Duodenases are a small subfamily of ruminant intestinal serine proteases that have undergone a remarkable diversification in cleavage specificity.

Ruminants have a very complex digestive system adapted for the digestion of cellulose rich food. Gene duplications have been central in the process of adapting their digestive system for this complex food source. One of the new loci involved in food digestion is the lysozyme c locus where cows have ten active such genes compared to a single gene in humans and where four of the bovine copies are expressed in the abomasum, the real stomach. The second locus that has become part of the ruminant digestive system is the chymase locus. The chymase locus encodes several of the major hematopoietic granule proteases. In ruminants, genes within the chymase locus have duplicated and some of them are expressed in the duodenum and are therefore called duodenases. To obtain information on their specificities and functions we produced six recombinant proteolytically active duodenases (three from cows, two from sheep and one from pigs). Two of the sheep duodenases were found to be highly specific tryptases and one of the bovine duodenases was a highly specific asp-ase. The remaining two bovine duodenases were dual enzymes with potent tryptase and chymase activities. In contrast, the pig enzyme was a chymase with no tryptase or asp-ase activity. These results point to a remarkable flexibility in both the primary and extended specificities within a single chromosomal locus that most likely has originated from one or a few genes by several rounds of local gene duplications. Interestingly, using the consensus cleavage site for the bovine asp-ase to screen the entire bovine proteome, it revealed Mucin-5B as one of the potential targets. Using the same strategy for one of the sheep tryptases, this enzyme was found to have potential cleavage sites in two chemokine receptors, CCR3 and 7, suggesting a role for this enzyme to suppress intestinal inflammation.

Inter-individual and inter-regional variations in enteric drug metabolizing enzyme activities: Results with cryopreserved human intestinal mucosal epithelia (CHIM) from the small intestines of 14 donors.

We have previously reported successful isolation and cryopreservation of human intestinal mucosa (CHIM) with retention of viability and drug metabolizing enzyme activities. Here we report the results of the quantification of drug metabolizing enzyme activities in CHIM from different regions of the small intestines from 14 individual donors. CHIM were isolated from the duodenum, jejunum, and ileum of 10 individuals, and from 10 consecutive 12-inch segments starting from the pyloric sphincter of human small intestines from four additional individuals. P450 and non-P450 drug metabolizing enzyme activities (CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A, UGT, SULT, FMO, MAO, AO, NAT1, and NAT2) were quantified via incubation with pathway-selective substrates. Quantifiable activities were observed for all pathways except for CYP2A6. Comparison of the duodenum, jejunum, and ileum in 10 donors shows jejunum had higher activities for CYP2C9, CYP3A, UGT, SULT, MAO, and NAT1. Further definition of regional variations with CHIM from ten 12-inch segments of the proximal small intestine shows that the segments immediately after the first 12-inch segment (duodenum) had the highest activity for most of the drug metabolizing enzymes but with substantial differences among the four donors. Our overall results demonstrate that there are substantial individual differences in drug metabolizing enzymes and that jejunum, especially the regions immediately after the duodenum, had the highest drug metabolizing enzyme activities.

The T2 Toxin Produced by Fusarium spp. Impacts Porcine Duodenal Nitric Oxide Synthase (nNOS)-Positive Nervous Structures-The Preliminary Study.

T2 toxin synthetized by Fusarium spp. negatively affects various internal organs and systems, including the digestive tract and the immune, endocrine, and nervous systems. However, knowledge about the effects of T2 on the enteric nervous system (ENS) is still incomplete. Therefore, during the present experiment, the influence of T2 toxin with a dose of 12 µg/kg body weight (b.w.)/per day on the number of enteric nervous structures immunoreactive to neuronal isoform nitric oxide synthase (nNOS-used here as a marker of nitrergic neurons) in the porcine duodenum was studied using the double immunofluorescence method. Under physiological conditions, nNOS-positive neurons amounted to 38.28 ± 1.147%, 38.39 ± 1.244%, and 35.34 ± 1.151 of all enteric neurons in the myenteric (MP), outer submucous (OSP), and inner submucous (ISP) plexuses, respectively. After administration of T2 toxin, an increase in the number of these neurons was observed in all types of the enteric plexuses and nNOS-positive cells reached 46.20 ± 1.453% in the MP, 45.39 ± 0.488% in the OSP, and 44.07 ± 0.308% in the ISP. However, in the present study, the influence of T2 toxin on the intramucosal and intramuscular nNOS-positive nerves was not observed. The results obtained in the present study indicate that even low doses of T2 toxin are not neutral for living organisms because they may change the neurochemical characterization of the enteric neurons.

