Interaction Between the a3 Region of Factor VIII and the TIL'E' Domains of the von Willebrand Factor.

The von Willebrand factor (VWF) and coagulation factor VIII (FVIII) are intricately involved in hemostasis. A tight, noncovalent complex between VWF and FVIII prolongs the half-life of FVIII in plasma, and failure to form this complex leads to rapid clearance of FVIII and bleeding diatheses such as hemophilia A and von Willebrand disease (VWD) type 2N. High-resolution insight into the complex between VWF and FVIII has so far been strikingly lacking. This is particularly the case for the flexible a3 region of FVIII, which is imperative for high-affinity binding. Here, a structural and biophysical characterization of the interaction between VWF and FVIII is presented with focus on two of the domains that have been proven pivotal for mediating the interaction, namely the a3 region of FVIII and the TIL'E' domains of VWF. Binding between the FVIII a3 region and VWF TIL'E' was here observed using NMR spectroscopy, where chemical shift changes were localized to two ß-sheet regions on the edge of TIL'E' upon FVIII a3 region binding. Isothermal titration calorimetry and NMR spectroscopy were used to characterize the interaction between FVIII and TIL'E' as well as mutants of TIL'E', which further highlights the importance of the ß-sheet region of TIL'E' for high-affinity binding. Overall, the results presented provide new insight into the role the FVIII a3 region plays for complex formation between VWF and FVIII and the ß-sheet region of TIL'E' is shown to be important for FVIII binding. Thus, the results pave the way for further high-resolution insights into this imperative complex.

Trefoil factor 3 in perinatal pancreas is increased by gestational low protein diet and associated with accelerated ß-cell maturation.

The endocrine pancreas expands markedly in the first postnatal days and the insulin producing ß-cells initiate a functional maturation preceded by a morphological change of the islets of Langerhans. Trefoil factor 3 (TFF3) is a secreted peptide expressed in intestinal epithelia, where it promotes migration, but its role in the pancreas is not characterized. The aim of this study was to examine the expression and function of TFF3 in perinatal rat pancreas, ex vivo cultured fetal rat pancreas and in the rat ß-cell line INS-1E. Control or gestational low-protein diet perinatal rat pancreas was harvested at embryonic day 20 (E20), day of birth (P0) and postnatal day 2 (P2). TFF3 mRNA was upregulated 4.5-fold at P0 vs. E20 and downregulated again at P2. In protein-undernourished pups induction of TFF3 at P0 was further increased to 9.7-fold and was increased at P2. TFF3 caused tyrosine phosphorylation of EGFR in INS-1E ß-cells, and purified recombinant TFF3 increased both attachment and spreading of INS-1E ß-cells. In ex vivo cultures of collagenase digested fetal rat pancreas, a model of perinatal ß-cell maturation, TFF3 increased cellular spreading as well as insulin mRNA levels. TFF3 also increased the expression of Pref1/Dlk1 that shares similarities in expression and regulation with TFF3. These results suggest that TFF3 may promote adhesion and spreading of cells to accelerate ß-cell maturation. This study indicates a functional role for TFF3 in pancreatic ß-cell maturation in the perinatal period, which is altered by low protein diet during gestation.

Molecular design and downstream processing of turoctocog alfa (NovoEight), a B-domain truncated factor VIII molecule.

Turoctocog alfa (NovoEight) is a third-generation recombinant factor VIII (rFVIII) with a truncated B-domain that is manufactured in Chinese hamster ovary cells. No human or animal-derived materials are used in the process. The aim of this study is to describe the molecular design and purification process for turoctocog alfa. A five-step purification process is applied to turoctocog alfa: protein capture on mixed-mode resin; immunoaffinity chromatography using a unique, recombinantly produced anti-FVIII mAb; anion exchange chromatography; nanofiltration and size exclusion chromatography. This process enabled reduction of impurities such as host cell proteins (HCPs) and high molecular weight proteins (HMWPs) to a very low level. The immunoaffinity step is very important for the removal of FVIII-related degradation products. Manufacturing scale data shown in this article confirmed the robustness of the purification process and a reliable and consistent reduction of the impurities. The contribution of each step to the final product purity is described and shown for three manufacturing batches. Turoctocog alfa, a third-generation B-domain truncated rFVIII product is manufactured in Chinese hamster ovary cells without the use of animal or human-derived proteins. The five-step purification process results in a homogenous, highly purified rFVIII product.

