Detection of actionable mutations in cytological specimens obtained by endoscopic ultrasound-guided fine needle aspiration with rapid onsite evaluation in pancreatic cancer.

BACKGROUND: It is not clear whether archived cytological specimens (ACSs) obtained with endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) with rapid onsite evaluation (ROSE) can be used for genomic profiling of tumors. We used ACSs to perform genomic analysis of specimens to identify oncogenic and druggable mutations. METHODS: A panel of 60 significantly mutated genes specific to pancreatobiliary cancer was created and used for genomic analysis of 113 specimens of 44 formalin-fixed paraffin-embedded (FFPE) tissues and 69 ACSs obtained by EUS-FNA with ROSE were included. The quantity and quality of DNA extracted from FFPE tissues and ACSs were compared. We also compared DNA from spray and touch ACSs. Next, genomic profiles were compared. We also evaluated detection of target gene mutations in each specimen. RESULTS: The amount of DNA in FFPE tissues was greater than in ACSs (P = 0.014), but the quality of DNA was comparable (P = 0.378). There was no quantitative or qualitative difference between spray and touch ACSs (P = 0.154 and P = 0.734, respectively). Oncogenic mutations were shared at 82 % in FFPE tissues and ACSs and 82 % in spray and touch ACSs. The sensitivity of genomic analysis in ACSs was 97 % (67 of 69), which was comparable to that of cytology (62 of 69, 90 %; P = 0.165), and was significantly higher than that of histology (32/44, 73 %; P < 0.001). Drug-matched mutations were identified in five of the 44 lesions (11 %). CONCLUSION: Genomic analysis of ACSs is useful in the prognosis of pancreatic cancer because detection of driver mutations is similar to detection in FFPE tissues.

Rapidly declining trend of signet ring cell cancer of the stomach may parallel the infection rate of Helicobacter pylori.

BACKGROUND: Studies indicate that gastric cancer (GC) incidence has decreased, whereas signet ring cell carcinoma (SRC) incidence has increased. However, recent trends in GC incidence are unclear. We used our hospital cancer registry to evaluate the changes in the incidence of GC, SRC, and non-SRC (NSRC) over time in comparison to changes in the H. pylori infection rates over time. METHODS: We identified 2532 patients with GC enrolled in our registry between January 2007 and December 2018 and statistically analyzed SRC and NSRC incidence. The H. pylori infection rate in patients with SRC was determined by serum anti-H. pylori antibody testing, urea breath test, biopsy specimen culture, and immunohistochemical analysis (IHC) of gastric tissue. Additionally, genomic detection of H. pylori was performed in SRCs by extracting DNA from formalin-fixed paraffin-embedded gastric tissue and targeting 16S ribosomal RNA of H. pylori. RESULTS: Overall, 211 patients had SRC (8.3%). Compared with patients with NSRC, those with SRC were younger (P <  0.001) and more likely to be female (P <  0.001). Time series analysis using an autoregressive integrated moving average model revealed a significant decrease in SRC (P <  0.001) incidence; NSRC incidence showed no decline. There was no difference in H. pylori infection prevalence between the SRC and NSRC groups. IHC and genomic methods detected H. pylori in 30 of 37 (81.1%) SRCs. CONCLUSIONS: Reduction in H. pylori infection prevalence may be associated with the decrease in the incidence of SRC, which was higher than that of NSRC.

Validation of Tokyo Guideline 2013 as Treatment of Acute Cholecystitis by Real World Data.

