Combined oral intake of short and long fructans alters the gut microbiota in food allergy model mice and contributes to food allergy prevention.

BACKGROUND: It has become clear that the intestinal microbiota plays a role in food allergies. The objective of this study was to assess the food allergy-preventive effects of combined intake of a short fructan (1-kestose [Kes]) and a long fructan (inulin ([Inu]) in an ovalbumin (OVA)-induced food allergy mouse model. RESULTS: Oral administration of fructans lowered the allergenic symptom score and alleviated the decreases in rectal temperature and total IgA levels and increases in OVA-specific IgE and IgA levels induced by high-dose OVA challenge, and in particular, combined intake of Kes and Inu significantly suppressed the changes in all these parameters. The expression of the pro-inflammatory cytokine IL-4, which was increased in the allergy model group, was significantly suppressed by fructan administration, and the expression of the anti-inflammatory cytokine IL-10 was significantly increased upon Kes administration. 16 S rRNA amplicon sequencing of the gut microbiota and beta diversity analysis revealed that fructan administration may induce gut microbiota resistance to food allergy sensitization, rather than returning the gut microbiota to a non-sensitized state. The relative abundances of the genera Parabacteroides B 862,066 and Alloprevotella, which were significantly reduced by food allergy sensitization, were restored by fructan administration. In Parabacteroides, the relative abundances of Parabacteroides distasonis, Parabacteroides goldsteinii, and their fructan-degrading glycoside hydrolase family 32 gene copy numbers were increased upon Kes or Inu administration. The concentrations of short-chain fatty acids (acetate and propionate) and lactate were increased by fructan administration, especially significantly in the Kes + Inu, Kes, and Inu-fed (Inu, Kes + Inu) groups. CONCLUSION: Combined intake of Kes and Inu suppressed allergy scores more effectively than single intake, suggesting that Kes and Inu have different allergy-preventive mechanisms. This indicates that the combined intake of these short and long fructans may have an allergy-preventive benefit.

Clinical and genetic features of cystic fibrosis in Japan.

Cystic fibrosis (CF) is an autosomal recessive disease caused by pathogenic variants in CF transmembrane conductance regulator (CFTR). While CF is the most common hereditary disease in Caucasians, it is rare in East Asia. In the present study, we have examined clinical features and the spectrum of CFTR variants of CF patients in Japan. Clinical data of 132 CF patients were obtained from the national epidemiological survey since 1994 and CF registry. From 2007 to 2022, 46 patients with definite CF were analyzed for CFTR variants. All exons, their boundaries, and part of promoter region of CFTR were sequenced and the presence of large deletion and duplications were examined by multiplex ligation-dependent probe amplification. CF patients in Japan were found to have chronic sinopulmonary disease (85.6%), exocrine pancreatic insufficiency (66.7%), meconium ileus (35.6%), electrolyte imbalance (21.2%), CF-associated liver disease (14.4%), and CF-related diabetes (6.1%). The median survival age was 25.0 years. The mean BMI percentile was 30.3%ile in definite CF patients aged < 18 years whose CFTR genotypes were known. In 70 CF alleles of East Asia/Japan origin, CFTR-dele16-17a-17b was detected in 24 alleles, the other variants were novel or very rare, and no pathogenic variants were detected in 8 alleles. In 22 CF alleles of Europe origin, F508del was detected in 11 alleles. In summary, clinical phenotype of Japanese CF patients is similar to European patients, but the prognosis is worse. The spectrum of CFTR variants in Japanese CF alleles is entirely different from that in European CF alleles.

[Cystic fibrosis with focal biliary cirrhosis and portal hypertension in Japan: a case report].

A 9-year-old Japanese girl was found to have persistently elevated hepatic enzymes, chronic bronchitis, chronic sinusitis, and poor weight gain beginning at 5 months of age. Chest computed tomography (CT) revealed diffuse bronchial wall thickening and peripheral bronchiectasis. Abdominal CT showed pancreatic atrophy, liver cirrhosis, a dilated splenic vein, and splenomegaly. Her sweat chloride concentration was 117mmol/l (normal, <60mmol/l). CFTR gene analysis revealed the presence of the Y517H variant on one allele and the 1540del10 variant one the other allele. These findings established a definitive diagnosis of cystic fibrosis (CF). While CF is the most common autosomal recessive genetic disorder among Europeans, it is quite rare in Southeast Asia including Japan. It is important that CF be considered in the work-up of children with chronic hepatic and respiratory disorders even if it is uncommon among children of a similar background.

