Adipose tissue derived stem cells: in vitro and in vivo analysis of a standard and three commercially available cell-assisted lipotransfer techniques.

INTRODUCTION: Autologous fat grafting is commonly used to correct soft-tissue contour deformities. However, results are impaired by a variable and unpredictable resorption rate. Autologous adipose-derived stromal cells in combination with lipoinjection (cell-assisted lipotransfer) seem to favor a long-term persistence of fat grafts, thus fostering the development of devices to be used in the operating room at the point of care, to isolate the stromal vascular fraction (SVF) and produce SVF-enhanced fat grafts with safe and standardized protocols. Focusing on patients undergoing breast reconstruction by lipostructure, we analyzed a standard technique, a modification of the Coleman's procedure, and three different commercially available devices (Lipokit, Cytori, Fastem), in terms of 1) ability to enrich fat grafts in stem cells and 2) clinical outcome at 6 and 12 months. METHODS: To evaluate the ability to enrich stem cells, we compared, for each patient (n=20), the standard lipoaspirate with the respective stem cell-enriched one, analyzing yield, immunophenotype and colony-forming capacity of the SVF cells as well as immunophenotype, clonogenicity and multipotency of the obtained adipose stem cells (ASCs). Regarding the clinical outcome, we compared, by ultrasonography imaging, changes at 6 and 12 months in the subcutaneous thickness of patients treated with stem-cell enriched (n=14) and standard lipoaspirates (n=16). RESULTS: Both methods relying on the enzymatic isolation of primitive cells led to significant increase in the frequency, in the fat grafts, of SVF cells as well as of clonogenic and multipotent ASCs, while the enrichment was less prominent for the device based on the mechanical isolation of the SVF. From a clinical point of view, patients treated with SVF-enhanced fat grafts demonstrated, at six months, a significant superior gain of thickness of both the central and superior-medial quadrants with respect to patients treated with standard lipotransfer. In the median-median quadrant the effect was still persistent at 12 months, confirming an advantage of lipotransfer technique in enriching improving long-term fat grafts. CONCLUSIONS: This comparative study, based on reproducible biological and clinical parameters and endpoints, showed an advantage of lipotransfer technique in enriching fat grafts in stem cells and in favoring, clinically, long-term fat grafts.

Specific mesothelial signature marks the heterogeneity of mesenchymal stem cells from high-grade serous ovarian cancer.

Mesenchymal stem/stromal cells (MSCs) are the precursors of various cell types that compose both normal and cancer tissue microenvironments. In order to support the widely diversified parenchymal cells and tissue organization, MSCs are characterized by a large degree of heterogeneity, although available analyses of molecular and transcriptional data do not provide clear evidence. We have isolated MSCs from high-grade serous ovarian cancers (HG-SOCs) and various normal tissues (N-MSCs), demonstrated their normal genotype and analyzed their transcriptional activity with respect to the large comprehensive FANTOM5 sample dataset. Our integrative analysis conducted against the extensive panel of primary cells and tissues of the FANTOM5 project allowed us to mark the HG-SOC-MSCs CAGE-seq transcriptional heterogeneity and to identify a cell-type-specific transcriptional activity showing a significant relationship with primary mesothelial cells. Our analysis shows that MSCs isolated from different tissues are highly heterogeneous. The mesothelial-related gene signature identified in this study supports the hypothesis that HG-SOC-MSCs are bona fide representatives of the ovarian district. This finding indicates that HG-SOC-MSCs could actually derive from the coelomic mesothelium, suggesting that they might be linked to the epithelial tumor through common embryological precursors.

Preliminary results from a crowdsourcing experiment in immunohistochemistry.

BACKGROUND: Crowdsourcing, i.e., the outsourcing of tasks typically performed by a few experts to a large crowd as an open call, has been shown to be reasonably effective in many cases, like Wikipedia, the Chess match of Kasparov against the world in 1999, and several others. The aim of the present paper is to describe the setup of an experimentation of crowdsourcing techniques applied to the quantification of immunohistochemistry. METHODS: Fourteen Images from MIB1-stained breast specimens were first manually counted by a pathologist, then submitted to a crowdsourcing platform through a specifically developed application. 10 positivity evaluations for each image have been collected and summarized using their median. The positivity values have been then compared to the gold standard provided by the pathologist by means of Spearman correlation. RESULTS: Contributors were in total 28, and evaluated 4.64 images each on average. Spearman correlation between gold and crowdsourced positivity percentages is 0.946 (p < 0.001). CONCLUSIONS: Aim of the experiment was to understand how to use crowdsourcing for an image analysis task that is currently time-consuming when done by human experts. Crowdsourced work can be used in various ways, in particular statistically agregating data to reduce identification errors. However, in this preliminary experimentation we just considered the most basic indicator, that is the median positivity percentage, which provided overall good results. This method might be more aimed to research than routine: when a large number of images are in need of ad-hoc evaluation, crowdsourcing may represent a quick answer to the need.

