Fibronectin-adherent peripheral blood derived mononuclear cells as Paclitaxel carriers for glioblastoma treatment: An in vitro study.

BACKGROUND: Glioblastoma (GBM) represents the most aggressive malignant brain tumor in adults, with a risible median life expectancy despite gold standard treatment. Novel drug-delivery methods have been explored. Here we evaluated the possibility to use mononuclear cells (MCs) belonging to the monocytic-dendritic lineage as drug-carrier. METHODS: MCs were obtained from 10 patients harboring a GBM, and from healthy volunteers, considered as controls. GBM tissue was also obtained from patients. MCs were cultured and the adherent population on fibronectin (FN-MCs), after immunocytochemistry and flow cytometry characterization, was loaded with Paclitaxel (FN-MCs-PTX). Antiproliferative and migration activity of FN-MCs-PTX was evaluated in two-dimensional (2D) and three-dimensional (3D) co-culture assays with red fluorescent U87 Malignant Glioma cells and primary GBM cells. Antiangiogenic properties of FN-MCs-PTX were tested on cultures with endothelial cells. RESULTS: Phenotypical characterization showed a high expression of monocytic-dendritic markers in GBM cells and FN-MCs. FN-MCs demonstrated to effectively uptake PTX and to strongly inhibit GBM growth in vitro (P <0.01). Moreover, tumor-induced migration of MCs, although partially affected by the PTX cargo, remained statistically significant when compared with unprimed cells and this was confirmed in a 3D Matrigel model (P <0.01) and in a Trans-well assay (P <0.01). FN-MCs-PTX also disclosed considerable antiangiogenic properties. DISCUSSION: Our results suggest that the fibronectin-adherent population of MCs isolated from peripheral blood can be an effective tool to inhibit GBM growth. Given the relative facility to obtain such cells and the short time needed for their culture and drug loading this approach may have potential as an adjuvant therapy for GBM.

Paclitaxel-releasing mesenchymal stromal cells inhibit the growth of multiple myeloma cells in a dynamic 3D culture system.

Multiple myeloma is an aggressive tumour able to suppress osteoblastogenesis probably mediated by bone marrow mesenchymal stromal cells (BM-MSCs) that can also support plasma cell growth/survival. The use of MSCs for multiple myeloma therapy is a controversial topic because of the contradictory results on the capacity of MSCs to inhibit or to promote cancer growth. Our previous studies demonstrated that MSCs could be loaded with Paclitaxel (PTX) and used to deliver the drug in situ in amount affecting tumour growth (in vitro and in vivo). Therefore, independently on the discussed action of MSCs in myeloma, MSCs could represent a 'trojan horse' to vehicle and deliver anti-tumour agents into bone marrow. This study confirms, by an in vitro 3D dynamic culture system, that PTX loaded BM-MSCs (PTXr-MSCs) are active on the proliferation of RPMI 8226, a human myeloma cell line. Our results demonstrated a dramatic suppression of myeloma cell growth by PTXr-MSCs, suggesting that drug loaded MSCs could be a tool to deliver drug into the bone marrow. Drug releasing MSCs provide a therapeutic approach to potentiate the existing treatments against a very aggressive malignancy as multiple myeloma. Copyright © 2016 John Wiley & Sons, Ltd.

HIV-1 matrix protein p17 promotes angiogenesis via chemokine receptors CXCR1 and CXCR2.

Vascular diseases supported by aberrant angiogenesis have increased incidence in HIV-1-infected patients. Several data suggest that endothelium dysfunction relies on action of HIV-1 proteins rather than on a direct effect of the virus itself. The HIV-1 matrix protein p17 is known to deregulate the biological activity of different immune cells. Recently, p17 was found to mimic IL-8 chemokine activity by binding to the IL-8 receptor CXCR1. Here we show that p17 binds with high affinity to CXCR2, a CXCR1-related receptor, and promotes the formation of capillary-like structures on human endothelial cells (ECs) by interacting with both CXCR1 and CXCR2 expressed on the EC surface. ERK signaling via Akt was defined as the pathway responsible for p17-induced tube formation. Ex vivo and in vivo experimental models confirmed the provasculogenic activity of p17, which was comparable to that induced by VEGF-A. The hypothesis of a major role for p17 in HIV-1-induced aberrant angiogenesis is enforced by the finding that p17 is detected, as a single protein, in blood vessels of HIV-1-patients and in particular in the nucleus of ECs. Localization of p17 in the nucleus of ECs was evidenced also in in vitro experiments, suggesting the internalization of exogenous p17 in ECs by mechanisms of receptor-mediated endocytosis. Recognizing p17 interaction with CXCR1 and CXCR2 as the key event in sustaining EC aberrant angiogenesis could help us to identify new treatment strategies in combating AIDS-related vascular diseases.

