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OBJECTIVES The aim of the present study was to investigate the segment- and time-related changes in rat short bowel syndrome and construct a 4-dimensional (4D) geometrical model of intestinal adaptation. METHODS Sprague-Dawley rats were divided into 3 groups: 2-day, 7-day, and 15-day postresection groups in which 75% of the jejunoileum was removed. Histological and morphometrical parameters in the remaining proximal to distal intestinal segments, from the jejunum to the distal colon, were comparatively evaluated in the groups. The data were used to construct a 4D geometric model in which villi were considered as cylinders, and their surface area was expressed as cylinder lateral area. RESULTS Major adaptive changes were observed in the ileum consisting of an increase in both the diameter of base and the height of villi. A parallel reduction in their number/mm was observed. The resulting ileal architecture was characterized by a limited number of large villi. An opposite pattern was observed in the jejunum whose postresection structure consisted of an increased number of villi. No changes were observed in the colon. Postresection restructuring was early and faster in the ileum than in the jejunum resulting in an increase in absorptive area of 81.5% and 22.5% in the ileum and jejunum, respectively. CONCLUSIONS Postresection adaptation is intestinal segment-specific because all of the major changes occur in the ileum rather than in the jejunum. Sparing ileal segments during resection may improve the outcome of patients undergoing extensive intestinal resection. Our 4D model can be used to test interventions aimed at optimizing postresection intestinal adaptation.

Guanylin receptor guanylate cyclase (GC-C) peaks in neonatal intestine and is involved in either enterocyte proliferation or chloride secretion. The latter is more potent when GC-C activator guanylin, or its analog Escherichia coli heat-stable enterotoxin (ST), is added to the mucosal rather than serosal side of intestinal monolayers. By using Ussing chambers, we investigated transepithelial ion transport and enterocyte proliferation and their mechanisms in response to the addition of guanylin or ST to the mucosal or serosal side of Caco-2 monolayers and in ileal specimens from neonates. GC-C activation showed a polar pattern of the effects. GC-C mucosal activation resulted in a potent cGMP-chloride secretion activation and in a marginal enterocyte proliferation. Conversely, serosal GC-C activation induced a potent enterocyte proliferation, through MAP kinase ERK 1/2. Finally, the inhibition of ERK1/2 enhanced the Isc increase in response to serosal but not to mucosal ST stimulation, indicating that ERK1/2 also acts as a brake of chloride secretion. These data suggest that the guanylin/GC-C system plays a key role in early postnatal intestinal adaptation exploiting the polar structure of enterocyte.

The role of angiotensin II (AII) in human preadipocyte physiology has been investigated in primary cultures from human adipose tissue. Receptor binding studies indicated that human preadipocytes express a high affinity AII binding site of the AT1 subtype, as binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific. As AII has previously been demonstrated to affect the cell cycle in adrenal and cardiac cells, the effect of AII on regulation of cycle progression was examined in human preadipocytes. Stimulation of preadipocytes with AII resulted in G1 phase progression of the cell cycle, as determined by flow cytometric analysis. AII treatment was associated with induction of expression of the messenger RNA for the cell cycle regulatory protein cyclin D1 in a dose-dependent manner. Pretreatment of cells with subtype-selective AT receptor ligands before AII stimulation indicated that the cyclin response was mediated via the AT1 receptor. The identity of the cells as preadipocyte was verified by culture in a defined differentiation medium, observing both leptin message expression and triglyceride accumulation by flow cytometry. These findings indicate that AII has early, receptor-mediated effects on cell cycle progression in human preadipocytes that may contribute to differentiation to the adipocyte phenotype.

