Indirect co-cultures of healthy mesenchymal stem cells restore the physiological phenotypical profile of psoriatic mesenchymal stem cells.

Psoriasis microenvironment, characterized by an imbalance between T helper type 1 (Th1)/Th17 and Th2 cytokines and also influences the mesenchymal stem cells (MSCs) phenotypical profile. MSCs from healthy donors (H-MSCs) can exert a strong paracrine effect by secreting active soluble factors, able to modulate the inflammation in the microenvironment. To evaluate the influence of H-MSCs on MSCs from psoriatic patients (PsO-MSCs), H-MSCs and PsO-MSCs were isolated and characterized. Indirect co-culture of H-MSCs with PsO-MSCs was performed; effects on proliferation and expression of cytokines linked to Th1/Th17 and Th2 pathways were assayed before and after co-culture. The results show that before co-culture, proliferation of PsO-MSCs was significantly higher than H-MSCs (P < 0·05) and the levels of secreted cytokines confirmed the imbalance of Th1/Th17 versus the Th2 axis. After co-culture of H-MSCs with PsO-MSCs, healthy MSCs seem to exert a 'positive' influence on PsO-MSCs, driving the inflammatory phenotypical profile of PsO-MSCs towards a physiological pattern. The proliferation rate decreased towards values nearer to those observed in H-MSCs and the secretion of the cytokines that mostly identified the inflammatory microenvironment that characterized psoriasis, such as interleukin (IL)-6, IL-12, IL-13, IL-17A, tumour necrosis factor (TNF)-α and granulocyte-macrophage colony-stimulating factor (G-CSF), is significantly lower in co-cultured PsO-MSCs than in individually cultured PSO-MSCs (P at least < 0·05). In conclusion, our preliminary results seem to provide an intriguing molecular explanation for the ever-increasing evidence of therapeutic efficacy of allogeneic MSCs infusion in psoriatic patients.

Pituitary adenomas, stem cells, and cancer stem cells: what's new?

PURPOSE: To clarify the existence of pituitary stem cells (SCs) both in the embryonic and the postnatal gland and the role for SCs in pituitary adenomas. METHODS: This work, which does not address the pathogenesis of pituitary adenomas, reviews the latest research findings and discoveries on SCs in pituitary and cancer SCs (CSCs) in pituitary adenomas and discusses the involvement of the EMT. RESULTS: Several groups using different approaches and techniques have demonstrated the existence of SCs and CSCs and as they are major players in pituitary adenoma onset. CONCLUSIONS: As in other benign and malignant tumors, the hypothesis that CSCs play a pivotal role in pituitary adenoma onset has been confirmed as well as the existence of a link between the epithelial-to-mesenchymal transition (EMT) process and CSC formation in epithelial tumors.

T helper (Th)1, Th17 and Th2 imbalance in mesenchymal stem cells of adult patients with atopic dermatitis: at the origin of the problem.

BACKGROUND: Atopic dermatitis (AD) is a chronic and inflammatory disease characterized by a marked imbalance of T helper (Th)2 vs. Th1/Th17 cells in the early phase of AD, whereas a mixed Th1/Th2 pattern of inflammation is usually found at the chronic stage. These features have not been extensively evaluated in undifferentiated skin cells of patients affected by AD. OBJECTIVES: To evaluate the relative expression of 22 genes encoding Th1, Th2 and Th17 cytokines and the secretion of the corresponding proteins in cutaneous mesenchymal stem cells (MSCs) isolated from skin of patients with AD (AD-MSCs) and their role in AD onset. METHODS: AD-MSCs were isolated, characterized and profiled by polymerase chain reaction array and enzyme-linked immunosorbent assay for the relative expression and secretion of cytokines involved in the Th1, Th2 and Th17 pathways. MSCs isolated from the skin of healthy people were used as controls (C-MSCs). RESULTS: AD-MSCs showed an upregulation of many Th1/Th17 cytokines [interleukin (IL)-6, IL-8, IL-12, IL-13, IL-17A, IL-17F, transforming growth factor-ß, interferon-γ], while Th2 chemokines (IL-2, IL-4, IL-5, IL-23A) were downregulated in AD-MSCs. Finally, some genes/proteins (CCL1, IL-17C, tumour necrosis factor-α) did not show variations between C-MSCs and AD-MSCs. CONCLUSIONS: The profile of MSCs obtained from patients with chronic AD retraces the Th1/Th17 cell environment observed in differentiated cells of chronic AD. This evidence could open a new scenario in the pathogenesis of AD, according to which the inflammatory process may involve MSCs early on.

