CMPF concentration, but did not correlate with IL-6 or TNF-α concentration. Multiple logistic regression analysis by stepwise selection identified plasma CMPF concentration and OATP1B1*15 allele as significant factors independently affecting plasma CP-I concentration at baseline and at the next visit, respectively. In conclusion, the present results suggest that inflammatory cytokines do not have clinically significant effects on OATP1B activity, whereas the effects of genetic polymorphisms and uremic toxins should be considered.

Undifferentiated pleomorphic sarcoma of the prostate in a young man.

INTRODUCTION: Prostate sarcoma is an extremely rare disease with a poor prognosis. Undifferentiated pleomorphic sarcoma has never been described in the prostate.Case presentation: A 27-year-old man complained of frequent urination and dysuria for several years. Various examinations were suggestive of prostate sarcoma. The pathological diagnosis was confirmed as prostate sarcoma via ultrasound-guided transrectal needle biopsy. Because the location of the tumor in the prostate was confirmed by magnetic resonance imaging, we performed robot-assisted radical prostatectomy. The final pathological diagnosis was undifferentiated pleomorphic sarcoma. Local recurrence occurred at the front of the rectum 2 months after surgery. Although chemotherapy and radiotherapy were initially effective, he died 18 months after surgery. CONCLUSION: Undifferentiated pleomorphic sarcoma of the prostate is believed to have a poor prognosis. When selecting the surgical procedure, functionality should be considered for individual cases with complete resection.

Effects of Sleeve Gastrectomy on Blood Pressure and the Renal Renin-Angiotensin System in Rats with Diet-Induced Obesity.

OBJECTIVE: Sleeve gastrectomy (SG) has been reported to decrease blood pressure (BP), although the reason has not been revealed. The present study aimed to establish the reason why SG decreases BP. METHODS: Male Sprague-Dawley rats were subjected to surgical (sham operation or SG) and dietary interventions (fed a normal diet or high-fat diet ad libitum or fed by pair-feeding [PF]). Systolic BP (SBP), urinary sodium excretion, and endocrine parameters were examined 4 weeks after surgery. RESULTS: Both SG and PF rats had reduced body weight compared with SO rats fed normal diet or high-fat diet ad libitum. SG rats exhibited a reduction in SBP compared with PF, which was associated with a reduction in renal renin, angiotensin II, and catechol-O-methyltransferase levels (P < 0.01 for each). SG increased plasma cholecystokinin (CCK) levels compared with PF (P < 0.0001 for each), whereas glucagon-like peptide 1 and peptide YY were not changed in fasting. Exogenous administration of CCK reduced renal catechol-O-methyltransferase (P = 0.0233), renin (P < 0.0001), and angiotensin II (P < 0.0001) levels and SBP (P = 0.0053). CONCLUSIONS: SG reduced SBP, at least in part, through suppression of sympathetic nerve action by elevation of CCK, which was followed by suppression of the intrarenal renin-angiotensin system.

Dysbiosis of the Gut Microbiota on the Inflammatory Background due to Lack of Suppressor of Cytokine Signalling-1 in Mice.

BACKGROUND: Both environmental and genetic factors have been implicated in the induction of autoimmune disease. Therefore, it is important to understand the pathophysiological significance of the gut microbiota and host genetic background that contribute to an autoimmune disease such as inflammatory bowel disease (IBD). We have previously reported that mice deficient for suppressor of cytokine signaling-1 (SOCS1), in which SOCS1 expression was restored in T and B cells on an SOCS1-/- background (SOCS1-/-Tg mice), developed systemic autoimmune diseases accompanied by spontaneous colitis. METHODS: To investigate whether the proinflammatory genetic background affects the gut microbiota, we used SOCS1-/-Tg mice as a model of spontaneous chronic colitis. Fecal samples were collected from SOCS1-/-Tg mice and SOCS1+/+Tg (control) mice at 1 and 6 months of age, and the fecal bacterial 16S ribosomal RNA genes were sequenced using the Illumina MiSeq platform. RESULTS: Gut microbial diversity was significantly reduced and the intestinal bacterial community composition changed in SOCS1-/-Tg mice in comparison with the control mice. Interestingly, the population of Prevotella species, which is known to be elevated in ulcerative colitis and colorectal cancer patients, was significantly increased in SOCS1-/-Tg mice regardless of age. CONCLUSION: Taken together, these results suggest that the proinflammatory genetic background owing to SOCS1 deficiency causes dysbiosis of the gut microbiota, which in turn generates a procolitogenic environment.

Preoperative weight loss program involving a 20-day very low-calorie diet for obesity before laparoscopic gastrectomy for gastric cancer.

