Development and validation of low-intensity pulsed ultrasound systems for highly controlled in vitro cell stimulation.

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500 kHz to 5 MHz) and for a low-frequency value (38 kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3 min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biological in vitro tests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Adipose stromal cells mediated switching of the pro-inflammatory profile of M1-like macrophages is facilitated by PGE2: in vitro evaluation.

OBJECTIVE: To define if adipose mesenchymal stromal cell (ASC) treatment mediated switching of the pro-inflammatory profile of M1-like macrophages as a means to develop a tailored in vitro efficacy/potency test. DESIGN: We firstly performed immunohistochemical analysis of CD68, CD80 (M1-like) and CD206 (M2-like) macrophages in osteoarthritic (OA) synovial tissue. ASC were co-cultured in contact and in transwell with activated (GM-CSF + IFNγ)-M1 macrophages. We analyzed IL1ß, TNFα, IL6, MIP1α/CCL3, S100A8, S100A9, IL10, CD163 and CD206 by qRT-PCR or immunoassays. Prostaglandin E2 (PGE2) blocking experiments were performed using PGE2 receptor antagonist. RESULTS: In moderate grade OA synovium we did not always find a higher percentage of CD80 with respect to CD206. M1-like-activated macrophage factors IL1ß, TNFα, IL6, MIP1α/CCL3, S100A8 and S100A9 were down-modulated both in contact and in transwell by ASC. However, in both systems ASC induced the typical M2-like macrophage markers IL10, CD163 and CD206. Activated-M1-like macrophages pre-treated with PGE2 receptor antagonist failed to decrease secretion of TNFα, IL6 and to increase that of IL10, CD163 and CD206 when co-cultured with ASC confirming a PGE2 specific role. CONCLUSIONS: We demonstrated that ASC are responsible for the switching of activated-M1-like inflammatory macrophages to a M2-like phenotype, mainly through PGE2. This evidenced that activated-M1-like macrophages may represent a relevant cell model to test the efficacy/potency of ASC and suggests a specific role of ASC as important determinants in therapeutic dampening of synovial inflammation in OA.

Specific inductive potential of a novel nanocomposite biomimetic biomaterial for osteochondral tissue regeneration.

Osteochondral lesions require treatment to restore the biology and functionality of the joint. A novel nanostructured biomimetic gradient scaffold was developed to mimic the biochemical and biophysical properties of the different layers of native osteochondral structure. The present results show that the scaffold presents important physicochemical characteristics and can support the growth and differentiation of mesenchymal stromal cells (h-MSCs), which adhere and penetrate into the cartilaginous and bony layers. H-MSCs grown in chondrogenic or osteogenic medium decreased their proliferation during days 14-52 on both scaffold layers and in medium without inducing factors used as controls. Both chondrogenic and osteogenic differentiation of h-MSCs occurred from day 28 and were increased on day 52, but not in the control medium. Safranin O staining and collagen type II and proteoglycans immunostaining confirmed that chondrogenic differentiation was specifically induced only in the cartilaginous layer. Conversely, von Kossa staining, osteocalcin and osteopontin immunostaining confirmed that osteogenic differentiation occurred on both layers. This study shows the specific potential of each layer of the biomimetic scaffold to induce chondrogenic or osteogenic differentiation of h-MSCs. These processes depended mainly on the media used but not the biomaterial itself, suggesting that the local milieu is fundamental for guiding cell differentiation. Copyright © 2013 John Wiley & Sons, Ltd.

Lack of anti-inflammatory and anti-catabolic effects on basal inflamed osteoarthritic chondrocytes or synoviocytes by adipose stem cell-conditioned medium.

