Arnaldo Caruso

Flow cytometric analysis of activation markers on stimulated T cells and their correlation with cell proliferation.

The expression of activation antigens, namely CD25, CD69, CD71, and HLA-DR on T cells from 15 healthy individuals stimulated with different mitogens and specific antigens was evaluated by immunofluorescence assay and flow cytometric analysis and compared with cell proliferation as a function of [3H]thymidine incorporation. CD69 was the earliest expressed antigen on stimulated cells, while HLA-DR was the latest. Regardless of the stimulus used, lymphocytes expressing CD25 and CD71 were always more numerous than cells expressing CD69 and HLA-DR. Variations in the proportion of CD4+ and CD8+ T cells expressing each activation marker were observed with different antigenic stimuli. The expression of each activation marker showed overall agreement with the [3H]thymidine incorporation assay in discriminating between positive and negative immune response. However, no correlation was observed between the percentage of CD25-, CD69-, CD71-, and HLA-DR-positive T cells and the amount of [3H]thymidine incorporation. Moreover, low doses of mitogens and antigens as well as short time of stimulation were sufficient to induce T cells to express activation antigens but not to proliferate. Our data show that results obtained by flow cytometry and [3H]thymidine incorporation may differ qualitatively, at least under certain conditions; this suggests that the 2 assays are complementary, and when combined, may gives a clearer understanding of events leading to efficient cell-mediated immune response.

Isolation and culture of human muscle-derived stem cells able to differentiate into myogenic and neurogenic cell lineages.

BACKGROUND Skeletal-muscle-derived stem cells seem to be a distinct population of immature progenitors of satellite cells, but their functional properties remain unclear, especially in human adult tissue. We investigated their differentiation in samples of skeletal muscle obtained from adults undergoing cardiovascular surgery. METHODS Samples were obtained from the brachioradialis muscle of 12 patients in whom the radial artery was the conduit for myocardial revascularisation. The stem cells were isolated by a procedure similar to that used for rat gastrocnemius and cultured in medium optimised for growth of neural stem cells. Cytometry was used for phenotypic characterisation and immunocytochemistry and RT-PCR to assess differentiation. Immunohistochemistry was used to examine engraftment of skeletal-muscle-derived stem cells into injured rat spinal cord. FINDINGS The skeletal-muscle stem cells consisted of two distinct types: one with the typical spindle morphology of satellite cells, the other of rounded cells. Some cultures could be maintained for longer than 6 months. The cells were mainly positive for desmin and to a lesser extent CD105, vimentin, and AC133/CD133, but negative for FLK-1/KDR, CD34, CD31, CD45, von Willebrand factor, Ve-cadherins, and BCL2. After in-vitro differentiation, the cells were able to organise skeletal-muscle fibres and stained positively for striated-muscle actin, smooth-muscle actin, and desmin. Moreover, they differentiated into astrocytes and neurons, as confirmed by positive staining for characteristic proteins. INTERPRETATION Adult human skeletal muscle includes a population of progenitor stem cells that can generate cells of the same lineage and cells with neurogenic properties. Muscle may therefore be a tissue source for the isolation of pluripotent stem cells for development of cell-based therapies for human myogenic and neurogenic diseases.

Expression of Inducible Nitric Oxide Synthase in Human Granulomas and Histiocytic Reactions

Inducible nitric oxide synthase (iNOS) is required in immune response against infections and is involved in granuloma formation in animals; in murine macrophages, iNOS is induced by lipopolysaccharide and interferon-gamma. In contrast, the role of iNOS in human immune response against infections is still questioned, and its expression in granulomas is poorly investigated. Using Western blotting and immunohistochemistry, we investigated iNOS expression in human lymph nodes with nonspecific reactions and in tissues containing granulomas caused by mycobacteria, Toxoplasma, Cryptococcus neoformans, Leishmania, Bartonella, noninfectious granulomas (sarcoidosis, foreign body), and other hystiocitic reactions (Kikuchis disease, Omenn syndrome). iNOS was undetectable in nonspecific reactive lymphadenitis, foreign-body granulomas, and Omenn syndrome, whereas it was strongly expressed in infectious granulomas, sarcoidosis, and Kikuchis diseases. Immunohistochemistry demonstrated that iNOS was selectively expressed by the epithelioid and multinucleated giant cells within the granulomas. Use of an anti-nitrotyrosine antibody, recognizing nitrosilated amino acid residues derived from nitric oxide production, revealed a consistent positivity within the cells expressing iNOS, thus suggesting that iNOS is functionally active. Detection of cytokines by reverse transcriptase-polymerase chain reaction demonstrated that tissues that were positive for iNOS, also expressed the Thl-type cytokine interferon-gamma mRNA, but not the Th2-type cytokine interleukin-4. Taken together, these results indicate that iNOS is involved in different human immune reactions characterized by histiocytic/granulomatous inflammation and associated with Th1-type cytokine secretion.

