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Pacing the gut in motility disorders.

Similar to cardiac pacing, gastrointestinal (GI) pacing is an attractive idea and may become a promising therapy, as the GI organs, like the heart, have their own natural pacemakers. Over the past 10 years, electrical stimulation of the gut has received increasing attention among researchers and clinicians. Several clinical studies have shown that gastric electrical stimulation (GES) with short pulses is able to reduce nausea and vomiting in patients with gastroparesis and that GES with long pulses is able to pace the intrinsic gastric slow waves and thus normalize gastric dysrhythmia. However, possible placebo effects cannot be ruled out, although recent animal studies have revealed various peripheral and central mechanisms involved with GES. Electrical stimulation of the small intestine, colon, or anal sphincter also has been reported for the treatment of dumping syndrome, constipation, and fecal incontinency. Similarly, there is a lack of placebo-controlled studies. In our opinion, pacing of the gut has great potential for the treatment of various GI motor disorders. However, none of the commercially available devices is designed for pacing the gut. The lack of well-suited devices and the invasive nature of gut pacing slow down the progress and clinical applications of gut pacing.

Journal Article↗

Aztreonam: the first monobactam.

A novel screening procedure led to isolation of the structurally unique, bacterially produced, monocyclic beta-lactam antibiotics early in 1979. These naturally occurring "monobactams" were not clinically useful as antibiotics because of their poor antibacterial properties. They were, however, found to interact with certain penicillin-binding proteins of bacteria and thus to interfere with the biosynthesis of bacterial cell walls. The focus of monobactam development then turned toward increasing the binding activity of the beta-lactam ring of the molecule. Aztreonam was the first compound to emerge that fulfilled the objectives of the program. It is relatively inactive against gram-positive and anaerobic bacteria but is extremely effective against aerobic gram-negative bacteria, even in low concentrations. In addition, it is highly resistant to enzymatic hydrolysis by beta-lactamases and demonstrates a high degree of stability against plasmid-mediated gram-negative lactamases. With the chromosomally mediated beta-lactamases, on the other hand, aztreonam can act either as an inhibitor or as a poor substrate. It is unique in that it does not induce production of chromosomally mediated enzymes. Interference with normal gut flora by the use of broad-spectrum antibiotics can result in decreased defense capacity and can lead to intestinal colonization by resistant pathogenic organisms. Therapy directed specifically against the invading pathogen is thus preferred. Such directed therapy is provided by aztreonam. Its narrow spectrum can, if necessary, be broadened by combining it with other antibiotics while continuing to maintain an alternative to the more generalized "shotgun" therapy with its attendant side effects such as disturbances of the natural gut flora, diarrhea, and the emergence of resistant bacteria.

Animals↗

Modulation of the hyperdynamic circulation of cirrhotic rats by nitric oxide inhibition.

The effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of nitric oxide (NO) biosynthesis on the splanchnic and systemic circulation, were investigated in rats with cirrhosis induced by carbon tetrachloride. Portal hypertension in these rats was accompanied by decreased arterial blood pressure and peripheral vascular resistance as well as by splanchnic vasodilation with increased portal venous inflow and decreased splanchnic resistance. Intravenous bolus administration of L-NMMA (25 mg/kg) significantly increased systemic blood pressure and decreased cardiac output. L-NMMA also significantly increased systemic and splanchnic vascular resistance; whereas blood flow to the stomach, small intestine, colon, pancreas, mesentery, spleen, and kidney was decreased significantly. L-NMMA did not alter the portal pressure or portosystemic shunting in these cirrhotic rats, yet portal vascular resistance increased, suggesting effects on the intrahepatic and collateral circulation. Pretreatment with L-arginine (300 mg/kg) prevented the hemodynamic changes induced by L-NMMA. These findings support the concept that local excess formation of NO contributes to changes in splanchnic circulation associated with portal hypertension in cirrhosis.

Animals↗

Independent release of peptide YY (PYY) into the circulation and ileal lumen of the awake dog.