Functional characteristics of L1156F-CFTR associated with alcoholic chronic pancreatitis in Japanese.

Although cystic fibrosis is rare in Japanese, measurement of sweat Cl(-) has suggested mild dysfunction of cystic fibrosis transmembrane conductance regulator (CFTR) in some patients with chronic pancreatitis. In the present study, we have investigated the association of CFTR variants and chronic pancreatitis in Japanese and the functional characteristics of a Japanese- and pancreatitis-specific CFTR variant, L1156F. Seventy patients with alcoholic chronic pancreatitis, 18 patients with idiopathic chronic pancreatitis, and 180 normal subjects participated. All exons and their boundaries and promoter region of the CFTR gene were sequenced. Human embryonic kidney-293 cells were transfected with three CFTR variants (M470V, L1156F, and M470V+L1156F), and the protein expression was examined. Xenopus laevis oocytes were injected with the CFTR variants, and bicarbonate (HCO3 (-)) transport activity was examined. CFPAC-1 cells were transfected with the CFTR variants and Cl(-)/HCO3 (-) exchange activity was examined. Six variants (E217G, I556V, M470V, L1156F, Q1352H, and R1453W) were identified in the coding region of the CFTR gene. Cystic fibrosis-causing mutations were not found. The allele frequencies of L1156F and Q1352H in alcoholic chronic pancreatitis (5.0 and 7.9%) were significantly (P < 0.01) higher than those in normal subjects (0.6 and 1.9%). L1156F was linked with a worldwide CFTR variant, M470V. Combination of M470V and L1156F significantly reduced CFTR expression to ∼60%, impaired CFTR-mediated HCO3 (-)/Cl(-) transport activity to 50-60%, and impaired CFTR-coupled Cl(-)/HCO3 (-) exchange activity to 20-30%. The data suggest that the Japanese-specific CFTR variant L1156F causes mild dysfunction of CFTR and increases the risk of alcoholic chronic pancreatitis in Japanese.

CFTR polymorphisms of healthy individuals in two Chinese cities--Changchun and Nanjing.

BACKGROUND AND AIM: Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a chloride channel, cause cystic fibrosis. In order to investigate the polymorphic backgrounds of CFTR genes of healthy populations in different Chinese cities (Changchun and Nanjing), we analyzed 119 blood samples (Changchun 64, Nanjing 55) of randomly selected healthy individuals for poly T, TG-repeats and M470V polymorphisms. We analyzed the differences of CFTR polymorphic distributions between the two Chinese cities from the south and the north. Methods Genomic DNA was extracted from whole blood. DNA fragments of CFTR gene were amplified by polymerase chain reaction (PCR). Poly-T and TG repeats were directly sequenced by auto sequencer (ABI 310). M470V was detected by a HphI restriction enzyme. RESULTS: The T7 allele was the most common haplotype in Changchun (0.938) and Nanjing (0.927) populations. The T5 allele was present in only 7 Changchun and 3 Nanjing subjects. The TG11 and TG12 alleles were dominant haplotypes in Changchun (TG11 0.500, TG12 0.453) and Nanjing (TG11 0.345, TG12 0.609). The frequency of the V470 allele was 0.633 in Changchun, which was higher than that in Nanjing (0.500) (p < 0.05). There were three major haplotypes: T7-TG11-V470, T7-TG12-M470 and T7-TG12-V470. The T7-TG11-V470 was the most common haplotype in Changchun (0.514), while T7-TG12-M470 was the most common haplotype in Nanjing (0.500). CONCLUSION: Though Changchun and Nanjing are in the same country, their polymorphic backgrounds of CFTR gene are very different. Most of the two populations have genotypes that cause lower CFTR function.

