Giovanna Bucci

Histological Adaptation of Orthotopic Ileal Neobladder Mucosa: 4-Year Follow-Up of 30 Patients

Objective: For 4 years we have monitored the histological evolution of ileal neobladders in a single cohort of 30 patients in order to systematically describe the histological changes occurring after surgery. The aim of the study was to evaluate the long-term evolution of many histological parameters with functional relevance as to the metabolic outcome of the reservoirs. Methods: Ileal samples were collected during surgery and by random biopsies during cystoscopy 6, 12, 18, 24, 36 and 48 months later. At each step qualitative and quantitative assessment of the histological and cytological conditions of the samples was carried out. Results: Morphological changes develop relatively early but the situation tends to level out in about 1 year. The morphological changes are topographically uneven and, although mucosal flattening becomes progressively prevalent, areas with shortened villi persist indefinitely. Goblet cells prevail over enterocytes and the secretive pattern shifts towards sialomucins. The overall replication rate decreases initially but tends to restore in 1 year. Dysplasia or atrophy were never recorded. Conclusions: The 4-year systematic follow-up revealed a typical histological adaptation pattern in the ileal neobladder without signs of dysplasia. The changes seem to be induced by the aggressive environment and develop in the time lag required for functional adaptation of the epithelium.

TRAIL up‐regulation must be accompanied by a reciprocal PKCε down‐regulation during differentiation of colonic epithelial cell: Implications for colorectal cancer cell differentiation

PKC isoenzymes play central roles in various cellular signalling pathways, participating in a variety of protein phosphorylation cascades that regulate/modulate cellular structure and gene expression. It has been firmly established that several isoforms of PKC have a role in the regulation of tumor necrosis factor‐related apoptosis inducing ligand (TRAIL) activity. Our interest in probing the role of the epsilon isoform of PKC in the colonic cell differentiation stems from the discovery that PKCε and TRAIL are involved in the differentiation of other cell types like hematopoietic stem cells. Although the role of PKCε and TRAIL in the gastrointestinal system is unclear, it has been observed that PKCε has oncogenic activity in colon epithelial cells (CEC), while TRAIL increases the death of intestinal epithelial cells during inflammation. Here we demonstrate a reciprocal expression of PKCε and TRAIL in human colon mucosa: CECs at the bottom of the colonic crypts show high levels of PKCε, being negative for TRAIL expression. On the contrary, luminal CECs are positive for TRAIL, while negative for PKCε. Indeed, TRAIL‐ and butyrate‐induced differentiation of the human colorectal cancer cell line HT29 requires the decrease of PKCε expression, whose absence in turn increases cell sensitivity to TRAIL‐induced apoptosis. Moreover, TRAIL preferentially promotes HT29 differentiation into goblet cells. Taken together, this data demonstrate that TRAIL and PKCε must be reciprocally regulated to ensure physiological CEC differentiation starting from the stem cell pool, and that the down‐regulation of PKCε is however critical for the differentiation and apoptosis of cancer cells. J. Cell. Physiol. 227: 630–638, 2012.

Skin, Inflammation and Sulfurous Waters: What is Known, What is Believed:

One could argue that balneotherapy and mud therapy would have not lasted 2,000 years or so If they were not effective. No doubt a long history cannot be taken per se as scientific proof of efficacy. Some empiricism is still present in the field: the concept of spa itself is quite confounding, whereas spring waters are used for leisure purposes but also for non-acute patient therapy and late phases of clinical recovery. These confounding elements ultimately feed the opinion of those who aprioristically reject any potential beneficial effect of balneotherapy: instead, it should at least generate questions that deserve scientific answers. Clinical practices sequentially integrating pharmacological therapy with those natural principles for which a sufficient scientific demonstration is available, would probably cut the costs of public health, generating widespread advantages for the community. Recently, it has become evident that mineral waters may have intrinsic pharmacological properties. Of the numerous salts dissolved in thermal waters that might show pharmacological properties, for certain hydrogen sulfide (H2S) contained in sulfurous waters is the one that has obtained greater scientific attention, to which should be added the extensive scientific effort recently dedicated to H2S as a cellular gasotransmitter, independently from its natural sources. Dermatology and cosmetics are among the most studied applications of sulfurous waters, around which, however, some empiricism still confounds opinions: we therefore considered that a state-of-the-art focus on this topic might be timely and useful for future studies.

