Proctocolectomy and ileal J-pouch anal anastomosis on the surgical treatment of familial adenomatous polyposis and ulcerative colitis: analysis of 49 cases

OBJECTIVE: To evaluate the results of ileal J-pouch anal anastomosis in ulcerative colitis and familial adenomatous polyposis. METHOD: Retrospective analysis of medical records of 49 patients submitted to ileal J-pouch anal anastomosis. RESULTS: Ulcerative colitis was diagnosed in 65% and familial adenomatous polyposis in 34%. Mean age was 39.5 years. 43% were male. Among familial adenomatous polyposis, 61% were diagnosed with colorectal cancer. Thirty-one percent of patients with ulcerative colitis was submitted to a previous surgical approach and 21% of these had toxic megacolon. Average hospital stay was 10 days. Post-operative complications occurred in 50% of patients with ulcerative colitis and 29.4% with familial adenomatous polyposis. Intestinal diversion was performed in 100% of ulcerative colitis and 88% of familial adenomatous polyposis. Pouchitis occurred in eight cases (seven ulcerative colitis and one FAP), requiring excision of the pouch in three ulcerative colitis. Mortality rate was 7.6%: two cases of carcinoma on the pouch and two post-operative complications. Late post-operative complications occurred in 22.4%: six familial adenomatous polyposis and five ulcerative colitis). Two patients had erectile dysfunction, and one retrograde ejaculation. One patient with severe perineal dermatitis was submitted to excision of the pouch. Incontinence occurred in four patients and two reported soil. Mean bowel movement was five times a day. CONCLUSION: Ileal J-pouch anal anastomosis is a safe surgery with acceptable morbidity and good functional results, if well indicated and performed in referral centers.

Resultados da cirurgia bariátrica e metabólica: gastrectomia vertical versus gastroplastia vertical com derivação em Y-de-Roux. Ensaio clínico prospectivo

Objetivos: Avaliar a equivalência da operação gastrectomia vertical com anel (GVA), em relação à operação gastroplastia vertical com anel e derivação gástrica em Y-de-Roux (DGA), na indução de perda ponderal e modificação da composição corporal em obesas mórbidas. Verificar os impactos laboratoriais e clínicos da GVA sobre as principais doenças associadas à obesidade mórbida, e a ocorrência de complicações, em comparação à DGA. Métodos: Ensaio clínico prospectivo não-randomizado, incluindo 65 mulheres obesas mórbidas, distribuídas em dois grupos, GVA (n = 33) e DGA (n = 32). Operadas consecutivamente, pelo mesmo cirurgião, por via laparotômica. Os parâmetros avaliados foram antropométricos; composição corporal, por meio de bioimpedância elétrica; laboratoriais; efeitos sobre as doenças pré-existentes e complicações. Resultados: Ocorreu perda de peso expressiva (p = 0,0000), redução do índice de massa corporal - IMC (p = 0,0000) e cintura abdominal (p = 0,0000) em ambos grupos. O índice cintura/quadril diminuiu (p = 0,0000) após ambas intervenções. A perda do excesso de IMC foi de 86,05% ± 14,2 no grupo GVA e 85,91 ± 15,71 no grupo DGA. A variação da gordura corporal foi de -35,84% ± 8,66 no grupo GVA e de -37,64% ± 9,62 no grupo DGA. A redução dos níveis de triglicerídios (p = 0,0222) foi mais expressiva no grupo DGA. O grupo DGA atingiu os alvos terapêuticos para o colesterol-LDL com maior freqüência (p = 0,0005), que o grupo GVA. Intolerância à glicose, diabetes mellitus tipo 2, hipertensão arterial sistêmica, esteatose hepática e síndrome metabólica, foram controladas de forma semelhante entre as técnicas. Anemia foi mais prevalente no grupo DGA (p=0,0033) e a esofagite erosiva, no grupo GVA (p = 0,0032). Não houve diferença na formação de cálculos biliares entre os grupos. Conclusões: A GVA é tão efetiva quanto a DGA em induzir perda ponderal e modificação favorável da composição corporal. A GVA é menos efetiva no controle da dislipidemia, em relação à DGA. GVA acarreta anemia em menor freqüência e, esofagite erosiva de maneira mais freqüente, que a DGA. GVA não é mais segura que a DGA, mas deve ser considerada intervenção bariátrica efetiva como segunda opção.