OBJECTIVE: To define whether good manufacturing practice (GMP)-clinical grade adipose stem cell (ASC)-derived conditioned medium (CM) is as effective as GMP-ASC in modulating inflammatory and catabolic factors released by both osteoarthritis (OA) chondrocytes or synoviocytes. METHODS: OA chondrocytes and synoviocytes were treated with ASC-CM or co-cultured with ASC. Inflammatory factors (IL6, CXCL1/GROα,CXCL8/IL8, CCL2/MCP-1, CCL3/MIP-1α and CCL5/RANTES) and proteinases, such as metalloproteinase (MMP13), a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS4, ADAMTS5) and their tissue metalloproteinase inhibitors (TIMP1, TIMP3) were evaluated by qRT-PCR or immunoassays. The involvement of prostaglandin E2 (PGE2) was also analyzed. RESULTS: Most ASC-CM ratios tested did not decrease IL6, CCL2/MCP-1, CCL3/MIP1-α, CCL5/RANTES on basal inflamed chondrocytes or synoviocytes in contrast to what we found using ASC in co-culture. CXCL8/IL8 and CXCL1/GROα were not decreased by ASC-CM on synoviocytes but were only partially reduced on chondrocytes. Moreover, ASC-CM was less efficient both on basal inflamed OA chondrocytes and synoviocytes in reducing proteinases, such as MMP13, ADAMTS4, ADAMTS5 and increasing TIMP1 and TIMP3 compared to ASC in co-culture. The different ratios of ASC-CM contain lower amounts of PGE2 which were not sufficient to reduce inflammatory factors. CONCLUSIONS: These data show that ASC-CM has a limited ability to decrease inflammatory and proteinases factors produced by OA chondrocytes or synoviocytes. ASC-CM is not sufficient to recapitulate the beneficial effect demonstrated using ASC in co-culture with inflamed OA chondrocytes and synoviocytes and shows that their use in clinical trials is fundamental to counteract OA progression.

Slug transcription factor and nuclear Lamin B1 are upregulated in osteoarthritic chondrocytes.

OBJECTIVE: To contribute to clarify molecular mechanisms supporting senescence and de-differentiation of chondrocytes in chondrocyte pathologies such as osteoarthritis (OA). Specifically, we investigated the relationship between the nuclear lamina protein Lamin B1 and the negative regulator of chondrogenesis Slug transcription factor in osteoarthritic chondrocytes. METHODS: Lamin B1 and Slug proteins were analyzed in cartilage explants from normal subjects and OA patients by immunohistochemical technique. Their expression was confirmed on isolated chondrocytes both at passage 0 and passage 2 (de-differentiated chondrocytes) by immunofluorescence and western blot. Subsequently, we explored the "in vivo" binding of Slug on LMNB1 promoter by chromatin immunoprecipitation assay (ChIP). RESULTS: In this study we demonstrated that nuclear lamina protein Lamin B1 and anti-chondrogenic Slug transcription factor are upregulated in cartilage and OA chondrocytes. Furthermore, we found that Slug is "in vivo" recruited by LMNB1 gene promoter mostly when chondrocytes undergo de-differentiation or OA degeneration. CONCLUSIONS: We described for the first time a potential regulatory role of Slug on the LMNB1 gene expression in OA chondrocytes. These findings may have important implications for the study of premature senescence, and degeneration of cartilage, and may contribute to develop effective therapeutic strategies against signals supporting cartilage damage in different subsets of patients.

Chemical-physical properties and in vitro cell culturing of a novel biphasic bio-mimetic scaffold for osteo-chondral tissue regeneration.

The requirements for a successful regeneration of an osteo-chondral defect could effectively be met by using a bi-layered composite scaffold, able to support proliferation and differentiation of mesenchymal stem cells, while providing a biochemical environment promoting the formations of the two distinct tissues. The novel strategy here presented consists of developing a bio-mimetic scaffolds obtained by the combination of two integrated organic compounds (type I collagen and chitosan) with or without bioactive Mg-doped hydroxyapatite (Mg-HA) nanocrystals, depending on the specific layer, reproducing cartilaginous or subchondral bone tissue. An innovative patented methodology for scaffolds production, called - pH-dependent 3-phasic assembling -, allowed to development of a highly homogenous and chemically stable scaffold, presenting a very good integration among all three components, as confirmed by extensive SEM and thermogravimetric analyses. A preliminary in vitro evaluation was also carried out by seeding bi-layered scaffold with human bone marrow stromal cells (h-MSCs), by giving particular emphasis to cell viability and distribution at day 0, 7 and 14. Cells were viable and uniformly colonized the whole scaffold until day 14, indicating that the scaffold contributed to the maintenance of cell behaviour.

Two years' experience of acute rejection monitoring of intestinal transplant recipients by real-time PCR assessment of granzyme B and perforin up-regulation: considerations on diagnostic accuracy.

