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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.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.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 %).Genomic analysis of ACSs is useful in the prognosis of pancreatic cancer because detection of driver mutations is similar to detection in FFPE tissues.

This study was performed to investigate the effect of the inclusion rate of corn silage (CS) in ensiled total mixed ration (TMR) on dry matter intake (DMI), nutrient digestibility, and ruminal fermentation in Japanese Wagyu steers. Four ensiled TMRs, in which the crude protein (CP) and total digestible nutrients (TDN) levels were adjusted to be similar, were prepared with 0, 15, 30, and 45% CS, respectively. Four Japanese Wagyu steers during the fattening period were used in a feeding trial with the four dietary treatments of the ensiled TMRs in a 4 × 4 Latin square design. Increasing the inclusion rate of CS resulted in higher pH values and lower lactate production in the ensiled TMR, although all TMR silages showed good fermentative characteristics, such as low pH value and high lactate content. The DMI linearly increase when the inclusion rate of CS in the TMRs increased. There were differences in the apparent digestibilities of CP, non-fibrous carbohydrate, acid detergent fiber, and neutral detergent fiber. The TDN contents were greater in the ensiled TMRs with 15 and 30% CS. The feeding of TMRs with higher inclusion rates of CS gave rise to slower fermentation in the rumen. Corn silage should be utilizable as a TMR material, and its inclusion at approximately 30% in a TMR is a safe upper limit for Japanese Wagyu steers during the fattening period.

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. 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. 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. 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.

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.

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 (pl0.05) 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: wheatgbarley ≥ricegmaize. 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.

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

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.

BackgroundStudies 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 theH. pyloriinfection rates over time.MethodsWe identified 2532 patients with GC enrolled in our registry between January 2007 and December 2018 and statistically analyzed SRC and NSRC incidence. TheH. pyloriinfection rate in patients with SRC was determined by serum anti-H. pyloriantibody testing, urea breath test, biopsy specimen culture, and immunohistochemical analysis (IHC) of gastric tissue. Additionally, genomic detection ofH. pyloriwas performed in SRCs by extracting DNA from formalin-fixed paraffin-embedded gastric tissue and targeting 16S ribosomal RNA ofH. pylori.ResultsOverall, 211 patients had SRC (8.3%). Compared with patients with NSRC, those with SRC were younger (P P P H. pyloriinfection prevalence between the SRC and NSRC groups. IHC and genomic methods detectedH. pyloriin 30 of 37 (81.1%) SRCs.ConclusionsReduction inH. pyloriinfection prevalence may be associated with the decrease in the incidence of SRC, which was higher than that of NSRC.

The objective of this study was to evaluate the effect of replacing corn with brown rice (BR) on dry matter intake (DMI), milk production, blood metabolites and energy balance (EB) in cows during the periparturient period. Twenty-four nonlactating multiparous Holstein cows in late gestation were studied from 3 week before the expected calving date through 6 week after parturition. The cows were divided into one of two dietary treatment groups based on parity and the expected calving date. The treatments were a diet containing steam-flaked corn and a diet containing steam-flaked BR. Prepartum DMI and body weight (BW) and postpartum DMI, BW, milk yield, milk protein and lactose yield were not affected by the dietary treatment. However, the milk fat content and fat yield were higher ( P = 0.02 and P = 0.01) for cows fed BR than for cows fed corn. Thus, cows fed BR-based total mixed ration (TMR) tended to have more negative EB in early lactation ( P = 0.06) compared with cows fed corn-based TMR. During the prepartum period, the plasma concentrations of glucose, insulin, β-hydroxybutyrate (BHBA), nonesterified fatty acids (NEFA), aspartate transaminase (AST) and Ca were not affected by dietary treatments. On the other hand, the postpartum plasma glucose and insulin concentrations were decreased ( P P = 0.02) and the BHBA and AST concentrations were increased ( P P = 0.02) by replacing corn with BR with no dietary effect on postpartum plasma NEFA and Ca concentrations. These results show that feeding BR instead of corn does not affect DMI or milk yield, but increases milk fat production and leads to a more negative EB state in early lactation. Replacing corn with BR during the periparturient period has adverse effects on the metabolism and energy status in early lactating cows.

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.

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. 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

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.

