Intrathecal Baclofen Therapy in Russia: National Register, the Effect of One-Year Usage.

Patients with severe muscular spasticity still represent the most complex and resistant to therapy group of neuro-rehabilitation patients. In a few years, in Russia, intrathecal baclofen therapy has appeared to be the most effective method for such spasticity. For the first time the authors developed and implemented in clinical practice "Prospective register to treat spastic states using intrathecal baclofen therapy in Russian Federation" aimed at therapy classification of spastic patients: to reveal management characteristics, assess treatment outcomes and frequency of occurrence of adverse events that will finally help specify the need for the method employment in real clinical practice. The article presents the findings of a one-year usage of Register, which enabled to make a preliminary evaluation of intrathecal baclofen therapy in Russia.

Novel goblet cell gene related to IgGFcgammaBP is regulated in adapting gut after small bowel resection.

The loss of functional small bowel surface area leads to a well-described adaptive response in the remnant intestine. To elucidate its molecular regulation, a cohort of cDNAs were cloned using a rat gut resection model and subtractive/differential hybridization cloning techniques. This study reports a novel cDNA termed "ileal remnant repressed" (IRR)-219, which shares 80% nucleotide identity with the 3'end of a human intestinal IgG Fc binding protein (IgGFcgammaBP) and is homologous to human and rat mucins. IRR-219 mRNA is expressed in intestine and colon only. At 48 h after 70% intestinal resection, mRNA levels decreased two- to fivefold in the adaptive small bowel but increased two- to threefold in the colon. Expression of IRR-219 was suppressed in adaptive small bowel as late as 1 wk after resection. IRR-219 expression is also regulated during gut ontogeny. In situ hybridization revealed IRR-219 expression in small intestinal and colonic goblet cells only. Its unique patterns of expression during ontogeny and after small bowel resection suggest distinctive roles in small bowel and colonic adaptation.

Vitamin A status modulates intestinal adaptation after partial small bowel resection.

BACKGROUND: Intestinal adaptation after loss of functional small bowel surface area is characterized by cellular hyperplasia and increased absorptive function. Interventions to enhance the adaptive response are needed to decrease the morbidity and mortality associated with short bowel syndrome. Retinoic acid was shown to stimulate crypt cell proliferation in the adapting remnant rat ileum by 6 hours after resection. Thus, vitamin A, which is required for normal epithelial cell proliferation and differentiation and which can modulate programmed cell death, may play an important role in the adapting intestine. On the basis of these observations, the effects of vitamin A deficiency on intestinal morphology, epithelial cell proliferation, and apoptosis in the adapting intestine after resection were investigated. METHODS: Weanling male Sprague-Dawley rats fed either a vitamin A-deficient or -sufficient diet for 58 days underwent 70% proximal small bowel resection. The deficient rats were divided into cohorts that were either maintained on the experimental diet after surgery or replenished with vitamin A 20 hours before surgery and switched to the control diet after surgery. RESULTS: Ten days after resection, vitamin A-deficient rats exhibited a markedly blunted adaptive response. The adaptive increase in villus height and crypt depth was absent in the deficient rats. However, adaptive increases in crypt cell proliferation were not attenuated by vitamin A deficiency, and there were no differences in apoptotic indices. CONCLUSIONS: Vitamin A deficiency inhibits the adaptive response to partial small bowel resection, supporting a role for vitamin A in the adaptive process. Changes in cellular proliferation or programmed cell death are not sufficient to account for this inhibition. This model system will be useful for examining the role of other mechanisms, such as changes in cell-cell and cell-extracellular matrix interactions, and rates of epithelial cell migration and cell extrusion.

Diagnosis and management of small intestinal diseases.

Advances in the diagnosis and management of small bowel diseases are the subject of this review. Topics covered include improving the specificity of breath tests for bacterial overgrowth; small bowel enteroscopy; early diagnosis of mesenteric ischemia; the use of polymerase chain reaction for diagnosing central nervous system involvement in Whipple's disease; progress in defining the cause and pathogenesis of chronic idiopathic intestinal pseudoobstruction and the Peutz-Jeghers syndrome; defining the role of gut barrier function in health and disease; the therapeutic role for bile acid-binding resins in diarrhea after refeeding in critically ill patients; use of genetic techniques and topical steroid therapy in treating graft-versus-host disease; and the beneficial effects of combination hormonal therapy in occult gastrointestinal blood loss resulting from angiodysplasia.

Regulation of PC4/TIS7 expression in adapting remnant intestine after resection.