Physico-chemical characterization and biological evaluation of two fibroin materials.

Silk fibroin fibres from two different sources, Bombyx mori pure‐breed silkworms and polyhybrid cross‐bred silkworm cocoons, were treated with formic acid under planar stirring conditions to prepare non‐woven nets. The treatment partially dissolved the fibres, which bound together and formed a non‐woven micrometric net with fibres coated by a thin layer of low molecular weight fibroin matrix. The starting fibres, net materials and fibroin coating layer were characterized in terms of amino acid composition, molecular weight and calorimetric properties. In vitro cell culture tests with rat fibroblasts were performed to investigate cell proliferation, morphology and spreading. Moreover, host‐rat fibroblasts were preseeded on the afore‐mentioned nets and implanted in the thorax of rats for histological analysis. In spite of the chemical differences between the two starting fibroins, the response of the said materials in vitro and in vivo were very similar. These results suggest that the outcome is likely correlated with the modification of the processing technique; that during the formation of the net, a thin gel layer of similar amino acid composition was formed on the fibroin fibres. Copyright

Ultrastructural basis for the efficiency of an ileal orthotopic neobladder 27 years after surgery

The morphological and functional basis of the excellent clinical outcome of ileal orthotopic neobladders are largely unknown. Only long-term follow-up studies will provide an adequate answer to this unsettled question. We have studied a patient who underwent this type of surgery over 27 years ago. Besides an important secretive adaptation we have found, at the ultrastructural level, that the monolayered epithelium does not show signs of true metaplasia and that changes had occurred in the intercellular junctions, namely that desmosomes are significantly increased. Although limited to a single case, these features, if confirmed by further observations, suggest a working hypothesis for the understanding of the definitive phenotypic adaptation of the ileal epithelium to the new aggressive environment.

PKC epsilon/PKC delta rate regulates human platelets production

Platelet production is the terminal step of the megakaryocytopoiesis process (1). We previously observed that PKCepsilon, a member of the Protein Kinase C family of serine/threonine kinases, expression varies during human MK differentiation and modulates MK maturation and platelet release (2). Besides, it is also critical for proplatelet formation in murine model (3). Here we utilized an in vitro model of human platelet production to investigate a potential role for PKCdelta, well known to medi- ate contrasting and even opposing effects as compared to PKCepsilon in several non-hematopoietic models (4), in MK differentiation and proplatelet formation. By western blot analysis we found that PKCdelta expression escalates during megakarocyte differentiation and remains elevated during the final step of this process, with an opposite kinetics as compared to PKCepsilon expression levels. Furthermore, by using specific shRNA we observed that PKCdelta inhibition affected both MK differentiation, in terms of cell survival and ploidy, and platelet production. The correlation between PKCdelta/PKCepsilon levels and MK differentiation was also con- firmed by pathological models. Indeed, primary myelofibrosis (PMF), characterized by an impaired MK differentiation, showed higher levels of PKCepsilon and reduced expression of PKCdelta. On the contrary, in essential thrombocytemia (ET), featured by a physiological increase of platelet number, PKCepsilon is virtually absent and PKCdelta is well represented. Finally, we demonstrated that the concurrent pharmacological inhibition of PKCepsilon and activation of PKCdelta are capable to build up platelet production in human. Collectively, these data indicate that novel PKCs are critical mediator of human proplatelet formation and the modulation of their expres- sion might be a future strategy to revise platelet production.