INTRODUCTION: The increased visceral fat in patients with obesity can increase the technical difficulty of surgery. This study was performed to evaluate a preoperative 20-day very low-calorie diet for obesity before laparoscopic gastrectomy for gastric cancer. METHODS: This prospective single-center study involved patients with obesity who were planning to undergo laparoscopic gastrectomy for gastric cancer. Obesity was defined according to the Japanese criteria: BMI ≥25 kg/m2 or waist circumference ≥85 cm in men and ≥90 cm in women. The patients underwent a preoperative 20-day very low-calorie diet and received nutritional counseling. Weight loss, body composition, visceral fat mass, and operative outcomes were evaluated. RESULTS: Thirty-three patients were enrolled from September 2013 to August 2015. Their median age was 71 years, and 78.8% were men. Their median bodyweight and BMI were 72.3 kg (range, 53.8-82.5 kg) and 26.0 kg/m2 (range, 23.5-31.0 kg/m2 ), respectively. The patients achieved a mean weight loss of 4.5% (95% confidence interval [CI]: 3.8-5.1), corresponding to 3.2 kg (95%CI: 2.7-3.7 kg). Body fat mass was significantly decreased by a mean of 2.5 kg (95%CI: 1.9-3.1), whereas skeletal muscle mass was unaffected (mean: -0.20 kg [95%CI: -0.55-0.15]). The visceral fat mass reduction rate was high as 16.8% (range, 11.6%-22.0%). All patients underwent laparoscopic gastrectomy as planned. Severe postoperative morbidity (Clavien-Dindo grade ≥III) was seen in only one patient (3.0%). CONCLUSION: The preoperative 20-day very low-calorie diet weight loss program is promising for the treatment of obesity before laparoscopic gastrectomy for gastric cancer.

Oral administration of antibiotics results in fecal occult bleeding due to metabolic disorders and defective proliferation of the gut epithelial cell in mice.

Antibiotics sometimes exert adverse effects on the pathogenesis of colitis due to the dysbiosis resulting from the disruption of gut homeostasis. However, the precise mechanisms underlying colitogenic effects of antibiotic-induced colitis are largely unknown. Here, we show a novel murine fecal occult bleeding model induced by the combinatorial treatment of ampicillin and vancomycin, which is accompanied by an enlarged cecum, upregulation of pro-inflammatory cytokines IL-6 and IL-12, a reduction in Ki-67-positive epithelial cell number and an increase in the apoptotic cell number in the colon. Moreover, gas chromatography-tandem mass analysis showed that various kinds of metabolites, including glutamic acid and butyric acid, were significantly decreased in the cecal contents. In addition, abundance of butyric acid producer Clostridiales was dramatically reduced in the enlarged cecum. Interestingly, supplementation of monosodium glutamate or its precursor glutamine suppressed colonic IL-6 and IL-12, protected from cell apoptosis and prevented fecal occult blood indicating that the reduced level of glutamic acid is a possible mechanism of antibiotic-induced fecal occult bleeding. Our data showed a novel mechanism of antibiotic-induced fecal occult bleeding providing a new insight into the clinical application of glutamic acid for the treatment of antibiotic-induced colitis.

Overview of Longitudinal Survey of Newborns in the 21st Century: Factors Affecting Infant Growth.

There are two major nationwide birth cohort studies in Japan, namely, the Longitudinal Survey of Newborns in the 21st Century conducted by the Ministry of Health, Labor and Welfare (MHLW) and the Japan Environment and Children's Study (JECS) conducted by the Ministry of Environment. The former was a longitudinal questionnaire survey focusing on environmental and socioeconomic factors for descriptive epidemiology conducted every year since 2001 by mail. The latter was based on 15 unit centers nationwide with environmental measurements and collection of biological samples for environmental risk evaluation. Both are prospective birth cohort studies whose findings will be expected as the basis for establishing health policies. The data obtained in the former study can be used for research with permission from MHLW. To date, there have been more than ten published studies using those data. We have reviewed these studies and introduced our preliminary findings on factors affecting infant growth. Employment before delivery, educational background of parents, household income, and smoking habit of both parents have been suggested to affect infant growth. We will analyze the associations between socioeconomic factors and infant growth trajectory to elucidate the most adequate intervention for children.

Splenic marginal zone lymphoma uncovered after a 10-year follow up as anemia of unknown cause.