The adaptive response of the small intestine to loss of functional surface area includes enhanced crypt cell proliferation and enterocyte differentiation. To better define the underlying molecular and cellular mechanisms, we have cloned rat genes that are specifically regulated in the adaptive gut after 70% small intestinal resection. One of these is the immediate early gene PC4/TIS7. Compared with sham-resected control ileum, PC4/TIS7 mRNA levels in the adaptive remnant ileum were markedly increased at 16 and 48 h but not 1 wk after resection. Greater augmentation of PC4/TIS7 mRNA levels occurred in the ileum compared with the duodenum and proximal jejunum. After resection, the changes in intestinal PC4/TIS7 mRNA levels also exceeded changes in extraintestinal levels. The demonstration by in situ hybridization that villus-associated, but not crypt, cells express PC4/TIS7 mRNA is consistent with a role in regulating cytodifferentiation. The pattern of expression in the Caco-2 cell line is also consistent with such a role. Although the precise function of PC4/TIS7 in adaptation remains unclear, the early and intestine-specific changes in mRNA levels after 70% resection suggest that it might augment the adaptive response by stimulating the production of differentiated enterocytes.

Characterization of rat epimorphin/syntaxin 2 expression suggests a role in crypt-villus morphogenesis.

The rodent intestinal mucosa undergoes a remarkable morphogenesis as the crypt-villus axis is formed. Endoderm-mesenchymal interactions play a critical role in this process. Epimorphin is a mesenchymal protein postulated to play a role in lung and skin morphogenesis. The rat homologue, syntaxin 2, belongs to a family of integral membrane proteins that function in vesicle docking and fusion. To clarify its role in fetal gut morphogenesis, epimorphin expression was examined during ontogeny, in an isograft model of ischemic injury and mucosal repair, and during intestinal adaptation after small bowel resection. Epimorphin/syntaxin 2 mRNA levels were increased in fetal gut during lumen formation and villus morphogenesis. mRNA levels remained elevated in the first 2 wk after birth and then declined at weaning. In situ hybridization showed epimorphin/syntaxin 2 mRNA in gestational day 14 (G14) and G15 intestinal mesenchymal cells and in the mucosal lamina propria during villus formation. Epimorphin/syntaxin 2 mRNA expression increased during villus repair in the isograft. In contrast, in the early stages of intestinal adaptation after small bowel resection, epimorphin/syntaxin 2 mRNA expression was suppressed in the adapting gut. We conclude the cell-specific and temporal patterns of epimorphin expression in the models used in this study suggest a role in the morphogenesis of the crypt-villus axis.

Advances in nutrition and gastroenterology: summary of the 1997 A.S.P.E.N. Research Workshop.

BACKGROUND: The 1997 A.S.P.E.N. Research Workshop was held at the annual meeting in San Francisco, on January 26, 1997. The workshop focused on advances in clinical and basic research involving the interface between nutrient and luminal gastroenterology. METHODS: Presentations on the genetic regulation of gastrointestinal development, the molecular biology of small intestinal adaptation, the effect of nutrition support on intestinal mucosal mass, the relationship between nutrition and gastrointestinal motility, nutrient absorption, and gastrointestinal tract substrate metabolism were made by the preeminent leaders in the field. RESULTS: The investigators presented an insightful analysis of each topic by reviewing data from their own laboratories and the published literature. CONCLUSIONS: This workshop underscored the important interactions between nutrition and luminal gastroenterology at the basic science, metabolic/physiologic, and clinical levels. The integration of presentations from the different disciplines provided a unique interaction of information and ideas to advance our understanding of nutrition and gastrointestinal tract.

Retinoic acid stimulates early cellular proliferation in the adapting remnant rat small intestine after partial resection.

Following loss of small bowel surface area, the remnant intestine undergoes a remarkable adaptive response. To define more fully the underlying molecular mechanisms, we have identified genes that are specifically induced in the adapting remnant after partial small bowel resection. Several of these, including cellular retinol binding protein II (CRBP II) and apolipoprotein (apo) AI, participate in vitamin A and lipid trafficking. The CRBP II and apo A-I promoters contain response elements for the nuclear retinoid X receptor RXR-alpha. It is well established that vitamin A is essential for normal cell growth, differentiation and maintenance of epithelial tissues and that CRBP II functions to facilitate intestinal vitamin A absorption and metabolism. On the basis of these considerations, changes in CRBP II and apo A-I mRNA levels could reflect a role for retinoids in modulating the intestinal adaptive response. To explore this hypothesis, we used a rat resection model of intestinal adaptation to examine the temporal patterns of CRBP II, apo A-I and RXR-alpha expression postresection. CRBP II and apo A-I mRNA levels were increased in the remnant intestine in distinct temporal patterns, whereas RXR-alpha expression was unchanged. To address directly the effects of vitamin A in adaptation, retinoic acid or vehicle was administered intravenously to rats immediately after 70% small bowel resection. Compared with vehicle, all-trans-retinoic acid significantly stimulated crypt cell proliferation in the adapting remnant intestine by 6 h after surgery. These data suggest that retinoic acid acts to modulate intestinal proliferation in the adapting small intestine after loss of functional small bowel surface area.