Pathology of upper tract urothelial carcinoma with emphasis on staging.

Classification of upper tract urothelial preneoplastic and neoplastic lesions mirrors that of the urinary bladder, with all lesions of the bladder urothelium being possible in the upper tract and vice versa. There are three major groups of non-invasive urothelial neoplasms: flat, papillary, and inverted. These three groups share a similar morphological spectrum of intraurothelial changes, ranging from hyperplasia to dysplasia to carcinoma in situ. However, they differ in terms of architectural growth pattern compared to the surrounding non-neoplastic mucosal surface. Infiltrating urothelial carcinoma is defined as a urothelial tumor that invades beyond the basement membrane. Unlike in non-invasive papillary urothelial neoplasms (pTa), the role of histologic grade in pT1 and higher stage tumors has been suggested to be of only relative importance. The vast majority of tumors of the upper urinary tract are urothelial carcinoma. More commonly seen, however, are foci of squamous differentiation and, less frequently, glandular differentiation. Pure urothelial carcinomas also display a wide range of variant morphologies, and recognition of these morphologies is important for diagnosis, classification, and prognosis.

Treatment effects in prostate cancer following traditional and emerging therapies.

Treatment options for prostate cancer consist of radical prostatectomy, hormonal therapy and radiation therapy. Hormonal and radiation therapy have well-known, often profound, effects on the histological appearance of benign and malignant prostate tissue. Novel therapies including focal ablative treatments, chemotherapies and targeted molecular therapies are beginning to emerge and pathologists will play a central role in documenting the effects of these treatments at the tissue level. As such, knowledge of treatment-related changes and access to clinical information are essential to ensure accurate interpretation and reporting of post-treatment prostate specimens by pathologists.

Karyometry and quantitative immunohistochemical analysis of the urothelium in tissue sections: a feasibility study based on chromatin remodeler DAXX immunostaining.

The aim of this study was to investigate the feasibility of applying a software traditionally used in the field of engineering to pathology, in particular to tissue sections from normal urothelium (NU) immuno-stained for the chromatin remodeler DAXX (death domain-associated protein). The study included 5 cases of NU. Images were recorded with a Nikon digital camera. The nuclear area and the intensity of nuclear staining were analyzed with a software package developed in LabVIEW environment. The nuclear size is 14.8 plus or minus 6.5 square microns. The nuclei in the cells adjacent to the stroma are slightly smaller than in the intermediate cells by a factor of 0.86. The mean nuclear area of the nuclei in the superficial cell layer in NU is identical to the nuclei in the intermediate cell layers. For each nucleus intensity of nuclear staining is calculated based on the gray value of the individual picture elements in the green color plane. The mean and standard deviation of nuclear gray value are 106 plus or minus 15. The mean value in the nuclei adjacent to the stroma is slightly greater by a factor 1.02 and 1.04 compared to the intermediate and superficial cell layers. In conclusion, this exploratory study shows that karyometry and quantitative immunohistochemical analysis can be done accurately by using a digital camera commonly available to pathologists and an image analysis software routinely used in the field of engineering.

Alterations of ROS pathways in scleroderma begin at stem cell level.