Impatto di rifaximina sul microbiota intestinale: selezione di bifidobatteri antibiotico resistenti

The ideal approach for the long term treatment of intestinal disorders, such as inflammatory bowel disease (IBD), is represented by a safe and well tolerated therapy able to reduce mucosal inflammation and maintain homeostasis of the intestinal microbiota. A combined therapy with antimicrobial agents, to reduce antigenic load, and immunomodulators, to ameliorate the dysregulated responses, followed by probiotic supplementation has been proposed. Because of the complementary mechanisms of action of antibiotics and probiotics, a combined therapeutic approach would give advantages in terms of enlargement of the antimicrobial spectrum, due to the barrier effect of probiotic bacteria, and limitation of some side effects of traditional chemiotherapy (i.e. indiscriminate decrease of aggressive and protective intestinal bacteria, altered absorption of nutrient elements, allergic and inflammatory reactions). Rifaximin (4-deoxy-4’-methylpyrido[1’,2’-1,2]imidazo[5,4-c]rifamycin SV) is a product of synthesis experiments designed to modify the parent compound, rifamycin, in order to achieve low gastrointestinal absorption while retaining good antibacterial activity. Both experimental and clinical pharmacology clearly show that this compound is a non systemic antibiotic with a broad spectrum of antibacterial action, covering Gram-positive and Gram-negative organisms, both aerobes and anaerobes. Being virtually non absorbed, its bioavailability within the gastrointestinal tract is rather high with intraluminal and faecal drug concentrations that largely exceed the MIC values observed in vitro against a wide range of pathogenic microorganisms. The gastrointestinal tract represents therefore the primary therapeutic target and gastrointestinal infections the main indication. The little value of rifaximin outside the enteric area minimizes both antimicrobial resistance and systemic adverse events. Fermented dairy products enriched with probiotic bacteria have developed into one of the most successful categories of functional foods. Probiotics are defined as “live microorganisms which, when administered in adequate amounts, confer a health benefit on the host” (FAO/WHO, 2002), and mainly include Lactobacillus and Bifidobacterium species. Probiotic bacteria exert a direct effect on the intestinal microbiota of the host and contribute to organoleptic, rheological and nutritional properties of food. Administration of pharmaceutical probiotic formula has been associated with therapeutic effects in treatment of diarrhoea, constipation, flatulence, enteropathogens colonization, gastroenteritis, hypercholesterolemia, IBD, such as ulcerative colitis (UC), Crohn’s disease, pouchitis and irritable bowel syndrome. Prerequisites for probiotics are to be effective and safe. The characteristics of an effective probiotic for gastrointestinal tract disorders are tolerance to upper gastrointestinal environment (resistance to digestion by enteric or pancreatic enzymes, gastric acid and bile), adhesion on intestinal surface to lengthen the retention time, ability to prevent the adherence, establishment and/or replication of pathogens, production of antimicrobial substances, degradation of toxic catabolites by bacterial detoxifying enzymatic activities, and modulation of the host immune responses. This study was carried out using a validated three-stage fermentative continuous system and it is aimed to investigate the effect of rifaximin on the colonic microbial flora of a healthy individual, in terms of bacterial composition and production of fermentative metabolic end products. Moreover, this is the first study that investigates in vitro the impact of the simultaneous administration of the antibiotic rifaximin and the probiotic B. lactis BI07 on the intestinal microbiota. Bacterial groups of interest were evaluated using culture-based methods and molecular culture-independent techniques (FISH, PCR-DGGE). Metabolic outputs in terms of SCFA profiles were determined by HPLC analysis. Collected data demonstrated that rifaximin as well as antibiotic and probiotic treatment did not change drastically the intestinal microflora, whereas bacteria belonging to Bifidobacterium and Lactobacillus significantly increase over the course of the treatment, suggesting a spontaneous upsurge of rifaximin resistance. These results are in agreement with a previous study, in which it has been demonstrated that rifaximin administration in patients with UC, affects the host with minor variations of the intestinal microflora, and that the microbiota is restored over a wash-out period. In particular, several Bifidobacterium rifaximin resistant mutants could be isolated during the antibiotic treatment, but they disappeared after the antibiotic suspension. Furthermore, bacteria belonging to Atopobium spp. and E. rectale/Clostridium cluster XIVa increased significantly after rifaximin and probiotic treatment. Atopobium genus and E. rectale/Clostridium cluster XIVa are saccharolytic, butyrate-producing bacteria, and for these characteristics they are widely considered health-promoting microorganisms. The absence of major variations in the intestinal microflora of a healthy individual and the significant increase in probiotic and health-promoting bacteria concentrations support the rationale of the administration of rifaximin as efficacious and non-dysbiosis promoting therapy and suggest the efficacy of an antibiotic/probiotic combined treatment in several gut pathologies, such as IBD. To assess the use of an antibiotic/probiotic combination for clinical management of intestinal disorders, genetic, proteomic and physiologic approaches were employed to elucidate molecular mechanisms determining rifaximin resistance in Bifidobacterium, and the expected interactions occurring in the gut between these bacteria and the drug. The ability of an antimicrobial agent to select resistance is a relevant factor that affects its usefulness and may diminish its useful life. Rifaximin resistance phenotype was easily acquired by all bifidobacteria analyzed [type strains of the most representative intestinal bifidobacterial species (B. infantis, B. breve, B. longum, B. adolescentis and B. bifidum) and three bifidobacteria included in a pharmaceutical probiotic preparation (B. lactis BI07, B. breve BBSF and B. longum BL04)] and persisted for more than 400 bacterial generations in the absence of selective pressure. Exclusion of any reversion phenomenon suggested two hypotheses: (i) stable and immobile genetic elements encode resistance; (ii) the drug moiety does not act as an inducer of the resistance phenotype, but enables selection of resistant mutants. Since point mutations in rpoB have been indicated as representing the principal factor determining rifampicin resistance in E. coli and M. tuberculosis, whether a similar mechanism also occurs in Bifidobacterium was verified. The analysis of a 129 bp rpoB core region of several wild-type and resistant bifidobacteria revealed five different types of miss-sense mutations in codons 513, 516, 522 and 529. Position 529 