Background The clonality of primary tumors is well elucidated. In contrast, the relation to metastatic foci is not well understood. We examined genomic clonality of primary tumors and metastatic foci. Method We studied 144 foci (34 primaries, 104 lymph nodes, 6 organ mets) from 10 patients with gastrectomy (c-Stage 3). We also searched the expression of MMR proteins. Results In 10 primaries, mutations were found in all (range 3-12, mean 6). Of 104 lymph nodes, 64 lymph nodes carrying mutations identical to corresponding primaries in all. In addition, all the genomic profiles were replicated in corresponding lymph nodes and visceral mets. Among 3 cases with dMMR, all metastatic foci showed identical phenotypes. Conclusion Surprisingly, significantly mutated genes (SMGs) profiles in all lymph nodes and visceral mets were identical to the primaries. In addition, MMR/MSI status was also identical. These results indicated proper selection of chemo-, molecular-, and immune-oncology treatments could be effectively applied to the spreaded mets. Internoduler ""de novo"" heterogeneity was so scarce. Often claimed tumor heterogeneity could be due to inappropriate selection of drugs and due to their pressure.

The effects of the substitution of brown rice (Oryza sativa L.; BR) for corn (Zea mays L.) in ensiled total mixed ration (TMR) that had a high proportion of grain on feed intake, lactation performance, ruminal fermentation, digestion, and N utilization were evaluated. Nine multiparous Holstein cows (51 ± 9 d in milk) were used in a replicated 3 × 3 Latin square design with 3 dietary treatments: a diet containing 0, 20, or 40% steam-flaked BR and 40, 20, or 0% steam-flaked corn (dry matter basis). Cows were fed ad libitum an ensiled TMR consisting of 40.7% alfalfa silage, 11.8% grass silage, 7.1% soybean meal, and 40.0% steam-flaked grain (dry matter basis). The ensiled TMR was prepared by baling fresh TMR, and then sealed by a bale wrapper and stored outdoors at 5 to 30 °C for over 6 mo. Dry matter intake and milk yield were lower for cows fed 40% BR than for cows fed 40% corn. The ruminal pH and total volatile fatty acid concentrations were not affected by dietary treatment. The ruminal ammonia-N concentration decreased as the percentage of BR in the diets was elevated. The proportion of acetate decreased, and that of propionate and butyrate increased with the increasing levels of BR. Plasma urea-N concentrations was lower and glucose and insulin concentrations were higher for cows fed 40% BR than for cows fed 40% corn. The whole-tract apparent digestibility of dry matter, organic matter, and starch increased, and the digestibility of neutral detergent fiber and acid detergent fiber decreased with the increasing BR level in the diet, with no dietary effect on crude protein digestion. As a proportion of N intake, the urinary N excretion was lower and the retention of N was higher for cows fed 40% BR than for cows fed 40% corn, with no dietary effect observed on N secretion in milk and fecal N excretion. These results show that substituting BR for corn decreases urinary N losses and improves N utilization, but causes adverse effects on milk production when cows are fed high-grain diets at 40% of dietary dry matter.

Mastitis is most critical disease in dairy cows and causes huge cost in the dairy industry. To prevent and treat it, it is important to understand the mechanisms of immune function in the mammary gland. Innate immunity is non-specific acute-response immune function. Some components of innate immunity in the mammary gland are found, e.g. Lingual Antimicrobial Peptide (LAP), Lactoferrin (LF). These components are found to be localized in the alveolar epithelium of mammary gland. LAP belongs to the β-defensin family, and plays a crucial role in killing a large variety of microorganisms. LF belongs to an iron-binding glycoprotein and has antibacterial activity. It is reported that LF has been localized immunohistochemically in mammary epithelial cells of lactating cows. Our previous study revealed that secretion of LAP into milk proceeded to that of LF after lipopolysaccharide (LPS) injection into the mammary gland. From this result, it is hypothesized that immunohistochemistry probably shows positive to either LF or LAP but not both in the alveolus vs epithelium in the mammary gland. Therefore, the aim of the present study is to investigate the immunolocalization of LAP and LF in the same bovine mammary tissue. Bovine mammary tissues were collected in the slaughterhouse and were fixed with neutralized formalin immediately. Paraffin sections (2-um thickness) were processed with antigen retrieval treatment followed by blocking with casein milk. Sections were cultured with LF antibody or LAP antibody. Immunoreaction products were visualized by incubation with a DAB. LAP and LF were localized in the cytoplasm of epithelial cell of alveolus. In some cases, LAP and LF were seen clearly in the same alveoli of section. In other cases, some epithelial cells were stained only LAP, but not LF, and other epithelial cells of alveolus were stained only LF, but not LAP. These results suggest the possibility that LAP and LF are differentially synthesized in the alveolar epithelium and may support our previous findings that their secretion occurs at the different time course.