Scleroderma is a chronic systemic autoimmune disease (primarily of the skin) characterized by fibrosis (or hardening), vascular alterations and autoantibodies production.There are currently no effective therapies against this devastating and often lethal disorder. Despite the interest for the immunomodulatory effects of mesenchymal stem cells (MSCs) in autoimmune diseases, the role of MSCs in scleroderma is still unknown. A pivotal role in scleroderma onset is played by oxidative stress associated with the accumulation of great amounts of reactive oxygen species (ROS). This study depicts some phenotypic and functional features of MSCs isolated from the skin of healthy and scleroderma patients; the ROS production and accumulation, the expression of ERK1/2 and the effects of the stimulation with PDGF, were analyzed in MSCs; results were compared to those observed in primary fibroblasts (Fbs) isolated from the same subjects. We found that the pro-oxidant environment exerted by scleroderma affects MSCs, which are still able to counteract the ROS accumulation by improving the antioxidant defenses. On the contrary, scleroderma fibroblasts show a disruption of these mechanisms, with consequent ROS increase and the activation of the cascade triggered by scleroderma auto-antibodies against PDGFR.

Microvessel density and VEGF, HIF-1α expression in primary oral melanoma: correlation with prognosis.

OBJECTIVE: To understand the role of angiogenesis and hypoxia in cancer progression of primary oral melanoma (POM). MATERIALS AND METHODS: Sixteen malignant primary melanomas were immunostained with markers CD34, VEGF and HIF-1α. Stained cells were counted in the invasive front and inside the tumour, and the differences were compared and correlated with histological parameters and disease-specific survival of the patients. RESULTS: Tumour invasive front showed increased MVD and increased vessel VEGF and HIF-1α expression compared with the intratumoural compartment. No such differences were seen in tumoural melanocytes of the two compartments. Positive correlations were observed between CD34 and VEGF, CD34 and HIF-1α and VEGF and HIF-1α expression in invasive front vessels. CD34 expression was statistically correlated with the level of infiltration. A significant trend to worse disease-free survival was also determined with increased invasive front vessel expression of CD34, VEGF and HIF-1α. CONCLUSIONS: Our data highlight the importance of the invasive margin in POM biology. The high angiogenic activity and endothelial VEGF and HIF-1α expression in invasive front vessels have a significant impact on patient survival and future agents targeted against VEGF pathway may represent a novel and effective therapeutic opportunity. Larger studies are needed to further address our findings.

Effect of biologic therapies targeting tumour necrosis factor-α on cutaneous mesenchymal stem cells in psoriasis.

BACKGROUND: Psoriasis is a Th1 immune-mediated, inflammatory disease, in which skin lesions appear many years before the related metabolic and cardiovascular comorbidities, according to the theory of the 'psoriatic march'. Inducible nitric oxide synthetase (iNOS), tumour necrosis factor (TNF)-α and vascular endothelial growth factor (VEGF) are directly implicated in determining both skin lesions and systemic involvement in psoriasis. Reactive oxygen species actively promote the secretion of inflammatory Th1 cytokines directly involved in the pathogenesis of psoriasis. OBJECTIVES: Evaluation of VEGF expression and production, nitric oxide (NO) production, iNOS expression, and the antioxidant response of mesenchymal stem cells (MSCs), both before and after 12 weeks of treatment with the TNF-α inhibitors adalimumab or etanercept. METHODS: Biochemical, morphological and immunohistochemical analyses were performed in MSCs isolated from nonlesional, perilesional and lesional skin of patients with psoriasis, before and after treatment. RESULTS: The treatments were able to reduce the expression and production of VEGF, the expression of iNOS and the production of NO in MSCs of patients with psoriasis. TNF-α inhibitors also reduced the oxidative damage in MSC membrane and proteins, several antioxidant systems responded to treatments with a general inhibition of activities (glutathione S-transferase and catalase) and these effects were also supported by a general decrease of total oxyradical scavenging capacity towards hydroxyl radicals and peroxynitrite. CONCLUSIONS: TNF-α inhibitors are able to change the physiopathological pathway of psoriasis, and our results suggest their therapeutic effects already take place at the level of MSCs, which probably represent the cells primarily involved in the 'psoriatic march'.