was a novel mutation site, not previously described, and position 522 appeared interesting for both the double point substitutions and the heterogeneous profile of nucleotide changes. The sequence heterogeneity of codon 522 in Bifidobacterium leads to hypothesize an indirect role of its encoded amino acid in the binding with the rifaximin moiety. These results demonstrated the chromosomal nature of rifaximin resistance in Bifidobacterium, minimizing risk factors for horizontal transmission of resistance elements between intestinal microbial species. Further proteomic and physiologic investigations were carried out using B. lactis BI07, component of a pharmaceutical probiotic preparation, as a model strain. The choice of this strain was determined based on the following elements: (i) B. lactis BI07 is able to survive and persist in the gut; (ii) a proteomic overview of this strain has been recently reported. The involvement of metabolic changes associated with rifaximin resistance was investigated by proteomic analysis performed with two-dimensional electrophoresis and mass spectrometry. Comparative proteomic mapping of BI07-wt and BI07-res revealed that most differences in protein expression patterns were genetically encoded rather than induced by antibiotic exposure. In particular, rifaximin resistance phenotype was characterized by increased expression levels of stress proteins. Overexpression of stress proteins was expected, as they represent a common non specific response by bacteria when stimulated by different shock conditions, including exposure to toxic agents like heavy metals, oxidants, acids, bile salts and antibiotics. Also, positive transcription regulators were found to be overexpressed in BI07-res, suggesting that bacteria could activate compensatory mechanisms to assist the transcription process in the presence of RNA polymerase inhibitors. Other differences in expression profiles were related to proteins involved in central metabolism; these modifications suggest metabolic disadvantages of resistant mutants in comparison with sensitive bifidobacteria in the gut environment, without selective pressure, explaining their disappearance from faeces of patients with UC after interruption of antibiotic treatment. The differences observed between BI07-wt e BI07-res proteomic patterns, as well as the high frequency of silent mutations reported for resistant mutants of Bifidobacterium could be the consequences of an increased mutation rate, mechanism which may lead to persistence of resistant bacteria in the population. However, the in vivo disappearance of resistant mutants in absence of selective pressure, allows excluding the upsurge of compensatory mutations without loss of resistance. Furthermore, the proteomic characterization of the resistant phenotype suggests that rifaximin resistance is associated with a reduced bacterial fitness in B. lactis BI07-res, supporting the hypothesis of a biological cost of antibiotic resistance in Bifidobacterium. The hypothesis of rifaximin inactivation by bacterial enzymatic activities was verified by using liquid chromatography coupled with tandem mass spectrometry. Neither chemical modifications nor degradation derivatives of the rifaximin moiety were detected. The exclusion of a biodegradation pattern for the drug was further supported by the quantitative recovery in BI07-res culture fractions of the total rifaximin amount (100 μg/ml) added to the culture medium. To confirm the main role of the mutation on the β chain of RNA polymerase in rifaximin resistance acquisition, transcription activity of crude enzymatic extracts of BI07-res cells was evaluated. Although the inhibition effects of rifaximin on in vitro transcription were definitely higher for BI07-wt than for BI07-res, a partial resistance of the mutated RNA polymerase at rifaximin concentrations > 10 μg/ml was supposed, on the basis of the calculated differences in inhibition percentages between BI07-wt and BI07-res. By considering the resistance of entire BI07-res cells to rifaximin concentrations > 100 μg/ml, supplementary resistance mechanisms may take place in vivo. A barrier for the rifaximin uptake in BI07-res cells was suggested in this study, on the basis of the major portion of the antibiotic found to be bound to the cellular pellet respect to the portion recovered in the cellular lysate. Related to this finding, a resistance mechanism involving changes of membrane permeability was supposed. A previous study supports this hypothesis, demonstrating the involvement of surface properties and permeability in natural resistance to rifampicin in mycobacteria, isolated from cases of human infection, which possessed a rifampicin-susceptible RNA polymerase. To understand the mechanism of membrane barrier, variations in percentage of saturated and unsaturated FAs and their methylation products in BI07-wt and BI07-res membranes were investigated. While saturated FAs confer rigidity to membrane and resistance to stress agents, such as antibiotics, a high level of lipid unsaturation is associated with high fluidity and susceptibility to stresses. Thus, the higher percentage of saturated FAs during the stationary phase of BI07-res could represent a defence mechanism of mutant cells to prevent the antibiotic uptake. Furthermore, the increase of CFAs such as dihydrosterculic acid during the stationary phase of BI07-res suggests that this CFA could be more suitable than its isomer lactobacillic acid to interact with and prevent the penetration of exogenous molecules including rifaximin. Finally, the impact of rifaximin on immune regulatory functions of the gut was evaluated. It has been suggested a potential anti-inflammatory effect of rifaximin, with reduced secretion of IFN-γ in a rodent model of colitis. Analogously, it has been reported a significant decrease in IL-8, MCP-1, MCP-3 e IL-10 levels in patients affected by pouchitis, treated with a combined therapy of rifaximin and ciprofloxacin. Since rifaximin enables in vivo and in vitro selection of Bifidobacterium resistant mutants with high frequency, the immunomodulation activities of rifaximin associated with a B. lactis resistant mutant were also taken into account. Data obtained from PBMC stimulation experiments suggest the following conclusions: (i) rifaximin does not exert any effect on production of IL-1β, IL-6 and IL-10, whereas it weakly stimulates production of TNF-α; (ii) B. lactis appears as a good inducer of IL-1β, IL-6 and TNF-α; (iii) combination of BI07-res and rifaximin exhibits a lower stimulation effect than BI07-res alone, especially for IL-6. These results confirm the potential anti-inflammatory effect of rifaximin, and are in agreement with several studies that report a transient pro-inflammatory response associated with probiotic administration. The understanding of the molecular factors determining rifaximin resistance in the genus Bifidobacterium assumes an applicative significance at pharmaceutical and medical level, as it represents the scientific basis to justify the simultaneous use of the antibiotic rifaximin and probiotic bifidobacteria in the clinical treatment of intestinal disorders.