Retinoic acid increases cellular retinol binding protein II mRNA and retinol uptake in the human intestinal Caco-2 cell line.

Cellular retinol binding protein II (CRBPII) is an abundant small intestinal protein that facilitates vitamin A trafficking and metabolism. The magnitude of retinol uptake and metabolism correlate to CRBPII levels in the human intestinal Caco-2 cell line. To investigate the importance of retinoic acid receptor response elements in the promoter of the CRBPII gene, retinoic acid regulation of CRBPII expression and vitamin A absorption was studied in differentiated Caco-2 cells. All-trans- or 9-cis-retinoic acid increased CRBPII mRNA levels two- to threefold. This was associated with a 50% increase in retinol absorption. Retinoic acid receptor beta and apolipoprotein A1 regulatory protein-1, two nuclear receptors that bind to the CRBPII promoter, were also induced, whereas other retinoid and orphan receptors were not. Thus, retinoic acid may regulate CRBPII expression directly or by selectively changing levels of nuclear receptors or other factors. These studies are the first to demonstrate that retinoic acid can modulate endogenous CRBPII mRNA levels and retinol absorption in Caco-2 cells and suggest that human intestinal vitamin A absorption may be regulated by retinoids.

Analysis of cloned cDNAs differentially expressed in adapting remnant small intestine after partial resection.

After partial resection, the remnant small intestine undergoes an adaptive response. Little is known about the molecular and cellular basis of intestinal adaptation. To identify genes transcriptionally regulated in response to loss of functional bowel surface area, we have isolated cDNAs differentially expressed in the adaptive ileum 48 h after 70% proximal small intestinal resection. A cDNA library constructed from the remnant ileum of rats subjected to resection was screened using subtractive hybridization techniques. Several groups of cDNA clones that were induced during intestinal adaptation were isolated. The first included liver fatty acid binding protein, apolipoprotein A-IV, cellular retinol binding protein II, and ileal lipid binding protein. These all encode proteins involved in the absorption, metabolism, and trafficking of nutrients. A second group included the catalytic subunit of protein phosphatase 1 delta, a 78-kDa glucose-regulated protein (grp 78; a glucose-regulated member of the 70-kDa heat-shock protein family), and several pancreatitis-associated proteins. A third group of induced genes contained novel cDNAs. To better characterize the adaptive response, the temporal, spatial, and cellular patterns of expression of several of these genes were analyzed with the use of immunohistochemical and in situ hybridization techniques. These studies indicate that during early adaptation, genes involved in nutrient trafficking, protein processing, and cell cycle regulation are transcriptionally regulated in the residual small intestine in distinct temporal and regional patterns consistent with a complex multifaceted response to intestinal resection.

Recent Dieulafoy's lesion bleeding and massive pulmonary emboli. Successful use of thrombolytic therapy.

A massive GI bleed secondary to a Dieulafoy's lesion developed in a 77-year-old woman. Fifteen days later, massive pulmonary emboli developed. We describe successful thrombolytic treatment of her pulmonary emboli without complicating GI hemorrhage.

Enterocytic gene expression in intestinal adaptation: evidence for a specific cellular response.

After massive small bowel resection, the remnant gut epithelium undergoes an adaptive response marked by an increase in villus height, crypt depth, and crypt cell production rate. Although morphological features of gut adaptation have been well characterized, the differentiation status and response of epithelial cells populating the adaptive villus is unclear. To address these issues, cell-specific and spatial patterns of expression of a set of enterocytic genes were characterized in rats after 70% small bowel resection. The liver and intestinal (I) fatty acid binding protein (FABP) and apolipoprotein A-I (apo A-I) and apo A-IV genes were studied because they exhibit unique regional and cell-specific patterns of expression in the developing and adult gut. At 48 h after surgery, apo A-IV and I-FABP mRNA levels were increased up to 3.5-fold in adaptive remnant ileum compared with sham-operated or sham-resected control ileum. In situ hybridization and immunohistochemical analyses revealed a marked increase in enterocytic apo A-IV mRNA and protein expression in the adaptive ileum, from villus base to tip but not in crypts. By 1 wk after resection, apo A-IV, but not I-FABP, mRNA levels remained elevated in remnant ileum, although duodenal I-FABP mRNA levels were still increased. In contrast, apo A-I mRNA levels were not significantly induced. These results indicate that the enterocyte can respond acutely to loss of small bowel surface area by increasing expression of several genes. This compensatory enterocytic response is spatially (from duodenum to ileum) and temporally regulated. These results suggest initiation of the adaptive response occurs by way of a complex set of molecular pathways involving villus and crypt cells.