Substrate Specificity Profiling of Peptidyl-Lys Metallopeptidase of Armillaria mellea by FRET Based Peptide Library.

Determining the substrate specificity of a protease is essential for developing assays, inhibitors and understanding the mechanisms of the enzyme. In this work, we have profiled the specificity of Peptidyl-Lys metallopeptidase, (LysN), of Armillaria mellea, by a synthetic fluorescence resonance energy transfer (FRET) positional-scanning library. The library was based on a reference sequence K(Abz)-S-A-Q-K-M-V-S-K(Dnp), where the fluorescent donor is 2-aminobenzamide and the quencher is N-2,4-dinitrophenyl. Each position was varied between 19 different amino acids one by one, to reveal the specificity of the protease. LysN exhibits strict specificity for lysine in S1', and has less specificity moving further away from the scissile bond. Additivity between the subsites was observed and the best substrate identified was K(Abz)-M-R-F-K-R-R-R-K(Dnp) with a kcat/KM of 42.6 µM/s. Based on a homology structure model the reference substrate was fitted into the active site using molecular dynamics to propose peptide-enzyme interactions.

Trefoil factors (TFFs) are increased in bronchioalveolar lavage fluid from patients with chronic obstructive lung disease (COPD).

Trefoil factors (TFFs) 1, 2 and 3 are small polypeptides that are co-secreted with mucin throughout the body. They are up-regulated in cancer and inflammatory processes in the gastrointestinal system, where they are proposed to be involved in tissue regeneration, proliferation and protection. Our aim was to explore their presence in pulmonary secretions and to investigate whether they are up-regulated in pulmonary diseases characterized by mucin hypersecretion. Bronchioalveolar lavage fluid was obtained from 92 individuals referred to bronchoscopy. The patients were grouped according to diagnosis and pulmonary function. The concentrations of TFF1, TFF2 and TFF3 were measured by ELISA. All three peptides were detected in bronchioalveolar lavage fluid. Patients with chronic obstructive pulmonary disease had concentrations two to three times above the levels in the healthy reference group, and patients with pulmonary malignancies had concentrations of TFF1 and TFF2 three times that of the reference group. The results suggest that TFFs are involved in tissue regeneration, proliferation and protection in lung diseases.

Trefoil factor peptides in serum and sputum from subjects with asthma and COPD.

OBJECTIVE: Trefoil factor peptides (TFF) are secreted onto mucosal surfaces together with mucins and occur in high concentrations in pulmonary secretions from patients with chronic obstructive pulmonary disease (COPD). In the present study, we aimed to explore the concentrations of the peptides in serum and sputum in patients with COPD. MATERIALS AND METHODS: Thirty-five individuals were included in the study, including 11 healthy individuals, 13 indivials with asthma and 11 individuals with COPD. TFF1, TFF2 and TFF3 were measured by enzyme-linked immunosorbent assay (ELISA) in sputum induced by hypertonic saline inhalation and in serum. Total protein content in sputum was also determined. RESULTS: In the sputum samples from COPD patients, we observed an eightfold higher concentration of TFF1 and a fivefold higher concentration of TFF3 compared with controls. In the serum samples from COPD patients, we observed three-, three- and twofold higher concentrations of TFF1, TFF2 and TFF3 respectively compared with controls. CONCLUSIONS: There is increased secretion of TFF peptides in the lungs of patients with COPD, as well as significant increases in serum levels. This suggests a role for TFF peptides in the pathogenesis of pulmonary diseases with mucus hypersecretion.

Implications for the offspring of circulating factors involved in beta cell adaptation in pregnancy.