L'infiammazione intestinale nell'animale sperimentale come modello per lo sviluppo di nuovi farmaci: ruolo della via tachichininergica nelle malattie infiammatorie intestinali

Background: Several lines of evidence showed that inflammation is associated with changes in the expression of tachykinins both in human and animal models. Tachykinins, including substance P (SP), are small peptides expressed in the extrinsic primary afferent nerve fibres and enteric neurons of the gut: they exert their action through three distinct receptors, termed NK1, NK2 and NK3. SP modulates intestinal motility and enteric secretion, acting preferentially through the NK1 receptor. SP neural network and NK1 receptor expression are increased in patients with inflammatory bowel disease, and similar changes were observed in experimental models of inflammation. The 2,4 Dinitrobenzene Sulphonic Acid (DNBS) model of colitis is useful to study innate immunity, non-specific inflammation and wound healing; it has been suggested that the transmural inflammation seen in this model resembles that found in Crohn’s disease and can therefore be used to study what cells and mediators are involved in this type of inflammation. Aim: To test the possible protective effect of the NK1 receptor antagonist SSR140333 on: 1) acute model of intestinal inflammation; 2) reactivation of DNBS-induced colitis in rats. Methods: Acute colitis was induced in male SD rats by intrarectal administration of DNBS (15 mg/rat in 50% ethanol). Reactivation of colitis was induced by intrarectal injections of DNBS on day 28 (7.5 mg/rat in 35% ethanol). Animals were sacrificed on day 6 (acute colitis) and 29 (reactivation of colitis). SSR140333 (10 mg/kg) was administered orally starting from the day before the induction of colitis for 7 days (acute colitis) or seven days before the reactivation of colitis. Colonic damage was assessed by means of macroscopic and microscopic scores, myeloperoxidase activity (MPO) and TNF-α tissue levels. Enzyme immunoassay was used to measure colonic substance P levels. Statistical analysis was performed using analysis of variance (one-way or two-way, as appropriate) with the Bonferroni’s correction for multiple comparisons. Results: DNBS administration impaired body weight gain and markedly increased all inflammatory parameters (p<0.01). Treatment with SSR140333 10 mg/kg significantly counteracted the impairment in body weight gain, decreased macroscopic and histological scores and reduced colonic myeloperoxidase activity (p<0.01). Drug treatment counteracted TNF-α tissue levels and colonic SP concentrations (acute model). Similar results were obtained administering the NK1 receptor antagonist SSR140333 (3 and 10 mg/kg) for 5 days, starting the day after the induction of colitis. Intrarectal administration of DNBS four weeks after the first DNBS administration resulted in reactivation of colitis, with increases in macroscopic and histological damage scores and increase in MPO activity. Preventive treatment with SSR140333 10 mg/kg decreased macroscopic damage score, significantly reduced microscopic damage score but did not affect MPO activity. Conclusions: Treatment with SSR140333 significantly reduced intestinal damage in acute model of intestinal inflammation in rats. The NK1 receptor antagonist SSR140333 was also able to prevent relapse in experimental colitis. These results support the hypothesis of SP involvement in intestinal inflammation and indicate that NK receptor antagonists may have a therapeutic potential in inflammatory bowel disease.