Granzyme B (GrB) and perforin are promising immunological markers to predict acute rejection of transplanted organs. Based on 2 years of experience with molecular monitoring on peripheral blood samples, we investigated the diagnostic accuracy of GrB/perforin gene up-regulation using real-time polymerase chain reaction (PCR) for prediction of acute cellular rejection (ACR) in intestinal transplantation recipients. Histology used as the reference standard. According to our definition of disease positivity (anything other than ACR score 0), GrB/perforin up-regulation showed 84% specificity but only 49% sensitivity. However, among the 26 false-negatives, 12 (46%) had an ACR score 1, which is indeterminate for rejection and no associated clinical manifestations; a further 10 (39%) had a score of 2 following rejection therapy (a confounder for GrB/perforin analysis). Thus only 4 (15%) false-negatives were actually associated with the onset of robust acute rejection. These data suggest that real-time PCR analysis for GrB/perforin up-regulation might play a role along with clinical criteria for detection of presymptomatic acute rejection episodes in intestinal recipients who require immediate endoscopy and pathological examination, especially during long-term follow-up.

Potential of real-time PCR assessment of granzyme B and perforin up-regulation for rejection monitoring in intestinal transplant recipients.

Granzyme B (GrB) and perforin are promising markers to predict acute rejection episodes of transplanted organs. Having recently reported that immunohistochemical expression of GrB/perforin correlates with histologically assessed acute cellular rejection (ACR) episodes in intestinal transplantation recipients, herein we have additionally explored the potential of real-time polymerase chain reaction (PCR) assessment of GrB/perforin gene up-regulation in peripheral blood mononuclear cells. Both immunohistochemical evaluation of GrB/perforin expression and real-time PCR assessment of up-regulation, which was defined as a 2-fold increase with respect to "basal" levels during maintenance immunosuppressive protocols, were performed among a population of 23 intestinal transplant recipients under routine surveillance, in addition to histological analysis of ACR. The ACR scores showed direct relationships both with GrB/perforin immunohistochemistry (IHC) scores (P < .001) and with gene up-regulation by real-time PCR (P = .004). Furthermore, real-time PCR upregulation was associated with the IHC score (P < .001). A preliminary analysis of diagnostic accuracy-performed to gain information to plan future studies-indicated that when using histological assessment as the reference technique, our current definition of PCR up-regulation provided good specificity (84%) but insufficient sensitivity (44%) for a noninvasive prediction of ACR. The results of this pilot study suggested that real-time PCR analysis of GrB/perforin upregulation may help therapeutic decision making, and have the potential for detection of presymptomatic rejection. More extensive studies must investigate strategies to improve the sensitivity of the analyses of GrB/perforin up-regulation.

Molecular monitoring of organ recipients from cancer-affected donors by detection of circulating tumor cells.

We have initiated regular molecular monitoring based on nested RT-PCR detection of circulating tumor cells for monitoring recipients of organs from cancer-affected donors in Italy (in the context of a "Donation Safety and Donated Organ Quality" project organized by the Centro Nazionale Trapianti). Five patients are being monitored. For two patients who each received a kidney from a single donor with prostate adenocarcinoma, RT-PCR was performed using PSA mRNA. For three recipients of organs (two livers and one kidney) from donors with renal cell carcinoma, RT-PCR was performed using cytokeratine 18 and 19 mRNA. Blood samples from five healthy subjects were used as negative controls. After a median monitoring time of 26 months (range 8 to 32), none of the regular 3-month assays has tested positive. This pilot study suggests that detection of circulating tumor cells by nested RT-PCR may provide a feasible molecular monitoring, which might assist decision making regarding other forms of clinical surveillance.

Granzyme B and perforin as predictive markers for acute rejection in human intestinal transplantation.

In human heart and kidney transplantations, granzyme B (GrB) and perforin have both been shown to be predictive markers for acute cellular rejection (ACR). We investigated the tissue expression and possible relationship of GrB and perforin to the clinical outcome, histopathology, and function of intestinal transplants. In 13 consecutive patients undergoing small intestine transplantation, histologic/immunohistochemical rejection monitoring was performed together with GrB and perforin immunostaining (score "0", 0%-10% positive lymphocytes; "1", 10%-25%; "2", 25%-50%; "3", >50%). Eleven patients are currently alive and well. All 11 had at least one episode of ACR: one patient had 6 episodes of severe ACR requiring retransplantation; the remaining 10 experienced only mild or moderate rejection. Both GrB and perforin were always co-expressed. A highly significant correlation was observed between GrB/perforin scores and histological severity of ACR (Pearson's coefficient, R < 0.0009). Interestingly, score 3 GrB/perforin immunostaining was recorded only in the context of severe ACR; all the histologically negative or "indeterminate" biopsies (n = 6) taken from a single affected patient showed GrB/perforin scores of 1 or 2. By contrast, none of the other tested histologically negative/"indeterminate" biopsies (n = 350), including those performed during graft stabilization, had raised GrB or perforin scores. We conclude that in intestinal transplantation recipients, a direct correlation seems to exist between histologically confirmed ACR and raised GrB/perforin immunohistochemical scores. Our findings suggest the need to investigate the possibility of predicting ACR by routine serum polymerase chain reaction (PCR) monitoring, which would reduce discomfort to patients.

