Confocal laser endomicroscopy for upper tract urothelial carcinoma: validation of the proposed criteria and proposal of a scoring system for real-time tumor grading.

PURPOSE: Confocal laser endomicroscopy (CLE) is a fluorescence-based fiber-optic imaging technique with the potential for intraoperative grading of upper tract urothelial carcinoma (UTUC). This study aims to (1) investigate the prevalence of the previously proposed CLE criteria for bladder cancer in papillary UTUC, (2) estimate the diagnostic value of CLE for UTUC grading and (3) propose a scoring system for a more quantifiable approach of CLE-based grading of UTUC. MATERIALS AND METHODS: Ureteroscopic CLE was performed in patients with UTUC. Following CLE imaging, co-localized biopsies were taken for histopathologic comparison. Postoperatively, two blinded raters assessed the CLE images. RESULTS: Fifty-three papillary UTUCs (34 low grade and 19 high grade) were imaged with CLE in 36 patients. All the previously described CLE criteria were identifiable in varying proportions. After excluding 10 non-diagnostic recordings (5 low grade and 5 high grade) due to insufficient image quality, the histopathologic grade was correctly identified with CLE in 26 low-grade UTUCs (90%) and in 12 high-grade UTUCs (86%). The most prevalent CLE criteria with the highest diagnostic potential were cellular organization, morphology and cohesiveness of cells. A scoring system was proposed with these criteria, which yielded similar diagnostic accuracies. CONCLUSIONS: Based on the previously proposed criteria, CLE enables accurate grading of papillary UTUC at a non-diagnostic rate of 19%. The most prevalent CLE criteria with the highest diagnostic potential for grading of papillary UTUC are cellular organization, morphology and cohesiveness of cells. The proposed scoring system may simplify the assessment of CLE images for UTUC grading but external validation is required.

A FIB induced boiling mechanism for rapid nanopore formation.

Focused ion beam (FIB) technology is widely used to fabricate nanopores in solid-state membranes. These nanopores have desirable thermomechanical properties for applications such as high-throughput DNA sequencing. Using large scale molecular dynamics simulations of the FIB nanopore formation process, we show that there is a threshold ion delivery rate above which the mechanism underlying nanopore formation changes. At low rates nanopore formation is slow, with the rate proportional to the ion flux and therefore limited by the sputter rate of the target material. However, at higher fluxes nanopores form via a thermally dominated process, consistent with an explosive boiling mechanism. In this case, mass is rapidly rearranged via bubble growth and coalescence, much more quickly than would occur during sputtering. This mechanism has the potential to greatly speed up nanopore formation.

Psychometric properties of two implementation measures: Normalization MeAsure Development questionnaire (NoMAD) and organizational readiness for implementing change (ORIC).

Background: Effective interventions need to be implemented successfully to achieve impact. Two theory-based measures exist for measuring the effectiveness of implementation strategies and monitor implementation progress. The Normalization MeAsure Development questionnaire (NoMAD) explores the four core concepts (Coherence, Cognitive Participation, Collective Action, Reflexive Monitoring) of the Normalization Process Theory. The Organizational Readiness for Implementing Change (ORIC) is based on the theory of Organizational Readiness for Change, measuring organization members' psychological and behavioral preparedness for implementing a change. We examined the measurement properties of the NoMAD and ORIC in a multi-national implementation effectiveness study. Method: Twelve mental health organizations in nine countries implemented Internet-based cognitive behavioral therapy (iCBT) for common mental disorders. Staff involved in iCBT service delivery (n = 318) participated in the study. Both measures were translated into eight languages using a standardized forward-backward translation procedure. Correlations between measures and subscales were estimated to examine convergent validity. The theoretical factor structures of the scales were tested using confirmatory factor analysis (CFA). Test-retest reliability was based on the correlation between scores at two time points 3 months apart. Internal consistency was assessed using Cronbach's alpha. Floor and ceiling effects were quantified using the proportion of zero and maximum scores. Results: NoMAD and ORIC measure related but distinct latent constructs. The CFA showed that the use of a total score for each measure is appropriate. The theoretical subscales of the NoMAD had adequate internal consistency. The total scale had high internal consistency. The total ORIC scale and subscales demonstrated high internal consistency. Test-retest reliability was suboptimal for both measures and floor and ceiling effects were absent. Conclusions: This study confirmed the psychometric properties of the NoMAD and ORIC in multi-national mental health care settings. While measuring on different but related aspects of implementation processes, the NoMAD and ORIC prove to be valid and reliable across different language settings.