The expression levels of CHI3L1 and IL15Rα correlate with TGM2 in duodenum biopsies of patients with celiac disease.

OBJECTIVE AND DESIGN: Celiac disease (CD) is an intestinal inflammatory disorder of the small intestine. Gliadins are a component of gluten and there are three main types (α, γ, and ω). Recent studies indicate that gliadin peptides are able to activate an innate immune response. IL15 is a major mediator of the innate immune response and is involved in the early alteration of CD mucosa. The chitinase molecules are highly expressed by the innate immune cells during the inflammatory processes. MATERIAL OR SUBJECTS: We analyzed several microarray datasets of PBMCs and duodenum biopsies of CD patients and healthy control subjects (HCs). We verified the modulation CHI3L1 in CD patients and correlated the expression levels to the IL15, IL15Rα, TGM2, IFNγ, and IFNGR1/2. Duodenal biopsy samples belonged to nine active and nine treated children patients (long-term effects of gliadin), and 17 adult CD patients and 10 adults HCs. We also selected 169 samples of PBMCs from 127 CD patients on adherence to a gluten-free diet (GFD) for at least 2 years and 44 HCs. RESULTS: Our analysis showed that CHI3L1 and IL15Rα were significantly upregulated in adult and children's celiac duodenum biopsies. In addition, the two genes were correlated significantly both in children than in adults CD duodenum biopsies. No significant modulation was observed in PBMCs of adult CD patients compared to the HCs. The correlation analysis of the expression levels of CHI3L1 and IL15Rα compared to TGM showed significant values both in adults and in children duodenal biopsies. Furthermore, the IFNγ expression levels were positively correlated with CHI3L1 and IL15Rα. Receiver operating characteristic (ROC) analysis confirmed the diagnostic ability of CHI3L1 and IL15Rα to discriminate CD from HCs. CONCLUSION: Our data suggest a role for CHI3L1 underlying the pathophysiology of CD and represent a starting point aiming to inspire new investigation that proves the possible use of CHI3L1 as a diagnostic factor and therapeutic target.

Sucrase-Isomaltase Deficiency as a Potential Masquerader in Irritable Bowel Syndrome.

BACKGROUND: Patients with irritable bowel syndrome (IBS) frequently have meal-related symptoms and can recognize specific trigger foods. Lactose intolerance is a well-established carbohydrate malabsorption syndrome that causes symptoms similar to IBS such as bloating, abdominal pain, and diarrhea. However, the prevalence of sucrase-isomaltase deficiency (SID) in this population is poorly defined. SID is a condition in which sucrase-isomaltase, an enzyme produced by brush border of small intestine to metabolize sucrose, is deficient. Just like lactase deficiency, SID causes symptoms of maldigestion syndromes including abdominal pain, bloating, gas, and diarrhea. In this study, we aim to determine the prevalence of SID in patients with presumed IBS-D/M and characterize its clinical presentation. METHODS: Patients with a presumed diagnosis of IBS-D/M based on symptoms of abdominal pain, diarrhea, and/or bloating who underwent esophagogastroduodenoscopy with duodenal biopsies and testing for disaccharidase deficiency were included. Patients with a history of inflammatory bowel disease, gastrointestinal malignancy, or celiac disease were excluded. Odds ratio was calculated for abdominal pain, diarrhea, and bloating in patients with versus without SID. RESULTS: A total of 31 patients with clinical suspicion for IBS-D/M were included with a median age of 46 years (IQR 30.5-60) and with 61% females. SID was present in 35% of patients. Among patients with SID, 63.6% had diarrhea, 45.4% had abdominal pain, and 36.4% had bloating. Patients with SID were less likely than controls to have abdominal pain (OR 0.16, 95% CI 0.03-0.81, p = 0.04) although no difference in diarrhea or bloating was found. Only two patients with SID underwent sucrose breath testing of which only one had a positive result. However, this patient also had a positive glucose breath test and may have had small intestinal bacterial overgrowth as a confounder. CONCLUSION: SID was found in 35% of patients with presumed IBS-D/M and should be considered in the differential diagnosis of patients presenting with abdominal pain, diarrhea, or bloating. Further studies should better characterize the clinical features of SID and investigate the effects of dietary modification in this group of patients.