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.

Some anthocyanins have been reported to exhibit antioxidant activity and to enhance antioxidant enzyme activity. The aim of this study was to determine the fermentative quality of purple rice, which is abundant in anthocyanin, and its effects on digestibility, ruminal fermentation and oxidative status markers in sheep. The whole crop silages of purple and control rice were prepared in drum silos after the materials were chopped and lactic acid bacteria was inoculated. These were followed by a feeding trial using eight wethers in a 2 × 2 crossover design. Both the purple and control rice silages showed excellent fermentative qualities. Although the purple rice silage contained abundant anthocyanin, it had inadequate nutritional values in comparison to the control rice silage, including higher levels of fiber fractions, lower nutrient digestibility and lower production of ruminal volatile fatty acids. The feeding of the purple rice silages led to an elevation in plasma activity of superoxide dismutase, which is an important antioxidant enzyme. The results of this study suggest that purple rice may become a source of anthocyanin to improve oxidative status in ruminants. To achieve that, however, a new variety of purple rice with good nutritional value is needed for practical use as a ruminant feed.

Anthocyanins are pigments found widely in plants. They have been reported to possess antioxidant activity and an enhancing effect on superoxide dismutase (SOD) activity in monogastric animals. Thus, anthocyanins could also affect the status of the antioxidative defense system in ruminants. This study aimed to investigate the effect of the supplementation of purple pigment from anthocyanin-rich corn on blood antioxidant activities and oxidation resistance in sheep. Twelve sheep were used in a crossover design with two dietary treatments: a diet with purple corn pigment (pigment treatment) and one without (control). The pigment was added to the diets at an anthocyanin concentration of 0.5%. Blood and urine samples were collected to determine the markers of oxidative status. A portion of the plasma was used for an oxidation resistance test, in which plasma was incubated with an oxidizer at 37 °C for 0, 4 and 8 h, and then the degree of oxidation was measured. The pigment addition to the diet caused a significant increase in the SOD activity of the plasma, although the total antioxidant capacity and glutathione concentration in the plasma of both treatment groups were similar. In the oxidation resistance test, the oxidation in the plasma from the pigment treatment group was significantly suppressed compared to that in the control group at 4 and 8 h after the onset of the incubation. These findings indicate that the intake of purple pigment from anthocyanin-rich corn increases the SOD activity and the potential of the antioxidant activity of plasma in sheep.

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.

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.

Immunolocalization of lingual antimicrobial peptide (LAP), a member of the β-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.

Specific immunoassay systems for intact human osteocalcin (I-OC) and its 26-residue propeptide have been newly developed to assess their usefulness as biochemical markers of bone metabolism. Using human cultured osteoblastic periosteal cells, we monitored 24 h secretion of these molecules from the osteoblastic cells and also examined the deposition of Ca, P, and I-OC on the extracellular matrix. At day 5, both I-OC and its propeptide were secreted by osteoblastic cells in a concentration-dependent manner by treatment with 1,25-(OH)2D3. This propeptide was not detected in the serum of adult subjects but was detected in the serum of normal children, which confirmed this in vitro result of propeptide secretion. The secretion of I-OC into medium transiently decreased at day 11, when the rapid accumulation of I-OC, Ca, and P, namely mineralization, was observed on the extracellular matrix of osteoblastic cells, although secretion of the propeptide constantly increased throughout the culture period. Therefore, the ratio of the amount of propeptide to I-OC in the supernatant markedly increased when mineralization started. These data demonstrate the superior specificity of propeptide as a marker of osteoblastic function in vitro compared with I-OC and that monitoring the changes in propeptide to I-OC ratios in the culture supernatant may be useful for predicting the timing of mineralization on the extracellular matrix of osteoblastic cells.

To estimate the response to hormone replacement therapy (HRT) by bone metabolic markers, 36 patients with postmenopausal osteoporosis or osteopenia were studied to assess the correlation between percent baseline changes in lumbar bone mineral density (BMD) after 12 months and those in various bone metabolic markers after 3, 6, and 12 months of HRT. All the patients were treated with 0.625 mg of conjugated estrogen and 2.5 mg of medroxyprogesterone per day and continued for 12 months. BMD was significantly increased up to 4.19 +/- 0.87% after 6 months and 4.93 +/- 1.27% after 12 months of HRT (p = 0.0001 by analysis of variance). In accordance with this, changes in the levels of osteocalcin (p = 0.041), alkaline phosphatase (p = 0.0001), N-terminal osteocalcin (p = 0.0001), urinary excretion of pyridinoline/Cr (p = 0.0001), and deoxypyridinoline/Cr (p = 0.0001) were significantly decreased, respectively. Among these bone metabolic markers, only the change in the serum N-terminal osteocalcin at 3 months (r = 0.557, p = 0.0022), at 6 months (r = 0.470, p = 0.0184), and at 12 months (r = 0.545, p = 0.0061) significantly correlated with the change in BMD 12 months after HRT. The elution profiles of immunoreactive osteocalcin-related molecules in serum fractionated by reverse-phase high performance liquid chromatography revealed that the N-terminal fragment as well as the intact osteocalcin molecule decreased after 3 months of HRT. These results demonstrate that N-terminal osteocalcin is a suitable predictor for estimating good responders to HRT in postmenopausal women.