Why was the study done?: Effective interventions need to be implemented successfully to achieve impact. Reliable measurement instruments are needed to determine if an implementation was successful or not. Two theory-based instruments exist for measuring the effectiveness of implementation strategies and monitor progress. The NoMAD measures aspects of normalization related to sense-making, willingness to implement, the work people do, and reflection. The Organizational Readiness for Implementing Change (ORIC) measures organization members' preparedness for implementing a change. What did we do?: This study examined whether the NoMAD and ORIC measure what they are supposed to measure. We translated the instruments from English to eight languages (Albanian, Danish, Dutch, French, German, Italian, and Spanish/Catalan) We applied various statistical methods to confirm the measurement properties, including correlations of scales, factor structures, test­retest reliability, consistency and floor and ceiling effects. 318 mental health professionals from nine countries participated in the study. What did we find?: For both instruments, total scores can be used as well as the subscale scores. Internal consistency for ORIC was high and for NoMAD adequate. Test­retest reliability was demonstrated, and floor and ceiling effects were rare. What does this mean?: NoMAD and ORIC are reliable instruments for measuring implementation processes and outcomes across mental health care settings in different countries and languages. They measure related but different aspects of implementation processes and outcomes. The measures are brief, and theory supported. However, more work is to be done on interpreting scores in relation to implementation success and regarding changes over time.

Clinical implementation of artificial-intelligence-assisted detection of breast cancer metastases in sentinel lymph nodes: the CONFIDENT-B single-center, non-randomized clinical trial.

Pathologists' assessment of sentinel lymph nodes (SNs) for breast cancer (BC) metastases is a treatment-guiding yet labor-intensive and costly task because of the performance of immunohistochemistry (IHC) in morphologically negative cases. This non-randomized, single-center clinical trial (International Standard Randomized Controlled Trial Number:14323711) assessed the efficacy of an artificial intelligence (AI)-assisted workflow for detecting BC metastases in SNs while maintaining diagnostic safety standards. From September 2022 to May 2023, 190 SN specimens were consecutively enrolled and allocated biweekly to the intervention arm (n = 100) or control arm (n = 90). In both arms, digital whole-slide images of hematoxylin-eosin sections of SN specimens were assessed by an expert pathologist, who was assisted by the 'Metastasis Detection' app (Visiopharm) in the intervention arm. Our primary endpoint showed a significantly reduced adjusted relative risk of IHC use (0.680, 95% confidence interval: 0.347-0.878) for AI-assisted pathologists, with subsequent cost savings of ~3,000 €. Secondary endpoints showed significant time reductions and up to 30% improved sensitivity for AI-assisted pathologists. This trial demonstrates the safety and potential for cost and time savings of AI assistance.

Enteropathogenic e.coli sustains iodoacetamide-induced ulcerative colitis-like colitis in rats: modulation of IL-1ß, IL-6, TNF-α, COX-2, and apoptosisi.

Pathogenic or non-pathogenic bacteria from flora may play a key role in inflammatory bowel disease (IBD) pathogenesis. However, a specific infectious agent causing IBD has not been identified. This study assessed the impact of enteropathogenic E. coli (EPEC) on the modulation of IL-1beta, IL-6, TNF- alpha, COX-2, BAX and Bcl-2 expression, in sustaining inflammation of a rat colitis model. Two hundred male Sprague-Dawley rats (4 groups) were inoculated weekly or bi-weekly for 70 days, with 1 percent methylcellulose (MC), (b) 6 percent iodoacetamide (IA) in 1 percent MC, (c) 4x108 CFU of EPEC, and (d) IA+EPEC. After a month, treatment was stopped in half of the animals in each group. IL-1beta, IL-6, TNF-alpha, COX-2, BAX and Bcl-2 expression were measured in colonic mucosa scrapings. IL-1beta, IL-6, TNF-alpha, and COX-2 were significantly increased in colonic mucosa of the IA+EPEC group and to a lesser but significant level in the IA group compared to controls, or EPEC alone, both in continued and discontinued treatment groups. Additionally, the BAX/Bcl-2 ratio decreased, indicating less apoptosis in the IA+EPEC group which exhibited more necrosis. These effects increased with experiment duration. This work provides new arguments favouring the role of bacteria in IBD pathogenesis.