Duodenal Infusions of Starch with Casein or Glutamic Acid Influence Pancreatic and Small Intestinal Carbohydrase Activities in Cattle.

BACKGROUND: Small intestinal starch digestion in ruminants is potentially limited by inadequate production of carbohydrases. Previous research has demonstrated that small intestinal starch digestion can be improved by postruminal supply of casein or glutamic acid. However, the mechanisms by which casein and glutamic acid increase starch digestion are not well understood. OBJECTIVES: The objective of this experiment was to evaluate the effects of duodenal infusions of starch with casein or glutamic acid on postruminal carbohydrase activities in cattle. METHODS: Twenty-two steers [mean body weight (BW) = 179 ± 4.23 kg] were surgically fitted with duodenal and ileal cannulas and limit-fed a soybean hull-based diet containing small amounts of starch. Raw cornstarch (1.61 ± 0.0869 kg/d) was infused into the duodenum alone (control), or with 118 ± 7.21 g glutamic acid/d, or 428 ± 19.4 g casein/d. Treatments were infused continuously for 58 d and then steers were killed for tissue collection. Activities of pancreatic (α-amylase) and intestinal (maltase, isomaltase, glucoamylase, sucrase) carbohydrases were determined. Data were analyzed as a randomized complete block (replicate group) design using the GLM procedure of SAS to determine effects of infusion treatment. RESULTS: Duodenal casein infusion increased (P < 0.05) pancreatic α-amylase activity by 290%. Duodenal glutamic acid infusion increased (P < 0.03) duodenal maltase activity by 233%. Duodenal casein infusion increased jejunal maltase (P = 0.02) and glucoamylase (P = 0.03) activity per gram protein by 62.9% and 97.4%, respectively. Duodenal casein infusion tended to increase (P = 0.10) isomaltase activity per gram jejunum by 38.5% in the jejunum. Sucrase activity was not detected in any segment of the small intestine. CONCLUSIONS: These results suggest that small intestinal starch digestion can be improved in cattle with increased small intestinal flow of casein through increases in postruminal carbohydrase activities.

Clinical Characteristics of Disaccharidase Deficiencies Among Children Undergoing Upper Endoscopy.

OBJECTIVES: The epidemiology and clinical significance of disaccharidase deficiencies have not been thoroughly characterized. Recent work suggests at least genetic sucrase-isomaltase deficiency is more prevalent than previously believed. Because lactase deficiency (LD) is well described, the present study focuses on the clinical characteristics of children with disaccharidase deficiencies determined by esophagogastroduodenoscopy. METHODS: Endoscopic records were reviewed from patients undergoing esophagogastroduodenoscopies with biopsies assayed for disaccharidase activity performed by 13 pediatric gastroenterologists during 5 years (2010-2014). Presenting symptoms, clinical and histological diagnosis, treatment, disaccharidase results, and demographic variables were obtained from medical and endoscopic records of those with maltase and sucrase deficiency (SD). RESULTS: Among 963 patients undergoing intestinal disaccharidase testing, 73 (7.6%) had SD on biopsy (enzyme activity <25 µmol ·â€Šmin ·â€Šg). Thirty-four (34/73; 47%) had normal duodenal histology and are the focus of this report. Four patients had SD without LD. Pan-disaccharidase deficiency was observed in 24 patients when maltase and palatinase assays were obtained (n = 646), and 11 had SD + LD when just those 2 enzymes were analyzed (n = 317). Those with SD without LD were younger 4.6 ±â€Š6.1 versus 14.1 ±â€Š3.6 years and uniformly presented with diarrhea. Patients with pan-disaccharidase deficiency or SD + LD primarily reported abdominal pain (33/35; 94%), diarrhea (16/35; 46%), nausea (14/35; 40%); and poor weight gain/weight loss (10/35; 29%); constipation, flatulence, and bloating were also noted. Maltase deficiency is less common (8/963; 0.8%), presenting with similar symptoms. CONCLUSIONS: Genetic sucrase-isomaltase deficiency often occurs together with lactase or pan-disaccharide deficiency. Disaccharidase deficiency should be considered a potential cause of abdominal pain and/or diarrhea in children and adolescents.