Effects of age and heart failure on human cardiac stem cell function.

Currently, it is unknown whether defects in stem cell growth and differentiation contribute to myocardial aging and chronic heart failure (CHF), and whether a compartment of functional human cardiac stem cells (hCSCs) persists in the decompensated heart. To determine whether aging and CHF are critical determinants of the loss in growth reserve of the heart, the properties of hCSCs were evaluated in 18 control and 23 explanted hearts. Age and CHF showed a progressive decrease in functionally competent hCSCs. Chronological age was a major predictor of five biomarkers of hCSC senescence: telomeric shortening, attenuated telomerase activity, telomere dysfunction-induced foci, and p21(Cip1) and p16(INK4a) expression. CHF had similar consequences for hCSCs, suggesting that defects in the balance between cardiomyocyte mass and the pool of nonsenescent hCSCs may condition the evolution of the decompensated myopathy. A correlation was found previously between telomere length in circulating bone marrow cells and cardiovascular diseases, but that analysis was restricted to average telomere length in a cell population, neglecting the fact that telomere attrition does not occur uniformly in all cells. The present study provides the first demonstration that dysfunctional telomeres in hCSCs are biomarkers of aging and heart failure. The biomarkers of cellular senescence identified here can be used to define the birth date of hCSCs and to sort young cells with potential therapeutic efficacy.

Transnational telepathology consultations using a basic digital microscope: experience in the Italy-Slovenjia INTERREG project "patient without borders".

BACKGROUND: In recent years, a number of technological advancements started to modify the long standing appearance and functionalities of traditional optical microscopes used in Pathology and other medical fields. In fact, at present many new tools for microscopical visualization exist that are based on digital imaging, robotization, and remote communication. Such tools are typically adopted in activities ranging from education to telediagnosis to remote consultation. Present paper describes the features of a basic digital microscope that has been tested to verify its performance for occasional remote consultation inside an international project between Italy and Slovenija, funded by Interreg initiative of the European Regional Development Fund. METHODS: The system is composed by a pair of digital microscopes (Leica DMD108, Leitz Microsystems, Wetzlar, Germany) associated to a high resolution videoconferencing systems (Tandberg 990, Lysaker, Norway). The systems are connected through the Internet. Sixty histology and cytology cases have been collaboratively diagnosed between two Pathology Institutes to verify the diagnostic performance of the system, regarding the image quality point of view as well as time needed for diagnosis. The system has also been tested for compatibility with standard videoconferencing software. RESULTS: No discrepancies between local and remote diagnoses have been identified, with diagnosis time reasonably close to typical microscope observation times. Time needed for most operations is not far from that needed on a traditional microscope, except for startup. CONCLUSIONS: The system can be considered usable as a standard microscope, and also for occasional remote consultations.

Myocyte turnover in the aging human heart.

RATIONALE: The turnover of cardiomyocytes in the aging female and male heart is currently unknown, emphasizing the need to define human myocardial biology. OBJECTIVE: The effects of age and gender on the magnitude of myocyte regeneration and the origin of newly formed cardiomyocytes were determined. METHODS AND RESULTS: The interaction of myocyte replacement, cellular senescence, growth inhibition, and apoptosis was measured in normal female (n=32) and male (n=42) human hearts collected from patients 19 to 104 years of age who died from causes other than cardiovascular diseases. A progressive loss of telomeric DNA in human cardiac stem cells (hCSCs) occurs with aging and the newly formed cardiomyocytes inherit short telomeres and rapidly reach the senescent phenotype. Our data provide novel information on the superior ability of the female heart to sustain the multiple variables associated with the development of the senescent myopathy. At all ages, the female heart is equipped with a larger pool of functionally competent hCSCs and younger myocytes than the male myocardium. The replicative potential is higher and telomeres are longer in female hCSCs than in male hCSCs. In the female heart, myocyte turnover occurs at a rate of 10%, 14%, and 40% per year at 20, 60, and 100 years of age, respectively. Corresponding values in the male heart are 7%, 12%, and 32% per year, documenting that cardiomyogenesis involves a large and progressively increasing number of parenchymal cells with aging. From 20 to 100 years of age, the myocyte compartment is replaced 15 times in women and 11 times in men. CONCLUSIONS: The human heart is a highly dynamic organ regulated by a pool of resident hCSCs that modulate cardiac homeostasis and condition organ aging.