Peptide YY (PYY) is a recently discovered polypeptide found in the endocrine cells of the distal small intestine, colon, and rectum. This study was designed to evaluate the postprandial release of PYY into the peripheral blood and the ileal lumen in response to different ingested nutrients. Six adult conditioned mongrel dogs had 25-cm distal ileal Thiry-Vella segments surgically constructed. After a 2-week postoperative recovery period the animals were fed meals consisting of protein (2 g/kg), fat (5 g/kg), or glucose (1.5 g/kg). Circulating and ileal intraluminal PYY concentrations were measured by a sensitive radioimmunoassay developed in our laboratory. Significant increases in circulating PYY levels were seen only after the fat meal in dogs. Ileal intraluminal PYY concentrations were significantly greater than those concentrations seen in the peripheral blood. A significant rise in ileal intraluminal PYY was seen only after the glucose meal without concomitant changes in circulating PYY. These findings document independent release mechanisms of PYY into the circulation and ileal lumen of the awake dog. We believe that the release of PYY into the canine circulation and into the ileal lumen after feeding may be dependent on the type of nutrient ingested and may serve to regulate the diverse physiologic roles of PYY in the gastrointestinal tract.

Animals↗

Tissue-differential expression of two distinct genes for human IMP dehydrogenase (E.C.1.1.1.205).

Human IMP dehydrogenase (E.C. 1.1.1.205) is recently regarded as a potent targeting enzyme for immunosuppressive drugs. Tissue differential expressions of human type I and type II IMP dehydrogenase were investigated in sixteen human adult organs (heart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas, spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocytes) and five human fetal organs (heart, brain, lung, liver, kidney) using Northern blot analysis. In all tissues examined in this study, the sizes of mRNAs of each isoform were identical, respectively. The 2.3 kb type II mRNA was shown predominantly, and the 3.5 kb type I mRNA level was lower than type II in most human tissues examined. In contrast, type I IMPDH gene expressed higher than type II in peripheral blood leukocytes, uniquely. We also demonstrated that both type I and type II IMPDH genes are widely distributed among various species by Southern blot analysis. Interestingly, type I IMPDH gene may have multiple gene families in primates.

Adult↗

Human monoclonal antibodies directed against ovarian carcinoma.

In order to obtain human monoclonal antibodies for immunodetection or treatment of ovarian carcinoma, we generated hybridomas by fusing peripheral blood lymphocytes of patients with ovarian carcinoma and the mouse myeloma cell line X63.Ag8.653. The patients were immunized prior to collection of peripheral blood lymphocytes with autologous tumor cells admixed with New Castle Disease Virus. Immunocytologic studies of hybridoma supernatant with tumor cells fixed with methanol and air-dried tumor cells indicated that all 14 antibodies reactive with tumor cells were directed against cytoplasmic or nuclear antigens. One hybridoma designated as 1B3 was very stable and secreted a specific IgM antibody. This cell line expanded in nude mice and the monoclonal antibody 1B3 was effectively purified from ascites or supernatant fluid. In experiments with tissue sections partly derived from the patient whose peripheral blood lymphocytes were used for fusion, biotinylated 1B3 recognized ovarian tumor cells. There was no significant cross-reaction against normal tissue from stomach, small intestine, colon, lung, kidney, endometrium, placenta, or lymph node. Mammary carcinoma preparations were also stained by 1B3 while normal breast tissue was negative.

Animals↗

Metabolism of i.v. administered 45 kDa epidermal growth factor in the rat.

The 45 kDa epidermal growth factor (EGF-(45 kDa)) has been purified from rat urine. We have investigated the distribution and the processing of i.v. injected 125I-labeled EGF-(45 kDa) in the rat. 2.5 min after the i.v. injection only 12% of the label remained in the blood. Most of the label was found in the liver (54%), in the kidneys (7%) and in the skin (4%). The submandibular glands, stomach, small intestine, colon, spleen and lungs contained 1% or less of the radioactivity. Some of the 125I-EGF-(45 kDa) was processed to 125I-EGF-(6 kDa) immunoreactivity in the liver and in the kidneys. The kidneys excreted 125I-EGF-(45 kDa) in the urine, but we were not able to demonstrate 125I-EGF-(6 kDa) in urine. In conclusion, this study shows that homologous EGF-(45 kDa) is cleared from the circulation of rats within a few minutes, mainly by the liver and the kidneys. In vivo both the liver and the kidneys are able to process some of the EGF-(45 kDa) to EGF-(6 kDa) immunoreactivity.

Animals↗

The human P2x1 receptor: molecular cloning, tissue distribution, and localization to chromosome 17.