BACKGROUND: The Tokyo Guidelines (TG; 2013) indicated that emergency cholecystectomy is an important early treatment option for acute cholecystitis; however, surgical intervention is not necessarily indicated in patients with advanced age. We evaluated percutaneous transhepatic gallbladder aspiration (PTGBA), percutaneous transhepatic gallbladder drainage (PTGBD), and the administration of antibiotics alone as treatment options for acute -cholecystitis. METHODS: From January 2010 to December 2017, 159 patients with acute cholecystitis were treated at our institution. The data from these patients were retrospectively analyzed. RESULTS: Of these 159 cases, 109 underwent PTGBA, 28 underwent PTGBD, and 22 were administered antibiotics alone. None of the 159 patients needed urgent (early) cholecystectomy, and all patients were discharged without mortality. PTGBA was unsuccessful in only 6 of 109 patients; PTGBD was performed in these 6 cases. Long-term follow-up was conducted in all cases. Of the 159 patients, 146 had gallbladder stones initially, while 13 had none at the time of presentation. Of these 146 patients with gallbladder stones, 84 underwent elective cholecystectomy, while 62 did not. Of the 84 patients who underwent elective cholecystectomy, 2 developed choledocholithiasis; of the 62 patients who did not undergo elective cholecystectomy, 5 developed choledocholithiasis and 2 developed acute cholecystitis. The incidences of choledocholithiasis and acute cholecystitis did not significantly differ between the 2 groups (p = 0.06). CONCLUSIONS: Despite the recommendations in the TG (2013), emergency cholecystectomy was not needed in any of the present patients with acute cholecystitis. Acute cholecystitis can be successfully treated with -PTGBA or PTGBD, which are simple procedures with good short- and long-term safety. These procedures are highly recommended for patients with acute cholecystitis, especially in the elderly population.

Anthocyanin and proanthocyanidin contents, antioxidant activity, and in situ degradability of black and red rice grains.

OBJECTIVE: An experiment was conducted to assess the antioxidant contents and activities of colored rice grains and to evaluate their nutritive characteristics in terms of chemical composition and in situ ruminal degradation. METHODS: Ten cultivars of colored rice grains (Oryza sativa L.) collected from several areas of Japan were studied, and control rice without pigment, maize, barley, and wheat grains were used as control grains. Their chemical compositions, pigment, polyphenol contents, total antioxidant capacity (TAC), and degradation characteristics were determined. RESULTS: The starch contents of the colored rice grains were in the range of 73.5% to 79.6%, similar to that of the control rice grain. The black and red rice grains contained anthocyanin (maximum: 5,045.6 µg/g) and proanthocyanidin (maximum: 3,060.6 µg/g) at high concentrations as their principal pigments, respectively. There were significantly (p<0.05) positive relationships among the pigment contents, polyphenol content, and TAC values in the colored and control rice grains, indicating that the increase in pigment contents also contributed to the increased polyphenol content and TAC values in the colored rice grains. The dry matter and starch degradation characteristics, as represented by c (fractional degradation rate of slowly degradable fraction) and by the effective degradability, of the colored rice grains and the control rice grain were ranked as follows among commonly used grains: wheat>barley ≥rice>maize. The colored rice grains also included the most-digestible starch, since their potential degradable fraction and actual degradability at 48 h incubation were almost 100%. CONCLUSION: Colored rice grains have high potential to be used as antioxidant sources in addition to starch sources in ruminants.

Factors Predicting Subsequent Hospitalization in Patients with Ulcerative Colitis: Total Colonoscopic Findings are the Strongest Predictor.

BACKGROUND/AIMS: Patients with ulcerative colitis suffer from long term impairment of quality of life, especially when subjected to repeated hospitalization. We aimed to identify factors that may predict future hospitalization. METHODOLOGY: We followed 139 consecutive patients with ulcerative colitis for average of 11.2 years (2.8 to 49.5 years) from the onset. Clinical and endoscopic stagings were determined by Japanese staging system, the extent of colitis by Montreal classification and endoscopic grading by Matts' grade. RESULTS: Overall hospitalization rate was 37% at 5 years, 47% at 10 years and 60% at 20 years from the onset. Of 5 parameters including demographic and staging scores, univariate analysis revealed clinical severity at onset (p = 0.003), total colonoscopic findings on severity (Matts' grade, p = 0.003), and total colonoscopic findings on sites of abnormality (p = 0.012) were significantly correlated with hospitalization. By multivariate analysis, total colonoscopic findings on sites of abnormality was the only baseline character significantly related to the need of hospitalization (p = 0.0007). In fact, 5/10/20 years hospitalization rates were only 18/26/33 percent for proctitis type, whereas those were 61/72/90 for total colitis type. CONCLUSIONS: The total colonoscopic finding on sites of abnormality at the onset is the only predictdr of hospitalization in patients with ulcerative colitis.

Factors contributing to the overall survival in patients with hepatocellular carcinoma treated by sorafenib.