OBJECTIVE: Several studies have shown an increase in beta cell mass during pregnancy. Somatolactogenic hormones are known to stimulate the proliferation of existing beta cells in rodents whereas the mechanism in humans is still unclear. We hypothesize that in addition to somatolactogenic hormones there are other circulating factors involved in beta cell adaptation to pregnancy. This study aimed at screening for potential pregnancy-associated circulating beta cell growth factors. SAMPLES: Serum samples from nonpregnant and pregnant women. METHODS: The effect of serum from pregnant women on the proliferation of rat beta cells was studied using [3H]thymidine incorporation and 5-ethynyl-2'-deoxyuridine proliferation assays. In addition, serum from pregnant and nonpregnant women was fractionated by gel filtration and high performance liquid chromatography. The fractionated serum was screened for mitogenic activity in INS-1E cells. Proteins and peptides in mitogenic active serum fractions were identified by amino acid sequencing and mass spectrometry. MAIN OUTCOME MEASURES: Presence of circulating beta cell proliferating factors. RESULTS: Late gestational pregnancy serum significantly increased proliferation of rat beta cells compared with early pregnancy and nonpregnancy. The mitogenic active serum fractions contained proteins and peptides derived from kininogen-1, fibrinogen-α, α1-antitrypsin, apolipoprotein-A1, placental lactogen, angiotensinogen and serum albumin. CONCLUSION: Pregnancy serum is able to stimulate proliferation of rat beta cells. We have identified several circulating factors that may contribute to beta cell adaptation to pregnancy. Further studies are needed to elucidate their possible role in glucose homeostasis in the mother and her offspring.

A variant form of the human deleted in malignant brain tumor 1 (DMBT1) gene shows increased expression in inflammatory bowel diseases and interacts with dimeric trefoil factor 3 (TFF3).

The protein deleted in malignant brain tumors (DMBT1) and the trefoil factor (TFF) proteins have all been proposed to have roles in epithelial cell growth and cell differentiation and shown to be up regulated in inflammatory bowel diseases. A panel of monoclonal antibodies was raised against human DMBT1(gp340). Analysis of lung washings and colon tissue extracts by Western blotting in the unreduced state, two antibodies (Hyb213-1 and Hyb213-6) reacted with a double band of 290 kDa in lung lavage. Hyb213-6, in addition, reacted against a double band of 270 kDa in colon extract while Hyb213-1 showed no reaction. Hyb213-6 showed strong cytoplasmic staining in epithelial cells of both the small and large intestine whereas no staining was seen with Hyb213-1. The number of DMBT1(gp340) positive epithelial cells, stained with Hyb213-6, was significantly up regulated in inflammatory colon tissue sections from patients with ulcerative colitis (p<0.0001) and Crohn's disease (p = 0.006) compared to normal colon tissue. Immunohistochemical analysis of trefoil factor TFF1, 2 and 3 showed that TFF1 and 3 localized to goblet cells in both normal colon tissue and in tissue from patients with ulcerative colitis or Crohn's disease. No staining for TFF2 was seen in goblet cells in normal colon tissue whereas the majority of tissue sections in ulcerative colitis and Crohn's disease showed sparse and scattered TFF2 positive goblet cells. DMBT1 and TFF proteins did therefore not co-localize in the same cells but localized in adjacent cells in the colon. The interaction between DMBT1(gp340) and trefoil TFFs proteins was investigated using an ELISA assay. DMBT1(gp340) bound to solid-phase bound recombinant dimeric TFF3 in a calcium dependent manner (p<0.0001) but did not bind to recombinant forms of monomeric TFF3, TFF2 or glycosylated TFF2. This implies a role for DMBT1 and TFF3 together in inflammatory bowel disease.

Evaluation of the metal binding sites in a recombinant coagulation factor VIII identifies two sites with unique metal binding properties.

Coagulation factor VIII is a glycosylated, non-covalent heterodimer consisting of a heavy chain (A1-A2-B domains) and a light chain (A3-C1-C2 domains). The association of the chains, and the stability and function of the dimer depend on the presence of metal ions. We applied X-ray fluorescence, X-ray crystallographic structure determination with anomalous signals at different wavelengths, and colorimetric measurements to evaluate the metal binding sites in a recombinant factor VIII molecule, turoctocog alfa. We identified a metal binding site in domain A3 dominated by Cu(+) binding and a site in domain A1 dominated by Zn(2+) binding.

A novel B-domain O-glycoPEGylated FVIII (N8-GP) demonstrates full efficacy and prolonged effect in hemophilic mice models.