A human urinary bladder cDNA library was screened with a rat P2x purinoceptor probe. A full-length cDNA was isolated, and like its rat homologue, the deduced protein consists of 399 amino acids (M(r) = 44980 Da), contains two hydrophobic, putative transmembrane, domains flanking a large presumed extracellular loop, and represents the P2x1 subtype of this multigene family (approximately 89% amino acid sequence identity to rat P2x1, but only 40-50% identity to rat P2x2-4). Expression of the P2x1 gene in human bladder was confirmed by Northern analysis, which demonstrated a major transcript of approximately 2.9 kb. Transcripts were also found in a variety of other tissues including adult peripheral leukocytes, pancreas, spleen, prostate, small intestine, colon, testis, and ovary, and in fetal liver. The gene encoding the human P2x1 receptor was localized to chromosome 17 by Southern analysis of DNAs isolated from a panel of somatic cell hybrids. The results support a role for P2x purinoceptors in the regulation of human bladder function.

Adult↗

Gastric inhibitory polypeptide (GIP) binding sites in rat brain.

Synthetic porcine gastric inhibitory polypeptide (GIP) was iodinated and purified by reverse-phase HPLC and used to localize saturable [125I]GIP binding sites by radioligand binding to frozen sections of rat brain followed by autoradiography. Saturable [125I]GIP binding sites were expressed in several brain regions including cerebral cortex, anterior olfactory nucleus, lateral septal nucleus, subiculum, inferior colliculus, and inferior olive. Saturable [125I]GIP binding was time dependent, reversible, high affinity, and specific for GIP. Scatchard analysis of equilibrium binding resulted in an estimated dissociation constant (Kd) of 16-62 pM for the rat brain [125I]GIP binding sites. Peptides with amino acid sequences similar to GIP such as secretin, vasoactive intestinal polypeptide (VIP), glucagon, and peptide histidine isoleucine (PHI) only partially inhibited saturable [125I]GIP binding at concentrations approximately 10,000-100,000-fold higher than GIP. Saturable [125I]GIP binding was not observed in other rat organs surveyed such as spinal cord, pituitary, stomach, small intestine, colon, pancreas, liver, heart, or skeletal muscle. We conclude that a saturable [125I]GIP binding site with the pharmacological properties of an authentic GIP receptor is expressed in certain regions of the rat brain.

Animals↗

Chromogranin A(210-301) is the major form of pancreastatin-like material in human gut extracts and endocrine tumors.

A radioimmunoassay of human pancreastatin was developed using a rabbit antiserum that selectively recognized the C-terminal amidated end of the peptide, and it was used for the identification of the molecular forms of pancreastatin in human gut (stomach, duodenum, small intestine, colon) and endocrine tumor extracts (liver metastasis of a gastrinoma and a medullary carcinoma of thyroid, one nonsecreting pancreatic tumor, one recurrence of a gut carcinoid, one vipoma and one insulinoma). In all gut extracts, a gel filtration chromatography revealed the presence of three peaks of pancreastatin-like immunoreactivity. The predominant form eluted with an apparent molecular weight higher than that of pancreastatin. This form was also predominant in the endocrine tumors analyzed, except in the insulinoma, where a lower molecular weight form predominated. The high molecular form was further purified from a liver metastasis of a gastrinoma. The pancreastatin-like immunoreactivity eluted in all the chromatographical systems (reverse-phase, ion exchange) as a single peak that was finally purified to homogeneity and sequenced. The sequence of the first 29 N-terminal amino acids was obtained unambiguously and corresponded to the sequence 210-238 of chromogranin A. Considering the selectivity of the assay used for peptide identification, this major form was identified as the fragment 210-301 of chromogranin A. It is likely that the predominant form of pancreastatin in human gut extracts and noninsular tumors is a 92 amino acid peptide.

Amino Acid Sequence↗

The HLA-G gene is expressed at a low mRNA level in different human cells and tissues.

Recently, HLA-G transgenic mice were shown to exhibit transgene transcription in several extraembryonic tissues. To determine whether HLA-G mRNAs are also expressed in other human tissues, we have undertaken Northern blot and RT-PCR assays using HLA-G locus-specific probe and primers. These studies demonstrate that the HLA-G gene is transcribed in a variety of cells and adult tissues obtained from different individuals (peripheral blood leukocytes, placenta, skin, spleen, thymus, prostate, testicle, ovary, small intestine, colon, heart, brain, lung, liver, and kidney), as well as in fetal tissues (heart, lung, liver, and kidney). The HLA-G mRNA level observed in most tissues is orders of magnitude lower than the level of classic class I genes in the same tissues. RT-PCR studies have demonstrated that alternative splicing of the HLA-G primary transcript is different from tissue to tissue and could be regulated in a tissue-specific fashion. Sequencing of keratinocyte transcripts has confirmed previous observations: (a) three different alternative splicing transcripts are produced (a full-length transcript, an mRNA lacking exon 3, and a transcript devoid of exon 3 and 4) and (b) HLA-G polymorphism is limited in the coding regions. In view of this wide HLA-G tissue distribution, a new hypothesis dealing with possible HLA-G function is proposed.

