Neurovascular alterations of muscularis propria in the human anterior vaginal wall in pelvic organ prolapse.

In the pathophysiology and progression of pelvic organ prolapse (POP), it has been demonstrated that there is a reorganisation of the muscularis propria of the anterior vaginal wall due to a phenotypic smooth muscle cell to myofibroblast switch. An abnormal deposition of collagen type III seems to be influenced by the involvement of advanced glycation end-products. The aim of the present study was to evaluate the hypothesis that this connective tissue remodelling could also be associated with neurovascular alterations of the muscularis in women with POP compared with control patients. We examined 30 women with POP and 10 control patients treated for uterine fibromatosis. Immunohistochemical analysis, using glial fibrillary acidic protein, S-100 protein, receptor tyrosine kinase, neurofilament and α-smooth muscle actin antibodies, was performed. S-100, receptor tyrosine kinase and neurofilament were also evaluated using Western blot analysis. We observed a decrease in all neurovascular-tested markers in nerve bundles, ganglia and interstitial cells of Cajal from POP samples as compared with controls. Even if the processes responsible for these morphological alterations are still not known, it is conceivable that collagen III deposition in the anterior vaginal wall affects not only the architecture of the muscle layer but could also modify the intramuscular neurovascularisation and account for an alteration of the neuromuscular plasticity of the layer.

PPAR-γ with its anti-inflammatory and anti-fibrotic action could be an effective therapeutic target in IBD.

OBJECTIVE: Intestinal fibrosis is a process characterized by an excessive deposition of Extracellular Matrix (ECM) proteins by activated myofibroblasts and represents a consequence of a chronic inflammation that usually occurs during Inflammatory Bowel Disease (IBD). The relationship between inflammation and fibrosis in IBD remains still unclear and nevertheless the recent pharmacological progresses, currently the only resolutive therapeutic strategy is surgery, especially when complications (stricture, stenosis and obstruction of intestinal tracts) appear. As many different cellular types and molecular mechanisms are implicated in the pathogenesis of IBD, the identification of molecules able to counteract this process could be crucial. MATERIALS AND METHODS: This is a literature review of several articles published on PubMed databases. RESULTS: A number of researches suggest that Proliferator-Activated Receptor-gamma (PPAR-γ) has both anti-inflammatory and anti-fibrotic effects in many organs. PPAR-γ has been demonstrated to be able to downregulate pro-inflammatory cytokines production such as Interleukin (IL)-4,-5,-6 but also to interfere with profibrotic molecules as Platelet-Derived Growth Factor (PDGF), IL-1 and Transforming Growth Factor Beta (TGF-ß), the main promoter of fibrosis. In preliminary clinical trials and in experimental models of intestinal fibrosis, natural and chemical PPAR-γ ligands have ameliorated the fibrotic process. CONCLUSIONS: Since PPAR-γ could play a crucial role in the development of the disease, the research of new molecules, capable of ameliorating both inflammation and fibrosis lesions, as PPAR-γ agonists, could represent a valid and effective therapeutic approach for the prevention and treatment of IBD and intestinal fibrosis.

Changes in muscularis propria of anterior vaginal wall in women with pelvic organ prolapse.

The objective of this study was to evaluate the morphological and immunohistochemical alterations of tissue removed from the upper third of anterior vaginal wall in a sample group of the female population presenting homogenous risk factors associated with Pelvic Organ Prolapse (POP). The case study consisted of 14 patients with POP and there were 10 patients in the control group. Patient selection was carried on the basis of specific criteria and all of the patients involved in the study presented one or more of the recognized POP risk factors. Samples were taken from POP patients during vaginal plastic surgery following  colpohysterectomy, and from control patients during closure of the posterior fornix following hysterectomy. Samples were processed for histological and  immunohistochemical analyses for Collagen I and Collagen III, α-Smooth Muscle Actin (α-SMA), Platelet-Derived-Growth-Factor (PDGF), matrix metalloproteinase 3 (MMP3), Caspase3. Immunofluorescence analyses for Collagen I and III and PDGF were also carried out. In prolapsed specimens our results show a disorganization of smooth muscle cells that appeared to have been displaced by an increased collagen III deposition resulting in rearrangement of the muscularis propria architecture. These findings suggest that the increase in the expression of collagen fibers in muscularis could probably due to a phenotypic switch resulting in the dedifferentiation of smooth muscle cells into myofibroblasts. These alterations could be responsible for the compromising of the dynamic functionality of the pelvic floor.