Vitamin A trafficking in Caco-2 cells stably transfected with cellular retinol binding proteins.

During intestinal vitamin A absorption, retinol is esterified by long-chain fatty acids and secreted in chylomicron particles. Stable transfectants of the human intestinal Caco-2 cell line overexpressing cellular retinol binding protein II (CRBP II) or coexpressing CRBP II and CRBP were established to study their role in intestinal vitamin A trafficking. Compared with control cell lines, retinol uptake increased up to twofold by overexpression of CRBP II and up to 2.9-fold by coexpression of CRBP and CRBP II. Retinyl ester synthesis was increased proportionate to the increase in retinol absorption in all cell lines. Retinyl ester secretion was directly correlated with retinyl ester synthesis in control and CRBP II-transfected cell lines. However, transfection with CRBP increased the proportion secreted. Expression of CRBP and CRBP II also affected the polarity of retinyl ester secretion by increasing the proportion secreted basolaterally. Thus these studies provide evidence that intestinal retinol uptake, retinyl ester synthesis, and retinyl ester secretion are correlated with levels of CRBP and CRBP II and that the effects of CRBP on retinyl ester secretion can be distinguished from those of CRBP II.

Cellular retinol-binding proteins are determinants of retinol uptake and metabolism in stably transfected Caco-2 cells.

The mammalian small intestine contains two related cellular retinol-binding proteins, CRBP and CRBP II, which are thought to have distinct functions. The human intestinal cell line, Caco-2, was used as a model system for testing the hypothesis that intracellular levels of these proteins directly modulate the absorption and subsequent metabolism of retinol in enterocytes. Immunoblot and Northern blot hybridization demonstrated that Caco-2 cells express CRBP II and cellular retinoic acid-binding protein I in increasing amounts as the cells become more differentiated but do not express detectable quantities of CRBP. Stably transfected cloned Caco-2 cell lines that over-express CRBP II or coexpress CRBP and CRBP II and a control cell line that contains the expression vector without an insert were established. Retinol uptake and retinyl ester synthesis were increased up to 2-fold by coexpression of CRBP or over-expression of CRBP II. No significant differences were detected in the pattern of retinyl esters synthesized from exogenous [3H]retinol. This suggests that the fatty acid pools utilized for retinol esterification were the same despite differences in the CRBP and CRBP II phenotype of the cell lines. There were no differences between apical and basolateral [3H]retinol uptake or metabolism for filter grown transfected or wild type cell monolayers. Thus, neither CRBP II nor CRBP appear to preferentially interact with luminal- or plasma-derived retinol. Notably, in a cell line which over-expressed CRBP, endogenous CRBP II was reduced by 5-10-fold compared with the wild type and control cell lines. These studies indicate that CRBP and CRBP II levels are determinants of intracellular retinol accumulation and esterification, and they suggest that CRBP-bound retinol or a metabolite can regulate the expression of CRBP II in the mammalian intestine.

Trafficking of exogenous fatty acids within Caco-2 cells.