Investigation of ErbB1 and ErbB2 expression for therapeutic targeting in primary liver tumours.

BACKGROUND: Molecular targets are needed for primary liver tumours. AIMS: ErbB1 and ErbB2 expression was analysed in neoplastic and surrounding tissue in surgical specimens from 52 hepatocellular carcinomas and 48 intrahepatic cholangiocarcinomas, randomly chosen from cases surgically treated in this institution. METHODS: ErbB1 and ErbB2 expression were evaluated immunohistochemically, the latter by Herceptest. Gene amplification of ErbB2 was tested by chromogenic in situ hybridisation. RESULTS: In normal/cirrhotic non-neoplastic tissue, the ErbB1 (but not ErbB2) antibody commonly stained normal hepatocytes and mature intrahepatic ducts. In neoplastic tissue, moderate/strong ErbB1 immunostaining occurred in 43/52 (85%) hepatocellular carcinomas and 39/48 (81%) intra-hepatic cholangiocarcinomas. With ErbB2 Herceptest, 0/52 (0%) hepatocellular carcinomas and 2/48 (4%) intra-hepatic cholangiocarcinomas had treatable scores of 2+/3+ (chromogenic in situ hybridisation confirmed gene amplification in the latter two cases only). Neither ErbB1 nor ErbB2 expression correlated with any of the main clinical-pathologic features or survival. CONCLUSIONS: Although not related to prognosis, ErbB1 could be a molecular target in a large percentage of patients with hepatocellular carcinoma or intrahepatic cholangiocarcinoma. Inclusion of anti-ErbB1 drugs such as ZD 1839 and c225 (and possibly also anti-ErbB2 drugs like Trastuzumab for a small subset of patients) in clinical trials is suggested.

Ovarian tissue banking and fertility preservation in cancer patients: histological and immunohistochemical evaluation.

OBJECTIVE: A combination of chemotherapy and radiotherapy in young females with cancer has greatly enhanced the life expectancy of these patients, even if these treatments have a highly deleterious effect on the ovary and cause a severe depletion of the follicular store. Cryopreservation of ovarian tissue before chemotherapy and/or radiotherapy, followed by autograft after remission or in in vitro maturation, could restore gonadal function and fertility. The aim of this study is to verify the efficiency of the ovarian tissue cryopreservation procedure by histological and immunohistochemical analyses. METHODS: Ovarian tissue was obtained by laparoscopy from 22 patients affected with different malignant diseases. Tissue specimens were frozen using a combination of PROH (1,2-propanediol) and sucrose as cryoprotectants, and the cryopreservation protocol used consisted of a slow freezing-rapid thawing program. Both fresh and frozen/thawed tissues were embedded in paraffin blocks for histological and immunohistochemical analyses. RESULTS: Good stromal and follicular morphology was found in fresh and frozen/thawed tissue. No significant differences were found in follicular density, distribution, and diameters in fresh and frozen/thawed tissue. Follicle immunohistochemical analysis showed a high percentage of negative staining for both estrogen receptor (ER) (100% both in fresh and frozen/thawed specimens) and progesterone receptor (PR) (97% versus 91%, respectively). Regarding the Ki67 protein, positive staining was found in both the granulosa cells and/or the oocytes (36% in fresh and 56% in frozen/thawed). For the Bcl2 protein, positive staining was observed in the follicle granulosa cells but not in the oocytes in 74% of the fresh and in 79% of the frozen/thawed specimens. For the stromal cells, ER showed a negative staining distribution in 97% of the fresh and 100% of the frozen/thawed specimens. The stroma staining distribution was diffuse/focal in fresh versus frozen/thawed specimens (50% versus 74% respectively) for PR, patch/focal (70% versus 80%, respectively) for Ki67 protein, and diffuse (55% versus 54%, respectively) for Bcl2. CONCLUSIONS: These results suggest that human ovarian tissue morphology, antigenicity, cellular proliferation, and anti-apoptotic index were well preserved by cryopreservation in PROH and sucrose.