A family history of type 2 diabetes increases risk factors associated with overfeeding.

AIMS/HYPOTHESIS: The purpose of the study was to test prospectively whether healthy individuals with a family history of type 2 diabetes are more susceptible to adverse metabolic effects during experimental overfeeding. METHODS: We studied the effects of 3 and 28 days of overfeeding by 5,200 kJ/day in 41 sedentary individuals with and without a family history of type 2 diabetes (FH+ and FH- respectively). Measures included body weight, fat distribution (computed tomography) and insulin sensitivity (hyperinsulinaemic-euglycaemic clamp). RESULTS: Body weight was increased compared with baseline at 3 and 28 days in both groups (p < 0.001), FH+ individuals having gained significantly more weight than FH- individuals at 28 days (3.4 +/- 1.6 vs 2.2 +/- 1.4 kg, p < 0.05). Fasting serum insulin and C-peptide were increased at 3 and 28 days compared with baseline in both groups, with greater increases in FH+ than in FH- for insulin at +3 and +28 days (p < 0.01) and C-peptide at +28 days (p < 0.05). Fasting glucose also increased at both time points, but without a significant group effect (p = 0.1). Peripheral insulin sensitivity decreased in the whole cohort at +28 days (54.8 +/- 17.7 to 50.3 +/- 15.6 micromol min(-1) [kg fat-free mass](-1), p = 0.03), and insulin sensitivity by HOMA-IR decreased at both time points (p < 0.001) and to a greater extent in FH+ than in FH- (p = 0.008). Liver fat, subcutaneous and visceral fat increased similarly in the two groups (p < 0.001). CONCLUSIONS: Overfeeding induced weight and fat gain, insulin resistance and hepatic fat deposition in healthy individuals. However, individuals with a family history of type 2 diabetes gained more weight and greater insulin resistance by HOMA-IR. The results of this study suggest that healthy individuals with a family history of type 2 diabetes are predisposed to adverse effects of overfeeding. TRIAL REGISTRATION: ClinicalTrials.gov NCT00562393 FUNDING: The study was funded by the National Health and Medical Research Council (NHMRC), Australia (no. #427639).

Expression and localisation of insulin receptor substrate 2 in normal intestine and colorectal tumours. Regulation by intestine-specific transcription factor CDX2.

BACKGROUND AND AIMS: Self-renewal and differentiation of intestinal epithelium is a tightly regulated process, whose perturbations are implicated in human colorectal tumourigenesis. The insulin/insulin-like growth factor (IGF) signalling pathway may play an important role in intestinal epithelium homeostasis. Insulin receptor substrate 2 (IRS2) is a poorly characterised component in this pathway. METHODS: Using complementary in vitro and in vivo human and murine models, expression (mRNA and protein levels), localisation (immunohistochemistry) and regulation of IRS2 were investigated in the normal intestine and colorectal tumours. In silico analysis of the human IRS2 promoter was performed together with reporter and chromatin immunoprecipitation assays. RESULTS: Significant IRS2 expression was detected in the intestine, with specific protein localisation in the villus region of the ileum and in the surface epithelium of the colon. In human HT29 and Caco2 cells, IRS2 mRNA levels increased with spontaneous and induced differentiation, together with CDX2 (caudal-related homeobox protein 2), P21 and KLF4 (Krüppel-like factor 4). Adenoviral infection with human CDX2 induced IRS2 expression in APC- (adenomatous polyposis coli) and beta-catenin-mutated cells. On the other hand, IRS2 downregulation was observed in differentiated enterocytes after adenoviral infection with short hairpin CDX2 (shCDX2), in the intestine of CDX2 heterozygous mice and in colorectal tumours of Apc(Min/+) and patients with familial adenomatous polyposis (FAP). The human IRS2 promoter region presents several CDX2-binding sites where CDX2 immunoprecipitated in vivo. IRS2 reporters were functionally activated via CDX2 and blocked via a dominant-negative CDX2 protein. CONCLUSIONS: Combining gain- and loss-of-function approaches, an intriguing scenario is presented whereby IRS2 is significantly expressed in the apical intestinal compartment and is directly controlled by CDX2 in normal intestine and tumours.