Frequent infusions of intravenous factor VIII (FVIII) are required to prevent bleeding associated with hemophilia A. To reduce the treatment burden, recombinant FVIII with a longer half-life was developed without changing the protein structure. FVIII-polyethylene glycol (PEG) conjugates were prepared using an enzymatic process coupling PEG (ranging from 10 to 80 kDa) selectively to a unique O-linked glycan in the FVIII B-domain. Binding to von Willebrand factor (VWF) was maintained for all conjugates. Upon cleavage by thrombin, the B-domain and the associated PEG were released, generating activated FVIII (FVIIIa) with the same primary structure and specific activity as native FVIIIa. In both FVIII- and VWF-deficient mice, the half-life was found to increase with the size of PEG. In vivo potency and efficacy of FVIII conjugated with a 40-kDa PEG (N8-GP) and unmodified FVIII were not different. N8-GP had a longer duration of effect in FVIII-deficient mouse models, approximately a twofold prolonged half-life in mice, rabbits, and cynomolgus monkeys; however, the prolongation was less pronounced in rats. Binding capacity of N8-GP on human monocyte-derived dendritic cells was reduced compared with unmodified FVIII, resulting in several-fold reduced cellular uptake. In conclusion, N8-GP has the potential to offer efficacious prevention and treatment of bleeds in hemophilia A at reduced dosing frequency.

Exogenous glucagon-like peptide-2 (GLP-2) prevents chemotherapy-induced mucositis in rat small intestine.

PURPOSE: Gastrointestinal mucositis is an unwanted and often dose-limiting side effect to most cancer treatments. Glucagon-like peptide-2 (GLP-2) is a peptide secreted from intestinal L-cells in response to nutrient intake. The peptide is involved in the regulation of apoptosis and proliferation in the intestine. We aimed to investigate the role of GLP-2 in experimental chemotherapy-induced mucositis. METHODS STUDY 1: Rats were given a single injection with 5-fluorouracil (5-FU) and killed in groups of five each day for 5 days. Blood samples were analysed for GLP-2 concentrations. The intestine was analysed for weight loss, morphometric estimates and proliferation. STUDY 2: Rats were treated with GLP-2 or control vehicle 2 days before a single injection of 5-FU or saline. The treatments continued until kill 2 days after. The intestine was investigated for influx of myeloperoxidase (MPO)-positive cells and morphometric estimates, such as villus height, as a marker of mucositis. RESULTS STUDY 1: Two days after chemotherapy, there was a rise in endogenous GLP-2, followed by a marked increase in proliferation. STUDY 2: Exogenous GLP-2 was able to protect the intestine from severe weight loss and completely prevented the reduction in villus height in the control rats. Furthermore, there was a significant decrease in influx of MPO-positive cells in the GLP-2-treated rats. CONCLUSION: GLP-2 is secreted from the intestine in response to intestinal injury, probably explaining the compensatory hyperproliferation after chemotherapy. Exogenous GLP-2 can protect the mucosa from chemotherapy-induced mucositis in rats.

Cocaine- and amphetamine-regulated transcript peptide (CART) in the telencephalon of the catfish, Clarias gariepinus: distribution and response to fasting, 2-deoxy-D-glucose, glucose, insulin, and leptin treatments.

The cocaine- and amphetamine-regulated transcript peptide (CART)-containing system in the forebrain of Clarias gariepinus was studied with immunocytochemistry. While the immunoreactivity was prominently seen in the neurons of the entopeduncular nucleus (EN) located in the ventral telencephalon, CART-immunoreactive fibers were widely distributed in the dorsal and ventral telencephalon. In view of the established role of CART in energy metabolism, we investigated the response of the CART immunoreactive system to positive and negative nutritional conditions. Neurons of the EN and fibers in the different areas of the telencephalon showed significant reduction in CART immunoreactivity following 48 hours food deprivation, or 2 hours following intracranial administration of 2-deoxy-D-glucose (2DG, 100 ng/g body weight, a metabolic antagonist of glucose). However, intracranial injection of glucose (100 ng/g body weight) resulted in a distinct increase in CART immunoreactivity in these components. In mammals, insulin and leptin have been recognized as adiposity agents that convey peripheral energy status-related information to brain. Intracranial administration of insulin (3 mU/fish) and leptin (10 ng/g body weight) significantly increased CART immunoreactivity in the EN neurons and in the fiber network within 2 hours. Superfusion of the EN-containing tissue fragments in the medium enriched in glucose, insulin, or leptin evoked a significant increase in CART immunoreactivity in the EN neurons, but 2DG reduced the immunoreactivity. We suggest that CART-containing neurons of the EN, and fibers in the telencephalon, may process the energy status-related information and contribute to satiety.