Demographic and Clinical Correlates of Mucosal Disaccharidase Deficiencies in Children With Functional Dyspepsia.

BACKGROUND: A subset of children with functional gastrointestinal disorders (FGIDs), which includes functional dyspepsia, may have duodenal disaccharidase deficiencies. OBJECTIVES: To determine the frequency, demographics, and clinical characteristics associated with duodenal disaccharidase deficiencies in children with functional dyspepsia. METHODS: Children ages 4 to 18 years undergoing esophagogastroduodenoscopy (EGD) evaluation for dyspepsia were enrolled in either a retrospective (study 1) or prospective (study 2) evaluation. Those with histologic abnormalities were excluded. Duodenal biopsies were obtained for disaccharidase enzyme analysis. In the retrospective study, both demographic and clinical characteristics were obtained via chart review. In the prospective study, parents completed the Rome II Questionnaire on Gastrointestinal Symptoms before the EGD. RESULTS: One hundred and twenty-nine children (n = 101, study 1; n = 28, study 2) were included. Mean age was 11.2 ±â€Š3.8 (SD) years in study 1 and 10.6 ±â€Š3.2 years in study 2. Forty-eight (47.5%) of subjects in study 1 and 13 (46.4%) of subjects in study 2 had at least 1 disaccharidase deficiency identified. All of those with a disaccharidase deficiency in both studies had lactase deficiency with 8 (7.9%) and 5 (17.9%) of those in studies 1 and 2, respectively, having an additional disaccharidase deficiency. The second most common disaccharidase deficiency pattern was that of pan-disaccharidase deficiency (PDD) in both studies. In study 1 (where both race and ethnicity were captured), self-identified Hispanic (vs non-Hispanic, P < 0.05) and non-white (vs white, P < 0.01) children were more likely to have lactase deficiency. Age, sex, and type of gastrointestinal symptom were not associated with presence or absence of a disaccharidase deficiency. CONCLUSIONS: Approximately half of children with functional dyspepsia undergoing EGD were identified as having a disaccharidase deficiency (predominantly lactase deficiency). Race/ethnicity may be associated with the likelihood of identifying a disaccharidase deficiency. Other clinical characteristics were not able to distinguish those with versus without a disaccharidase deficiency.

13C-Labeled-Starch Breath Test in Congenital Sucrase-isomaltase Deficiency.

BACKGROUND AND HYPOTHESES: Human starch digestion is a multienzyme process involving 6 different enzymes: salivary and pancreatic α-amylase; sucrase and isomaltase (from sucrose-isomaltase [SI]), and maltase and glucoamylase (from maltase-glucoamylase [MGAM]). Together these enzymes cleave starch to smaller molecules ultimately resulting in the absorbable monosaccharide glucose. Approximately 80% of all mucosal maltase activity is accounted for by SI and the reminder by MGAM. Clinical studies suggest that starch may be poorly digested in those with congenital sucrase-isomaltase deficiency (CSID). Poor starch digestion occurs in individuals with CSID and can be documented using a noninvasive C-breath test (BT). METHODS: C-Labled starch was used as a test BT substrate in children with CSID. Sucrase deficiency was previously documented in study subjects by both duodenal biopsy enzyme assays and C-sucrose BT. Breath CO2 was quantitated at intervals before and after serial C-substrate loads (glucose followed 75 minutes later by starch). Variations in metabolism were normalized against C-glucose BT (coefficient of glucose absorption). Control subjects consisted of healthy family members and a group of children with functional abdominal pain with biopsy-proven sucrase sufficiency. RESULTS: Children with CSID had a significant reduction of C-starch digestion mirroring that of their duodenal sucrase and maltase activity and C-sucrase BT. CONCLUSIONS: In children with CSID, starch digestion may be impaired. In children with CSID, starch digestion correlates well with measures of sucrase activity.