A contemporary update on pathology reporting for urinary bladder cancer.

Providing the best management for patients with bladder cancer relies on close cooperation among uro-oncologists and pathologists. The pathologist is involved in the diagnosis and assessment of prognostic and therapeutic factors in bladder biopsies, transurethral resection (TUR) and cystectomy specimens. The pathologist must report accurately the key features using terms that are well understood by clinicians. Adequate clinical information is important to pathologists in deciding the best approach in handling and processing the surgical specimens.

Immunohistochemical expression and localization of somatostatin receptors in normal prostate, high grade prostatic intraepithelial neoplasia and prostate cancer and its many faces.

Data on the immunohistochemical expression and localization of the five somatostatin receptors (SSTRs) have been obtained by our group in separate studies concerning the many faces of prostate cancer (PCa), its precursor high grade prostatic intraepithelial neoplasia (HGPIN) and normal epithelium (Nep). This publication highlights the key findings, with special reference to: normal prostate epithelium; untreated HGPIN and PCa, both clinically and incidentally detected; PCa with NE differentiation; HGPIN and PCa following complete androgen ablation (CAA); and hormone refractory (HR) PCa. Taken together, the data obtained in these investigations demonstrate that SSTR profiling in individual patients with HGPIN and the multifaceted PCa is feasible and is of relevance to better tailor the somatostatin analogue-based treatment.

In vitro evaluation of mesenchymal stem cell isolation possibility from different intra-oral tissues.

Mesenchymal stem cells (MSCs) are of great interest for the regeneration of tissues and organs. Bone marrow is the first sources of MSCs, but in the recent years there has been interest in other tissues for the isolation of these pluripotent cells. In this study, we investigated the features of MSCs isolated from different oral regions in order to evaluate their potential application in the regeneration of damaged maxillofacial tissues. Sampling from human periodontal ligament, dental pulp, maxillary periosteum as well as bone marrow were collected in order to obtain different stem cell populations. Cells were morphologically and immunophenotipically characterized. Their proliferation potential and their ability to differentiate in osteoblasts were also assessed. All tested cell population showed a similar fibroblast-like morphology and superimposable immunophenotype. Slight differences were observed in proliferation and differentiation potential. Cells isolated from human periodontal ligament, dental pulp, maxillary periosteum had the characteristics of stem cells. Considering their peculiar feature they may alternatively represent interesting cell sources in stem cell-based bone/periodontal tissue regeneration approaches.

The mesenchymal stem cell profile in psoriasis.

BACKGROUND: The expression of inducible nitric oxide synthase (iNOS) and vascular endothelial growth factor (VEGF) and the level of total oxyradical scavenging capacity have been evaluated extensively in the cutaneous cells of patients with psoriasis. As yet, no indications are available about the undifferentiated cells, the mesenchymal stem cells (MSCs), isolated from skin. OBJECTIVES: To isolate MSCs in patients with psoriasis and to compare them with those obtained from atopic and healthy subjects, in order to analyse whether MSCs show some typical psoriatic profiles and to understand whether pathophysiological events leading to psoriasis start early at the stem cell level. METHODS: MSCs isolated from seven patients with psoriasis, seven patients with acute atopic dermatitis and seven healthy subjects were characterized by fluorescence-activated cell sorting analysis. VEGF and nitric oxide (NO) content was measured in conditioned medium, the expression of VEGF and iNOS was analysed by immunohistochemistry, and the total oxyradical scavenging capacity towards peroxynitrite was tested. RESULTS: VEGF content was highest in the medium conditioned by psoriatic perilesional MSCs, whereas NO concentration was maximally increased in medium conditioned by MSCs isolated from lesional psoriatic skin. The ability to neutralize the oxidizing effects of peroxynitrite was lower for MSCs isolated from lesional psoriatic skin compared with other MSCs, except for MSCs of lesional atopic skin. CONCLUSIONS: The microenvironment in psoriasis differs from those of atopic dermatitis and healthy skin; it could induce resident MSCs to produce angiogenic and proinflammatory mediators which lead to a reduction in the antioxidant capacity of these cells, contributing to the development of skin lesions in psoriasis.