Espressione e ruolo funzionale di interleuchina-33 e del suo recettore, ST2, nelle malattie infiammatorie croniche intestinali

IL-33 is a novel member of the IL-1 family and ligand for the IL-1 receptor-related protein, ST2. Recent evidence suggests that the IL-33/ST2 axis plays a critical role in several autoimmune and inflammatory disorders; however, its role in inflammatory bowel disease (IBD) has not been clearly defined. We characterized IL-33 and ST2 expression and modulation following conventional anti-TNF therapy in Crohn’s disease and ulcerative colitis (UC) patients, and investigated the role of IL-33 in SAMP1/YitFc (SAMP) mice, a mixed Th1/Th2 model of IBD. Our results showed a specific increase of mucosal IL-33 in active UC, localized primarily to intestinal epithelial cells (IEC) and colonic inflammatory infiltrates. Importantly, increased expression of full-length IL-33, representing the most bioactive form, was detected in UC epithelium, while elevated levels of cleaved IL-33 were present in IBD serum. ST2 isoforms were differentially modulated in UC epithelium and sST2, a soluble decoy receptor with anti-inflammatory properties, was also elevated in IBD serum. Infliximab (anti-TNF) treatment of UC decreased circulating IL-33 and increased sST2, while stimulation of HT-29 IEC confirmed IL-33 and sST2 regulation by TNF. Similarly, IL-33 significantly increased and correlated with disease severity, and potently induced IL-5, IL-6 and IL-17 from mucosal immune cells in SAMP mice. Taken together, the IL-33/ST2 system plays an important role in IBD and experimental colitis, is modulated by anti-TNF therapy, and may represent a specific biomarker for active UC.

Ruolo di interleuchina-33 sull'espressione di geni profibrotici e sull'ipertrofia dei miofibroblasti nella fibrosi intestinale

Background: Intestinal fibrosis is a serious complication of IBD, with more than a third of Crohn’s disease (CD) patients developing a fibrostenosing phenotype with formation of strictures that will require surgical intervention. Remarkably, SAMP1/YitFc (SAMP) mice, a spontaneous model of CD, develop gut fibrosis; similar to IBD patients, the pathophysiology of SAMP fibrosis is unknown. IL-33 is a member of the IL-1 cytokine family and increased expression is associated with IBD. Emerging evidence suggests its potential role in liver and cutaneous fibrosis, as well as myofibroblast-associated colonic ulcerations . Aim: The aim of this study was to evaluate the role of IL-33 as a potential mediator of profibrotic events leading to intestinal fibrosis and possible stricture formation. Methods: A detailed histologic time course study, with collagen-specific Masson trichrome staining and IHC for ST2 (IL-33 receptor), was performed on SAMP and control AKR (parental strain) mice. qRT-PCR was done on full-thickness ilea for the profibrogenic genes, collagen (coll)-1, coll-3, connective tissue growth factor (CTGF) and insulin-like growth factor 1 (IGF-1). Exogenous IL-33 (33 μg/kg, i.p.) or vehicle was administered daily for 7d to SAMP and AKR mice (N=6/exp group), and ileal tissues evaluated as above. Finally, microarray analysis was performed on full-thickness ilea from SAMP and AKR mice, and IL-33 stimulated subepithelial myofibroblasts (SEMFs). Results: SAMP mice displayed ileal skip lesions with randomly distributed strictures, preceded by typical pre-stricture dilations of the ileum. Ileal wall was visibly thickened with hypertrophy of the serosa, muscularis mucosa, muscularis propria, within which intense collagen deposition was observed, and inflammatory infiltrates in segments showing strictures. Interestingly, intense ST2 staining was present within the inflamed lamina propria of SAMP, notably localized to SEMFs. Fibrosis was first observed at 20 wks, and reached its peak by 50 wks of age. mRNA expression of coll-1 (4.74±0.69-fold; P=0.001), coll-3 (4.92±1.05-fold; P=0.01), IGF1 (12.9±3.45; P=0.006), and CTGF (3.29±0.69; P=0.004) was dramatically elevated in SAMP vs. AKR ilea. IL-33 treatment of AKR mice induced a marked increase in muscle fiber/myofibroblast cellularity and hypertrophy of the muscularis propria (4.13±0.74-fold; P<0.0001), and mRNA expression of coll-1 (5.16±0.89-fold; P=0.0009), coll-3 (1.97±0.14-fold; P=0.01), IGF-1 (9.32±2.27-fold; P=0.004), and CTGF (1.43±0.31-fold; P=0.006) vs. vehicle controls. Microarray data from SAMP ilea and IL-33-treated SEMFs confirmed these trends, displaying a global increase in profibrogenic gene expression. Conclusion: These data suggest an important role for IL-33 in intestinal fibrosis, and may represent a potential target for the treatment of IBD-associated fibrosis and stricture formation.