BACKGROUND/AIMS: In both SHARP and Asia-Pacific Study, sorafenib was proved to improve the overall survival of the patients with hepatocellular carcinoma. However, factors contributing to the improvement of overall survival of the patients treated by sorafenib have not been fully evaluated. In this study, patient-derived, background liver disease-derived and tumor-derived factors before treatment were evaluated whether they have contributed to the improvement of the overall survival. METHODOLOGY: Forty-seven cases with HCC treated by sorafenib between Sept 2009 and Feb 2011 were included in this analysis. The survival of these cases was analyzed by Kaplan-Meier Method. Factors used for univariate analysis were two patient-derived parameters, two background liver disease-derived, five tumor-derived. Factors related to the over-all survival were analyzed by multivariate analysis using Cox regression model. RESULTS: In the multivariate analysis, only background liver disease-derived parameter Child-Pugh class A vs. B, (p=0.007, HR=0.21 (0.07-0.65)) was significant. No other parameters including tumor-derived factors were statistically significant by multivariate analysis. CONCLUSIONS: We undertook the statistical analysis on the three categories. Surprisingly, no tumor derived parameter contributed to the overall survival. Background liver disease-derived parameter rather than tumor-derived parameter was found to define the prognosis of patients with advanced HCC treated by sorafenib.

The Use of Electronic Medical Records-Based Big-Data Informatics to Describe ALT Elevations Higher than 1000 IU/L in Patients with or without Hepatitis B Virus Infection.

Hepatitis B virus (HBV) infection is one of the serious health problems in the world as HBV causes severe liver diseases. Moreover, HBV reactivation has occasionally been observed in patients with resolved HBV infection and patients using immunosuppression and anticancer drugs. Large-scale hospital data focused on HBV infection and severe liver function were analyzed at our hospital, located in an urban area adjacent to Tokyo, the capital city of Japan. A total of 99,932 individuals whose blood samples were taken at 7,170,240 opportunities were analyzed. The HBV surface antigen (HBsAg)-positive group had a more frequent prevalence of patients with higher transaminase elevations than the HBsAg-negative group. However, among the HBsAg-negative group, patients who were positive for anti-HBV surface antibody and/or anti-HBV core antibody, had more severe liver conditions and fatal outcomes. More careful attention should be paid to alanine transaminase (ALT) elevations higher than 1000 IU/L in patients who had current and previous HBV infection.

Molecular subtype switching in early-stage gastric cancers with multiple occurrences.

BACKGROUND: Multiple gastric cancers at the same time (synchronous) or recurrence after 1 year (metachronous) are frequently encountered. Since their genetic profiles were not well elucidated, we molecularly subtyped the genetic events of synchronous and metachronous early-stage gastric cancers. METHODS: We studied mismatch repair (MMR) genes in 84 tumors from 31 patients (15 synchronous and 16 metachronous) by immunohistochemistry. We performed microsatellite instability analysis and targeted sequencing of 58 significantly mutated genes (SMGs) in 35 tumors from thirteen patients. Genomic data from TCGA were used for comparisons with advanced-stage cancers. RESULTS: Among the 31 patients, at least one deficient-MMR (dMMR) tumor was observed in eight (26%). Of eight patients, seven showed a mixture of proficient-MMR (pMMR) and dMMR tumors. The one case with only dMMR had six recurrent tumors within 2 years. To further subtype, we sequenced 58 SMGs in 35 samples (25 pMMR and 10 dMMR) from thirteen patients. In 35 samples, 163 mutations were identified, but none matched in almost cases, strongly indicating different clonal origins, whether synchronous or metachronous occurrences. Of the 25 pMMR cases, 1 belonged to Epstein-Barr virus (EBV), 24 belonged to chromosomal instability (CIN) subtypes. Of the thirteen cases, repetitive CIN, a mixture of CIN and MSI, a mixture of CIN and EBV, and repetitive MSI were observed in nine (70%), two (15%), one (8%) and one (8%), respectively. CONCLUSIONS: Despite multiple tumors occurring in the same patient simultaneously or several years apart, clonal origin was totally different. 'Switching' or 'mixing' of dMMR and pMMR, EBV or CIN occurred, which had clinical relevance with regard to immunotherapy.

Deficiency of mismatch repair genes is less frequently observed in signet ring cell compared with non-signet ring cell gastric cancer.