Ex vivo Culture of Duodenal Biopsies from Patients with Dermatitis Herpetiformis Indicates that Transglutaminase 3 Antibody Production Occurs in the Gut.

Coeliac disease and dermatitis herpetiformis (DH) are characterized by autoantibodies targeting transglutaminase (TG)2 and TG3, respectively. Previous studies show that TG2 antibodies are produced in the gut and can be assessed in organ culture of small-intestinal biopsies from patients with coeliac disease. Thus far, no studies have investigated TG3 antibodies in organ culture of biopsies from patients with DH, or exploited the method in DH. The aim of this study was to investigate TG3 and TG2 antibody responses in serum and small-intestinal biopsies from patients with DH with active disease, and from those in remission. The majority of patients with DH were negative for both serum and organ culture medium TG2-targeting antibodies. Surprisingly, patients with active DH secreted TG3 antibodies into the culture medium despite seronegativity. In patients secreting high levels of TG3 antibodies into the culture medium, we also detected TG3-antibody-positive cells in the small-intestinal mucosa. These findings suggest that TG3 antibodies can be investigated in the organ culture system and that their secretion occurs in the small intestine, especially in active DH.

Intestinal Production of Anti-Tissue Transglutaminase 2 Antibodies in Patients with Diagnosis Other Than Celiac Disease.

It has been hypothesized that gluten-dependent production of anti-tissue-transglutaminase 2 (anti-TG2) antibodies may occur only at an intestinal level. We have investigated intestinal production of anti-TG2 antibodies in 136 patients with normal serum levels of anti-TG2 antibodies and normal duodenal mucosa. Intestinal deposits of anti-TG2 antibodies were evaluated by immunofluorescence and anti-TG2 antibodies released in organ culture supernatants measured by ELISA. Intestinal antibody libraries were obtained from 10 subjects. Immunohistochemistry for CD25⁺, CD3⁺, and TCR-γδ⁺ was assessed in subjects with positive (n = 32) and negative (n = 31) intestinal anti-TG2 antibodies. Globally 33/136 (24%) seronegative patients produced anti-TG2 autoantibodies at an intestinal level. Antibody libraries analysis confirmed the anti-TG2 antibodies mucosal production in all (n = 8) positive subjects. Lamina propria CD25⁺ cell count was significantly (p < 0.05) higher in patients with intestinal anti-TG2. Moreover, 13/32 (41%) of them showed high TCR-γδ⁺/CD3⁺ ratios. Intestinal anti-TG2 antibody production does not show absolute specificity for CD. It is seen more often in association with inflamed mucosa. Further investigations are necessary to prove the possible role of dietary gluten.

Correlation of Tissue Transglutaminase with Modified Marsh Grading in Celiac Disease: A Prospective Cohort Study.

OBJECTIVE: To find out correlation between serum anti-tissue transglutaminase immunoglobulin-A (tTGA) levels and Marsh grading on duodenal histopathology in Celiac disease (CD). METHODS: In a prospective cohort study, a total of 52 symptomatic patients between age group of 2-18 y were enroled. All enroled patients were subjected to upper GI endoscopy by an experienced endoscopist. Two biopsies each from the bulb (D1) and second part (D2) of the duodenum were taken and Marsh grading was performed by a single experienced pathologist. Serum tTGA levels were also performed to find out correlation between serum tTGA levels and Marsh grading. RESULTS: The mean age of the patients was 8.21 ± 3.45 y (Range: 2-16 y). Anemia was the most common non-gastrointestinal (GI) sign and was present in 73% of the cases. However the authors could not find out any significant association between Marsh grading and hemoglobin levels (r = 0.32, p > 0.05). Serum tTGA levels were found to be positively correlated with Marsh grading (Spearmen correlation coefficient ρ = 0.74, p 0.000). Significant differences were found in tTGA levels between different Marsh gradings (ANOVA test) (p 0.000). Receiver-operator curve (ROC) analysis cut-off value of serum tTGA for predicting villous atrophy was 178.8 (nine times of cut-off value) with sensitivity of 100% and specificity of 85.7%. CONCLUSIONS: Serum tTGA levels can be used to predict villous atrophy and biopsy may be avoided in strongly suspected cases with more than 9 times of cut-offs.

l-Glutamine supplementation promotes an improved energetic balance in Walker-256 tumor-bearing rats.