Multiple levels of control of the stage- and region-specific expression of rat intestinal lactase.

To elucidate the mechanisms leading to the functional regionalization of the digestive epithelium, lactase expression was analyzed at the protein, mRNA and gene levels, along the intestinal tract at various stages of the rat postnatal development. In the colon of neonates, the transient expression of mRNA and enzyme correlated well with gene transcription. In contrast to the colon, complex patterns were observed in the small intestine. In suckling animals, the mRNA was present at a high level despite the progressive decline of enzyme activity. Crypts were devoid of mRNA and the transcript mainly accumulated in the lower half of the villi. From weaning onwards, a functional regionalization of the epithelium was defined, characterized by the modification of the longitudinal distribution of lactase mRNA. Indeed the transcript remained abundant in the distal duodenum, jejunum and proximal ileum, but decreased in the proximal duodenum and became virtually absent in the distal ileum. Concomitantly, the mRNA and enzyme distribution along the villi changed in the different segments of the small intestine. Patterns similar to those described in sucklings were retained in the adult jejunum. In contrast, mRNA and enzyme could no longer be detected in the distal ileum, while mosaicism appeared in the proximal duodenum. In vitro transcription assays carried out with isolated nuclei suggested that the decay of lactase mRNA in the proximal duodenum at weaning was associated with a decreasing rate of transcription of the gene. However active gene transcription was retained in the nuclei of the adult jejunum and ileum. The loss of mRNA in the adult distal ileum despite the maintenance of active transcription did not result from an intragenic block of pre-RNA elongation, as shown by transcription assays carried out at various positions of the lactase gene. In addition, we looked for the ontogenic decline of lactase protein despite the maintenance of a high amount of mRNA in the jejunum, and it became evident that the fraction of mRNA present in polysomes was constant with age. Taken together, these data indicate that lactase constitutes an unusual marker of development and of functional regionalization of the intestinal tract which exhibits a complex time- and space-specific pattern of gene, mRNA, and protein expression. The distinct patterns occurring in the duodenum, jejunum, ileum, and the colon of pre- and postweaned rats depend on a combination of transcriptional, posttranscriptional, and posttranslational levels of regulation. and are associated with a different mRNA distribution along villi in each intestinal segment.

Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development.

In rodents, the intestinal tract progressively acquires a functional regionalization during postnatal development. Using lactase-phlorizin hydrolase as a marker, we have analyzed in a xenograft model the ontogenic potencies of fetal rat intestinal segments taken prior to endoderm cytodifferentiation. Segments from the presumptive proximal jejunum and distal ileum grafted in nude mice developed correct spatial and temporal patterns of lactase protein and mRNA expression, which reproduced the normal pre- and post-weaning conditions. Segments from the fetal colon showed a faint lactase immunostaining 8-10 d after transplantation in chick embryos but not in mice; it is consistent with the transient expression of this enzyme in the colon of rat neonates. Heterotopic cross-associations comprising endoderm and mesenchyme from the presumptive proximal jejunum and distal ileum developed as xenografts in nude mice, and they exhibited lactase mRNA and protein expression patterns that were typical of the origin of the endodermal moiety. Endoderm from the distal ileum also expressed a normal lactase pattern when it was associated to fetal skin fibroblasts, while the fibroblasts differentiated into muscle layers containing alpha-smooth-muscle actin. Noteworthy, associations comprising colon endoderm and small intestinal mesenchyme showed a typical small intestinal morphology and expressed the digestive enzyme sucrase-isomaltase normally absent in the colon. However, in heterologous associations comprising lung or stomach endoderm and small intestinal mesenchyme, the epithelial compartment expressed markers in accordance to their tissue of origin but neither intestinal lactase nor sucrase-isomaltase. A thick intestinal muscle coat in which cells expressed alpha-smooth-muscle actin surrounded the grafts. The results demonstrate that: (a) the temporal and positional information needed for intestinal ontogeny up to the post-weaning stage results from an intrinsic program that is fixed in mammalian fetuses prior to endoderm cytodifferentiation; (b) this temporal and positional information is primarily carried by the endodermal moiety which is also able to change the fate of heterologous mesodermal cells to form intestinal mesenchyme; and (c) the small intestinal mesenchyme in turn may deliver instructive information as shown in association with colonic endoderm; yet this effect is not obvious with nonintestinal endoderms.