Reproductive phase-related variations in cocaine- and amphetamine-regulated transcript (CART) in the olfactory system, forebrain, and pituitary of the female catfish, Clarias batrachus (Linn.).

The annual sexual cycle of Clarias batrachus is divisible into resting (December-January), preparatory (February-April), prespawning (May-June), spawning (July- August), and postspawning (September-November) phases. The gonosomatic indices rose steadily through the preparatory and prespawning phases, peaked in the spawning phase, and were greatly reduced during the postspawning and resting phases. A clear pattern of change was also identified in the immunocytochemical profile of the luteinizing hormone (LH) cells in the pituitary. These changes were correlated with the cocaine- and amphetamine-regulated transcript (CART)-immunoreactive system in the forebrain and pituitary. In the olfactory bulb, CART immunoreactivity in the terminal fields of the mitral cell layer, granule cells, and medial olfactory tracts gradually decreased during the resting through prespawning phases. However, it was considerably augmented during spawning (P < 0.001) and showed highest activity in the postspawning phase (P < 0.001). A different pattern was noticed in the fibers and/or neurons of the lateral part of ventral telencephalic area, the entopeduncular nucleus, and the dorsal part of the nucleus preopticus periventricularis. In these areas, intense immunoreactivity seen in preparatory phase, declined during prespawning (P < 0.01) then through spawning, and was partially augmented during the postspawning and resting phases (P < 0.05). A similar pattern was also seen in the nucleus preglomerulosus lateralis and medialis, nucleus dorsalis posterioris of thalamus, lobobulbar nucleus, and the nucleus of posterior recess. CART was transiently expressed in LH cells in the pituitary during the preparatory period. We suggest that the CART system may play a role in triggering the brain-pituitary-ovary axis at the onset of the preparatory phase.

The intestinotrophic peptide, GLP-2, counteracts the gastrointestinal atrophy in mice induced by the epidermal growth factor receptor inhibitor, erlotinib, and cisplatin.

PURPOSE: Erlotinib, an epidermal-growth-factor receptor inhibitor, belongs to a new generation of targeted cancer therapeutics. Gastrointestinal side-effects are common and have been markedly aggravated when erlotinib is combined with cytostatics. We examined the effects of erlotinib alone and combined with the cytostatic, cisplatin, on the gastrointestinal tract and examined whether glucagon-like peptide-2 (GLP-2), an intestinal hormone with potent intestinotrophic properties, might counteract the possible damaging effects of the treatments. EXPERIMENTAL DESIGN: Groups of ten mice were treated for 10 days with increasing doses of erlotinib alone or in combination with cisplatin and/or GLP-2. Weight and length of the gastrointestinal organs were determined and histological sections were analyzed with morphometric methods as well as BrdU- and ApopTag-staining to determine mitotic and apoptotic activity. RESULTS: Erlotinib was found to induce small-intestinal and colonic growth inhibition through an increased apoptotic activity but had no effect on mitotic activity. The combined treatment with cisplatin synergistically aggravated the intestinal growth inhibition. Erlotinib, and especially the combination therapy, increased the weight of the stomach contents considerably. Concomitant treatment with GLP-2 counteracted the intestinal mucosal atrophy induced both by erlotinib alone and combined with cisplatin through a reduction of the apoptotic activity. There was no influence on the mitotic activity. CONCLUSIONS: The findings demonstrate that the intestinal mucosal damage induced by erlotinib alone and in combination with cisplatin can be counteracted by GLP-2 treatment, which might suggest a role for GLP-2 in the treatment of the gastrointestinal side-effects caused by these cancer therapeutics.

Association of cocaine- and amphetamine-regulated transcript and neuropeptide Y in the forebrain and pituitary of the catfish, Clarias batrachus: a double immunofluorescent labeling study.