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.

The fermentative quality and quantitative change in anthocyanin of anthocyanin-rich corn (Zea mays L.) during storage and in vitro ruminal fermentation were studied. The anthocyanin-rich corn silages in bag silo, drum silo and round bale had good fermentative qualities, such as low pH ( 5% DM) and butyric acid-free, and its quality was maintained for more than 370 d. The amount of anthocyanin in the anthocyanin-rich corn decreased after ensiling by about 45% (from 3.34 to 1.88 mg/g DM), but stayed constant after day 60. The in vitro incubation of the anthocyanin-rich corn with ruminal fluid revealed little degradation of anthocyanin. These results indicate that the anthocyanin had no negative effect on silage fermentation, and the anthocyanin-rich corn silage is utilizable for practical use as a feedstuff. Our results also demonstrate alteration of the anthocyanin content during storage, and show that anthocyanin-rich corn is a suitable antioxidant source for ruminants because of the high stability of the anthocyanin in ruminal fluid.

An experiment was conducted to examine feed intake, apparent digestibility, nitrogen balance, ruminal fermentation and blood components of wethers fed diets containing increasing levels of wet green tea grounds (WGTG). The experimental design was a 4 x 4 Latin square with four wethers in four 15-day periods. Wethers were allowed access to diets ad libitum, and allotted to one of four treatments in which WGTG replaced 0% (no WGTG added, CTG), 5% (low level, LBG), 10% (medium level, MTG), and 15% (high level, HTG) of total mixed ration (TMR) dry matter (DM) as wet brewers grains (WBG). All TMR silages were ensiled for 120 days and, irrespective of the WGTG addition, they were well preserved with a high lactic acid content, low pH and ammonia-N contents. There were no differences among treatments in feed intake, with the exception of ether extract intake (P = 0.032). Digestibilities for LTG and MTG treatments were not different from CTG. However, the organic matter, crude protein, acid detergent fiber and energy digestibilities for HTG treatment were lower than the CTG (P < 0.05). As the level of WGTG increased, nitrogen intake did not differ, but fecal nitrogen increased (P = 0.002), while urinary nitrogen decreased (P < 0.001). No differences among treatments were found in pH level and volatile fatty acids concentrations. However, the ruminal ammonia-N of the HTG silage was lower than for the CTG silage at all times (P < 0.05). Increasing concentrations of WGTG in the TMR silage decreased (P = 0.019) plasma urea nitrogen content. Therefore, the possible mixing proportion of WGTG for TMR silages can be 10% of the diet DM.

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.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.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.The total colonoscopic finding on sites of abnormality at the onset is the only predictdr of hospitalization in patients with ulcerative colitis.

The effects of the lengths of growth of round bale whole-crop corn silage on its nutritive value, the methane production, chewing time, and ruminal fermentation were evaluated in three Holstein steers (599.3 ± 31.5 kg). Corn forages were harvested, baled and wrapped on the same day at 129 (D129), 118 (D118) and 107 (D107) days from planting. All steers were fed the three corn silages along with urea at maintenance energy requirements during three 14-day periods in a 3 × 3 Latin square design. A balance trial was conducted to investigate whole tract digestibility by the collection of total feces and urine over a 7-day test period. For 2 days of the test period, a head hood-type respiration chamber was used to measure methane production. Ruminal fluid was collected immediately before and 3 h after, the morning feeding on the last day of the test period. There was no spoilage in any of the silages (n = 9). The average fresh weight of the bales was 326.4 ± 10.7 kg. The DM content of the corn silage was highest in D118 and lowest in D129 (P