Angiotensin II (AII) receptor binding assays were performed in rat adipocytes from three separate anatomic depots. Fat cells were isolated by collagenase digestion, and plasma membranes were prepared from the epididymal, mesenteric, and retroperitoneal fat depots of male Sprague-Dawley rats at 100 days of age. Binding of 125I-labeled [Sar1,Ile8]AII was rapid, saturable, and specific in membranes from all depots, identifying a receptor with a similar affinity of approximately 1 nM. Site-associated differences in receptor number were observed, with epididymal and mesenteric fat cell membranes exhibiting significantly more receptors than retroperitoneal fat cells when binding was expressed per unit of membrane protein. When corrected for cell volume, the number of receptors per cell ranked epididymal > retroperitoneal > mesenteric. Inhibitory constants for the peptide agonists AII and AIII and the peptide antagonist [Sar1,Ala8]AII indicated similar affinities in all three depots. Because the receptor has been classified pharmacologically into two subtypes, the AT1 selective antagonist losartan, and the AT2 selective antagonist PD 123,319 were used to classify the adipocyte receptor, indicating an AT1 subtype with an affinity for losartan in the mesenteric and retroperitoneal adipocytes that was significantly greater than the epididymal. Similar studies were performed in adipocyte membranes obtained from human omental and subcutaneous adipose tissue, revealing the presence of an AII receptor in both depots with an affinity of approximately 10 nM for losartan. These data indicate site-specific differences in AII receptor number in fat cell membranes from rats and the existence of human adipocyte AII receptors, suggesting that the adipocyte is significant for the peripheral metabolism of components of the renin-angiotensin system.

Nutrition has a key role in the modulation of the developing intestine in early infancy, and nutrients are able to modulate several intestinal functions including nutrient absorption, ion transport, cell growth and differentiation, motility and immunomodulation. Such modulation is exerted in part through a direct interaction between a single nutrient and the enterocyte. Two functions – ion transport and cell growth – are closely connected and appear to be modulated by nutrients. This is supported by the finding that a single nutrient (e.g. zinc) stimulates ion absorption and also promotes enterocyte growth. Interestingly, intracellular signalling for either effect is similar and involves mitogen-activated protein kinase. Other nutrients also modulate enterocyte function and there is evidence that their effect may depend on their side of action (apical or basolateral) and concentration. Knowledge of the interaction between nutrients and enterocytes may be exploited to obtain clinical effects using nutrition as a long-term treatment for intestinal and non-intestinal conditions.

The colon is actively implicated in intestinal infections not only as a target of enteric pathogens and their products but also as a target organ for treatment. In the presence of diarrhea, both of osmotic and secretory nature, the colon reacts with homeostatic mechanisms to increase ion absorption. These mechanisms can be effectively exploited to decrease fluid discharge. A model of intestinal infections using rotavirus (RV) in colonic cells was set up and used to define a dual model of secretory and osmotic diarrhea in sequence. Using this model, antidiarrheal drugs were tested, namely zinc and the enkephalinase inhibitor racecadotril. Zinc was able to decrease the enterotoxic activity responsible for secretory diarrhea. It also inhibited the cytotoxic effect of RV. The mechanism of zinc was related at least in part to the activation of MAPK activity, but also a direct antiviral effect was observed. Racecadotril showed a potent and selective inhibition of active secretion, being particularly effective in the first phase of RV diarrhea. The use of drugs active at the colonic level, therefore, offers effective options to treat intestinal infections in childhood. In addition, the colon is the natural site of colonic microflora, a target of probiotic therapy, which is the first line of approach recommended by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition to treat infectious diarrhea.

Vascular endothelial cell growth factor (VEGF), also known as vascular permeability factor, is an endothelial cell mitogen which stimulates angiogenesis. Here we report that a previously identified receptor tyrosine kinase gene, KDR, endodes a receptor for VEGF. Expression of KDR in CMT-3 (cells which do not contain receptors for VEGF) allows for saturable 125I-VEGF binding with high affinity (KD = 75 pM). Affinity cross-linking of 125I-VEGF to KDR-transfected CMT-3 cells results in specific labeling of two proteins of Mr = 195 and 235 kDa. The KDR receptor tyrosine kinase shares structural similarities with a recently reported receptor for VEGF, f t , in a manner reminiscent of the similarities between the α and β forms of the PDGF receptors.