Microvascular infiltration has limited clinical value for treatment and prognosis in hepatocellular carcinoma.

BACKGROUND: Microvascular infiltration (MVI) is considered a necessary step in the metastatic evolution of hepatocellular carcinoma (HCC), but its prognostic value after liver resection (LR) is uncertain. We studied the clinical value of MVI compared to the Milan criteria in a consecutive series of patients submitted to radical LR. METHODS: A total of 441 patients were retrospectively evaluated. MVI and the Milan criteria were analyzed and compared as prognostic factors for overall and disease-free survival (DFS). RESULTS: MVI was present in 189 patients (42.8 %). Grading, satellitosis, size of cancer, and alfa fetoprotein value were significantly related to MVI, which was present in 34.3 and 53.2 % of Milan+ and Milan- patients, respectively (p = 0.00001). Both MVI and the Milan criteria were associated with a lower overall and DFS, but only the Milan criteria were associated with the rate of early recurrence and the feasibility of a curative treatment of the recurrence. The application of MVI parameters to patients classified by the Milan criteria further selects the outcome in Milan+ patients (5-year survival rate of 54.1 and 67.9 %, respectively, in the presence or absence of MVI) but not in Milan- patients. CONCLUSIONS: MVI is related to survival after LR for HCC, but the clinical value of this information is limited. In Milan+ patients, the absence of MVI selects the cases with better prognosis. In the presence of a liver recurrence, the Milan criteria related to the primary HCC show a better prognostic accuracy and have clinical relevance in the decision-making process.

Mesenchymal stromal cells primed with Paclitaxel attract and kill leukaemia cells, inhibit angiogenesis and improve survival of leukaemia-bearing mice.

Current leukaemia therapy focuses on increasing chemotherapy efficacy. Mesenchymal stromal cells (MSCs) have been proposed for carrying and delivery drugs to improve killing of cancer cells. We have shown that MSCs loaded with Paclitaxel (PTX) acquire a potent anti-tumour activity. We investigated the effect of human MSCs (hMSCs) and mouse SR4987 loaded with PTX (hMSCsPTX and SR4987PTX) on MOLT-4 and L1210, two leukaemia cell (LCs) lines of human and mouse origin, respectively. SR4987PTX and hMSCsPTX showed strong anti-LC activity. hMSCsPTX, co-injected with MOLT-4 cells or intra-tumour injected into established subcutaneous MOLT-4 nodules, strongly inhibited growth and angiogenesis. In BDF1-mice-bearing L1210, the intraperitoneal administration of SR4987PTX doubled mouse survival time. In vitro, both hMSCs and hMSCsPTX released chemotactic factors, bound and formed rosettes with LCs. In ultrastructural analysis of rosettes, hMSCsPTX showed no morphological alterations while the attached LCs were apoptotic and necrotic. hMSCs and hMSCsPTX released molecules that reduced LC adhesion to microvascular endothelium (hMECs) and down-modulated ICAM1 and VCAM1 on hMECs. Priming hMSCs with PTX is a simple procedure that does not require any genetic cell manipulation. Once the effectiveness of hMSCsPTX on established cancers in mice is proven, this procedure could be proposed for leukaemia therapy in humans.

The long bone deformity of osteogenesis imperfecta III: analysis of structural changes carried out with scanning electron microscopic morphometry.