Contribution of intestinal smooth muscle to Crohn's disease fibrogenesis.

Mesenchymal cells transdifferentiation and extracellular matrix deposition are involved in the fibrotic process of Crohn's disease (CD). Mesenchymal smooth muscle cells (SMCs) de-differentiation, driven by Platelet-derived growth factor (PDGF) that counteracts Transforming growth factor (TGF-ß) has been studied in vascular muscle. The role of SMCs in intestinal fibrogenesis is still not clearly elucidated. Aim of the study was to evaluate the possible myogenic contribution to CD fibrotic process through the comparative analysis of histological, morphometric and molecular alterations occurring in human smooth muscle. Full thickness specimens were obtained from CD (non-involved and stenotic tracts) and healthy (control) ileum. Tissues were processed for histological and immunohistochemical (IHC) analyses and SMCs were isolated from the muscularis propria for morphofunctional and molecular (qPCR) analyses. CD stenotic ileum showed a significant increased thickness of all layers compared to CD non-involved and control ileum. IHC revealed an overexpression of α-smooth muscle actin and collagens I-III throughout all intestinal layers only in stenotic tracts. The two growth factors, PDGF and TGF-ß, showed a progressive increase in expression in the muscle layer from CD non-involved to stenotic tracts. Freshly isolated SMCs presented alterations in CD non-involved tracts that progressively increased in the stenotic tracts consisting in a statistical increase in mRNA encoding for PDGF-ß and collagen III, paralleled to a decrease in TGF-ß and Tribbles-like protein-3 mRNA, and altered morphofunctional parameters consisting in progressive decreases in cell length and contraction to acetylcholine. These findings indicate that intrinsic myogenic alterations occur in CD ileum, that they likely precede stricture formation, and might represent suitable new targets for anti-fibrotic interventions.

Localization of ανß6 integrin-TGF-ß1/Smad3, mTOR and PPARγ in experimental colorectal fibrosis.

A simultaneous action of several pro-fibrotic mediators appears relevant in the development of fibrosis. There are evidences that transforming growth factor-ß (TGF-ß)/Smad3 pathway forms with αvß6 integrin, mammalian target of Rapamycin (mTOR) and peroxisome proliferator-activated receptor-γ (PPARγ) a complex signalling network with extensive crosstalk and strong effects on fibrosis development. The present study evaluated the expression of TGFß, Smad3, αvß6 integrin, mTOR and PPARγ in 2,4,6-trinitrobenzenesulphonic acid (TNBS)-induced colorectal fibrosis in Smad3 wild-type (WT) and null mice. Smad3 WT mice treated with TNBS developed a marked colorectal fibrosis and showed a concomitant up-regulation of TGFß, Smad3, αvß6 and mTOR and a reduction of PPARγ expression. On the other hand, Smad3 Null mice similarly treated with TNBS did not develop fibrosis and showed a very low or even absent expression of TGFß, Smad3, αvß6 and mTOR and a marked over-expression of PPARγ. At the same time the expression of α-smooth muscle actin (a marker of activated myofibroblasts), collagen I-III and connective tissue growth factor (a downstream effector of TGFß/Smad3-induced extracellular matrix proteins) were up-regulated in Smad3 WT mice treated with TNBS compared to Null TNBS-treated mice. These preliminary results suggest a possible interaction between these pro-fibrotic molecules in the development of intestinal fibrosis.

Prevention of colonic fibrosis by Boswellia and Scutellaria extracts in rats with colitis induced by 2,4,5-trinitrobenzene sulphonic acid.