Do DNA-methylation and histone acetylation play a role in clear cell renal carcinoma? Analysis of radical nephrectomy specimens in a long-term follow-up.

We investigated global methylation and histone acetylation in 50 conventional clear cell renal carcinomas (RCC), treated with radical nephrectomy, to assess their possible role as diagnostic biomarkers. The features considered in this study were patient age, tumor size and grade, percentage and intensity of 5-methylcytosine (5mc) and Acetyl-Histone (Lys 9) expression in tumor tissue. All considered parameters were correlated with patient specific survival. The mean percentage of global cellular methylation in tumoral tissue was significantly higher compared to normal peritumoral tissue (p<0.0001), while the intensity of cellular methylation was significantly higher in normal tissue than in tumoral tissue (p=0.001). The mean percentage of histone cellular acetylation in tumoral tissue was significantly lower compared to normal peritumoral tissue (p=0.0005), while the intensity of mean acetylation in neoplastic tissue was similar to the normal tissue. The percentage of global DNA methylation was significantly higher in grades 3 and 4 tumors (p=0.033). Global DNA methylation and histone acetylation in tumoral tissue did not correlate with survival. Fuhrman grade was statistically significant for prognosis (p=0.031). In conclusion, global hypermethylation and histone hypoacetylation play an important role in RCC carcinogenesis; Fuhrman grade is still considered the most important factor for patient survival; 5mc can have a role as markers of aggressiveness.

Neurogenic potential of mesenchymal-like stem cells from human amniotic fluid: the influence of extracellular growth factors.

Amniotic fluids contain human stem cells, among which mesenchymal stem cells could be isolated. These cells have multipotent differentiation ability and no tumorigenic potential after transplantation in mice. These features make them good candidates for in vitro studies and for therapeutic purposes. The aim of this study was to isolate mesenchymal stem cell-like cultures from different amniotic fluids in order to study in vitro their neurogenic potential and assess if this process could be reproducible and standardized. We focused attention on the possible differential effects of soluble growth factors. Immunophenotypical and molecular characterization showed that the 31 amniotic fluid-derived cultures expressed mesenchymal markers as well as some stemness properties. These cells also appeared to be responsive to purines or acetylcholine showing an intracellular calcium increase, also reported for mesenchymal stem cells derived from other sources. Interestingly, in the presence of retinoic acid, these cells assumed a neuronal-like morphology. In addition, functional and molecular analyses revealed that retinoic acid-treated cells showed immature electric functional properties, the expression of neuronal markers and stemness genes. In conclusion, even if further investigations are required, the results presented here contribute to support the finding that amniotic fluid contains cells able to differentiate in vitro towards neural-like lineage in the presence of retinoic acid. The ability of retinoic acid to induce a possible neuronal progenitor culture makes the model useful to study a possible in vivo transplantation of these cells and to contribute to define the protocols for cell therapy.

Osteoinduction properties of different growth factors on cells from non-union patients: in vitro study for clinical application.