Type IV collagen mRNA accumulates in the mesenchymal compartment at early stages of murine developing intestine.

The expression of type IV collagen mRNA during mouse intestinal morphogenesis was examined by in situ hybridization using a cDNA probe corresponding to mRNA for alpha 1 (IV) chain. Type IV collagen mRNA is detected in the embryonic mesenchymal cells at early stages of development (12 d of gestation). A segregation of mesenchymal cells expressing high levels of type IV collagen mRNA in close vicinity of the epithelium occurs just before villus formation. During villus outgrowth, type IV collagen mRNA, still confined to mesenchyme-derived tissues, is progressively restricted to the mucosal connective tissue (the lamina propria) and to a lesser extent to the muscular layers. In the adult, the amount of messenger is quite low as compared to the level found in the developing intestine and the in situ hybridization signal, indistinguishable from the background, is uniform throughout the whole intestinal wall. At all developmental stages no detectable specific hybridization signal is virtually observed over the epithelium cell layer. These results show that high amounts of the type IV collagen messenger are detected during phases of intensive morphogenetic events. Furthermore, they reinforce the notion already gained previously (Simon-Assmann et al. 1988) that the mesenchymal compartment is the principal endogenous source of type IV collagen. They also indicate that the continuous migration of epithelial cells along the basement membrane of intestinal villi in the mature organ is not accompanied by a significant remodeling of the collagen IV network.

Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit, laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, an homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. In the present study, we demonstrate that the differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, we analyzed a series of Caco2 clones in which the production of laminin-alpha1 chain is differentially inhibited by antisense RNA. We found a positive correlation between the level of Cdx2 expression, that of endogenous laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (a) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium, (b) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (c) that Cdx2 expression is modulated by basement membrane components. Hence, we conclude that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cell differentiation.

A multiscale crater function model for ion-induced pattern formation in silicon.

The ion-induced formation of nanometer-scale ripples on semiconductors, long known as the sputter erosion surface instability, is explained using a coupled atomistic-continuum framework. Molecular dynamics simulations of individual medium energy ion impacts on an amorphous silicon target show that the average effect of an incident ion is to leave an ångström-scale crater-like impression on the surface, complete with a crater rim. The summation of many such impacts on a micron-scale surface, combined with the smoothing effect of surface diffusion, leads to the formation of surface ripples aligned perpendicular to the projected ion beam direction. The same numerical approach can be used to evaluate the standard analytical model for this process, known as the Bradley-Harper model. Both Bradley-Harper surface evolution and the atomistically determined crater function surface evolution are computed over time under conditions similar to those for known experimental data. The results show that the surface mass rearrangement associated with the finite atomistic crater rims explains a key experimental observation, ripple amplitude saturation, which cannot be accurately explained using the Bradley-Harper model or any other known numerical or analytical model for the sputter erosion surface instability.

Shock-induced bubble jetting into a viscous fluid with application to tissue injury in shock-wave lithotripsy.

Shock waves in liquids are known to cause spherical gas bubbles to rapidly collapse and form strong re-entrant jets in the direction of the propagating shock. The interaction of these jets with an adjacent viscous liquid is investigated using finite-volume simulation methods. This configuration serves as a model for tissue injury during shock-wave lithotripsy, a medical procedure to remove kidney stones. In this case, the viscous fluid provides a crude model for the tissue. It is found that for viscosities comparable to what might be expected in tissue, the jet that forms upon collapse of a small bubble fails to penetrate deeply into the viscous fluid "tissue." A simple model reproduces the penetration distance versus viscosity observed in the simulations and leads to a phenomenological model for the spreading of injury with multiple shocks. For a reasonable selection of a single efficiency parameter, this model is able to reproduce in vivo observations of an apparent 1000-shock threshold before wide-spread tissue injury occurs in targeted kidneys and the approximate extent of this injury after a typical clinical dose of 2000 shock waves.

Sprouty2 inhibits BDNF-induced signaling and modulates neuronal differentiation and survival.

Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.

The effects of high-intensity intermittent exercise training on fat loss and fasting insulin levels of young women.