Signet ring cell (SRC) gastric cancer at advanced stage has poor prognosis. While a recent study reported nearly one-third of SRC cases contain tumors with deficient mismatch repair (MMR) genes, other studies in SRC have been inconclusive. To re-analyze the results, we performed immunohistochemical staining of MLH1, MSH2, MSH6 and PMS2 proteins in 38 SRC gastric tumors compared with 109 non-SRC (NSRC) tumors from 94 patients. In contrast to the previous study, all SRC gastric tumors normally expressed MMR proteins, whereas 22 of 109 of NSRC (20%) showed deficient MMR proteins. To reinforce our results, we referred to the Cancer Genome Atlas (TCGA) genomic database and found that only 6 (6%) of 99 samples with diffuse gastric tumors showed deficient MMR, whereas 64 (21%) of 304 in intestinal gastric tumors showed deficient MMR. Our results as well as the TCGA database indicated that MMR genes are infrequently inactivated in SRC gastric cancer. These findings indicate that SRC patients may not be the best candidates for immuno-oncology therapy.

The efficacy and safety of lenvatinib for advanced hepatocellular carcinoma in a real-world setting.

BACKGROUND/PURPOSE: Lenvatinib (an inhibitor of vascular endothelial growth factor (GF) receptors 1-3, fibroblast GF receptors 1-4, platelet-derived GF receptor α, rearranged during transfection, and stem cell factor receptor) was non-inferior to sorafenib in a phase 3 (REFLECT) trial of advanced hepatocellular carcinoma. This study examined the efficacy and safety of lenvatinib in a real-world setting. METHODS: This was a retrospective, multicenter, observational study. Inclusion and exclusion criteria were based on the phase 3 trial, and participants were observed for at least 12 weeks. Therapeutic effect was determined using the modified Response Evaluation Criteria In Solid Tumors (m-RECIST) at the 8th week. Patients received oral lenvatinib 12 mg/day (body weight > 60 kg) or 8 mg/day (body weight < 60 kg). Dose interruptions followed by reductions for lenvatinib-related toxicities were permitted. Grades of adverse events (AEs) complied with the Common Terminology Criteria for Adverse Events version 4.0. RESULTS: All 16 patients included in this study had prior treatment history, and a median 3.9 years had passed since the first treatment. Fatigue, hypertension, and proteinuria were the most frequent AEs, and were higher than Grade 2. AEs could be controlled by appropriate dose reduction, interruption, and symptomatic treatment according to the protocol. In the m-RECIST evaluation at the 8th week, 0, 6, 8, and 1 patients had achieved complete response, partial response, stable disease, and progressive disease, respectively. The objective response rate was 40%. CONCLUSION: Lenvatinib treatment could be accomplished with safety and good response in a real-world setting.

Influence of NUDT15 variants on hematological pictures of patients with inflammatory bowel disease treated with thiopurines.

AIM: The single nucleotide polymorphism (SNP) c.415C>T in exon 3 of NUDT15 affects thiopurine-induced leukopenia in Asian patients with Crohn's disease. Meanwhile, three additional genetic variants of NUDT15 were reported in patients with acute lymphoblastic leukemia. We evaluated the effects of these additional genetic variants of NUDT15 in patients with inflammatory bowel disease (IBD) treated with thiopurines. METHODS: Ninety-six Japanese patients with IBD were enrolled. Genotyping for the NUDT15 and TPMT genes was performed using Custom TaqMan SNP genotyping assays or Sanger sequencing. The changes in white blood cell (WBC) count, mean corpuscular volume (MCV), platelet count, hemoglobin, CRP, amylase, albumin, AST, ALT, and ESR were evaluated. RESULTS: Genetic variants of exon 1 and exon 3 of NUDT15 were identified in 24 of 96 patients (25.0%). C.52G > A and c.36_37insGGAGTC in exon 1 were found in three patients each. All three patients with c.36_37insGGAGTC in exon 1 were heterozygotes of p.Arg139Cys in exon 3. Eighteen patients had p.Arg139Cys in exon 3 alone. The WBC count gradually decreased after initiation of thiopurine treatment in the mutated cases (n = 24), and was significantly lower at 6, 8, 10, and 16 wk (P = 0.0271, 0.0037, 0.0051, and 0.0185, respectively). The WBC counts were also evaluated in patients with and without prednisolone treatment. In the patients with prednisolone treatment, the WBC count tended to show a greater decrease in the mutated cases, with significant differences at 8 and 10 wk (P = 0.012 and 0.029, respectively). In the patients without prednisolone treatment, the WBC count was significantly lower at 2, 4, 8, and 14 wk in mutated cases (P = 0.0196, 0.0182, 0.0237 and 0.0241, respectively). MCV increased after starting thiopurine treatment in the mutated cases, and was significantly higher at 10 wk (P = 0.0085). Platelet count, hemoglobin, CRP, amylase, albumin, AST, ALT and ESR did not differ significantly between the wild-type and mutated cases. TPMT mutations were not found in any of the patients. CONCLUSION: Mutations in exon 1 of NUDT15 also affect thiopurine-induced leukopenia in patients with IBD. To discuss thiopurine-induced leukopenia in more detail, investigation of SNPs in both exon 1 and exon 3 of NUDT15 is needed.