Detection of a large heterozygous deletion and a splicing defect in the CFTR transcripts from nasal swab of a Japanese case of cystic fibrosis.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in CFTR (CF transmembrane conductance regulator). Although CF is the most common hereditary disease in Caucasians, it is rare in Asian populations. Common disease-causing mutations of CFTR in Caucasians are rarely identified in Japanese patients with CF. In the present study, CFTR transcripts from nasal swab were analyzed in a Japanese boy, in addition to conventional PCR and direct sequence of all exons, their boundaries and promoter region of the CFTR gene. The boy was diagnosed with CF by chronic respiratory infection and the elevated sweat chloride level. None of the disease-causing mutations of CFTR was detected by the conventional analysis. Cloning and sequence of the CFTR transcripts revealed a heterozygous deletion spanning exons 16, 17a and 17b. The deletion was confirmed by multiplex ligation-dependent probe amplification and the direct sequence of the junction fragment obtained from the genomic DNA by primer walking, which revealed the mutation c.2908+1085_3367+260del7201. We also identified a splicing defect: deletion/skipping of exon 1 in the CFTR transcript from the other allele. The analysis of CFTR transcripts from nasal swab is recommended in the genetic analysis of CF in Japanese.

High glucose inhibits HCO3(-) and fluid secretion in rat pancreatic ducts.

Cellular mechanisms underlying the impairment of pancreatic fluid and electrolyte secretion in diabetes were examined using interlobular ducts isolated from rat pancreas. Fluid secretion was assessed by monitoring changes in luminal volume. HCO3(-) uptake across the basolateral membrane was estimated from the recovery of intracellular pH following an acid load. Exposure to high glucose concentrations inhibited fluid secretion and reduced the rate of basolateral HCO3(-) uptake in secretin-stimulated ducts isolated from normal rats. In ducts isolated from streptozotocin-treated diabetic rats, fluid secretion and basolateral HCO3(-) uptake were also severely impaired but could be largely reversed by incubation in normal-glucose solutions. Sodium-dependent glucose cotransporter 1 (SGLT1), glucose transporter (GLUT)1, GLUT2, and GLUT8 transcripts were detected by reverse transcriptase polymerase chain reaction in isolated ducts. Raising the luminal glucose concentration in microperfused ducts caused a depolarization of the membrane potential, consistent with the presence of SGLT1 at the apical membrane. Unstimulated ducts filled with high-glucose solutions lost luminal fluid by a phlorizin-sensitive mechanism, indicating that pancreatic ducts are capable of active glucose reabsorption from the lumen via SGLT1. In ducts exposed to high glucose concentrations, continuous glucose diffusion to the lumen and active reabsorption via SGLT1 would lead to elevation of intracellular Na+ concentration and sustained depolarization of the apical membrane. These two factors would tend to inhibit the basolateral uptake and apical efflux of Cl(-) and HCO3(-) and could therefore account for the impaired fluid and electrolyte secretion that is observed in diabetes.

Glucose transport in interlobular ducts isolated from rat pancreas.

Pancreatic duct cells express Na(+)-dependent glucose transporter, SGLT1 and Na(+)-independent glucose transporters, GLUT1, GLUT2, and GLUT8. We examined transepithelial glucose transport by pancreatic duct. Interlobular ducts were isolated from rat pancreas. During overnight culture both ends of the duct segments sealed spontaneously. The lumen of the sealed duct was micropunctured and the luminal fluid was replaced by HEPES-buffered solution containing 10.0 mM or 44.4 mM glucose. The bath was perfused with HEPES-buffered solution at 37 degrees C containing 10.0 or 44.4 mM glucose. Transepithelial differences in osmolality were balanced with mannitol. Glucose transport across ductal epithelium was measured by monitoring changes in luminal volume. When the lumen was filled with 44.4 mM glucose, with either 10.0 or 44.4 mM glucose in the bath, the luminal volume decreased to 65 approximately 70% of the initial volume in 15 min. Luminally-injected phlorizin, an inhibitor of SGLT1, abolished the decrease in luminal volume. With 10.0 mM glucose in the lumen and 44.4 mM glucose in the bath, the luminal volume did not change significantly. Luminal application of phlorizin under identical condition led to an increase in luminal volume. The data suggest that both active and passive transport mechanisms of glucose are present in pancreatic ductal epithelium.