Dietary fatty acids (FAs) crossing the apical plasma membrane of small intestinal enterocytes are targeted to different metabolic pathways than serum FAs crossing the basolateral membrane. This apparent compartmentalization of FA metabolism in enterocytes was further investigated using a model human enterocyte-like intestinal cell line. [3H]Oleic acid bound to bovine serum albumin (BSA) was added to the apical or basolateral surfaces of confluent monolayers of Caco-2 cells growing on uncoated polycarbonate filters. In other experiments, [3H]oleic acid incorporated into micelles with taurocholate (+/- 2-monoacylglycerol) was added apically. Caco-2 cells absorbed oleic acid bound to BSA from both the apical and basolateral surfaces at the same rate. Oleic acid in micellar solution was absorbed more efficiently than oleic acid bound to BSA. Regardless of its site or mode of presentation, the majority of the incorporated oleic acid was found in triglycerides. Only a small fraction was subjected to beta-oxidation or esterification into phospholipids. Most of the incorporated oleic acid was still retained intracellularly at 24 h. The polarity of triglyceride secretion was influenced by the experimental conditions. Triglyceride secretion was not significantly polarized when oleic acid-BSA was presented apically. However, the ratio of basolateral to apical secretion at 24 h was 9:1 for oleic acid-BSA presented basolaterally. For oleic acid in taurocholate micelles there was a trend toward polarity of secretion to the apical media (apical to basolateral ratio = 2:1). The inclusion of 2-monoacylglycerol in oleic acid-taurocholate micelles did not augment triglyceride synthesis or secretion. These differences indicate that compartmentation of FA metabolism in Caco-2 cells is influenced by the site of FA presentation. Northern and Western blot hybridization studies indicated that the liver fatty acid-binding protein but not the intestinal fatty acid-binding protein gene is expressed in these cells. The absence of this latter 15 kDa protein indicates that it is not required by Caco-2 cells for the synthesis of triglycerides or for the polarized export of triglyceride. These studies indicate that the Caco-2 cell line will be a useful model system for studying the polarization of FA trafficking/metabolism in enterocytes and defining the role of intracellular fatty acid binding proteins in these processes.

Fluorine nuclear magnetic resonance analysis of the ligand binding properties of two homologous rat cellular retinol-binding proteins expressed in Escherichia coli.

Comparative 19F NMR studies were performed on rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBPII) to better understand their role in intracellular retinol metabolism within the polarized absorptive epithelial cells (enterocytes) of the intestine. Efficient incorporation of 6-fluorotryptophan (6-FTrp) into these homologous proteins was achieved by growing a tryptophan auxotroph of Escherichia coli, harboring prokaryotic expression vectors with either a full-length rat CRBPII or CRBP cDNA on defined medium supplemented with the analog. It is possible to easily distinguish resonances corresponding to 6-FTrp-apoCRBP, 6-FTrp-CRBP-retinol (or retinal), 6-FTrp-apoCRBPII, and 6-FTrp-CRBPII-retinol (or retinal). We were thus able to use 19F NMR spectroscopy to monitor transfer of all-trans-retinol and all-trans-retinal between CRBPII and CRBP in vitro. Retinol complexed to CRBPII is readily transferred to CRBP, whereas retinol complexed to CRBP is not readily transferred to CRBPII. We estimated that the Kd for CRBP-retinol is approximately 100-fold less than the Kd for CRBPII-retinol. Transfer of all-trans-retinal occurs readily from CRBPII to CRBP and from CRBP to CRBPII. Results from competitive binding studies with retinol and retinal indicated that there is a much larger difference between the affinities of CRBP for retinol and retinal than between the affinities of CRBPII for these two ligands. However, the differences in binding specificities reflect differences in how the two proteins interact with retinol, rather than with retinal. 19F NMR analysis of recombinant isotopically labeled proteins represents a sensitive new and useful method for monitoring retinoid flux between the CRBPs in vitro.

Developmental changes in the expression of genes involved in cholesterol biosynthesis and lipid transport in human and rat fetal and neonatal livers.

Cloned cDNAs encoding a number of enzymes involved in cholesterol biosynthesis as well as extracellular and intracellular lipid transport were used to compare the developmental maturation of these biologic functions in the fetal and neonatal rat and human liver. The results of RNA blot hybridization analyses indicate that steady-state levels of rat HMG-CoA synthase, HMG-CoA reductase and prenyl transferase mRNAs are highest in late fetal life and undergo precipitous (up to 80-fold) co-ordinate reductions immediately after parturition. These changes reflect the ability of the fetal rat liver to produce large quantities of cholesterol as well as the repression of this function during the suckling period in response to exogenous dietary cholesterol. Striking co-ordinate patterns of HMG-CoA synthase, reductase and prenyl-transferase mRNA accumulation were also observed in four extrahepatic rat tissues (brain, lung, intestine and kidney) during the perinatal period. The concentrations of all three mRNAs in the 8-week-old human fetal liver are similar to those observed throughout subsequent intrauterine development with less than 2-fold changes noted between the 8th through 25th weeks of gestation. Analysis of the levels of human apo AI, apo AII, apo B and liver fatty acid binding protein mRNAs during this period and in newborn liver specimens also indicated less than 2-3-fold changes. These observations suggest that the 8-week human liver has achieved a high degree of biochemical differentiation with respect to functions involved in lipid metabolism/transport which may be comparable to that present in 19-21 day fetal rat liver. Further analysis of human and rat fetal liver RNAs using cloned cDNAs should permit construction of a developmental time scale correlating hepatic biochemical differentiation to be constructed between these two mammalian species.