The aim of this research was to investigate the effect of peppermint feeding on dry matter intake, nutrient digestibility, ruminal fermentation and milk production in early lactating cows. Four Holstein cows were offered a diet with 5% dried peppermint and four Holstein cows remained on a diet without 5% of dried peppermint on a dry matter basis. The addition of peppermint to feed did not affect dry matter intake, although the eating time of feed was increased by mixing the feed with peppermint. There were no significant differences in the nutrient digestibilities between the two treatments. The ruminal ammonia and volatile fatty acids concentrations were similar in the two treatments, however, peppermint ingestion by cows led to a decrease in ruminal pH. The lowered pH value was within the stable pH condition range. No significant differences in the treatments were observed in milk production or milk composition except for the milk fat content. These results suggest that feeding peppermint to early lactating cows had little effect on their dry matter intake, nutrient digestibility, ruminal fermentation and milk production.

The effects of green tea (Camellia sinensis) waste silage and supplemental polyethylene glycol (PEG) on rumen fermentation and blood components were studied in cattle. Six Holstein steers were fed three diets in a 3x3 Latin square design, replicated twice. One diet was a control with no added silage, and the other two diets were supplemented (20% of the dry matter) with green tea waste silage either with (PEG) or without PEG (tea). Most of the fermentation parameters including major volatile fatty acids (VFA) were not affected by the diet treatments. The concentrations of high density lipoprotein cholesterol in the PEG group and urea nitrogen in the tea and PEG groups were greater than those in the control before morning feeding. The plasma 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid equivalent concentration was not different before morning feeding, but 3 h after morning feeding, its concentrations in both the tea and PEG groups were higher than in the control. Although the concentration of plasma vitamin A in the animals was not affected by feeding green tea waste silage, the concentrations of plasma vitamin E were significantly higher in the tea and PEG groups than in the control, both before and 3 h after morning feeding. The results from the present study suggest that feeding diets containing 20% of the dietary dry matter as green tea waste silage to Holstein steers has no negative impact on their ruminal fermentation, and increases their plasma antioxidative activity and concentration of vitamin E.

Blood flow to the gravid and nongravid uterine horns of four multiparous Holstein cows (mean +/- SD, BW=641.8 +/- 95.4 kg; age=4.8 +/- 1.2 years; parity=3.0 +/- 1.2) was measured on days 225, 248, and 266 of gestation. Surgery was conducted on day 214.5 +/- 4.0 of gestation through the flank of the standing cows. Transit-time ultrasonic flow probes (diameter 12 or 14 mm) were fitted surgically around the uterine arteries of each cow. Surgery was completed within two hours of anesthesia, and the animals recovered rapidly following surgery. Uterine blood flow (UBF, l/min) was recorded at 10 sec intervals for approximately 23.5 hours; these values were averaged to determine UBF. The mean gravid UBF was significantly (P0.05) greater than the nongravid UBF in this study. The range of the gravid and nongravid UBFs varied from 3.61 to 14.05 and 0.72 to 6.54 l/min, respectively. There were no changes (P0.1) in the mean gravid and nongravid UBFs from day 225 to 266 of gestation.

茶系飲料製造残渣の飼料特性を明らかにする研究の一環として，乳酸菌および繊維分解酵素添加緑茶飲料製造残渣サイレージの乾物（DM）および粗タンパク質（CP）のウシのルーメン内における分解性を，数種飼料と比較検討した．緑茶飲料製造残渣サイレージのルーメン内における消失率は高く，DMおよびCPの消失率は浸漬24時間でそれぞれ82.0％と91.7％であった．また，DMおよびCPのルーメン内分解パラメータでは，易分解性区分割合a（％）が31.5と21.5，難分解性区分割合b（％）が55.0と75.7，b区分の分解速度定数c（/h）が0.089と0.092と推定された．ルーメン内通過速度定数（/h）を0.05と仮定し，ルーメン内分解パラメータから計算されたDMの有効分解率は66.8％であり，大豆粕，豆腐粕およびアルファルファヘイキューブと差がなかった．また，CPの有効分解率は70.6％であり，大豆粕と差が認められなかった．さらに，両分解率ともビール粕とビートパルプより有意に高かった（P＜0.01）．これらの結果から，緑茶飲料製造残渣を乳酸菌および繊維分解酵素添加サイレージに調製した場合，そのルーメン内における消失速度は速く，CPの有効分解率は大豆粕並みであると考えられた．

The characteristic smell of cow milk was suppressed when herbs were consumed by lactating dairy cows. But it is unclear whether or not peppermint ingestion affects the nutritional and milk production parameters in lactating dairy cows. The objective of this study was to examine the effect of peppermint feeding to lactating dairy cows on nutrient digestibility, energy metabolism, ruminal fermentation and milk production. Eight Holstein cows were given a diet supplemented with or without 5% of dried peppermint per diet on a dry matter basis. The digestion of nutrients from cows fed the diet with peppermint was significantly lower than that of the control group. Energy loss as methane and methane released from cows receiving the peppermint treatment was significantly lower than that in the control cows. Peppermint feeding to cows resulted in the promotion of thermogenesis. However, ruminal fermentation and milk production were not affected by peppermint feeding. In conclusion, peppermint ingestion by lactating dairy cows reduces the nutrient digestibility and methanogenesis, and changes energy metabolism.