Dieulafoy lesion in a two-year-old boy: a case report.

BACKGROUND: Massive gastrointestinal bleeding in children, mostly caused by esophageal varices secondary to chronic liver disease, is uncommon. Dieulafoy lesion in the gastrointestinal tract is a rare but important cause of gastrointestinal bleeding; massive bleeding from this lesion can be fatal unless adequate treatment is promptly initiated. We report a case of gastric Dieulafoy lesion in a 2-year old successfully treated with endoscopic hemoclipping. CASE PRESENTATION: A 2-year-old Japanese boy was admitted to our department with sudden massive hematemesis. He had no significant past medical illness, and he was well just before the episode of hematemesis. A clinical examination revealed anemia (hemoglobin, 8.0 g/dl). The rapidly progressive anemia associated with massive hematemesis indicated the presence of an active bleeding in his upper gastrointestinal tract. We performed emergency gastroscopy under general anesthesia. The gastroscopy revealed the presence of an abnormal visible vessel with an adherent clot on the lower body of his stomach. No mucosal abnormality surrounding the lesion was noted; the lesion was thus diagnosed as Dieulafoy lesion. One hemostatic clip was placed on the Dieulafoy lesion and excellent hemostasis was obtained. He recovered without blood transfusion and was discharged 4 days post-endoscopy. He has recovered well with no recurrence of hematemesis. CONCLUSIONS: Dieulafoy lesion is rare cause of sudden massive gastrointestinal bleeding in children. Nevertheless, it should be considered a differential diagnosis, even in babies. With advances in gastrointestinal endoscopy, as both a diagnostic and therapeutic modality, laparotomy secondary to gastrointestinal bleeding from Dieulafoy lesion has decreased in pediatric cases. Our case report demonstrates the feasibility of endoscopic hemoclipping for gastric Dieulafoy lesion in a child.

Milk production, nutrient digestibility and nitrogen balance in lactating cows fed total mixed ration silages containing steam-flaked brown rice as substitute for steam-flaked corn, and wet food by-products.

The objective of this study was to evaluate the effect of substituting brown rice grain for corn grain in total mixed ration (TMR) silage containing food by-products on the milk production, whole-tract nutrient digestibility and nitrogen balance in dairy cows. Six multiparous Holstein cows were used in a crossover design with two dietary treatments: a diet containing 30.9% steam-flaked corn (corn TMR) or 30.9% steam-flaked brown rice (rice TMR) with wet soybean curd residue and wet soy sauce cake. Dietary treatment did not affect the dry matter intake, milk yield and compositions in dairy cows. The dry matter and starch digestibility were higher, and the neutral detergent fiber digestibility was lower for rice TMR than for corn TMR. The urinary nitrogen (N) excretion as a proportion of the N intake was lower for rice TMR than for corn TMR with no dietary effect on N secretion in milk and fecal N excretion. These results indicated that the replacement of corn with brown rice in TMR silage relatively reduced urinary N loss without adverse effects on feed intake and milk production, when food by-products such as soybean curd residue were included in the TMR silage as dietary crude protein sources.

Digestibility, ruminal fermentation, nitrogen balance and methane production in Holstein steers fed diets containing soy sauce cake at 10 or 20%.