We evaluated the effects of supplementation with oral l-glutamine in Walker-256 tumor-bearing rats. A total of 32 male Wistar rats aged 54 days were randomly divided into four groups: rats without Walker-256 tumor, that is, control rats (C group); control rats supplemented with l-glutamine (CG group); Walker-256 tumor rats without l-glutamine supplementation (WT group); and WT rats supplemented with l-glutamine (WTG group). l-Glutamine was incorporated into standard food at a proportion of 2 g/100 g (2%). After 10 days of the experimental period, the jejunum and duodenum were removed and processed. Protein expression levels of key enzymes of gluconeogenesis, that is, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase, were analyzed by western blot and immunohistochemical techniques. In addition, plasma corticosterone, glucose, insulin, and urea levels were evaluated. The WTG group showed significantly increased plasma glucose and insulin levels ( p < 0.05); however, plasma corticosterone and urea remained unchanged. Moreover, the WTG group showed increased immunoreactive staining for jejunal phosphoenolpyruvate carboxykinase and increased expression of duodenal glucose-6-phosphatase. Furthermore, the WTG group presented with less intense cancer cachexia and slower tumor growth. These results could be attributed, at least partly, to increased intestinal gluconeogenesis and insulinemia, and better glycemia maintenance during fasting in Walker-256 tumor rats on a diet supplemented with l-glutamine.

Oral administration of Saccharomyces boulardii alters duodenal morphology, enzymatic activity and cytokine production response in broiler chickens.

The present study evaluated the effects of Saccharomyces boulardii on duodenal digestive enzymes, morphology and cytokine induction response in broiler chicken. A total of 200 birds were allotted into two groups (n = 100) and each group divided into five replications (n = 20). The control group was fed basal diet in addition to antibiotic (virginiamycin 20 mg/kg), and treatment group received (1 × 108  colony-forming units/kg feed) S. boulardii in addition to basal diet lasting for 72 days. The results compared to control group revealed that adenosine triphosphatase, gamma glutamyl transpeptidase, lipase and trypsin activities were higher, while, no significant improvement was observed in amylase activities in the duodenum of the treatment group. Moreover, morphological findings showed that villus height, width and number of goblet cells markedly increased. Additionally, transmission electron microscopy visualized that villus height, width and structural condensation significantly increased in the treatment group. The immunohistological observations showed increased numbers of immunoglobulin A (IgA)-positive cells in the duodenum of the treatment group. Meanwhile, cytokine production levels of tumor necrosis factor-α, interleukin (IL)-10, transforming growth factor-ß and secretory IgA markedly increased, and IL-6 statistically remained unchanged as compared to the control group. These findings illustrated that initial contact of S. boulardii to the duodenum has significant impact in improving enzymatic activity, intestinal morphology and cytokine response in broiler chicken.

Duodenal Disaccharidase Activities During and After Weaning off Parenteral Nutrition in Pediatric Intestinal Failure.

OBJECTIVES: Data on factors affecting absorptive function in children with intestinal failure (IF) are sparse. We evaluated duodenal disaccharidase activities and inflammation in relation to parenteral nutrition (PN) and intestinal resection in pediatric onset IF. METHODS: Disaccharidase (maltase, sucrase, and lactase) activities and histologic inflammation were evaluated from duodenal biopsies in 58 patients during PN (n = 23) or full enteral nutrition (n = 40) and in 43 matched controls. The first and the last postresection biopsies were analyzed separately after 4.3 (1.2-9.7) years and 6.5 (2.3-12.4) years, respectively. RESULTS: During PN, maltase and sucrase activities were 1.6-fold lower and mucosal inflammation more frequent (22% vs 3%) when compared to matched controls (P < 0.05 for both). In patients on full enteral nutrition, activities of maltase and sucrase were significantly higher than that in patients receiving PN and comparable to those of matched controls. Postresection time correlated positively (r = 0.448 and r = 0.369) and percentage length of the remaining small intestine inversely (r = -0.337 and r = -0.407) with maltase and sucrase activity in patients on full enteral nutrition (P < 0.05 for all), whereas proportional length of remaining colon correlated positively with maltase and lactase activity (r = 0.424-0.544, P < 0.05) in patients receiving PN. CONCLUSIONS: In children with IF, PN dependency associated with decreased duodenal maltase and sucrase activities and mucosal inflammation, which may disturb intestinal absorptive function. Localization and extent of intestinal resection and post-resection time correlated with duodenal disaccharidase activities.