混合飼料（TMR）中の緑茶飲料製造残渣サイレージ（TGS）配合割合の違いが泌乳牛の飼料摂取量，ルーメン発酵および乳生産に及ぼす影響を調べた．ホルスタイン種泌乳牛4頭を用い，TMR中TGSを乾物比で0，5，10および15％配合とするTGS0，TGS5，TGS10およびTGS15の4区を設け，4×4ラテン方格法による飼養試験を実施した．乾物摂取量は，TGS0に比べTGS5で変化がなかったものの，TGS10で減少する傾向にあり，TGS15で有意に減少した（P＜0.05）．乳量は，TGS0とTGS5に比べTGS15で有意に低下した（P＜0.01）．乳タンパク質率と乳脂率は，4区間に差はなかったが，乳糖率は，TGS15で有意に低かった（P＜0.05）．ルーメン内容液のpHおよび総VFA濃度に区間差はなかったが，プロピオン酸のモル比率はTGS15で，アンモニア態窒素濃度はTGS10とTGS15でそれぞれ有意に低かった（P＜0.05）．以上の結果から，TGSは，乳量30kg程度の泌乳牛用TMRの素材として利用でき，乾物比5％程度の配合であれば乳量，乳成分に影響しないと考えられた．

Seven Holstein cows (BW = 639.4 +/- 28.8 kg, age = 62.6 +/- 6.9 mo, parity = 3.1 +/- 0.4 yr) (mean +/- SE) were used to measure uterine blood flow using a transit time ultrasonic flow meter. Ultrasonic flow probes were implanted (d 218 +/- 4 of gestation) around both uterine arteries (n = 3) or the gravid uterine artery (n = 4) to measure variations in uterine blood flow through this vessel in response to postural change (standing and lying). Implantation of the ultrasonic blood flow probe was successful in all 7 cows. Uterine blood flow was measured for 22.9, 23.1, and 21.4 h/d on d 226, 248, and 269 of gestation, respectively. Cows exhibited normal durations of gestation (272.8 +/- 1.6 d), gave birth to normal healthy calves (birth weight = 41.5 +/- 3.0 kg), and did not retain their placenta. Uterine blood flow to the gravid horn during lying was greater than during standing on d 226 (standing vs. lying, 8.04 vs. 8.79 L/min, respectively), d 248 (9.87 vs. 11.06 L/min), and d 269 (8.15 vs. 9.74 L/min) of gestation. Cows spent nearly 50% of their time lying during our observations at all gestational ages. Greater blood supply to the gravid uterus was observed during lying than during standing in this study.

1.無給与牛と比較して,レモングラス給与牛,クローブ給与牛はラジカル捕捉活性および総ポリフェノール量が有意に増加した.増加の程度は給与量が多いほど大きかった.2.ハーブ牛乳のラジカル捕捉活性とポリフェノール量の間には高い相関が見られた.また,ポリフェノールがラジカル捕捉活性に大きく寄与していた.3.レモングラス給与牛ではシトラールが,クローブ給与牛ではオイゲノールがそれぞれ乳中に検出されたが,これらの精油成分はラジカル捕捉活性にほとんど寄与していなかった.