Cocaine- and amphetamine-regulated transcript (CART) and neuropeptide Y (NPY) are involved in the regulation of food intake, body weight, pituitary hormones, and reproduction. While CART and NPY occupy overlapping fields in the brain of mammals, little is known about the interaction between these peptide-containing systems in other vertebrates. We explored neuroanatomical associations between CART and NPY in the olfactory system, forebrain and pituitary of the catfish, Clarias batrachus, using double immunofluorescence method. NPY-containing fascicles from olfactory receptor neurons innervated the olfactory glomeruli and mitral cell layer in close association with CART-containing terminal fields. Distinct CART- or NPY-containing fibers were seen in the medial olfactory tract. In the dorsal telencephalon, CART- and NPY-immunoreactive axons were closely associated in area dorsalis telencephali/pars lateralis dorsalis (Dld), and posterioris (Dlp). In the ventral telencephalon, while most of the cells of nucleus entopeduncularis (NE) showed the presence of CART as well as NPY, a few cells with only NPY-immunoreactivity were observed. Similarly, a CART and NPY colocalized cell population was prominent in the preoptic area (POA); and a small population of cells with NPY-immunoreactivity was also evident. Other areas where CART and NPY were colocalized included fibers in the tuberal area, inferior lobe, neurohypophysis, proximal pars distalis and pars intermedia of the pituitary. No association between CART and NPY was observed in the thalamus and habenular ganglion. These results suggest that CART- and NPY-peptidergic systems may interact in NE, POA, tuberal area, certain telencephalic areas and pituitary and jointly process information relating to reproduction, feeding and neuroendocrine regulation.

Importance of cocaine- and amphetamine-regulated transcript peptide in the central nucleus of amygdala in anxiogenic responses induced by ethanol withdrawal.

We studied the involvement of cocaine- and amphetamine-regulated transcript peptide (CART) in the central nucleus of amygdala (CeA), lateral bed nucleus of the stria terminalis (BNSTl) and nucleus accumbens shell (AcbSh) in generation of ethanol withdrawal symptoms, with particular focus on anxiety-like behavior using a social interaction test. Administration of CART (54-102) into the lateral ventricle (50 and 100 ng) and bilaterally in the CeA (10 and 20 ng) caused a significant reduction in social interaction, suggesting an anxiogenic action of the peptide. Chronic ethanol treatment for 15 days followed by withdrawal precipitated an anxiogenic response at 24 h that was attenuated by intracerebroventricular (5 mul) and intra-CeA (1 mul) administration of antibodies against CART (1 : 500 dilution). An immunocytochemistry protocol was employed to study the response of the endogenous CART system in the CeA following chronic ethanol withdrawal. At 0 h ethanol withdrawal, CART immunoreactivity was apparent in few fibers and the profile was similar to that in the pair-fed control rats. Twenty-four hours following ethanol withdrawal, a highly significant increase (P<0.001) in CART immunoreactivity was noticed in the CeA, which returned to normal 48 and 72 h post-withdrawal. Similar doses of CART or CART antibody injected bilaterally into the BNSTl or AcbSh produced no response in the social interaction test. Furthermore, the CART immunoreactivity profile did not change at the post-withdrawal time points in each of these brain sites. We suggest that CART may mediate the early signs of anxiety-like behavior induced by ethanol withdrawal within the neuroanatomical framework of the CeA.

Glucagon-like peptide-2 improves both acute and late experimental radiation enteritis in the rat.

PURPOSE: Acute and/or chronic radiation enteritis can develop after radiotherapy for pelvic cancers. Experimental and clinical observations have provided evidence of a role played by acute mucosal disruption in the appearance of late effects. The therapeutic potential of acute administration of glucagon-like peptide-2 (GLP-2) against acute and chronic intestinal injury was investigated in this study. METHODS AND MATERIALS: Intestinal segments were surgically exteriorized and exposed to 16.7 or 19 Gy X-rays. The rats were treated once daily with vehicle or a protease-resistant GLP-2 derivative for 14 days before irradiation, with or without 7 days of GLP-2 after treatment. Macroscopic and microscopic observations were made 2 and 15 weeks after radiation exposure. RESULTS: In the control animals, GLP-2 induced an increase in intestinal mucosal mass, along with an increase in villus height and crypt depth. GLP-2 administration before and after irradiation completely prevented the acute radiation-induced mucosal ulcerations observed after exposure to 16.7 Gy. GLP-2 treatment strikingly reduced the late radiation damage observed after 19 Gy irradiation. Microscopic observations revealed an improved organization of the intestinal wall and an efficient wound healing process, especially in the smooth muscle layers. CONCLUSION: GLP-2 has a clear therapeutic potential against both acute and chronic radiation enteritis. This therapeutic effect is mediated through an increased mucosal mass before tissue injury and the stimulation of still unknown mechanisms of tissue response to radiation damage. Although these preliminary results still need to be confirmed, GLP-2 might be a way to limit patient discomfort during radiotherapy and reduce the risk of consequential late effects.