BACKGROUND: Currently, no effective preventive measures or medical therapies are available for intestinal fibrosis and, thus, surgery remains the only available strategy in the management of fibrostenotic enteropathies, especially Crohn's disease. The aim of this study was to evaluate the efficacy of a combined therapy of anti-inflammatory Boswellia and antifibrotic Scutellaria extracts on the development of colonic fibrosis in rats. MATERIALS AND METHODS: Chronic colonic inflammation-associated fibrosis was induced in rats by intracolonic administration of 2,4,5-trinitrobenzene sulphonic acid (TNBS). Sixty-four healthy male Sprague-Dawley rats were assigned to five groups: 8 controls, 14 TNBS, 14 TNBS orally treated with Boswellia extracts (50 mg kg(-1) day(-1)), 14 TNBS orally treated with Scutellaria extracts (150 mg kg(-1) day(-1)), and 14 TNBS orally treated with both Boswellia (50 mg kg(-1) day(-1)) and Scutellaria extracts (150 mg kg(-1) day(-1)). The colon was removed after 21 days of treatment and assessed by macroscopic, histological, morphometric and immunohistochemical analyses. For immunohistochemical analysis, alpha-smooth muscle actin (alpha-SMA), collagen types I-III, connective tissue growth factor (CTGF), transforming growth factor-beta1 (TGF-beta1), Smad3, Smad7 and CD3 antibodies were used. RESULTS: Combined oral administration of Boswellia and Scutellaria significantly improved the course and macroscopic findings of TNBS-induced chronic colitis assessed by disease activity index, colon weight, length, adhesions, strictures, dilatation, thickness, oedema, ulcerations and extension of damage. The histological severity of the colonic fibrosis was also notably improved by the treatment and associated with a significant reduction in the colonic expression of alpha-SMA, collagen I-III, CTGF, TGF-beta1, Smad3, and Smad7. CONCLUSIONS: These data demonstrate that the prophylactic administration of anti-inflammatory Boswellia and antifibrotic Scutellaria extracts is effective in preventing colonic fibrosis in TNBS-induced colitis. Their antifibrotic mechanism of action seems to be mediated by the inhibition of TGF-beta1/Smad3 pathway.

Smad3-null mice lack interstitial cells of Cajal in the colonic wall.

BACKGROUND: Transforming growth factor-beta (TGF-beta)/Smad's signalling pathway plays a pivotal role in organogenesis, oncogenesis, inflammation, repair and fibrosis. The aim of this study was to evaluate the morphology of muscle layers and the density and distribution of interstitial cells of Cajal (ICC) in the colon of Smad3 knockout mice. MATERIALS AND METHODS: Eighteen Smad3 wild-type mice and 12 null mice were sacrificed at age 4 months and the colons were collected for histology (Haematoxilin-Eosin, Masson thrichrome and Gomori silver staining), morphometry and immunohistochemistry (IHC) analysis. For IHC we used the c-Kit, alpha-smooth muscle actine (alpha-SMA), vimentin, desmin and neuronal cocktail (S-100, NSE, neurofilament 200) antibodies. RESULTS: When sacrificed, 40% of the null mice showed different degrees of colon dilatation when compared with the wild-type. Histological and morphometric evaluation revealed a significant reduction in muscle layer thickness of the colon in all the null mice when compared with the wild-type. Immunohistochemistry evaluation showed a marked reduction, or even absence, of c-Kit immunoreactivity, which identifies ICC, in the colon of all the null mice, compared with the wild-type. CONCLUSIONS: Smad3 null mice showed a marked reduction, or even absence, of ICC in the colon together with a concomitant reduction of intestinal smooth muscle layer thickness. This data could account for the colonic dilation observed in approximately 40% of the Smad3 null mice. Alteration of intestinal smooth muscle layers and ICC could also be involved in the resistance of the Smad3 null mice to develop colonic fibrosis.

Slime production by clinical isolates of Blastoschizomyces capitatus from patients with hematological malignancies and catheter-related fungemia.

In order to expand the present knowledge of the pathogenic potential of Blastoschizomyces capitatus in central venous catheter (CVC)-related bloodstream infections, six strains of the organism recovered from three leukemic patients with CVC-related fungemia in different years were investigated. Isolates and control strains were tested for their genetic relatedness and for their ability to produce slime in glucose-containing solutions. DNA restriction enzyme analysis revealed that all strains of B. capitatus were identical, whereas slime production assays and examination of ex vivo material showed that they were able to produce large amounts of slime. Slime production may therefore play a relevant pathogenic role in cases of CVC-related fungemia caused by B. capitatus.

[MDR (multidrug resistance) in hepatocarcinoma clinical-therapeutic implications]. / MDR (multidrug resistance) nell'epatocarcinoma: implicazioni clinico-terapeutiche.