Two feeding trials were performed to evaluate the effect of soy sauce cake (SSC) on digestibility, ruminal fermentation and nitrogen (N) balance as well as methane production in Holstein steers. Six animals received a diet without or with the SSC at 10% (Experiment 1) and 20% (Experiment 2) in a 2 × 2 crossover design with a balance trial and respiratory exchange measurement. The SSC feeding showed a strongly inhibitory effect on methane production when its proportion was elevated up to 20%. The digestibility of nutrients with the 10% SSC treatment was similar to that of the control, whereas that for the 20% SSC treatment was significantly decreased in comparison with the control. The 20% SSC treatment also inhibited ruminal fermentation. A lower N partition to urine and a higher N partition to retention were observed in the animals receiving the 20% SSC treatment. These results suggested that SSC feeding at 20% suppressed methane production and changed the N balance; however, the feeding level of 20% caused deterioration in some productive aspects, such as nutrient digestibility and ruminal fermentation. In addition, a feeding level of SSC at 10% of the diet should be considered a reasonable level in cattle.

Chronic heat stress up-regulates leptin and adiponectin secretion and expression and improves leptin, adiponectin and insulin sensitivity in mice.

Heat stress (HS) induces adaptive responses that are responsible for alterations of carbohydrate and lipid metabolism. This study aimed to evaluate the effects of chronic heat treatment on the expression and secretion of leptin and adiponectin, important regulators of energy homeostasis, food intake and insulin action. C57BL/6 mice were subdivided into three groups (24 mice each). The first group was kept under control conditions (C: 22±2 °C). The second group was exposed to HS (35±1 °C). The third group was kept under control conditions and was food restricted (FR). The HS group had higher rectal temperature than the C and FR groups and lower food intake than the C group. Hspa1 (Hspa1a) gene expression in adipose tissue, muscle and liver was higher under HS than FR and C. Heat treatment resulted in decreased blood glucose and non-esterified fatty acids; increased leptin, adiponectin and insulin secretion; and greater glucose disposal. Leptin, adiponectin, leptin and adiponectin receptors, insulin receptor substrate-1 and glucose transporter mRNAs were up-regulated in HS mice. This study provides evidence that HS improves leptin and adiponectin signalling in adipose tissue, muscle and liver. Heat stress was responsible for improving insulin sensitivity and glucose uptake in peripheral tissues, probably mediated by adipokines. Changes in the adipokine levels and sensitivity to them may be considered as an adaptive response to heat.

Effect of replacing corn with brown rice in a total, mixed, ration silage on milk production, ruminal fermentation and nitrogen balance in lactating dairy cows.

Nine multiparous Holstein cows were used in a replicated 3 × 3 Latin square design to determine the effects of substituting corn grain with brown rice (BR) grain in total mixed ration (TMR) silage on milk yield, ruminal fermentation and nitrogen (N) balance. The TMR silages were made from the ensiling of TMR containing (dry matter basis) 50.1% forage in rice silage and corn silage combination, and 49.9% concentrate. The grain portion of the diets contained 31.2% steam-flaked corn, 31.2% steam-flaked BR or an equal mixture of corn and BR. Dietary treatments did not affect dry matter intake, milk yield and milk fat, protein and lactose yields. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The urinary N excretion decreased linearly (P < 0.01) in response to increased levels of BR, with no dietary effect on N intake, N secretion in milk and fecal N excretion. Our results indicate that steam-flaked BR is a suitable replacement for steam-flaked corn in dairy cow diets, and that it can be included in rations to a level of at least 31.2% of dry matter without adverse effects on milk production, when cows were fed rice silage and corn silage-based diets.

Effect of anthocyanin-rich corn silage on digestibility, milk production and plasma enzyme activities in lactating dairy cows.

Anthocyanin in purple corn (Zea mays L.) has been reported to show several functional and biological attributes, displaying antioxidant, antiobesity and antidiabetic effects in monogastric animals. The objective of this study was to investigate the effect of feeding anthocyanin-rich corn (Zea mays L., Choko C922) silage on digestibility, milk production and plasma enzyme activities in lactating dairy cows. The cows were fed diets based on the control corn or the anthocyanin-rich corn silage (AR treatment) in a crossover design. The anthocyanin-rich corn silage-based diet had a lower starch content, nutrient digestibility and total digestible nutrients content when compared to the control diet. The milk yield, lactose and solids-not-fat contents in the AR-treatment cows were lower than in the control cows. The feeding of the anthocyanin-rich corn silage led to a reduction in aspartate aminotransferase (AST) activity and an increase in superoxide dismutase (SOD) activity in the plasma. These data suggest that the anthocyanin-rich corn has a lowering effect on AST activity with concomitant enhancement of SOD activity in lactating dairy cows. However, a new variety of anthocyanin-rich corn with good nutritional value is needed for practical use as a ruminant feed.