The wedges of the mid-diaphyseal osteotomies carried out to correct the femoral and/or tibial native deformity in type III osteogenesis imperfecta (OI III) were used to study the remodeling patterns and lamellar organization at the level of the major deformity. Histology and scanning electron microscopy (SEM) morphology showed abnormal cortical remodeling characterized by the failure to form a cylinder of compact bone with a regular marrow canal. Atypical, flattened, and large resorption lacunae with a wide resorption front on one side and systems of parallel lamellae on the opposite side were observed, resembling those formerly reported as drifting osteons. SEM morphometry documented a higher percentage of nonossified vascular/resorption area (44.3 %) in OI than in controls (13.6 %), a lower density of secondary osteons, and lower values for the parameters expressing the individual osteon size. The mean osteon total area, the mean central canal area, and the mean osteon bone area of two selected, randomized populations of secondary osteons were significantly higher (p < 0.001, p = 0.028, and p < 0.001, respectively) in control bones than in OI. The mean ossified matrix area was not significantly different, but the mean secondary osteon number and mean density were higher in controls (both p < 0.001). Osteon wedges were carried out to correct the native deformity of OI III and morphologic analysis suggested that the abnormal remodeling pattern (with "drifting osteons") may result from the altered load and tensile stresses on the deformed tubular bones.

Transforming growth factor-beta1 induces microvascular abnormalities through a down-modulation of neural cell adhesion molecule in human hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a very angiogenic and malignant cancer. Conventional chemotherapy is poorly effective because of the abnormal structural organization of HCC-infiltrating vessels. In previous work, we demonstrated that HCC angiogenesis is driven by transforming growth factor beta-1(TGF-ß1)/CD105 axis, stimulating liver-derived microvascular endothelial cells (Ld-MECs) migration. As TGF-ß1 also affects mural cells (MCs) recruitment and maturation, we asked whether it may contribute to HCC-induced vascular abnormalities. HCC and adjacent non-neoplastic liver (nNL) biopsies obtained from 12 patients were analyzed by immunohistochemistry for angiogenic markers CD105, TGF-ß1, CD44 and vascular endothelial growth factor-a (VEGFa) and for MC markers NG2, α-smooth muscle actin (αSMA) and neural cell adhesion molecule (NCAM). The same markers were also investigated by immunocytochemistry on cultured HCC-derived stromal cells (HCC-StCs) and nNL-derived StCs (nNL-StCs) isolated from the same liver biopsies. Angiogenic factors released by StCs were analyzed by ELISA and the interaction between StCs and Ld-MECs by adhesion assay. Compared with nNL, HCC biopsies showed increased angiogenic markers and αSMA that was localized in vessels. By contrast, NG2 and NCAM were substantially localized in tumor cells but absent in vessels and stroma. Cultured HCC-StCs showed less expression of NG2, αSMA and NCAM. They also demonstrated a lower capacity to release angiogenic factors and adhered on Ld-MECs. HCC-StCs and nNL-StCs treated with TGF-ß1 or with of HepG2 (a human hepatoma cell line) derived conditioned medium (CM), down-modulated NCAM expression, whereas anti-NCAM antibodies significantly reduced the adhesion of StCs to Ld-MECs. By further blocking TGF-ß1 with anti-TGF-ß1 antibodies or with Ly-364947 (a specific inhibitor TGF-ß1-receptor) adhesion to Ld-MECs and NCAM expression respectively was partially restored. TGF-ß1 contributes to HCC-induced vascular alterations by affecting the interaction between HCC-StCs and Ld-MECs through a down-modulation of NCAM expression.

Diagnostic implications of L1, p16, and Ki-67 proteins and HPV DNA in low-grade cervical intraepithelial neoplasia.

The expressions of p16, Ki-67, and L1 proteins and human papillomavirus DNA were investigated using polymerase chain reaction (HPV/PCR) and catalyzed signal-amplified colorimetric DNA in situ hybridization (CSAC/ISH) as potential molecular markers for the diagnosis and transforming potential of low cervical intraepithelial neoplasia (CIN1). Ki-67 and p16 protein expression increased linearly from control cases to more dysplastic cases (CIN1, CIN2, and CIN3), peaking in squamous cell carcinoma cases (P<0.05). In contrast, L1 expression was inversely correlated with malignant transformation. Patients with CIN1 were divided into 4 groups: L1p16, L1p16, L1p16, and L1p16, and the immunohistochemical results were combined with HPV/PCR, L1/PCR, and high-risk E6/E7 genome and CSAC/ISH data. Malignant transformation correlated with L1p16 patients (100% of CIN2, CIN3, and squamous cell carcinoma cases) and was evident in approximately 23% of CIN1 cases. In addition, the presence of HPV/DNA was evident in 52% of CIN1 cases, and within the L1p16 group. In 4 of 7 cases, the high-risk E6/E7 HPV genome was present and in 1 case it was integrated into the host DNA, as confirmed using CSAC/ISH. In patients with CIN1, investigating the presence of HPV/DNA using PCR and the presence of the high-risk E6/E7 genome is necessary to distinguish high-risk oncogenic patient groups from low-risk groups. This study highlights the importance of combining immunohistochemical analysis with HPV/PCR and CSAC/ISH to identify patients with CIN1 with a risk of neoplastic progression.