Strain differences in intestinal toxicity of warfarin in rats.

Intestinal hemorrhage characterizes effectiveness of warfarin (WF) as rodenticide and is among adverse effects of therapy in humans. Having in mind genetic variations in the effectiveness of WF in wild rats and in the doses required for therapeutic effect, strain differences in the intestinal toxicity of oral warfarin in rats were examined in this study. High WF dose (3.5mg/l) led to mortality in Albino Oxford (AO) rats, with no lethality in Dark Agouti (DA) rats. Higher values of prothrombin time were noted at low WF dose (0.35mg/l) in the former strain. Leukocyte infiltration in intestine noted at this dose in both strains was associated with oxidative injury and more pronounced anti-oxidative response in AO rats. Suppression of mesenteric lymph node cell proliferation and IFN-γ and IL-10 production in AO rats and lack of these effects in DA rats, represent different strategies to protect vulnerable intestine from harmful immune responses.

A Novel Zak Knockout Mouse with a Defective Ribotoxic Stress Response.

Ricin activates the proinflammatory ribotoxic stress response through the mitogen activated protein 3 kinase (MAP3K) ZAK, resulting in activation of mitogen activated protein kinases (MAPKs) p38 and JNK1/2. We had a novel zak-/- mouse generated to study the role of ZAK signaling in vivo during ricin intoxication. To characterize this murine strain, we intoxicated zak-/- and zak+/+ bone marrow-derived murine macrophages with ricin, measured p38 and JNK1/2 activation by Western blot, and measured zak, c-jun, and cxcl-1 expression by qRT-PCR. To determine whether zak-/- mice differed from wild-type mice in their in vivo response to ricin, we performed oral ricin intoxication experiments with zak+/+ and zak-/- mice, using blinded histopathology scoring of duodenal tissue sections to determine differences in tissue damage. Unlike macrophages derived from zak+/+ mice, those derived from the novel zak-/- strain fail to activate p38 and JNK1/2 and have decreased c-jun and cxcl-1 expression following ricin intoxication. Furthermore, compared with zak+/+ mice, zak-/- mice have decreased duodenal damage following in vivo ricin challenge. zak-/- mice demonstrate a distinct ribotoxic stress-associated phenotype in response to ricin and therefore provide a new animal model for in vivo studies of ZAK signaling.

Lysosomal Acid Lipase Hydrolyzes Retinyl Ester and Affects Retinoid Turnover.

Lysosomal acid lipase (LAL) is essential for the clearance of endocytosed cholesteryl ester and triglyceride-rich chylomicron remnants. Humans and mice with defective or absent LAL activity accumulate large amounts of cholesteryl esters and triglycerides in multiple tissues. Although chylomicrons also contain retinyl esters (REs), a role of LAL in the clearance of endocytosed REs has not been reported. In this study, we found that murine LAL exhibits RE hydrolase activity. Pharmacological inhibition of LAL in the human hepatocyte cell line HepG2, incubated with chylomicrons, led to increased accumulation of REs in endosomal/lysosomal fractions. Furthermore, pharmacological inhibition or genetic ablation of LAL in murine liver largely reduced in vitro acid RE hydrolase activity. Interestingly, LAL-deficient mice exhibited increased RE content in the duodenum and jejunum but decreased RE content in the liver. Furthermore, LAL-deficient mice challenged with RE gavage exhibited largely reduced post-prandial circulating RE content, indicating that LAL is required for efficient nutritional vitamin A availability. In summary, our results indicate that LAL is the major acid RE hydrolase and required for functional retinoid homeostasis.