Characterization of novel probiotics and prebiotics

The role of the human gut microbiota in impacting host’s health has been widely studied in the last decade. Notably, it has been recently demonstrated that diet and nutritional status are among the most important modifiable determinants of human health, through a plethora of presumptive mechanisms among which microbiota-mediated processes are thought to have a relevant role. At present, probiotics and prebiotics represent a useful dietary approach for influencing the composition and activity of the human gut microbial community. The present study is composed of two main sections, aimed at elucidating the probiotic potential of the yeast strain K. marxianus B0399, as well as the promising putative prebiotic activity ascribable to four different flours, naturally enriched in dietary fibres content. Here, by in vitro studies we demonstrated that K. marxianus B0399 possesses a number of beneficial and strain-specific properties desirable for a microorganism considered for application as a probiotics. Successively, we investigated the impact of a novel probiotic yoghurt containing B. animalis subsp. lactis Bb12 and K. marxianus B0399 on the gut microbiota of a cohort of subjects suffering from IBS and enrolled in a in vivo clinical study. We demonstrated that beneficial effects described for the probiotic yoghurt were not associated to significant modifications of the human intestinal microbiota. Additionally, using a colonic model system we investigated the impact of different flours (wholegrain rye and wheat, chickpeas and lentils 50:50, and barley milled grains) on the intestinal microbiota composition and metabolomic output, combining molecular and cellular analysis with a NMR metabolomics approach. We demonstrated that each tested flour showed peculiar and positive modulations of the intestinal microbiota composition and its small molecule metabolome, thus supporting the utilisation of these ingredients in the development of a variety of potentially prebiotic food products aimed at improving human health.

Decorso delle coliti severe: un confronto tra colite ulcerosa e di Crohn

Pochi sono i dati disponibili sul decorso clinico della malattia di Crohn del colon severa(CD). L'obiettivo è quello di descrivere il decorso clinico della colite di Crohn severa (CC) in una coorte di pazienti isolata con CD del colon o ileocolica, e di confrontarlo con il decorso clinico di pazienti affetti da colite ulcerosa severa (UC). 34 pazienti con CC severa sono stati identificati retrospettivamente nella nostra coorte di 593 pazienti ricoverati (2003-2012) attraverso la valutazione di CDAI score e HBI. 169 pazienti con UC severa sono stati identificati retrospettivamente in una coorte di 449 pazienti ricoverati (2003-2012) attraverso la valutazione del score di Lichtiger e di Truelove-Witts. Abbiamo valutato questi risultati: risposta agli steroidi, risposta ai farmaci biologici, tasso di colectomia acuta, tasso di colectomia durante il follow-up, megacolon e tasso di infezione da citomegalovirus. Non abbiamo trovato differenze significative nella risposta agli steroidi e biologici, della percentuale di infezione da citomegalovirus e di megacolon, mentre il tasso di colectomia in acuto è risultato essere maggiore nei pazienti con CC rispetto ai pazienti con UC; anche la differenza tra i tassi di colectomia alla fine del follow-up è risultata non significativa. Con l'analisi univariata la giovane età alla diagnosi è associata ad un aumentato rischio di colectomia in assoluto (p = 0,024) e in elezione (p = 0.022), ma non in acuto. Il tasso globale di colectomia nei pazienti con CC severa è superiore a quella dei pazienti con UC severa , ma questo dato non è supportato da una diversa risposta clinica alla terapia steroidea o terapia di salvataggio con biologici. Il vero decorso clinico della colite di Crohn severa necessita di essere chiarito da studi prospettici che includano un numero maggiore di pazienti con questo sottogruppo di malattia.

Beschleunigte postoperative Rekonvaleszenz bei Coloneingriffen durch Kaugummikauen und Periduralkatheter