Limited liver resection: a good indication for the treatment of hepatocellular carcinoma in elderly patients.

OBJECTIVE: Hepatocellular carcinoma is often diagnosed in elderly people. METHODS: One hundred and seventy-five patients older than 70 years were operated on for hepatocellular carcinoma (Group 1). The results were compared with 276 resected patients younger than 70 (Group 2) and to 108 aged patients with chronic liver disease without hepatocellular carcinoma (Group 3). RESULTS: Hepatocellular carcinoma in the elderly is more frequently associated with hepatitis C virus, less frequently capsulated and less frequently diagnosed by screening programs than in young patients. After resection, no difference was noted in post-operative complications and in mortality rates (3.2%); major hepatic resection in cirrhosis carried a high risk of death (22%). Five years survival was 42%, comparable with the young surgical patients but significantly lower than the medical patients in Group 3. Recurrence of hepatocellular carcinoma was the main reason of death, but it was suitable for a radical treatment in 37.6% of cases, including surgery, with a mean survival of 31 months. CONCLUSIONS: Liver resection is a valid option for the treatment of hepatocellular carcinoma in the elderly; major resections in cirrhotic old patients must be reserved for selected cases. Recurrence may be suitable of a radical approach, including surgery.

A review of the actual knowledge of the processes governing growth and development of long bones.

Autoptic samples of human bones (from 8 weeks of gestation to 12 years of age) and a second group of serial, skeletal x-rays (required for pathologies not related to bone dysplasia in children from 4 months to 17 years of age) provided the material for the analysis of the physes normal growth mechanism presented in this review. Before the appearance of the ossification centers epiphyseal growth rests exclusively on chondrocytes proliferation (interstitial growth), without any detectable differentiated cellular organization. When endochondral ossification starts a defined spatial disposition of chondrocytes and a corresponding organization of the intercellular matrix is set up, so that it is possible to identify a growth vector corresponding to the columns of piled chondrocytes with direction from hypertrophic toward the proliferative cell layers. The complexity of the tubular bones growth process is well represented by the spatial arrangement of the growth vectors. In the late epiphyseal growth another mechanism is active in addition to endochondral ossification, namely, articular cartilage interstitial growth and subchondral remodelling. The knowledge of the normal mode of organization of the physis and its temporal sequence can help to better understand of the deviaton from the normal development of metaphyseal and epiphyseal dysplasias.

Intrahepatic cholangiocarcinoma and combined hepatocellular-cholangiocarcinoma: a Western experience.

BACKGROUND: Intrahepatic cholangiocarcinoma (ICC) is an unusual tumour. METHODS: The clinicopathological data of 67 patients with ICC and combined hepatocellular-cholangiocarcinoma (HCC-ICC) are presented. RESULTS: HCV-HBV infection was present in 37.3% and chronic liver disease in 38.7% of cases, a rate higher than in the normal population; in these patients the cancer was small, often asymptomatic and of combined type. Liver resection was performed in 51 patients; at 1, 3 and 5 years, overall survival was 87.9%, 59.0%, and disease-free survival was 47.7% and 78.8%, 51.4%, and 46.7%, respectively. The better results were in the group of cirrhotic patients in whom ICC was diagnosed by a screening program for HCC (5-year survival 76.6%). Nodal metastasis showed negative prognostic value for both overall and disease-free survival; in N+ patients mean survival was 14.7 months after liver resection and lymph node dissection. CONCLUSION: Viral infection and cirrhosis may be considered risk conditions for ICC and combined HCC-ICC; in resected patients survival was good. Nodal metastases must not be considered a contraindication for liver resection.

Germline and somatic c-met mutations in multifocal/bilateral and sporadic papillary renal carcinomas of selected patients.