Prognostic stratification of stage IIIA pN2 non-small cell lung cancer by hierarchical clustering analysis of tissue microarray immunostaining data: an Alpe Adria Thoracic Oncology Multidisciplinary Group study (ATOM 014).

INTRODUCTION: Stage IIIA non-small cell lung cancer (NSCLC) with ipsilateral mediastinal lymph node metastases (N2) is a heterogeneous disease with differing prognoses. In this study, we retrospectively investigated the prognostic value of the expression of 10 molecular markers in 87 patients with stage IIIA pN2 NSCLC treated with radical surgery. METHODS: Primary tumor tissue microarrays (TMAs) were constructed and sections used for immunohistochemical analysis of epidermal growth factor receptor, ErbB-2, c-kit, cyclooxygenase-2, survivin, bcl-2, cyclin D1, cyclin B1, metalloproteinase (MMP)-2, and MMP-9. Univariate and multivariate analyses and unsupervised hierarchical clustering analysis of clinical pathologic and immunostaining data were performed. RESULTS: Bcl-2 (p < 0.0001) and cyclin D1 (p = 0.015) were more highly expressed in squamous cell carcinoma (SCC), whereas MMP-2 (p = 0.009), MMP-9 (p = 0.005), and survivin (p = 0.032) had increased expression in other histologic subtypes. In univariate analysis, SCC histology and cyclin D1 expressions were favorable prognostic factors (p = 0.015 and p < 0.0001, respectively); by contrast, MMP-9 expression was associated with worse prognosis (p = 0.042). In multivariate analysis, cyclin D1 was the only positive prognostic factor (p < 0.0001). Unsupervised hierarchical clustering analysis of TMA immunostaining data identified five distinct clusters. They formed two subsets of patients with better (clusters 1 and 2) and worse (clusters 3, 4, and 5) prognoses, and median survival of 51 and 10 months, respectively (p < 0.0001). The better prognosis subset mainly comprised patients with SCC (80%). CONCLUSIONS: Hierarchical clustering of TMA immunostaining data using a limited set of markers identifies patients with stage IIIA pN2 NSCLC at high risk of recurrence, who may benefit from more aggressive treatment.

Influence of iodide excess and interferon-gamma on human primary thyroid cell proliferation, thyroglobulin secretion, and intracellular adhesion molecule-1 and human leukocyte antigen-DR expression.

BACKGROUND: The effect of iodide on thyroid cell proliferation and function in vivo or in cultured thyroid cells has been previously reported and is still controversial. The aim of this study was to clarify these conflicting results by examining if prolonged high iodide exposition with or without interferon (IFN)-gamma has an effect on human primary thyroid cell proliferation, thyroglobulin (Tg) production, and intercellular adhesion molecule-1 (ICAM-1) and human leukocyte antigen (HLA)-DR expression. METHODS: Primary human thyroid cells were used. Cells were cultured in Coon's modified Ham's F-12 medium supplemented with 5% fetal calf serum in monolayer conditions to induce proliferation and were aggregated for molecular expression and Tg production analysis. HLA-DR and ICAM-1 expression were measured by flow cytometry and Tg by immunometric assay. RESULTS: Potassium iodide (KI) was more potent in arresting primary human thyroid cell proliferation as compared to sodium iodide and the effect was mediated by its action at G0/G1 and G2/M phases of the cell cycle. There were no signs of apoptosis or necrosis. An excess of KI alone did not change the expression of HLA-DR and Tg production, but gradually increased ICAM-1. Low-dose IFN-gamma and excess KI in combination transiently inhibited HLA-DR expression, while ICAM-1 was expressed at a higher level than with IFN-gamma alone. Tg production was moderately increased with low-dose IFN-gamma. However, a combination of high-dose KI with low-dose IFN-gamma significantly decreased Tg secretion, compared with IFN-gamma alone. CONCLUSIONS: Augmented ICAM-1 in the presence of iodide excess and low-dose IFN-gamma could induce secretion of proinflammatory cytokines and lymphocytic infiltration in the thyroid gland. Decreased Tg production in the presence of KI excess and IFN-gamma could explain the development of hypothyroidism after adding iodide in a diet of subjects that already have lymphocytic infiltration and/or mild inflammation in the thyroid gland.

Are PSA density and PSA density of the transition zone more accurate than PSA in predicting the pathological stage of clinically localized prostate cancer?