A 75-year-old man was referred to our hospital for evaluation of persistent anemia. Despite repeated diagnostic tests, including bone marrow aspiration, the cause of his anemia remained unknown. On each occasion, computed tomography had revealed neither swollen lymph nodes nor splenomegaly. After a 10-year follow-up period, he was admitted with general fatigue and had developed splenomegaly as well as the anemia. Bone marrow biopsy revealed increased abnormal lymphocytes with short villi that were positive for CD11c, CD19, CD20, and kappa chain, but not for CD5, CD10, CD23, or cyclin D1, according to flow cytometry. The bone marrow biopsy sample showed nodular proliferation of small to medium-sized abnormal lymphocytes. Based on these findings, the patient was diagnosed as having splenic marginal zone lymphoma, a rare indolent B-cell neoplasm. Although his splenomegaly diminished after eight cycles of weekly rituximab monotherapy, the anemia did not improve, and abnormal lymphocytes remained detectable in his bone marrow. The patient was then treated with bendamustine monotherapy for six cycles, after which the anemia resolved, and he has since been in good condition. Although rare, it is important to consider splenic marginal zone lymphoma during the differential diagnosis of patients with a long history of anemia of unknown cause.

PRG4 expression in myxoid liposarcoma maintains tumor cell growth through suppression of an antitumor cytokine IL-24.

PRG4 is one of the downstream molecules of the myxoid liposarcoma (MLS)-specific fusion oncoproteins TLS-CHOP and EWS-CHOP. Exogenous PRG4 expression increases the tumorigenicity of cells injected in nude mice. The molecular functions of PRG4 in tumorigenesis and/or tumor progression of MLS cells, however, still remain unclear. In this report, we demonstrated that siRNA-mediated knockdown of PRG4 suppressed the growth of the MLS-derived cell lines 1955/91 and 2645/94. In addition, PRG4 knockdown promoted adipocytic differentiation in 1955/91 cells. Thus, PRG4 may play essential roles in MLS cell growth and have potential as a therapeutic target. On the other hand, our previous study has revealed that TLS-CHOP suppresses expression of an anti-tumor cytokine IL-24, contributing to tumor cell survival. In this study, we found that double knockdown of PRG4 and IL-24 did not inhibit MLS cell growth, and single knockdown of PRG4 remarkably increased IL-24 expression. These results suggest that the growth inhibitory effect of PRG4 knockdown is caused by induction of IL-24 expression, and PRG4 may contribute to maintain MLS cell growth through repression of IL-24 expression.

Large-vessel involvement in granulomatosis with polyangiitis successfully treated with rituximab: A case report and literature review.

Granulomatosis with polyangiitis (GPA) is primary necrotizing vasculitis, which predominantly affects small to medium vessels. Herein, we describe a case of a 60-year-old female with GPA who developed inflammatory wall thickening localized in the aortic arch, upper abdominal aorta, and pulmonary artery. The wall thickening in the large vessels and other GPA lesions such as lung nodules and orbital mass had failed to respond to high-dose glucocorticoids combined with cyclophosphamide; however, all were successfully treated with rituximab. Our literature review identified 24 cases of large-vessel involvement associated with GPA. Luminal stenosis, occlusion, or wall thickening were observed in 8, periaortitis in 11, and aneurysms in 5 cases. The most commonly affected vessel was the abdominal aorta (12 cases), followed by the thoracic aorta (6 cases), subclavian artery (4 cases), and internal carotid artery (4 cases). Glucocorticoids were used in 23 cases, 20 of which received combination therapy with cyclophosphamide. Surgical or endovascular therapies were performed in 10 cases with aneurysmal dilatation. This is the first case showing the potential efficacy of rituximab for refractory large-vessel involvement associated with GPA.

Streptococcal Toxic Shock Syndrome: Life Saving Role of Peritoneal Lavage and Drainage.

PURPOSE: We encountered a case where an infection with group A streptococcus (GAS; ie, Streptococcus pyogenes) initially caused primary peritonitis and then subsequently caused streptococcal toxic shock syndrome. The patient's life was likely saved by an emergency laparotomy followed by extensive peritoneal lavage and drainage. CASE PRESENTATION: A 40-year-old woman was admitted to the Emergency Department for lower abdominal pain and numbness in the extremities. She presented with systemic inflammatory response syndrome. An emergency laparotomy was performed, and ascites that resembled pus and general peritonitis were noted. Peritoneal lavage and drainage were performed, and GAS was isolated from peritoneal fluid. Gram staining of cervical polyp specimens revealed Gram-positive bacteria. CONCLUSIONS: The patient was diagnosed with streptococcal toxic shock syndrome due to an ascending GAS infection originating from vagina.

Staple-Line Reinforcement of the Duodenal Stump With Intracorporeal Lembert's Sutures in Laparoscopic Distal Gastrectomy With Roux-en-Y Reconstruction for Gastric Cancer.