Ein wesentliches Problem der elektiven Colonchirurgie ist die postoperative Darmträgheit. Sie wird von verschieden Faktoren wie der Manipulation am Darm, Ausschüttung von inflamatorischen Substanzen und durch perioperativ notwendige Medikamente wie morphinhaltige Analgetika ausgelöst und aufrechterhalten. Sie führt zu körperlichem Unwohlsein, einem geblähten Abdomen und gegebenenfalls zu Schmerzen. Die Patienten leiden unter Übelkeit und Erbrechen, fühlen sich müde, abgeschlagen und haben einen höheren Schmerzmittelverbrauch. Hierdurch wird die Motivation zur Mobilisation herabgesetzt und führt zu längerem Aufenthalt im Krankenbett, wodurch wiederum die Darmträgheit gefördert, die Rekonvaleszenz verlängert und die Patientenzufriedenheit gemindert wird. Eine längere Immobilisation steigert die Komplikationshäufigkeit, vor allem für thrombo-embolische Ereignisse.rnrnDurch die Einführung des Periduralkatheters (PDK) und der laparoskopischen Operationstechnik in die Colonchirurgie sind die Voraussetzungen zur Frühmobilisation verbessert worden.rnDeshalb und durch den wachsenden wirtschaftlichen Druck zu kürzeren Rekonvaleszenzzeiten und zur Optimierung der Bettenauslastung, sind in den letzten Jahren eine Vielzahl von Konzepten zur beschleunigten postoperativen Rekonvaleszenz erarbeitet worden, die unter dem Schlagwort „Fast Track“ bekannt geworden sind.rnWesentliche Punkte sind hierbei die längst mögliche Erhaltung des körpereigenen Gleichgewichts durch Verzicht auf Darmsäuberung und längere präoperative Nüchternheit. Intraoperativ werden flüssigkeitsrestriktive Infusionsschemata genutzt und auf eine Minimierung der intravenösen Dosierung von morphinhaltigen Analgetika geachtet. Postoperativ gehören die Frühmobilisation sowie die „Frühenterale –Ernährung“ zu den wichtigsten Merkmalen.rnDie Implementierung eines solchen Konzeptes als Standard in den laufenden Betrieb ist sehr aufwendig und teuer. Die individuelle Verträglichkeit der „Frühenteralen – Ernährung“ ist sehr unterschiedlich und wird oft von den Patienten aufgrund der postoperativen Darmträgheit nicht gut toleriert.rnrnEin neuer Ansatz hierfür ist das sogenannte „sham feeding“, also das Vortäuschen von Nahrungsaufnahme durch Kaugummikauen, das helfen soll die Paralyse des Darms postoperativ schneller, komplikationsärmer, sicherer und kostengünstiger zu beheben.rnrnZiel der vorliegenden Studie war es, die Effekte von Kaugummikauen sowie der Kombination von Kaugummikauen und Verwendung eines PDK in Hinblick auf postoperative Rekonvaleszenz, Darmtätigkeit und Patientenzufriedenheit bei unseren Patienten zu vergleichen, um einen Benefit des „sham feedings“ aufzuzeigen. Gleichzeitig sollten alle Einflußgrößen wie Mobilisation, Kostverträglichkeit, Schmerzmittelbedarf und Komplikationen mit erfasst werden, um die jeweilige Effektivität jeweils besser beurteilen zu können.rnDie vorliegende Arbeit soll die Frage beantworten, ob Patienten mit colonresizierenden Eingriffen postoperativ von „sham feeding“ durch kaugummikauen profitieren und ob dies standardmäßig genutzt werden sollte.rn

Identifizierung neuer potentieller Biomarker für das Kolonkarzinom sowie dessen Vorstufen

Zur Detektion von neuen potentiellen Biomarkerkandidaten für das KRK sowie dessen Vorstufen wurde Blutplasma von gesunden Kontrollen und von Patienten mit kolorektalen Adenomen und Karzinomen untersucht. Mit Hilfe immunoaffinitäts-basierter Chromatographiesäulen wurde das Blutplasma mit einer automatisierten Methode von hoch und moderat abundanten Proteinen entfernt und daraufhin massenspektrometrisch analysiert. Hierfür wurde eine labelfreie quantitative und datenunabhängige Methode angewendet. Nach Auswertung der Daten kristallisierten sich mehrere potentielle Biomarkerkandidaten heraus, wobei das Augenmerk auf solchen Proteinen lag, die sowohl bei Adenompatienten als auch bei Karzinompatienten vermehrt im Plasma vorlagen. Nach Western Blot Experimenten und einer weiteren Validierung mittels eines ELISAs an einem größeren Patientenset zeigte sich, dass die beiden Proteine AMBP und CRP als potentielle Biomarker zur Detektion von Adenomen und Karzinomen herangezogen werden konnten. Receiver Operating Charakteristik (ROC)-Analysen ergaben einen AUC-Wert von 0,79 für AMBP und 0,77 für CRP für das Erkennen von Adenomen und Karzinomen. Dies resultierte in einer Sensitivität von 72,5% für AMBP bzw. 60% für CRP bei einer Spezifität von 80%. Durch die Kombination beider Markerproteine erhielt man sogar eine noch höhere Sensitivität von 80% bei 80% Spezifität für das Aufdecken kolorektaler Neoplasien. Ebenso zeigte sich in der histochemischen Analyse, dass AMBP direkt von intestinalen Epithelzellen exprimiert wird.