Alternative Splicing↗

Effects of 8-week exposure of the B6C3F1 mouse to dietary deoxynivalenol (vomitoxin) and zearalenone.

Weanling female B6C3F1 mice were fed semi-purified diets containing 0, 0.5, 2.0, 5.0, 10.0 or 25.0 ppm (mg/kg) deoxynivalenol (DON) over 8 wk and were assessed for effects on feed intake, body-weight gain, terminal organ weights, histopathology, haematology and serum immunoglobulin levels. To determine whether DON effects were potentiated by the oestrogen zearalenone (ZEA), a mycotoxin frequently found to occur with DON in cereals, two additional groups of mice were fed diets containing either 10 ppm ZEA or 10 ppm ZEA plus 5 ppm DON. The rate of body-weight gain was significantly reduced (P less than 0.01) for all mice consuming feed containing 2.0 ppm or more of DON, whereas only the mice ingesting the diet containing 25 ppm DON showed a significantly decreased (P less than 0.01) rate of feed consumption. Gross and histopathological evaluation of thymus, spleen, liver, kidney, uterus, small intestine, colon, heart, brain, lungs and bone marrow from control and all mycotoxin-exposed mice revealed that these tissues were normal in appearance and in histological architecture. DON-amended diets did however, cause dose-dependent decreases in the terminal organ weights recorded (thymus, spleen, liver, kidney and brain). In the DON-treated groups, statistically significant dose-dependent decreases in the counts of total circulating white blood cells were associated with an increase in polymorphonuclear neutrophils and a decrease in lymphocytes and monocytes. Dietary DON caused a dose-dependent decrease in serum IgM but, in contrast, a dose-dependent increase in serum IgA. In none of the above instances was 10 ppm ZEA shown to act synergistically or antagonistically with 5 ppm DON. Since dietary DON at levels as low as 2.0 ppm exerted significant effects on the growing B6C3F1 female mouse, future approaches should include studies of the mechanisms by which this mycotoxin affects nutrient utilization and modifies the normal immune response.

Animals↗

Expression of the gene encoding the tyrosine kinase-deficient human insulin receptor in transgenic mice.

Defects in the insulin receptor (IR) in diabetic patients have been given much attention. To address the role of such defects, we generated a transgenic (TG) mouse carrying the cDNA encoding a tyrosine-kinase (TK)-deficient human IR (hIR), under the control of the native promoter. The TG mouse expressed the transgene (TG) mRNA in the liver, as identified in Northern blots. Analyses of various tissues by reverse transcription-polymerase chain reaction revealed that expressions of the TG mRNA in brain, heart, kidney, lung, stomach, skeletal muscle and adipose tissue were higher than those seen with the endogenous mouse IR (mIR), but expression in small intestine, colon, spleen, testis and ovary were approximately half those seen with the endogenous mIR. In the liver, the expression of the TG was about one tenth that of the endogenous mIR. In analyses of insulin binding and IR autophosphorylation, using a human-specific anti-IR antibody, the TK-deficient hIR was synthesized in the tissues of the TG mice. Despite the expression of TK-deficient hIRs in various tissues, including the major insulin-target tissues, muscle and adipose tissues, of the TG mice, no glucose intolerance was observed as assessed by the intraperitoneal glucose tolerance test, before and after sucrose feeding for 55 weeks. Our results suggest that a higher expression of the mutated IR, especially in the liver which is another major insulin-target tissue, or additional pathogenic factors, environmental or genetic, might be required for glucose intolerance.

Animals↗

Inheritance of resistance to oedema disease in the pig: experiments with an Escherichia coli strain expressing fimbriae 107.