Hoxc6 is overexpressed in gastrointestinal carcinoids and interacts with JunD to regulate tumor growth.

BACKGROUND & AIMS: The molecular alterations that underlie carcinoid tumor pathogenesis remain poorly defined. The homeobox gene HOXC6 was highly up-regulated in human gastrointestinal carcinoid tumors, and we sought to define its pathogenic role. METHODS: The functional and physical properties of Hoxc6 were investigated by establishing carcinoid cells that stably overexpressed Hoxc6 or were deficient in Hoxc6. Cellular proliferation assays, luciferase reporter assays, Western blotting, immunoprecipitation, DNA affinity precipitation, and DNA microarray studies were performed. RESULTS: Expression of Hoxc6 in cultured human BON1 carcinoid cells enhanced their proliferation, and knock-down of Hoxc6 inhibited their growth. Hoxc6 activated the oncogenic activator protein-1 signaling pathway through a physical interaction with JunD. Mutations in the homeodomain of Hoxc6 blocked this interaction and inhibited proliferation of carcinoid cells. Of note, Hoxc6 induced the expression of genes that characteristically are up-regulated in carcinoid tumors, including neurotensin and connective tissue growth factor. CONCLUSIONS: A novel molecular pathway has been identified that links Hoxc6 with oncogenic signaling through the activator protein-1 pathway in carcinoid tumorigenesis.

Is genetic analysis helpful for diagnosing chronic pancreatitis in its early stage?

Diagnosis of chronic pancreatitis in its early stage is an extremely difficult task. If the genetic predispositions are identified, it may help make possible the earlier diagnosis of chronic pancreatitis or the detection of patients at risk. There are two major hypotheses about the pathogenesis of chronic pancreatitis known as the "necrosis-fibrosis" and "pancreatic stone protein" hypotheses. Recent molecular and genetic evidence suggests that both pathways contribute to the pathogenesis of chronic pancreatitis. Chronic pancreatitis may be caused by either increased proteolytic activity [the cationic trypsinogen (PRSS1) gene] or decreased protease inhibition (the pancreatic secretory trypsin inhibitor (PSTI) gene]. The impaired pancreatic duct function [cystic fibrosis transmembrane conductance regulator (CFTR) gene] may also be involved in the pathogenesis of the disease. Except for PRSS1 mutations, the known genetic risk for chronic pancreatitis is not high. The high individual variability and low incidence of chronic pancreatitis suggest that yet unidentified genetic and environmental factors are important. Further genetic analysis is necessary for understanding the pathogenesis of chronic pancreatitis, which may be helpful for the earlier diagnosis of the juvenile- or young-onset disease.

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig.

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and alpha-helical regions of PACAP-27 are required for initiating gallbladder contraction.

Hypoxic regulation of vascular endothelial growth factor through the induction of phosphatidylinositol 3-kinase/Rho/ROCK and c-Myc.

The induction of vascular endothelial growth factor (VEGF) is an essential feature of tumor angiogenesis. Hypoxia is a potent stimulator of VEGF expression, and hypoxia-inducible factor-1 (HIF-1) is considered to be critical for this induction. However, we have previously demonstrated that induction of VEGF by hypoxia was preserved when HIF-1alpha was silenced. We sought to better define the molecular basis of this HIF-1-independent regulation. In colon cancer cells, hypoxia stimulated multiple K-ras effector pathways including phosphatidylinositol 3-kinase. VEGF promoter deletion studies identified a novel promoter region between -418 and -223 bp that was responsive to hypoxia in a PI3K/Rho/ROCK-dependent manner. Electrophoretic mobility shift assays identified a fragment between -300 and -251 bp that demonstrated a unique shift only in hypoxic conditions. Inhibition of PI3K or ROCK blocked the formation of this complex. A binding site for c-Myc, a target of ROCK, was identified at -271 bp. A role for c-Myc in the hypoxic induction of VEGF was demonstrated by site-directed mutagenesis of the VEGF promoter and silencing of c-Myc by small interfering RNA. Collectively, these findings suggest an alternative mechanism for the hypoxic induction of VEGF in colon cancer that does not depend upon HIF-1alpha but instead requires the activation of PI3K/Rho/ROCK and c-Myc.