Although the FGFs have been subject to extensive biological studies, only limited progress has been made so far in determining the critical elements of structure-activity relationships in the FGFs. Among the recognized structural elements with potential to affect the biological activity of FGFs are the cysteine residues, and the heparin- and receptor-binding domains. These features have been studied using a variety of experimental approaches, but the available data are inconclusive. For example, ambiguity regarding the presence of a disulfide structure in FGFs was not resolved until the availability of x-ray crystal structure data. Furthermore, the functionally important heparin- and receptor-binding domains have been poorly characterized, with some interpretations being controversial. In this report, we describe a novel fragment of basic FGF (bFGF) with high biological activity [Ser78,96-bFGF(70-153)]. This fragment was generated by pronase treatment of heparin-bound recombinant Glu3,5Ser78,96-bFGF mutant and is active in vitro at an ED50 of about 100 ng/ml. The structure of the fragment and the manner by which it was generated provide additional insight into important aspects of structure-activity relationships in FGFs. Specifically, we conclude that (a) the cysteines in our bFGF mutant do not form a disulfide bond, (b) the high-affinity heparin binding of bFGF critically depends on an intact 3-dimensional structure of the growth factor rather than on specific heparin-binding sequence domains, and (c) the bFGF sequence between residues 70 and 122 is important for high biological activity.

CMC-544 is CD22-specific cytotoxic immunoconjugate of calicheamicin that has demonstrated significant anti-tumor activity in phase I clinical trial in B-NHL patients (Blood2005;106:230). In this trial, thrombocytopenia was reported as the most common dose-limiting adverse event. The present non-clinical study attempted to provide insights into the observed thrombocytopenia associated with CMC-544 treatment in B-NHL patients. In vitro, CMC-544 neither bound to nor caused aggregation of platelets in platelet-rich plasma of human or murine origin. When administered to mice, a single dose of CMC-544 resulted in a 50% to 75% reduction in the number of circulating platelets with a nadir on day 3 or 4. The platelet levels returned to normal at the latest by day 12. Similar effects were also observed in cynomolgus macaques with nadirs on day 4 or 5. CMC-544 dose not cross-react with either murine or cynomolgus macaque CD22. Unconjugated anti-CD22 antibody, G5/44 (humanized IgG4 antibody) had no effect on circulating platelets. CMC-544-induced thrombocytopenia in mice was associated with a concomitant reduction in circulating levels of thrombopoietin (TPO) with a nadir on day 4. In contrast, treatment with carboplatin, a cytotoxic chemotherapeutic agent known to cause thrombocytopenia, caused thrombocytopenia in mice but with a nadir on day 11. Unlike that with CMC-544, carboplatin-induced thrombocytopenia was associated with an increase in the levels of circulating TPO. Thus, CMC-544-induced thrombocytopenia appears to be mechanistically and kinetically distinct from that associated with conventional cytotoxic chemotherapeutic agents. In this murine model, pretreatment with human IGIV (Carimune) failed to prevent CMC-544-associated thrombocytopenia. However, subcutaneous pretreatment of mice with oprelvekin (Neumega/rhIL-11; 250 ug/kg) significantly reduced the magnitude of the CMC-544-associated thrombocytopenia. When evaluated in cynomolgus macaques, daily subcutaneous pretreatment with oprelvekin (125 ug/kg) for 5 days completely prevented CMC-544-associated thrombocytopenia and also suppressed elevations in serum hepatic aminotransferases associated with the CMC-544 treatment. Concurrent administration of oprelvekin with CMC-544 failed to prevent the initial thrombocytopenia but did accelerate complete recovery of platelets counts to the pretreatment levels and also suppressed elevations in serum hepatic aminotransferases. These results suggest that thrombocytopenia associated with CMC-544-treatment may in part be due to CMC-544-induced hepatic changes, including decreased TPO production. This study further provides evidence that oprelvekin not only stimulates megakaryocytes to produce platelets but is also hepatoprotective, reducing the degree of CMC-544-mediated hepatotoxicity. We have presented preclinical evidence that the prophylactic use of oprelvekin can ameliorate CMC-544-induced thrombocytopenia and thus, supports the clinical evaluation of oprelvekin administered with CMC-544 to augment its therapeutic benefit in patients with B-non-Hodgkin’s lymphoma.