PKCε regulates vessel formation by peri-vascular adipose tissue (PVAT) cells

Vessel formation is crucial in tumour growth and tissue regeneration. Protein kinase C (PKC) e has a well-known role on hematopoietic and mesenchymal progenitor cell differentiation and proliferation (Gobbi et al. 2013). Although PKCe has a demonstrated role in vascular restenosis, data on PKCe and vascular progenitor differentiation are still lacking. The aim of this work was to study the role of PKCe in vessel formation by adult adipose tissue cell progenitors. We, first, isolated the vessel progenitors from the adipose tissue localized between aortic arch and pulmonary artery of adult mice by collagenase/elastase digestion followed by magnetic immunoselection of Sca1+ cells (Passmann et al. 2008). We, then, tested their capability to form vessels in collagen gels and to differentiate to endothelial and smooth muscle lineage after treatment with PKCe specific activator and inhibitor peptides. The functional experiments showed that the pharmacological activation of endogenous PKCe abrogated tubule formation with a concomitant decrease of smooth alpha-actin (SMA) and platelet endothelial cell adhesion molecule (PECAM) together with the up-regulation of p-PAK1 expression. In vivo transient over-expression of PKCe significantly reduced SMA and PECAM expression levels in vessel wall cells. Together our data suggests that PKCe may affect vessel wall remodelling balancing the “phenotypic switching” (Salmon et al. 2013) between the proliferative and the differentiated state of smooth muscle and endothelial progenitor mesenchymal cells.

PKCε expression is required during proplatelet formation in murine model

Megakaryocytes (MK) remodel their cytoplasm into long proplatelet extensions to generate platelets [1]. We have previously demonstrated that PKCepsilon expression is strictly regulated during megakaryocytopoiesis (MKpoiesis), and its forced expression in the late phases of MK differentiation impairs platelet production [2,3]. However, our preliminary data suggest that PKCepsilon positive platelets may be released around the acute event of myocardial infarction, affecting their aggregation potential. Primary fetal liver (FL) cells isolated from CD1 pregnant mice are the preferential model to study the platelet formation mechanism [4]. Therefore, here we focused on the mouse PKCepsilon positive model to elucidate the role of PKCepsilon in MK maturation. Our data show that not only PKCepsilon expression increases during mouse MK differentiation, but also its forced down-regulation strongly reduces pro-platelet formation. Therefore, PKCepsilon is strongly required for murine proplatelet production. On the basis of these results and other known model systems, we show that PKCepsilon has a relevant role in the completion of platelet release.

Impaired cytokine expression in hydrogen sulphide-treated keratinocytes

Hydrogen sulfide is a component of several natural compounds known to be effective in many inflammatory pathologies. We have recently demonstrated that: i) exogenous H2S can delay the onset of apoptosis of granulocytes in vitro [Lab Invest 2006;86:391]; ii) H2S exerts a subset-specific toxicity on peripheral blood lymphocytes in terms of cell survival and cytokine production [J Cell Physiol 2007;213:826]; iii) H2S impairs adhesion and proliferation of human keratinocytes by preventing the activation of Raf-MEK-ERK signaling pathway. Moreover, performing in vivo experiments we have observed that NaHS-treatment reduced pERK levels in the epidermis of patients affected by psoriasis [Lab Invest 2009;89:994]. Psoriasis, a chronic inflammatory skin condition that is characterized by hyperproliferation and abnormal differentiation of epidermal keratinocytes, and also by neutrophil and lymphocyte infiltration of epidermal and dermal layers. On the basis of the biochemical analyses and in vitro studies, it has become evident that IL-8 greatly contributes to the major pathologic changes seen in psoriasis. Furthermore, others cytokines have an emerging role in the pathogenesis of this disease: in particular, IL-17 and IL-22 activate keratinocytes, interfere with keratinocyte and lymphocyte differentiation and proliferation, promote the secretion of inflammatory cytokines. The signaling pathway downstream IL-17 receptor appears to involve ERK activation: however additional molecular mediators may be recruited as PI3K and Akt. On this basis, we have now studied the ability of NaHS to affect IL-8 transcription and secretion in human normal keratinocyte NCTC cell line. IL-8 expression was analyzed both at transcriptional and protein level by RT-PCR and ELISA, respectively. Studies have been performed in resting and IL-17 or IL-22-stimulated cell cultures. The ability of NaHS to suppress IL-8 expression in vivo was also studied by immunohistochemistry on biopsies from a patient affected by psoriasis, treated for 1 week with NaHS. Results show that NaHS, blocking ERK activation, interferes with IL-8 production. Sulfurs are able to penetrate the skin, and a sulfur-rich balneotherapy is known to be effective in the treatment of psoriasis. These data suggest that H2S might be useful to limit the proliferation of keratinoblasts in skin diseases like psoriasis, dermatitis, skin iperplasia and basaliomas, where a functional modification of keratinocyte behavior plays a major pathogenetic role.