Papillary renal carcinoma (PRC) comprises about 10% of all kidney epithelial tumors. Familiar/hereditary papillary renal carcinomas (HPRCs) have been described, but the majority of cases seem to be sporadic. HPRC is characterized by the predisposition to develop bilateral, multifocal renal tumors. Activating mutations in the tyrosine kinase domain (TK) of the hepatocyte growth factor (HGF) receptor, c-met, have been identified in both hereditary and sporadic PRC. The main aim of this study was to examine a family with no history of PRC in which the proband was a female patient affected by multiple and bilateral PRC at early onset. DNA mutation analysis has been performed by direct sequencing of exons 14-21 of c-met gene which include the TK domain. The proband displayed the germline c-met missense mutation g.3522G--> A in exon 16. Two other family members were found to carry the same mutation. The mutation analysis extended to 15 selected patients, allowed to identify the first case of an Italian patient affected by PRC displaying the somatic missense mutation g.3997 T-->C curved arrow C located in exon 19 of c-met. The mutation frequency of the selected-based population of PRC patients in this report was 12.5%. Furthermore, the phosphorylated c-met expression detected by immunohistochemistry in PRCs with germline/somatic or no c-met mutation, supports the concept that c-met activation may occur in PRC oncogenesis by c-met mutations and/or c-met over-expression.

IL-6 Promotes compensatory liver regeneration in cirrhotic rat after partial hepatectomy.

Major hepatic resection in cirrhotic patients is associated with impaired liver regeneration and failure, leading to high peri-operative mortality. In this work, the causes of defective regeneration in cirrhotic liver and the utility of IL-6 treatment were investigated in an experimental model combining cirrhosis and partial hepatectomy in the rat. Relative to normal controls, decompensated cirrhotic animals showed decreased survival, while compensated cirrhotic animals showed similar survival but reduced hepatic DNA synthesis and newly regenerated liver mass amount. Defective liver regeneration was associated with a decrease in STAT3 and NF-kB activation, consistent with an increased accumulation of their respective inhibitors PIAS3 and IkBalpha, and with a decreased induction of Bcl-xL. Treatment with recombinant IL-6 enhanced survival of decompensated cirrhotic animals, while it did not affect survival of compensated cirrhotic animals but sustained liver regeneration, by restoring STAT3 and NF-kB activation and Bcl-xL induction to the levels found in normal controls. The pro-growth effects exerted by IL-6 treatment in cirrhotic liver were attained also at low, pharmacologically acceptable doses. In conclusion, our results suggest that IL-6 treatment may be therapeutic in major resection of cirrhotic liver.

Expression of p16, p53 and Ki-67 proteins in the progression of epithelial dysplasia of the oral cavity.

BACKGROUND: The overexpression of the protein products of genes associated with the cell cycle tumour protein53 (p53), cyclin-dependent kinase inhibitor 2A (p16) and antigen identified by monoclonal antibody Ki-67 (Ki-67) is apparently of great significance. This study evaluated the immunohistochemical expression of these proteins in precancerous lesions and in carcinoma of the oral cavity. MATERIALS AND METHODS: The nuclear expression of p53 and Ki-67 and nuclear and/or cytoplasmic expression of p16 protein was examined in 54 biopsy specimens from the oral cavity obtained over a period of 3 years. The samples included 18 cases of normal/hyperplastic mucosa, 25 cases of dysplasia and 11 cases of invasive squamous cell carcinoma. The specimens were grouped into three categories: 1 = no or mild dysplasia, 2 = moderate or severe dysplasia, and 3 = invasive carcinoma. RESULTS: p16 was negative in all the group 1 specimens, while both p53 and Ki-67, when present, were limited to the cells of the basal layer. In the group 2 specimens, the number of p16-, p53-, and Ki-67-positive cells increased as the grade of dysplasia progressed. In group 3 (invasive carcinomas), p53 and p16 expression occurred respectively in 81.8% and 54.5% of cases, while Ki-67 was elevated in all the cases. CONCLUSION: The expression of the cell-cycle proteins p16 and p53 in the dysplastic epithelium, in association with Ki-67, may represent significant markers to recognize evolution of precancerous disease in the oral cavity and to improve identification of the degree of dysplasia.

The nature of cartilage stippling in chondrodysplasia punctata: histopathological study of Conradi-Hünermann syndrome.