Low p27 expression is an independent predictor of survival for patients with either hilar or peripheral intrahepatic cholangiocarcinoma.

PURPOSE AND EXPERIMENTAL DESIGN: The prognosis for intrahepatic cholangiocarcinoma (ICC) depends mainly on the feasibility of complete surgical resection. In the absence of demonstrated biological predictors of survival, we evaluated the prognostic value of the cyclin-dependent kinase inhibitor p27 by immunohistochemistry in a series of routine specimens from 47 ICC patients, 22 with the hilar and 25 with the peripheral subtype. Proliferation rate was also evaluated in the same cases by the MIB1 index. Tumors were scored as high, low, and negative p27 expressers (> or =50%, <50%, and no positive nuclei, respectively). RESULTS: High, low, and negative p27 expression was recorded in 18 (38%), 17 (36%), and 12 (26%) cases, respectively. No significant correlation was found between p27 expression and gender, age, tumor grade, tumor location, vascular or perineural invasion, or proliferative index. Tumors with low or absent p27 expression were associated with poor survival compared with the high-expresser group. Kaplan-Meier curves comparing different p27 expression levels with survival showed highly significant separation (P < 0.0001, log-rank test). With univariate Cox proportional hazard regression, only p27 score among all of the parameters was found to influence survival (P = 0.0003). CONCLUSION: We conclude that in ICC, low or absent p27 expression can predict poor survival, regardless of tumor location, pathological features, and tumor proliferation. Immunohistochemical detection of p27 on routine sections may provide the first biological prognostic marker for ICC, thus influencing the therapeutic strategies for these patients.

Expression of Epstein-Barr virus-encoded RNA and biological markers in Italian nasopharingeal carcinomas.

Nasopharingeal carcinoma (NPC) is frequently associated with Epstein-Barr virus (EBV), but little is known about its pathogenesis in Western countries, where its incidence is low and EBV is not endemic. We studied 26 Italian cases of NPC: 24 of the non-keratinizing subtype, and 2 keratinizing. We used immunohistochemistry to evaluate p53, bcl-2 and Ki-67 protein expression, and in situ hybridization (ISH) to assess bcl-2 mRNA and the EBV transcript, EBER1, with bcl-2 protein and mRNA expression being compared in serial sections. As regards bcl-2, the protein or mRNA was expressed in 15/26 (58%) and 17 (65%) cases respectively, with co-expression being found only in 9 (35%) cases; 23 (88%) cases were positive for one or the other. EBER1 was detected in 17/26 (65%) cases, of which 80% and 71% coexpressed bcl-2 protein or mRNA, respectively. Only 9/26 (35%) cases had a high Ki67 proliferative index; 3/26 (12%) overexpressed p53. This study of NPC in Italy confirms the importance of EBV and bcl-2 in Western cases. The discrepancy between bcl-2 mRNA and protein expression invites investigation into a possible internal bcl-2 regulation system.

Solitary fibrous tumour of the urinary bladder with expression of bcl-2, CD34, and insulin-like growth factor type II.

We describe a solitary fibrous tumour of the urinary bladder wall removed from a 50-year-old man with a history of pelvic pain, dysuria, and urinary bleeding. Anamnesis revealed a weight increase during the preceding 3 months, but no apparent episodes of biochemical hypoglycaemia or hormonal abnormalities. The patient is alive and well 18 months after surgery. Pathological examination revealed a 6.5-cm well-circumscribed nodular mass composed of uniform spindle cells arranged in bundles and fascicles with varying amounts of collagen and a typical haemangiopericytoma-like vascular pattern. The tumour cells were positive for bcl-2, CD34, and vimentin and ultrastructurally showed mesenchymal-myofibroblastic traits. These cells produced insulin-like growth factor type II mRNA as demonstrated by non-isotopic in situ hybridization. This rare case with a solitary fibrous tumor suggests that insulin-like growth factor type II could join CD34 and bcl-2 as markers for postoperative differential diagnosis.

Hepatoid adenocarcinoma of the rectum arising in ulcerative colitis: report of a case.

We report a case of intestinal hepatoid adenocarcinoma, confirmed by albumin m-RNA in situ hybridization, with subsequent metastatic spread to the liver in a male with a long-standing history of ulcerative colitis. This novel finding strongly suggests that ulcerative colitis can lead not only to conventional adenocarcinomas but also to hepatoid adenocarcinoma and highlights the mimicry of hepatocellular carcinoma by metastatic hepatoid adenocarcinoma liver nodules.