OBJECTIVE: To determine the effects of a 15-week high-intensity intermittent exercise (HIIE) program on subcutaneous and trunk fat and insulin resistance of young women. DESIGN AND PROCEDURES: Subjects were randomly assigned to one of the three groups: HIIE (n=15), steady-state exercise (SSE; n=15) or control (CONT; n=15). HIIE and SSE groups underwent a 15-week exercise intervention. SUBJECTS: Forty-five women with a mean BMI of 23.2+/-2.0 kg m(-2) and age of 20.2+/-2.0 years. RESULTS: Both exercise groups demonstrated a significant improvement (P<0.05) in cardiovascular fitness. However, only the HIIE group had a significant reduction in total body mass (TBM), fat mass (FM), trunk fat and fasting plasma insulin levels. There was significant fat loss (P<0.05) in legs compared to arms in the HIIE group only. Lean compared to overweight women lost less fat after HIIE. Decreases in leptin concentrations were negatively correlated with increases in VO(2peak) (r=-0.57, P<0.05) and positively correlated with decreases in TBM (r=0.47; P<0.0001). There was no significant change in adiponectin levels after training. CONCLUSIONS: HIIE three times per week for 15 weeks compared to the same frequency of SSE exercise was associated with significant reductions in total body fat, subcutaneous leg and trunk fat, and insulin resistance in young women.

Different effects of the Cdx1 and Cdx2 homeobox genes in a murine model of intestinal inflammation.

AIMS: The CDX1 and CDX2 homeoproteins are intestine-specific transcription factors regulating homeostasis. We investigated their relevance in experimentally-induced intestinal inflammation. METHODS: The response to intestinal inflammation induced by dextran sodium sulfate (DSS) was compared in wild type, Cdx1(-/-) and Cdx2(+/-) mice. Intestinal permeability was determined in wild type and Cdx2(+/-) mice. Protein-protein interactions were investigated by co-immunoprecipitation and GST-pulldown, and their functional consequences were assessed using Luciferase reporter systems. RESULTS: Heterozygous Cdx2(+/-) mice, but not Cdx1(-/-) mice, were hypersensitive to DSS-induced acute inflammation as all these mice showed blood in the stools at day 1 of DSS treatment. Hypersensitivity was associated to a 50% higher intestinal permeability. In Cdx2(+/-) mice, the colonic epithelium was repaired during the week after the end of DSS treatment, whereas two weeks were required for wild type animals. Subsequently, no colonic tumour was observed in Cdx2(+/-) mice subjected to 5 repeated cycles of DSS, in contrast to the 2.7 tumours found per wild type mouse. Based on the fact that Smad3(+/-) mice, like Cdx2(+/-) mice, better repair the damaged intestinal epithelium, we found that the CDX2 protein interacts with SMAD3, independently of SMAD4, resulting in a 5-fold stimulation of SMAD3 transcriptional activity. CDX1 also interacted with SMAD3 but it inhibited by 10-fold the SMAD3/SMAD4-dependent transcription. CONCLUSION: The Cdx1 and Cdx2 homeobox genes have distinct effects on the outcome of a pro-inflammatory challenge. This is mirrored by different functional interactions of the CDX1 and CDX2 proteins with SMAD3, a major element of the TGFbeta signalling pathway.

Advanced three-dimensional culture of equine intestinal epithelial stem cells.

BACKGROUND: Intestinal epithelial stem cells are critical to epithelial repair following gastrointestinal injury. The culture of intestinal stem cells has quickly become a cornerstone of a vast number of new research endeavours that range from determining tissue viability to testing drug efficacy for humans. This study aims to describe the methods of equine stem cell culture and highlights the future benefits of these techniques for the advancement of equine medicine. OBJECTIVES: To describe the isolation and culture of small intestinal stem cells into three-dimensional (3D) enteroids in horses without clinical gastrointestinal abnormalities. STUDY DESIGN: Descriptive study. METHODS: Intestinal samples were collected by sharp dissection immediately after euthanasia. Intestinal crypts containing intestinal stem cells were dissociated from the underlying tissue layers, plated in a 3D matrix and supplemented with growth factors. After several days, resultant 3D enteroids were prepared for immunofluorescent imaging and polymerase chain reaction (PCR) analysis to detect and characterise specific cell types present. Intestinal crypts were cryopreserved immediately following collection and viability assessed. RESULTS: Intestinal crypts were successfully cultured and matured into 3D enteroids containing a lumen and budding structures. Immunofluorescence and PCR were used to confirm the existence of stem cells and all post mitotic, mature cell types, described to exist in the horse intestinal epithelium. Previously frozen crypts were successfully cultured following a freeze-thaw cycle. MAIN LIMITATIONS: Tissues were all derived from normal horses. Application of this technique for the study of specific disease was not performed at this time. CONCLUSIONS: The successful culture of equine intestinal crypts into 3D "mini-guts" allows for in vitro studies of the equine intestine. Additionally, these results have relevance to future development of novel therapies that harness the regenerative potential of equine intestine in horses with gastrointestinal disease (colic).