Our purpose was to summarize current knowledge on "multidrug resistance", or MDR, an intrinsic or acquired cross resistance to a variety of structurally and functionally unrelated drugs, still representing one of the major problems in the therapy of cancer and other diseases. MDR depends on various mechanisms, the best known being the activity of ABC transport proteins, mainly Pgp, MDR1 gene product,and MRPs; but also other transporters can cause resistance, for example TAP, a peptide transporter, CFTR, cystic fibrosis transmembrane regulator, ABCG2, or breast cancer resistance protein (BCRP) and LRP, lung resistance protein. MDR has been detected in nearly all types of cancer, because it affects many organs and can occur against a wide number of drugs; it is frequent even in other diseases, such as epilepsy and HIV. We focused on MDR phenomenon in HCC, one of the commonest tumors in the world, and one of the most resistant to pharmacological treatment. This characteristic might be partly determined by a link between MDR and angiogenic phenotypes. The relationship between MDR in hepatocellular carcinoma and the effectiveness of therapeutic treatments has been particularly examined. Finally, the importance to overcome the strong chemoresistance of hepatocellular carcinoma with methods alternative to drugs, namely gene therapy, which makes use of antisense oligonucleotides and anti-MDR1 ribozymes, has been pointed out.

Peyer's patches epithelium in the rat: a morphological, immunohistochemical, and morphometrical study.

The epithelial layer covering lymphoid follicles of Peyer's patches consists of cells with a different surface morphology. Some of these cells have been described as a distinct cytotype, the so-called M cells. In order to resolve the controversy on the specific morphological and biochemical markers of M cells, structural, ultrastructural, and morphometrical study of the epithelium covering the rat Peyer's patches were performed. Peyer's patches from healthy rats were processed for light microscopy, immunohistochemistry, in situ nick-end labeling (TUNEL), and scanning and transmission electron microscopy. A morphometric study was also performed to evaluate microvillus density, length, and number of lysosomes in different areas of the epithelium. Peyer's patches were covered by simple columnar/cubical dome epithelium (DE). Scarce goblet cells and a large number of enterocytes were observed. Ultrastructural observations revealed that the DE showed cells with different morphology. The density and length of microvilli and the lysosome number varied along the whole dome without significant differences. The DE cells characterized by short and disorganized microvilli appeared always in close spatial relationship with lymphocytes. In conclusion, the concept that distinct cell types (enterocytes and M cells) can be identified in the rat DE does not appear to be valid based on morphological criteria. It seems correct to consider that in rat Peyer's patches the presence of scarce goblet cells and a large number of enterocytes showing dynamic morphofunctional modifications is related to the functional state and/or to cell cycle.

Dextran sulfate sodium (DSS) colitis in rats: clinical, structural, and ultrastructural aspects.

Aim of this study was to assess the structural, ultrastructural, immunohistochemical, and clinical aspects in Sprague-Dawley rats with dextrane sulfate sodium (DSS)-induced colitis. Colitis was induced in Sprague-Dawley rats by seven days of DSS oral administration followed by seven days of tap water only (for one, two and three cycles). Controls were fed with water only. Segments of proximal, mid-, and distal colon of each animal were adequately prepared for light and scanning electron microscope observations. The severity of the lesions was scored histologically. For immunohistochemical study, a cocktail of S-100, NSE, and antineurofilament antibodies was used. Symptoms such as weight, feces consistency, diarrhea, hematochezia were recorded daily. From a clinical point of view symptoms appeared significantly later after the first cycle than after the second and third cycles and lasted significantly longer in the second and third cycles. Treated rats showed a slower weight gain rate by 20% compared to controls, and the whole colon length appeared to be significantly shorter after colitis induction compared to controls. Structural observations by light microscopy showed prominent involvement of the distal colon. Immunohistochemical study of both submucosal and myoenteric nerve plexuses was similar to controls. Scanning electron microscope observations of the colonic mucosal surface in colitis rats showed a complete subversion of its architecture, characterized by dilatations of gland crypt openings, dropout of goblet cells, and inhomogeneous distribution or lack of microvilli. These were most evident after the third cycle. In conclusion, experimental DSS colitis in SD rats appeared to be highly reproducible and shared most features with human UC, not only from a structural and clinical but also from an ultrastructural point of view.