This report compares the effect of rhBMPs and PRG on cells derived from human non-union sites. Treatment of non-union continues to be a challenging task for the trauma surgeon often resulting in unsatisfactory results and long-term morbidity. Over the past two decades, the possibility to use growth factors in bone regeneration has been investigated. In this study we compared the in vitro capability of two recombinant human bone morphogenetic proteins (rhBMP-2 and rhBMP-7) and activated platelet-rich plasma (PRG) to stimulate proliferation and/or differentiation of cells derived from non-union patients. Cells derived from the lesion sites, osteoblasts and mesenchymal stem cells (MSCs) derived from other bone sites of the same patients were used. Treatment with rhBMP-7 or rhBMP-2 showed an improvement in the expression of osteoblastic markers (osteonectin and osteocalcin) in cells derived from human non-union sites. This enhancement was more marked in MSCs, while no significant changes were observed in osteoblast cultures. The PRG treatment produced in all analysed samples a considerable increase in cell proliferation without affecting cell differentiation. On the basis of our results, for an effective biological treatment of non-unions, small amounts of autologous bone marrow (MSCs) are necessary in the lesion site in order to provide both growth factors and a sufficient number of responsive cells. Finally, our results prove that sequential timing administration of PRG and rhBMPs may be used in new therapeutic strategy.

Determining the temperature of petroleum formation from the kinetic properties of petroleum asphaltenes

Knowledge of the timing and location of petroleum formation is important in assessing the extent of available reserves in hydrocarbon-forming basins. This can be predicted from the thermal history of a basin and the kinetic parameters that characterize the thermal breakdown of kerogen in source rocks. At present, the kinetic parameters of kerogen breakdown are experimentally determined using immature rock samples from basin margins, but questions remain about the accuracy of this approach, especially when significant variability is observed within individual source units. Here we show that the kinetics of hydrocarbon generation from petroleum asphaltenes can be used to determine the temperature conditions of the actual source rock at the time of expulsion of the sampled petroleum. This relationship reflects the structural similarity of asphaltenes to the parent kerogen. We expect that our approach may be used as a comparatively simple alternative method for assessing the petroleum generation characteristics of a given basin, which will allow for better estimates of the available oil resources and the risks associated with their exploration.

Functional characterization of calcium-signaling pathways of human skin-derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) derived from adult human tissues are able to differentiate into various specialized cell types. In research, they can therefore be used like embryonic cells but without the ethical restrictions. Among the various human tissues, skin as a source is characterized by great accessibility and availability using noninvasive procedures and is without the risk of oncogenesis after transplantation. The recent isolation of MSCs has shown the lack of knowledge regarding their specific features, including the calcium-signaling pathways. METHODS: In this study, we isolated MSCs from human skin biopsies (S-MSCs) and characterized them phenotypically and their calcium-signaling pathways by the means of Ca2+ imaging and video microscopic experiments. RESULTS: The cytofluorimetric analysis of the expression of surface markers on S-MSCs revealed that they express the normal pattern present on MSCs. Interestingly, these cells appeared to be successfully cryopreserved at early passages. Calcium imaging on single S-MSCs shows that these cells did not display significant spontaneous activity or a response to a depolarizing agent. However, ATP or acetylcholine-induced intracellular calcium increase via ionotropic or metabotropic receptors, respectively. CONCLUSION: The results presented here reveal that S-MSCs show morphological and functional features that make them useful as an in vitro model to study cell differentiation.

Localization of dystrophin COOH-terminal domain by the fracture-label technique.

The precise localization of dystrophin in the skeletal muscle cell should contribute to a better understanding of the yet unclear functional role of this protein, both in normal and in Duchenne muscular dystrophy. Immunocytochemical studies did not give conclusive results on the localization of dystrophin with respect to the sarcolemma and to the cytoskeletal components. To improve the reliability of the electron microscopic immunocytochemical localization of dystrophin, a mAb against the COOH-terminus of the molecule has been used in association with the fracture-label technique, which, causing a partition of the membrane in protoplasmic and exoplasmic halves, allows a more precise dystrophin localization. The results obtained indicate that dystrophin is associated with the protoplasmic half of the plasmalemma, and the observation that it does not randomly follow the partition of the membrane is consistent with a stable association with the cytoskeleton.