The chondrodysplasia punctatas (CDP) are a group of genetic diseases presenting with the common hallmark of epiphyseal calcific deposits. A 21.3 week female fetus with a clinical and molecular diagnosis of X-linked dominant CDP (CDPX2 or Conradi-Hunermann syndrome) has been investigated with particular attention to the morphology of the calcific deposits (stipplings). A variable combination of normal endochondral ossification centers, calcific deposits, and a mixture of these two were observed in the lumbar spine. The calcific deposits were characterized by a growing phase, corresponding to the expansion of calcification, a steady phase, and a resorbing/remodeling phase, when calcification was progressively reduced through different biological mechanisms. The pathological mechanism of cartilage matrix calcification seemed to influence the morphology and the distribution pattern of vessels inside the epiphysis. These findings fit well with the radiographic observations of progressive regression of stippling with advancing age of the child and also give a morphological correlation.

Study of Endochondral Ossification in Human Fetalcartilage Anlagen of Metacarpals: Comparative Morphology of Mineral Deposition in Cartilage and in the Periosteal Bone Matrix.

The progression of mineral phase deposition in hypertrophic cartilage and periosteal bone matrix was studied in human metacarpals primary ossification centers before vascular invasion began. This study aimed to provide a morphologic/morphometric comparative analysis of the calcification process in cartilage and periosteal osteoid used as models of endochondral ossification. Thin, sequential sections from the same paraffin inclusions of metacarpal anlagen (gestational age between the 20th and 22nd weeks) were examined with light microscopy and scanning electron microscopy, either stained or heat-deproteinated. This process enabled the analysis of corresponding fields using the different methods. From the initial CaPO4 nucleation in cartilage matrix, calcification progressed increasing the size of focal, globular, randomly distributed deposits (size range 0.5-5 µm), followed by aggregation into polycyclic clusters and finally forming a dense, compact mass of calcified cartilage. At the same time, the early osteoid calcification was characterized by a fine granular pattern (size range 0.1-0.5 µm), which was soon compacted in the layer of the first periosteal lamella. Scanning electron microscopy of heat-deproteinated sections revealed a rod-like hydroxyapatite crystallite pattern, with only size differences between the early globular deposits of the two calcifying matrices. The morphology of the early calcium deposits was similar in both cartilage and osteoid, with variations in size and density only. However, integration of the reported data with the actual hypotheses of the mechanisms of Ca concentration suggested that ion transport was linked to the progression of the chondrocyte maturation cycle (with recall of H2 O from the matrix) in cartilage, while ions transport was an active process through the cell membrane in osteoid. Other considered factors were the collagen type specificity and the matrix fibrillar texture. Anat Rec, 301:571-580, 2018. © 2017 Wiley Periodicals, Inc.

Melanoma contains CD133 and ABCG2 positive cells with enhanced tumourigenic potential.

The failure to eradicate most cancers and in particular melanoma may be as fundamental as a misidentification of the target. The identification of cancer stem/initiating cells within the tumour population with a crucial role for tumour formation may open new pharmacological perspectives. Our data show three main novelties for human melanoma: firstly, melanoma biopsy contains a subset of cells expressing CD133 (CD133+) and the latter is able to develop a Mart-1 positive tumour in NOD-SCID mice. Secondly, the WM115, a human melanoma cell line, has been found to express both CD133 and ABCG2 markers. This cell line grows as floating spheroids, expresses typical progenitors and mature neuronal/oligodendrocyte markers and is able to transdifferentiate into astrocytes or mesenchymal lineages under specific growth conditions. As in xenografts generated with CD133+ biopsy melanoma cells, those produced by the cell line displayed lower levels of CD133 and ABCG2. Thirdly, the WM115 cells express the most important angiogenic and lymphoangiogenic factors such as notch 4, prox1 and podoplanin which can cooperate in the development of the tumourigenic capability of melanoma in vivo. Therefore, in this study, we demonstrate the presence of stem/initiating subsets in melanoma both in biopsy and in an established melanoma cell line grown in vitro and in xenografts. Interestingly, considering that melanoma gives metastasis primarily through lymphatic vessels, herein, we demonstrated that a melanoma cell line expresses typical lymphoangiogenic factors.