PURPOSE: To assess whether PSA density (PSAD) and PSA density of the transition zone (PSADTZ) are more accurate than PSA alone in predicting the pathological stage of prostate cancer. MATERIALS AND METHODS: One hundred and nine consecutive patients with clinically localized prostate cancer and preoperative PSA values over the whole range, treated with radical retropubic prostatectomy and limited pelvic lymph node dissection were included in this prospective study. Total prostate and transition zone volumes were measured by transrectal ultrasound using the prolate ellipsoid method. PSA, PSAD, and PSADTZ were compared to percentage of positive biopsy cores (% PC), biopsy and surgical Gleason score, and pathological stage, using univariate and multivariate analysis. RESULTS: Pathological stage was pT2a, pT2b, pT3a, and pT3b in 25.6%, 37.7%, 25.6%, and 11.1% of patients, respectively. Lymph node metastases were found in 4.6% of patients. PSA, PSAD, and PSADTZ were significantly related to % PC, biopsy, and surgical Gleason score and pathological stage (P < 0.001), and were equally able to predict higher pathological stage, i.e., seminal vesicle invasion and lymph node metastases. Only by adding % PC in multivariate analysis was it possible to discriminate intra- from extracapsular tumors. CONCLUSIONS: The results of the present study demonstrate that PSAD and PSADTZ failed to outperform PSA in preoperative stage prediction of prostate cancer, possibly because the formula used to calculate them does not eliminate the contribution to total PSA of the nonmalignant portion of the gland.

Multipotent cells can be generated in vitro from several adult human organs (heart, liver, and bone marrow).

The aims of our study were to verify whether it was possible to generate in vitro, from different adult human tissues, a population of cells that behaved, in culture, as multipotent stem cells and if these latter shared common properties. To this purpose, we grew and cloned finite cell lines obtained from adult human liver, heart, and bone marrow and named them human multipotent adult stem cells (hMASCs). Cloned hMASCs, obtained from the 3 different tissues, expressed the pluripotent state-specific transcription factors Oct-4, NANOG, and REX1, displayed telomerase activity, and exhibited a wide range of differentiation potential, as shown both at a morphologic and functional level. hMASCs maintained a human diploid DNA content, and shared a common gene expression signature, compared with several somatic cell lines and irrespectively of the tissue of isolation. In particular, the pathways regulating stem cell self-renewal/maintenance, such as Wnt, Hedgehog, and Notch, were transcriptionally active. Our findings demonstrate that we have optimized an in vitro protocol to generate and expand cells from multiple organs that could be induced to acquire morphologic and functional features of mature cells even embryologically not related to the tissue of origin.

Prevention of postoperative recurrence of Crohn's disease by infliximab.

The prevention of the recurrence of Crohn's disease after surgery remains difficult. The monoclonal antibody anti-TNF-alpha, infliximab, is very effective in inducing and maintaining the remission of uncomplicated, active Crohn's disease. We present here the case of a 23-year-old white woman who underwent resection for a sigmoid stricture caused by Crohn's disease. Surgery removed the involved colon, and pathology confirmed the stricture to be fibrotic. Two weeks after the operation she was given infliximab at the dose of 5 mg/kg body weight and followed in time. Since then, she has been disease free for approximately 4 years after surgery on clinical, radiological and endoscopic/histological grounds (Crohn's Disease Activity Index < or = 110 on all occasions). Up to now, she has had no increase in inflammatory indices, no anaemia and no abnormal blood tests. In contrast, all of five control patients operated in the same period with colonic or ileocolonic resection for symptomatic strictures and treated with mesalamine or no medication developed endoscopic or clinical recurrence (abdominal pain or diarrhoea) by year 3. This is the first case, to our knowledge, in which infliximab has been successfully used to prevent the postsurgical recurrence of Crohn's disease, an event so far considered to be inescapable. We believe that, with this aim in mind, clinical trials with this drug are warranted.

Neovascularization and mast cells with tryptase activity increase simultaneously with pathologic progression in human endometrial cancer.

OBJECTIVE: In vitro and in vivo studies have linked mast cell (MC) degranulation and activation with angiogenesis and neovascularization. This assumption is partially supported by the close anatomic association between MC and the vasculature and the recruitment of these cells during tumor growth. The aim of this study was to correlate the extent of angiogenesis with the number of MC expressing tryptase in human endometrial adenocarcinoma. STUDY DESIGN: Tissues from human endometrial hyperplasia and endometrial adenocarcinoma were investigated immunohistochemically, using 2 murine monoclonal antibodies against the endothelial cell marker CD31 and the MC marker tryptase. RESULTS: Angiogenesis, measured as microvessel counts, was highly correlated with MC tryptase-positive cell counts and that these parameters increase in agreement with tumor progression. CONCLUSION: These results suggest that angiogenesis in endometrial cancer increases with tumor progression and that angiogenic tryptase secreted by host MC cooperate in its induction.