PURPOSE: We report a duodenal stump reinforcement procedure in laparoscopic distal gastrectomy with Roux-en-Y reconstruction. METHODS: We retrospectively reviewed the data of 223 patients who underwent laparoscopic distal gastrectomy with Roux-en-Y reconstruction for gastric cancer. We compared 2 groups: group NR (not reinforced, n=102, June 2009 to December 2011) when we did not perform reinforcement of the duodenal stump, and group R (reinforced, n=121, January 2012 to July 2014) when we did the reinforcement. The duodenum was divided with an endoscopic linear stapler. In group R, the duodenal staple line was reinforced by hand-sewn Lembert's sutures. RESULTS: There were no significant differences between group NR and R in patients' characteristics. Duodenal stump leakage occurred in 2 patients in group NR (2.0%). By contrast, in R group, no patients had duodenal stump leakage or fistula. CONCLUSIONS: Duodenal stump leakage can be avoided by using reinforcement with Lembert's sutures.

Expression of stanniocalcin 1 in thyroid side population cells and thyroid cancer cells.

BACKGROUND: Mouse thyroid side population (SP) cells consist of a minor population of mouse thyroid cells that may have multipotent thyroid stem cell characteristics. However the nature of thyroid SP cells remains elusive, particularly in relation to thyroid cancer. Stanniocalcin (STC) 1 and 2 are secreted glycoproteins known to regulate serum calcium and phosphate homeostasis. In recent years, the relationship of STC1/2 expression to cancer has been described in various tissues. METHOD: Microarray analysis was carried out to determine genes up- and down-regulated in thyroid SP cells as compared with non-SP cells. Among genes up-regulated, stanniocalcin 1 (STC1) was chosen for study because of its expression in various thyroid cells by Western blotting and immunohistochemistry. RESULTS: Gene expression analysis revealed that genes known to be highly expressed in cancer cells and/or involved in cancer invasion/metastasis were markedly up-regulated in SP cells from both intact as well as partial thyroidectomized thyroids. Among these genes, expression of STC1 was found in five human thyroid carcinoma-derived cell lines as revealed by analysis of mRNA and protein, and its expression was inversely correlated with the differentiation status of the cells. Immunohistochemical analysis demonstrated higher expression of STC1 in the thyroid tumor cell line and thyroid tumor tissues from humans and mice. CONCLUSION: These results suggest that SP cells contain a population of cells that express genes also highly expressed in cancer cells including Stc1, which warrants further study on the role of SP cells and/or STC1 expression in thyroid cancer.

[A Case of Local Recurrence of Bile Duct Cancer Completely Responding to Chemoradiotherapy with S-1].

An 80-year-old man with common bile duct cancer was treated by pancreaticoduodenectomy with D2 lymph node dissection in October 2005. The patient presented with frequent episodes of bloody-mucous rectal discharge in July 2009. An abdominal CT demonstrated local recurrence at the hepatoduodenal ligament. We treated him with concurrent chemoradiotherapy (CRT) with single-dose S-1 chemotherapy. After 6 months, we diagnosed a complete response (CR) by follow-up CT. The patient was treated with S-1 for 3 years after the diagnosis of a CR. He is alive without disease 6 years after the diagnosis of the recurrence. Concurrent CRT with S-1 chemotherapy may be the therapy of choice for recurrence of bile duct cancer.

[A Case of Gastric Carcinoid Associated with Hypergastrinemia Treated with LATG].

A 67-year-old man had a gastric polyp diagnosed on screening. Atrophic changes in the upper gastric mucosa were seen on upper gastrointestinal endoscopy. In addition, endoscopy revealed in the middle area of the stomach wall a 10 mm polyp that was diagnosed as a carcinoid tumor through biopsy. Blood serum gastrin was elevated at 2,800 pg/mL.We diagnosed a Rindi Type 1 gastric carcinoid. The patient was planned to be treated with surgical laparoscopy assisted distal gastrectomy (LADG); however, the procedure was changed to intraoperative laparoscopy assisted total gastrectomy (LATG). Chromogranin-positive tumor pathological findings in the mucous membrane submucosa and in the muscularis mucosae endocrine cell micronest (ECM) were widespread. There was no obvious vascular invasion. After the surgery, the serum gastrin level normalized and the patient remains alive.

Rheumatic manifestations and an epipharyngeal mass accompanied by myelodysplastic syndrome.

We herein report two cases of myelodysplastic syndrome with rheumatic manifestations. (Case 1) A 70-year-old man presented with fever, arthritis and bone pain and developed cranial nerve palsy caused by an epipharyngeal mass. Steroid therapy led to a prolonged remission of the febrile condition and mass lesion. (Case 2) An 82-year-old male was treated for intractable polyarthritis and fever with steroid therapy, and serious side effects resulted in lethal pneumonia. We herein describe the entire course of steroid therapy in these two cases. Various rheumatic manifestations in myelodyaplastic syndrome often require empirical steroid therapy. It was effective for the soft tissue mass in Case 1, in which indolent lymphoma could not be denied, and was only partially effective for Case 2 of the febrile and putatively benign conditions, suggesting heterogeneous nature of rheumatic complications in myelodysplastic syndrome.