Differential immunolocalization between lingual antimicrobial peptide and lactoferrin in mammary gland of dairy cows.

Lingual antimicrobial peptide (LAP), one of the ß-defensins in bovines, and lactoferrin (LF) are synthesized in mammary epithelium and have bactericidal and bacteriostatic functions. However, it is not known whether they have similar expression patterns. Therefore, the present study was undertaken to compare (1) immunolocalization of LAP and LF in the mammary gland and (2) changes in concentration of these two components in milk after lipopolysaccharide (LPS) challenge. Bovine mammary tissues without LPS challenge were collected and their sections were immunostained with antibodies to LAP or LF. Milk from our previous study was collected every hour up to 12h and twice daily from d 1 to 7 after LPS challenge (the day of infusion was considered as d 0). These milk samples were measured for LAP but not LF in our previous report. Therefore, concentration of LF was measured by enzyme immunoassay in the present study. Epithelial cells of some alveoli showed immunopositive reaction for LF, but negative for LAP. Conversely, some alveoli were LAP positive in their epithelial cells but LF negative. Many alveoli had immunoreactions for neither LAP nor LF. The concentration of LAP in milk was elevated significantly at 3h after LPS infusion compared with pre-infusion values and remained at a high level until 12h. However, LF concentration in milk remained low at d 0 and increased at d 2. These results suggest that LAP and LF were mostly differentially localized in the alveolar epithelium in mammary glands. The different spatial expressions between them may be associated with their different temporal expression mechanisms.

Differential localization of lingual antimicrobial peptide in the digestive tract mucosal epithelium of calves.

Immunolocalization of lingual antimicrobial peptide (LAP), a member of the ß-defensin family, in the digestive tract of calves was investigated to determine its distribution in the digestive tract of Holstein-Friesian calves. Various regions of the digestive tract were collected from slaughtered calves, and fixed in 10% formalin in PBS. Paraffin sections were stained with anti-LAP antibody, followed by visualization of immunoreactions products utilizing the avidin-biotin complex method. Expression of LAP mRNA was analyzed with reverse transcription-PCR. Immunoreactive LAP was localized in the stratum corneum of the stratified squamous epithelium of the tongue, esophagus, rumen, reticulum and omasum but not in their basal layer and lamina propria. In the gastric glands of the abomasum, only chief cells showed LAP positive reaction at the apical side of their cytoplasm. Lamina propria and Peyer's patch of the ileum had some leukocyte-like cells immunopositive for LAP. Weak immunoreaction of LAP was also detected in the mucosal epithelium of the intestinal gland of the cecum, colon and rectum. All regions of digestive tract showed LAP mRNA expression with PCR. These results indicate differential localization of LAP in the mucosal epithelium of digestive tracts in calves. The LAP expressed in stratum corneum of stratified squamous epithelium and chief cells of abomasum specifically may play role in the innate immune function in these tissues.

Immunolocalization of lingual antimicrobial peptide (LAP) in the bovine mammary gland.

Lingual antimicrobial peptide (LAP), a member of the beta-defensin family in cows, is involved in the innate immune system and plays a crucial role in killing a large variety of microorganisms. The aim of the present study was to demonstrate the immunolocalization of LAP in the mammary glands of cows. A LAP antibody was raised in a rabbit by immunity with a synthetic 11 amino acid sequence out of a 42-amino acid sequence of the mature form of LAP. The specificity of the LAP antibody was checked using a competitive immunoassay and Western blotting. Paraffin sections of the mammary gland were immunostained with LAP antibody. In the competitive immunoassay, an increase of synthetic LAP concentration suppressed the optical density. Western blotting analysis for LAP revealed the presence of the LAP peptide in mammary alveolar tissue. When the mammary gland was immunostained with LAP antibody, epithelial cells of both infected and non-infected alveoli were immunopositive. These results indicate that LAP is localized in the epithelium of non-infected as well as infected alveolus in the mammary gland in cows.