A pixel-based autofocusing technique for digital histologic and cytologic slides.

The present paper describes a method for autofocusing specifically studied for the acquisition of digital slides, i.e. full histologic and cytologic slides, utilising low-cost equipment. At first, experimentations with some of the most used focus measures and algorithms have been made, in order to choose the most suitable for histologic and cytologic images. Then, a study of the specific features of digital slides has been preliminarily carried out in order to understand the constraints of the domain. These included the capability of autofocusing in an unattended way on thousands of microscope fields, while fast performance is not a strict requirement. Based on the findings, an algorithm based on a dynamic focus position space, with a variance-based focus measure has been adapted to the specific situation. A qualitative and quantitative evaluation of the proposed algorithm allowed us to show that the proposed algorithm is suitable for the acquisition of digital slides, and furthermore it can be implemented starting from a basic microscope with an inexpensive motorised stage. The algorithm is currently implemented into a complete digital slide acquisition system, which is in turn being used for a Quality Assurance Programme in cervicovaginal cytologic screening.

Desmoplastic melanoma of the nail.

Desmoplastic melanoma represents a variant of melanoma that is difficult to diagnose because 71% of patients have amelanotic skin lesions. In the acral region of the limbs, the clinical diagnosis is more difficult, especially in cases in which there are not clear, rapidly growing, pigmented nail streaks. Histopathological identification of desmoplastic melanoma is confusing because of the intense fibrous reaction in the dermis and minimal, atypical melanocytic proliferation at the dermal-epidermal junction. For these reasons, it is still misdiagnosed unfortunately as a variety of entities, including simple scar, fibrohistiocytic neoplasms, neural tumors, and superficial fibromatoses-with potentially devastating consequences. In equivocal cases, the use of immunohistochemistry (in particular S-100 and neuron-specific enolase) may be helpful in establishing the diagnosis. Because of the high local recurrence rate for desmoplastic melanoma of the finger, amputation is recommended in an effort to gain effective tumor control. Lymph node dissection may be reserved for patients with positive axillary nodes.

Introducing videoconferencing into educational oncopathology seminars: technical aspects, user satisfaction and open issues.

We used set-top videoconferencing equipment connected by ISDN at 384 kbit/s for six educational seminars held between the University of Udine (the local site) and the National Cancer Institute in Aviano (the remote site), 60 km away. User satisfaction was evaluated by questionnaire. The median length of seminars was 58 min (range 48-61 min), followed by a 20 min (15-26 min) discussion. Eighty-two users answered the questionnaire (a 43% response rate): 56 in Udine (a median of 11 per seminar) and 26 in Aviano (a median of 5 per seminar). Answers to the questions were similar at the two sites. Videoconferencing did not affect the users' experience of attending the seminars, as both interest and clarity were similar at the local and remote site. The results suggested that videoconferencing is a viable method for delivering seminars in oncopathology, where image quality is important.

Chimerism of the transplanted heart.

BACKGROUND: Cases in which a male patient receives a heart from a female donor provide an unusual opportunity to test whether primitive cells translocate from the recipient to the graft and whether cells with the phenotypic characteristics of those of the recipient ultimately reside in the donor heart. The Y chromosome can be used to detect migrated undifferentiated cells expressing stem-cell antigens and to discriminate between primitive cells derived from the recipient and those derived from the donor. METHODS: We examined samples from the atria of the recipient and the atria and ventricles of the graft by fluorescence in situ hybridization to determine whether Y chromosomes were present in eight hearts from female donors implanted into male patients. Primitive cells bearing Y chromosomes that expressed c-kit, MDR1, and Sca-1 were also investigated. RESULTS: Myocytes, coronary arterioles, and capillaries that had a Y chromosome made up 7 to 10 percent of those in the donor hearts and were highly proliferative. As compared with the ventricles of control hearts, the ventricles of the transplanted hearts had markedly increased numbers of cells that were positive for c-kit, MDR1, or Sca-1. The number of primitive cells was higher in the atria of the hosts and the atria of the donor hearts than in the ventricles of the donor hearts, and 12 to 16 percent of these cells contained a Y chromosome. Undifferentiated cells were negative for markers of bone marrow origin. Progenitor cells expressing MEF2, GATA-4, and nestin (which identify the cells as myocytes) and Flk1 (which identifies the cells as endothelial cells) were identified. CONCLUSIONS: Our results show a high level of cardiac chimerism caused by the migration of primitive cells from the recipient to the grafted heart. Putative stem cells and progenitor cells were identified in control myocardium and in increased numbers in transplanted hearts.