Inheritance of resistance to intestinal colonization with E. coli causing oedema disease is hypothesized to be under the control of one locus consisting of two alleles with susceptibility (S) dominating resistance (s). This mode of inheritance was investigated by mating pigs, resistant and susceptible to the disease, and examining the offspring. Weaned piglets were repeatedly inoculated orally with 5 x 10(5) CFU per pig per day of a streptomycin resistant strain of E. coli serotype O139:K12(B):H1:F(107) and susceptibility determined by daily semiquantitative cultural examination of rectal swabs. Using results obtained from offspring, 5 boars were retrospectively assigned the genotype ss, 1 was assigned Ss, and 2 were assigned SS. Nine sows were designated ss, 8 classified Ss and 4 SS. Ninety two pigs resulted from matings regarded as ss x ss; 89 (97%) of these were resistant to colonization and oedema disease. Of the 168 pigs from Ss x ss matings, 83 (49%) were resistant, while only 13 (9%) of 146 pigs from matings with at least one SS parent were classified resistant. The results are compatible with inheritance being controlled by one locus and with susceptibility dominating resistance to oedema disease.

Amoxicillin↗

Mutagenicity of three aromatic amines in the presence of fractions from various tissues.

The mutagenic activity of 2-aminofluorene, 4-aminobiphenyl and 3,2'-dimethylaminobiphenyl towards Salmonella typhimurium strain TA1538 was tested in the presence of post-mitochondrial fractions of liver, kidney, intestine, colon and testicle from untreated and pretreated rats. The metabolic activation capacity of these various fractions was very different. In the experimental conditions, no relationship was found between the mutagenic activity and the carcinogenicity of the three aromatic amines.

Aminobiphenyl Compounds↗

Effect of mucin on Campylobacter jejuni association and invasion on HEp-2 cells.

We report the development of an in vitro assay using HEp-2 cell monolayers in which cell association of five C. jejuni strains and E. coli K12 C600 were tested under different experimental conditions. Both total cell associated colony forming units (CFU) and the CFU protected from gentamicin killing (i.e. internalized bacteria) were determined. Pretreatment of HEp-2 cells with mucin (100 micrograms/ml) enhanced internalization of four fresh isolates of C. jejuni by 3.2 to 20.7-fold, depending on the strain, but not internalization of C. jejuni K105 (a stock culture) or E. coli C600. Mucin also increased the total number of cell associated C. jejuni fresh isolates by 2 to 3.4-fold, the four strains yielding similar results. We conclude from these studies that mucin, which has been shown to be a prerequisite of intestinal colonization by C. jejuni, also promotes infectivity.

Campylobacter fetus↗

Epitope differences in toxin-coregulated pili produced by classical and El Tor Vibrio cholerae O1.

A toxin-coregulated pilus (TCP), that is important for intestinal colonization of Vibrio cholerae O1, may be produced by vibrios of both classical and EI Tor biotypes. By comparing TCP produced by various strains of the two biotypes in immunoblotting and enzyme-linked immunosorbent assays (ELISA) using monoclonal antibodies (mAbs) and polyclonal antisera against TCP from classical vibrios, we have found biotype-related epitope differences in TCP. Our results indicate that TCP of classical strains has an epitope in the TcpA-subunit (20.5 kDa) that is missing in EI Tor TcpA, and an additional epitope that is more strongly expressed in classical TcpA. A polyclonal antiserum reacted strongly with TcpA from strains of both biotypes in immunoblotting suggesting both the presence of major shared TcpA epitopes and that the low or absent reactivity of EI Tor TcpA with the mAbs was not due to lower production of TcpA by EI Tor strains. Whereas all the TcpA-positive classical strains inhibited the binding of polyclonal antiserum and mAbs to solid phase-bound TCP-positive bacteria in an inhibition ELISA, practically no inhibition was observed with TcpA-positive EI Tor strains. This together with findings in immunoelectron microscopy studies that TCP 'bundles' were only detected on classical strains, suggest that TCP is poorly expressed on EI Tor vibrios.

Blotting, Western↗

High levels of anti-Campylobacter-flagellin IgA antibodies in breast milk.

Several studies have shown the beneficial role of immunity against enteric Campylobacter sp. Circulating antibodies against flagellin and possibly maternal antibodies seem to protect against intestinal colonization by Campylobacter. Milk from women in Central Africa (n = 120) and in France (n = 31) were investigated for the presence of anti-flagellin antibodies. Results showed that most French and all African milk possessed anti-flagellin IgA but not IgG. The titres of anti-flagellin IgA were elevated in colostrum and decreased in later milk. Specific activities were constant during lactation, suggesting constant antigenic stimulation. The fact that IgA but not IgG are elicited against flagellin could be due to an infection limited to the mucosal surface.

Adult↗