Interleukin-6 sustains hepatic regeneration in cirrhotic rat.

BACKGROUND/AIMS: In the liver IL-6 displays growth-inducing and pro-survival activities. We studied the pro-proliferative and protective mechanisms of IL-6 treatment in a model of liver cirrhosis in wild type rat, investigating the theoretical basis for a potential pharmacologic role of IL-6 in cirrhosis. METHODOLOGY: We analyzed IL-6 receptors levels in cirrhotic liver. We also studied the activation of signaling pathways downstream IL-6 receptors by analyzing the DNA-binding activity of transcription factors STAT3, AP-1, HNF-1 and NF-kappaB and the phosphorylation status of AKT and eNOS. We also analyzed hepato-cell proliferation, by determining BrdU incorporation into DNA, and liver mass expansion. RESULTS: We show that liver cells from cirrhotic animals have increased expression of the IL-6 receptor alpha/gp80. In addition, we show that in cirrhosis the main molecular pathways downstream the receptors are intact and that IL-6 activates STAT3, AP-1 and NF-kappaB transcription factors, induces AKT and eNOS phosphorylation and increases hepato-cell proliferation and liver mass expansion in a dose-dependent manner. CONCLUSIONS: Our data demonstrate that the theoretical basis exists for the therapeutic employment of IL-6 in liver cirrhosis.

Growth and shaping of metacarpal and carpal cartilage anlagen: application of morphometry to the development of short and long bone. A study of human hand anlagen in the fetal period.

A histological and morphometric analysis of human metacarpal and carpal anlagen between the 16th and 22nd embryonic weeks was carried out with the aim of studying the establishment of the respective anlage architecture. No differences in the pattern of growth were documented between the peripheral and central zones of the metacarpal epiphyses and those of the carpals. The regulation of longitudinal growth in long bone anlagen occurred in the transition zone between the epiphysis and the diaphysis (homologous to the metaphyseal growth plate cartilage in more advanced developmental stage of the bone). Comparative zonal analysis was conducted to assess the chondrocyte density, the mean chondrocyte lacunar area, the paired chondrocyte polarity in the orthogonal longitudinal and transverse planes, and the lacunar shape transformation in the metacarpal. In transition from epiphysis to diaphysis chondrocyte density decreased and mean lacunar area increased. No significant differences in the chondrocyte maturation cycle were observed between proximal/distal metacarpal epiphyses and the carpal anlagen. The number of paired chondrocyte oriented along the growth vector was significantly higher in both proximal/distal transition zones between epiphysis and diaphysis. Human metacarpals shared with experimental models (like mice and nonmammal tetrapods) an early common chondrocyte maturation cycle but with a different timing due to the slower embryonic and fetal developmental rate of human anlagen.

U94 of human herpesvirus 6 down-modulates Src, promotes a partial mesenchymal-to-epithelial transition and inhibits tumor cell growth, invasion and metastasis.

U94, the latency gene of human herpesvirus 6, was found to inhibit migration, invasion and proliferation of vascular endothelial cells (ECs). Because of its potent anti-migratory activity on ECs, we tested the capability of U94 to interfere with the individual steps of the metastatic cascade. We examined the U94 biological activity on the human breast cancer cell line MDA-MB 231, as a model of highly aggressive cancer cell. Here we show that the expression of U94 delivered by an HSV-1-based amplicon promoted down-modulation of Src and downstream molecules linked to cell motility and proliferation. Indeed, U94 expression strongly inhibited cell migration, invasiveness and clonogenicity. We investigated the effects of U94 in a three-dimensional rotary cell-culture system and observed the ability of U94 to modify tumor cell morphology by inducing a partial mesenchymal-to-epithelial transition. In fact, despite U94 did not induce any expression of the epithelial marker E-cadherin, it down-modulated different mesenchymal markers as ß-catenin, Vimentin, TWIST, Snail1, and MMP2. In vivo data on the tumorigenicity of MDA-MB 231 displayed the capability of U94 to control tumor growth, invasiveness and metastasis, as well as tumor-driven angiogenesis. The antitumor U94 activity was also confirmed on the human cervical cancer cell line HeLa. The ability of U94 to inhibit cell growth, invasion and metastasis opens the way to a promising field of research aimed to develop new therapeutic approaches for treating tumor and cancer metastasis.