Hemodynamic parameters change earlier than tissue oxygen tension in hemorrhage

BACKGROUND: Untreated hypovolemia results in impaired outcome. This study tests our hypothesis whether general hemodynamic parameters detect acute blood loss earlier than monitoring parameters of regional tissue beds. MATERIALS AND METHODS: Eight pigs (23-25 kg) were anesthetized and mechanically ventilated. A pulmonary artery catheter and an arterial catheter were inserted. Tissue oxygen tension was measured with Clark-type electrodes in the jejunal and colonic wall, in the liver, and subcutaneously. Jejunal microcirculation was assessed by laser Doppler flowmetry (LDF). Intravascular volume was optimized using difference in pulse pressure (dPP) to keep dPP below 13%. Sixty minutes after preparation, baseline measurements were taken. At first, 5% of total blood volume was withdrawn, followed by another 5% increment, and then in 10% increments until death. RESULTS: After withdrawal of 5% of estimated blood volume, dPP increased from 6.1% +/- 3.0% to 20.8% +/- 2.7% (P < 0.01). Mean arterial pressure (MAP), mean pulmonary artery pressure (PAP) and pulmonary artery occlusion pressure (PAOP) decreased with a blood loss of 10% (P < 0.01). Cardiac output (CO) changed after a blood loss of 20% (P < 0.05). Tissue oxygen tension in central organs, and blood flow in the jejunal muscularis decreased (P < 0.05) after a blood loss of 20%. Tissue oxygen tension in the skin, and jejunal mucosa blood flow decreased (P < 0.05) after a blood loss of 40% and 50%, respectively. CONCLUSIONS: In this hemorrhagic pig model systemic hemodynamic parameters were more sensitive to detect acute hypovolemia than tissue oxygen tension measurements or jejunal LDF measurements. Acute blood loss was detected first by dPP.

TNF suppresses acute intestinal inflammation by inducing local glucocorticoid synthesis

Although tumor necrosis factor (alpha) (TNF) exerts proinflammatory activities in a variety of diseases, including inflammatory bowel disease, there is increasing evidence for antiinflammatory actions of TNF. In contrast, glucocorticoids (GCs) are steroid hormones that suppress inflammation, at least in part by regulating the expression and action of TNF. We report that TNF induces extraadrenal production of immunoregulatory GCs in the intestinal mucosa during acute intestinal inflammation. The absence of TNF results in a lack of colonic GC synthesis and exacerbation of dextran sodium sulfate-induced colitis. TNF seems to promote local steroidogenesis by directly inducing steroidogenic enzymes in intestinal epithelial cells. Therapeutic administration of TNF induces GC synthesis in oxazolone-induced colitis and ameliorates intestinal inflammation, whereas inhibition of intestinal GC synthesis abrogates the therapeutic effect of TNF. These data show that TNF suppresses the pathogenesis of acute intestinal inflammation by promoting local steroidogenesis.

Lipopolysaccharide induces intestinal glucocorticoid synthesis in a TNFalpha-dependent manner

Stringent control of immune responses in the intestinal mucosa is critical for the maintenance of immune homeostasis and prevention of tissue damage, such as observed during inflammatory bowel disease. Intestinal epithelial cells, primarily thought to form a simple physical barrier, critically regulate intestinal immune cell functions by producing immunoregulatory glucocorticoids on T-cell activation. In this study we investigated whether stimulation of cells of the innate immune system results in the induction of intestinal glucocorticoids synthesis and what role TNF-alpha plays in this process. Stimulation of the innate immune system with lipopolysaccharide (LPS) led to an up-regulation of colonic steroidogenic enzymes and the induction of intestinal glucocorticoid synthesis. The observed induction was dependent on macrophage effector functions, as depletion of macrophages using clodronate-containing liposomes, but not absence of T and B cells, inhibited intestinal glucocorticoid synthesis. LPS-induced glucocorticoid synthesis was critically dependent on TNF-alpha as it was significantly decreased in TNF-alpha-deficient animals. Both TNF receptor-1 and -2 were found to be equally involved in LPS- and T-cell-induced intestinal GC synthesis. These results describe a novel and critical role of TNF-alpha in immune cell-induced intestinal glucocorticoid synthesis.

Enhanced activity of meprin-?, a pro-migratory and pro-angiogenic protease, in colorectal cancer

Meprin-α is a metalloprotease overexpressed in cancer cells, leading to the accumulation of this protease in a subset of colorectal tumors. The impact of increased meprin-α levels on tumor progression is not known. We investigated the effect of this protease on cell migration and angiogenesis in vitro and studied the expression of meprin-α mRNA, protein and proteolytic activity in primary tumors at progressive stages and in liver metastases of patients with colorectal cancer, as well as inhibitory activity towards meprin-α in sera of cancer patient as compared to healthy controls. We found that the hepatocyte growth factor (HGF)-induced migratory response of meprin-transfected epithelial cells was increased compared to wild-type cells in the presence of plasminogen, and that the angiogenic response in organ-cultured rat aortic explants was enhanced in the presence of exogenous human meprin-α. In patients, meprin-α mRNA was expressed in colonic adenomas, primary tumors UICC (International Union Against Cancer) stage I, II, III and IV, as well as in liver metastases. In contrast, the corresponding protein accumulated only in primary tumors and liver metastases, but not in adenomas. However, liver metastases lacked meprin-α activity despite increased expression of the corresponding protein, which correlated with inefficient zymogen activation. Sera from cancer patients exhibited reduced meprin-α inhibition compared to healthy controls. In conclusion, meprin-α activity is regulated differently in primary tumors and metastases, leading to high proteolytic activity in primary tumors and low activity in liver metastases. By virtue of its pro-migratory and pro-angiogenic activity, meprin-α may promote tumor progression in colorectal cancer.

Impact of CD39 and purinergic signalling on the growth and metastasis of colorectal cancer

Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.