A finger sweat chloride test for the detection of a high-risk group of chronic pancreatitis.

OBJECTIVES: Mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene are associated with chronic pancreatitis in Caucasians. We developed a simple method for measuring finger sweat chloride concentration to test whether CFTR dysfunction underlies chronic pancreatitis in Japan where cystic fibrosis (CF) is rare. METHODS: We studied 25 patients with chronic (21 alcoholic and 4 idiopathic) pancreatitis and 25 healthy volunteers. Sweat chloride concentrations were measured by a finger sweat chloride test. We analyzed DNA for 20 common CFTR mutations in Europeans, 9 CF-causing mutations in Japanese, and 2 polymorphic loci, a poly-T tract and (TG) repeats, at intron 8. RESULTS: Thirteen patients (52%) had sweat chloride levels >60 mmol/L, a level consistent with CF, while only 4 (16%) healthy subjects exceeded this level. The 29 CF mutations and the 5T allele were detected in neither the patients nor controls. The (TG) 12 allele was common in both the patients (58%) and controls (48%). The (TG) 12/12 genotype was common in alcoholic pancreatitis (29%) compared with the (TG) 11/11 (10%). Patients with the (TG) 12/12 genotype had significantly higher sweat chloride concentrations than the controls. CONCLUSION: CFTR dysfunction as evidenced by a finger sweat chloride test is present in about half of Japanese patients with chronic pancreatitis, suggesting that this test may be useful for detecting the high-risk group. A higher proportion of the (TG) 12 allele may be a genetic background for elevated sweat chloride concentrations in Japanese patients.

Effect of lactase preparations in asymptomatic individuals with lactase deficiency--gastric digestion of lactose and breath hydrogen analysis.

We compared two lactase preparations derived from Aspergillus orizae (AOL) and Penicillinase multicolor (PML) for stability in the stomach and overall enzymatic activity in 10 asymptomatic subjects with lactase deficiency. The subjects were given 10,000 FCC units of either AOL or PML 30 min prior to or simultaneously with 300 ml of milk. Gastric juice was withdrawn through a nasogastric tube immediately after and every 15 min for 60 min, and breath was sampled before and every 15 min for 6 h after the milk ingestion. When lactase was given simultaneously with the milk, gastric juice lactase activity and galactose concentration were significantly higher than the control levels. When lactase preparations were given 30 min prior to the milk, neither lactase activity nor galactose was detected in the gastric juice. The pH of the gastric juice was about 6.0 after the milk ingestion. Breath hydrogen did not increase when milk was ingested simultaneously with enzymes, but did increase if enzymes were given 30 min prior to milk ingestion. There were no significant differences in lactase activity, galactose concentration in gastric juice, or breath hydrogen when AOL and PML were compared. In conclusion, with exogenous lactase, digestion of lactose begins in the stomach when pH is raised to 6.0 by the buffering action of milk. Lactase preparations are effective assessed by breath hydrogen analysis in asymptomatic individuals with lactase deficiency if the enzymes are given simultaneously with milk.

Cystic fibrosis and related diseases of the pancreas.

The discovery of the gene for cystic fibrosis (CF), the cystic fibrosis transmembrane conductance regulator (CFTR), brought about a new era in the study of this disease. Identification of the molecular target has yielded a flood of data that add to our understanding of the pathogenesis, diagnosis and treatment of CF. The CFTR protein is a cAMP-regulated Cl(-) channel with multiple functions in epithelial cells. In the exocrine pancreas the CFTR plays a key role in the apical Cl(-), HCO(3)(-), and water transport in duct cells. The severe loss of functions, caused by mutations of the CFTR gene, leads to pathological lesions of the pancreas. Over 1200 CFTR mutations and polymorphisms have been identified and their diversity may explain the high level of heterogeneity in the CF phenotype. Mutation analyses of the CFTR gene have revealed a spectrum of CFTR-related diseases that do not fit the classical CF picture but are associated with dysfunction of CFTR, such as chronic pancreatitis.