Fluorescence in situ hybridization in 1 mL of selective urine for the detection of upper tract urothelial carcinoma: a feasibility study.

Kidney-sparing surgery of upper tract urothelial carcinoma (UTUC) requires a stringent follow-up with frequent ureteroscopies. Triage testing could reduce the number of follow-up ureteroscopies and hence minimize the invasiveness of follow-up. The use of urine-based markers for triage seems appealing but should be feasible with selective urine from outpatient cystoscopy to maximize the reduction of invasiveness. In this study, the feasibility of UroVysion® fluorescence in situ hybridization (FISH) for the detection of UTUC in 1 mL of selective urine is investigated. Ten consecutive patients with biopsy-proven UTUC and five patients with negative diagnostic ureteroscopy findings were included in this case-control study. During ureteroscopy, 1 mL of selective urine was collected passively with a ureteral splint for Urovysion® FISH. The FISH rater was blinded to any clinical information. The results of FISH were compared to the findings of concomitantly collected selective urine cytology and the patients' UTUC status. FISH was feasible in all samples with a sensitivity of 90% and a specificity of 80% for UTUC. In comparison, selective cytology resulted in a diagnostic yield of 87% with a sensitivity of 80% and a specificity of 67%. In conclusion, UTUC detection is feasible with FISH in 1 mL of passively collected selective urine. Thus from a technical point of view, FISH could be used as an outpatient triage test to decide if follow-up ureteroscopy is necessary after kidney-sparing surgery of UTUC. Evaluation of the diagnostic accuracy of FISH for the suggested pathway deserves further attention.

The route of estrogen replacement therapy confers divergent effects on substrate oxidation and body composition in postmenopausal women.

The route of estrogen replacement therapy has a major impact on the growth hormone (GH)/insulin-like growth factor-I (IGF-I) axis. Estrogen administration by the oral, but not the transdermal route, reduces IGF-I and increases GH levels in postmenopausal women. To investigate whether these perturbations have metabolic consequences, we compared the effects of 24 wk each of oral (Premarin 1.25 mg) and transdermal (Estraderm 100TTS) estrogen on energy metabolism and body composition in 18 postmenopausal women in an open-label randomized crossover study. Energy expenditure, lipid oxidation (lipid(ox)), and carbohydrate oxidation (CHOox) were measured by indirect calorimetry in the fasted and fed state before and after 2 and 6 mon treatment. Lean body mass, fat mass, and total body bone mineral density were measured by dual X-ray absorptiometry before and after 6 mon treatment. Mean (+/-SE) Luteinizing hormone levels fell to comparable levels during oral and transdermal estrogen, and bone mineral density was significantly increased by both treatments. Mean IGF-I was significantly lower during oral estrogen (77+/-7 versus 97+/-7 microg/liter, P < 0.05) treatment. Lipid(ox) 30-60 min after a standardized meal was significantly lower (36+/-5 versus 54+/-5 mg/min, P < 0.01) and CHOox higher (147+/-13 versus 109+/-12 mg/min, P < 0.05) with oral compared with transdermal estrogen. Oral estrogen resulted in a 1.2+/-0.5 kg (P < 0.05) increase in fat mass and a 1.2+/-0.4 kg (P < 0.01) decrease in lean mass compared with transdermal estrogen. Lean body mass (0.4+/-0.2 kg) and fat mass (0. 1+/-0.4 kg) did not change significantly during transdermal estrogen. In summary, when compared with the transdermal route, oral estrogen reduces lipid(ox), increases fat mass, and reduces lean body mass. The route of estrogen therapy confers distinct and divergent effects on substrate oxidation and body composition. The suppression of lipidox during oral estrogen therapy may increase fat mass although the fall in IGF-I may lead to a loss of lean body mass. The route-dependent changes in body composition observed during estrogen replacement therapy may have important implications for postmenopausal health.