TFF3 and EGF induce different migration patterns of intestinal epithelial cells in vitro and trigger increased internalization of E-cadherin.

BACKGROUND/AIMS: TFF3, a member of the TFF (trefoil factor family) peptides, and epidermal growth factor (EGF) actively support the repair of mucosal barriers, particularly during restitution. The aim of this study was to compare the motogenic effects of TFF3 and EGF. METHODS: The influence of recombinant human TFF3 (dimeric form) and EGF on the migration of IEC-18 cells was characterized in an in vitro restitution model (scratch wound assay) with the help of time-lapse video microscopy, morphometry, and immunocytochemistry including confocal laser scanning microscopy. RESULTS: TFF3- and EGF-treated cells re-populated the wounded area via different migration patterns; TFF3 treatment resulted in the formation of continuous sheets of migrating cells with only a few gaps. In contrast, EGF-treated cells formed a network of migrating cells (often with a fibroblast-like morphology) with numerous gaps and only punctual contacts. TFF3 and EGF treatment also changed the localization of E-cadherin indicating endocytotic recycling and/or degradation of E-cadherin. CONCLUSION: TFF3, in contrast to EGF, enhanced a collective cell migration ensuring a precise coverage of the re-populated area avoiding gaps.

Pharmacokinetics of trefoil peptides and their stability in gastrointestinal contents.

Trefoil factor family (TFF) peptides are considered promising for therapeutic use in gastrointestinal diseases, and there is a need to explore the fate of injected TFF and the stability of the peptides in the gastrointestinal tract. We studied the pharmacokinetics of intravenously (i.v.) administered hTFF2 in mice and rats and of hTFF3 administered i.v., intramuscularly, intraperitoneally, and subcutaneously in mice, and estimated by ELISA the decay of the peptides added to rat and human gastrointestinal contents. We found that i.v. injected hTFF2 and hTFF3 were cleared from the circulation within 2-3h, exhibiting comparable pharmacokinetic profiles. In contents from the rat stomach, hTFF levels remained unchanged for up to 6 days. In the small and large intestine of rats, the hTFF levels decreased markedly after 4 and 1h, respectively. In small intestinal contents from humans, the levels remained stable for more than 24h. We conclude that systemically administered hTFF2 and hTFF3 are rapidly eliminated from the circulation and that the stability of hTFF2 and hTFF3 in GI contents appeared higher in the gastric and small intestinal milieu than in the large intestine and feces, suggesting a higher stability toward gastric acid and digestive enzymes than toward microbial degradation.

Immunohistochemical localization of cocaine- and amphetamine-regulated transcript peptide in the brain of the catfish, Clarias batrachus (Linn.).

The organization of cocaine- and amphetamine-regulated transcript peptide (CARTp, 54-102) immunoreactivity was investigated in the brain of the catfish, Clarias batrachus. CARTp-immunoreactivity was observed in several granule cells of the olfactory bulbs, in dot-like terminals around mitral cells, and in the fibers of the medial olfactory tracts. While several groups of discrete cells in the telencephalon showed CARTp-immunoreactivity, the immunostained fibers were widely distributed in the area dorsalis and ventralis telencephali. Immunoreactivity was seen in several periventricular and a few magnocellular neurons, and in a dense fiber network throughout the preoptic area. Varying degrees of immunoreactive fibers were seen in the periventricular region in the thalamus, hypothalamus, and pituitary. Some neurons in the nucleus preglomerulosus medialis and lateralis, central nucleus of the inferior lobes, nucleus lobobulbaris of the posterior tuberculum, and nucleus recessus posterioris showed distinct CARTp-immunoreactivity. Considerable immunoreactivity was seen in the optic tectum, rostral torus semicircularis, central pretectal area, and granule cells of the cerebellum. While only isolated immunoreactive cells were seen at three distinct sites in the metencephalon, a fiber network was seen in the facial and vagal lobes and periventricular and ventral regions of the medulla oblongata. The pattern of the CARTp distribution in the brain of C. batrachus suggests that it may play an important role in the processing of sensory information, the regulation of hormone secretion by hypophysial cell types, and motor and vegetative function. Finally, as in other animal species, CARTp seems to play a role in the processing of gustatory information.