ウシにおける緑茶飲料製造残渣サイレージの栄養価を求めると共に窒素およびエネルギー出納に及ぼすポリエチレングルコール（PEG）の影響を調べた．チモシー乾草，圧ぺんトウモロコシと大豆粕を組成とした基礎飼料のみを給与（対照区），乾物（DM）比で基礎飼料の20％を緑茶飲料製造残渣サイレージに置換（茶殻区）およびPEGを茶殻区の飼料中タンパク質量の1/5となるように添加（PEG区）する3試験区を設けた．各区にホルスタイン種去勢ウシ2頭ずつを割り当て，3×3のラテン方格法による消化試験を実施した．間接方法により求められた緑茶飲料製造残渣サイレージのDM中可消化養分総量（TDN），可消化エネルギー（DE）および代謝エネルギー（ME）は，それぞれ69.8％，13.0MJ/kgと10.5MJ/kgであった．PEG区では，茶殻区に比べ粗脂肪（EE）の消化率が有意に減少した（P＜0.01）ものの，粗タンパク質（CP）の消化率が有意に上昇した（P＜0.05）．PEGに添加により，糞への窒素排出量は有意に減少した（P＜0.05）が，尿中への窒素排出量が増加したため，体内窒素蓄積量は増加しなかった．以上の結果から，緑茶飲料製造残渣サイレージのウシでの栄養価が明らかとなり，PEGの添加により緑茶飲料製造残渣サイレージに由来するタンニンとタンパク質の結合が解離する可能性が示唆された．

緑茶飲料製造残渣サイレージの給与が泌乳牛の採食量，飼料消化率，ルーメン発酵，血液性状および乳生産に及ぼす影響について検討した．緑茶飲料製造残渣サイレージを飼料乾物中に10％含む飼料（茶殻区）または含まない飼料（対照区）を給与する2試験区を設け，各区に乾乳直前のホルスタイン種乳牛4頭ずつを割り当て，馴致期を7日間，予備期を7日間および本試験期を8日間とする消化試験を実施した．採食量および乾物消化率に両区間で有意差を認めなかったが，粗タンパク質（CP）の消化率は茶殻区で低い傾向を示した．ルーメン内容液のpH，プロトゾア数および総揮発性脂肪酸（VFA）濃度に区間差はなかったが，酪酸のモル比率とアンモニア態窒素濃度は茶殻区で有意に低かった（P＜0.05）．血漿中のグルコースとコレステロール濃度に区間差はみられなかったが，茶殻区で尿素態窒素濃度が有意に低く（P＜0.05），トリグリセリド濃度が高い傾向を示した．乳量（対照区25.1kg ; 茶殻区24.5kg），乳脂率（対照区4.2％ ; 茶殻区4.1％）および乳タンパク質率（対照区3.6％ ; 茶殻区3.5％）に区間差はなかったが，乳糖率（対照区4.4％ ; 茶殻区3.9％）は茶殻区で有意に低かった（P＜0.05）．以上の結果から，緑茶飲料製造残渣サイレージは，乳量25kg前後の泌乳牛に対し飼料乾物中に10％程度の給与ならば，ルーメン発酵，血液性状，乳量および乳成分に大きな影響を与えずに利用できる可能性が示唆された．

We describe an immuno-polymerase chain reaction (immuno-PCR) assay for the detection of human angiotensinogen using identical first and second polyclonal antibodies. The reporter DNA was initially generated by PCR amplification using a biotinylated primer, and was bound with streptavidin to biotinylated second antibody. Human recombinant angiotensinogen sandwiched by antibodies was detected by amplifying the reporter DNA using PCR. To reduce the effect of nonspecific amplification, the optimal concentrations of streptavidin and DNA label were determined to be 0.1 mg/l and 0.5 ng/l, respectively. The detection limit of the immuno-PCR assay was 0.1 ng/l, an approximately 2.5 ×10 5 -fold improvement compared with a conventional enzyme-linked immunosorbent assay. These results indicate that a highly sensitive immuno-PCR for human angiotensinogen can be developed even with identical first and second polyclonal antibodies.

Protein C is one of the most important antithrombotic components. After activation by the thrombin-thrombomodulin complex on endothelial cells, activated protein C (APC) inactivates factors Va and VIIIa, which leads to the inhibition of thrombin formation. We examined the association of plasma levels of APC with the responsiveness to coronary thrombolytic therapy of the infarct-related coronary artery in patients with acute myocardial infarction (AMI). Plasma levels of APC, thrombin-antithrombin III complex (TAT), and plasminogen activator inhibitor (PAI) activity were measured in 32 consecutive AMI patients who underwent coronary angiography followed by thrombolytic therapy, and compared to the measurements in 23 control subjects. On admission, APC levels (ng/mL) were significantly elevated in patients with AMI, as compared with controls (2.5+/-0.4 vs. 1.2+/-0.2, 1.3+/-0.2, respectively, p0.01). At discharge, plasma levels in AMI patients decline to values not significantly different from those in controls. (1.2+/-0.2, 1.3+/-0.2, respectively). TAT levels (ng/mL) were different among the groups in a fashion similar to that of APC (14.1+/-3.1 on admission vs. 3.3+/-0.4 at discharge, 1.8+/-0.1 in the control subjects, respectively, p0.01). PAI activity levels (IU/mL) were higher on admission than at discharge and higher than the control subjects (19.7+/-1.8 vs. 10.5+/-1.0, 5.4 +/- 0.7, respectively, p0.01). Thirty-two patients with AMI were classified into two groups according to the results of thrombolysis: the success group (24 patients) and the failure group (eight patients). APC levels were higher in the failure group than in the success group (5.1+/-0.7 vs. 1.6+/-0.2, p0.01). TAT levels were also higher in the failure group than in the success group (30.8+/-9.6 vs. 8.6+/-1.7, p0.01). PAI activity levels (IU/mL) were lower in the failure group than in the success group (13.5+/-3.1 vs. 21.7+/-2.1, p0.05). There were correlations between APC and TAT levels both on admission (r=0.75, p0.0001) and at discharge (r=0.71, p0.0001). Elevated APC was thought to correlate with increased thrombin generation in patients with AMI. This study demonstrated that there was a significant relation between plasma APC level and the responsiveness to thrombolytic therapy of the infarct artery. This study may also indicate that increased thrombin generation is a cause of the resistance to thrombolytic therapy.

As a novel method for measuring bone resorption, an immunoassay system for human N-terminal osteocalcin (N-OC) was developed to determine if osteocalcin molecules are released from bone degraded by osteoclasts in vitro. The assay system employed a monoclonal antibody to the first 20 N-terminal residues of osteocalcin as the solid phase and polyclonal antibodies against these same residues as an enzyme conjugate. This assay system could detect the N-terminal portion of osteocalcin formed during the degradation of the human osteocalcin molecule by trypsin or cathepsin D. Osteoclasts were isolated from human alveolar bone and cultured on human bone slices; the osteocalcin content in the media from these cultures was measured by this N-OC assay method. The N-terminal fragment of osteocalcin was the major form of osteocalcin released during osteoclastic bone resorption along with small amounts of intact osteocalcin. Interleukin-1 (IL-1β) and interleukin-6 (IL-6), stimulators of osteoclastic bone resorption, increased the N-OC level by 1.5 fold compared with the level of N-OC in osteoclast cultures in the absence of stimulators. In contrast, treatment of the cultures with calcitonin or an inhibitor of cathepsin D (E-64), both of which inhibit osteoclastic bone resorption, decreased the amount of N-OC in the culture supernatants. These results suggest that the N-terminal fragment of osteocalcin is released during osteoclastic bone resorption and that its level may serve as an index of bone resorption in vitro.

A monoclonal antibody (10B) against the N-terminal sequence of human osteocalcin was selected to characterize its epitope and species specificity. The cross-reactivity of 10B with human and rat osteocalcin demonstrated that the reactivities of 10B with both human and rat osteocalcins were very similar. The pin technology method was used to determine the epitope and clearly demonstrated that the epitope recognized by 10B was localized to residues 12–16, the sequence of which is identical in rat and human osteocalcin molecules. This monoclonal antibody was found to be useful for designing region-specific sandwich immunoassay systems for human and rat N-terminal osteocalcin (N-OC) molecules using rabbit anti-hN20 and anti-rN20 polyclonal antibodies, respectively. The osteocalcin levels in serum determined by this N-OC method were stable during prolonged storage at 25°C and the method could be usefully applied in the development of immunoassay systems for many osteocalcin molecules from many other species.

Pulmonary surfactant protein A (SP-A) is known to be a major phospholipid-associated glycoprotein in pulmonary surfactant, which is specific to the lung. In this study, the SP-A concentrations in sera of patients with various lung diseases were determined using an enzyme-linked immunosorbent assay. Patients with idiopathic pulmonary fibrosis (IPF) and pulmonary alveolar proteinosis (PAP) exhibited prominently high concentrations of serum SP-A compared to those of other lung diseases and healthy volunteers, although there were significant increases in serum SP-A concentrations in patients with pulmonary tuberculosis, chronic pulmonary emphysema, diffuse panbronchiolitis and bacterial pneumonia compared to those of healthy volunteers. Successive measurement in 2 patients with IPF showed that serum SP-A levels reflect the disease activity of IPF. In patients with IPF, serum SP-A concentrations were significantly correlated with those of serum lactate dehydrogenase, whereas there were no significant correlations of serum SP-A concentrations with erythrocyte sedimentation rate, arterial oxygen saturation, vital capacity and carbon monoxide diffusing capacity. Determination of serum SP-A will contribute to diagnosing IPF and PAP, and may reflect the disease activity of IPF.