Human vasculogenesis ex vivo: embryonal aorta as a tool for isolation of endothelial cell progenitors.

Vasculogenesis, the de novo formation of new blood vessels from undifferentiated precursor cells or angioblasts, has been studied with experimental in vivo and ex vivo animal models, but its mechanism is poorly understood, particularly in humans. We used the aortic ring assay to investigate the angioforming capacity of aortic explants from 11- to 12-week-old human embryos. After being embedded in collagen gels, the aorta rings produced branching capillary-like structures formed by mesenchymal spindle cells that lined a capillary-like lumen and expressed markers of endothelial differentiation (CD31, CD34, von Willebrand factor [vWF], and fms-like tyrosine kinase-1 [Flk-1;[sol;vascular endothelial growth factor receptor 2 [VEGFR2]). The cell linings of these structures showed ultrastructural evidence of endothelial differentiation. The neovascular proliferation occurred primarily in the outer aspects of aortic rings, thus suggesting that the new vessels mainly arose from immature endothelial precursor cells localized in the outer layer of the aortic stroma, ie, a process of vasculogenesis rather than angiogenesis. The undifferentiated mesenchymal cells (CD34+/CD31−), isolated and cultured on collagen-fibronectin, differentiated into endothelial cells expressing CD31 and vWF. Furthermore, the CD34+/CD31+ cells were capable of forming a network of capillary-like structures when cultured on Matrigel. This is the first reported study showing the ex vivo formation of human microvessels by vasculogenesis. Our findings indicate that the human embryonic aorta is a rich source of CD34+/CD31− endothelial progenitor cells (angioblasts), and this information may prove valuable in studies of vascular regeneration and tissue bioengineering.

Human Metapneumovirus Infection in Young Children Hospitalized With Acute Respiratory Tract Disease : Virologic and Clinical Features

Background: Human metapneumovirus (hMPV) is an emerging virus associated with acute respiratory tract infections (ARIs) in young children. Objectives: To evaluate virologic and clinical features of hMPV infection during 2 consecutive winter-spring seasons. Methods: Nasal washes were obtained from children younger than 5 years of age hospitalized for ARI. Specimens were tested for hMPV by reverse transcription-polymerase chain reaction. The hMPV F gene amplification products were sequenced, and phylogenetic trees were constructed. Results: A high incidence of hMPV infection (25.3%) was observed during the 2005–2006 winter-spring season, whereas a much lower rate of infection (4.7%) during the following season was found. Phylogenetic analysis revealed that, during the 2 seasons, 60.4% of the hMPV detected were A2a, 22.9% were A2b, 4.2% were B1, and 12.5% were B2. hMPV A1 strains were not detected in any tested specimen. Clinical diagnosis was bronchiolitis in 57.1%; pneumonia in 25%; and a upper respiratory tract illness in 17.8%. Bronchiolitis was more frequent in children less than 1 year of age (80%) than in children more than 1 year of age (30.8%) (P < 0.05). When hMPV was found frequently, the hMPV spread overlapped with that of respiratory syncytial virus (RSV) and hMPV/RSV coinfections were common events (19 of 39; 48.7%). hMPV/RSV-coinfected children developed pneumonia more frequently than hMPV-infected patients (57.9% versus 20%) but no differences in disease severity (gauged by duration of hospitalization and requirement of oxygen) were observed. Conclusions: These results provide further evidence of the importance of hMPV as a pathogen associated with ARI in young children. Involvement of hMPV/RSV coinfection in cases of pneumonia is suspected.

Endothelin-1 Stimulates Lymphatic Endothelial Cells and Lymphatic Vessels to Grow and Invade

The lymphatic vasculature is essential for tissue fluid homeostasis and cancer metastasis, although the molecular mechanisms involved remain poorly characterized. Endothelin-1 (ET-1) axis plays a crucial role in angiogenesis and tumorigenesis. Here, we first report that ET-1 acts as a lymphangiogenic mediator. We performed in vitro and in vivo studies and show that lymphatic endothelial cells produce ET-1, ET-3, and express the endothelin B receptor (ET(B)R). In these cells, ET-1 promotes proliferation, invasiveness, vascular-like structures formation, and phosphorylation of AKT and p42/44 mitogen-activated protein kinase through ET(B)R. In normoxic conditions, ET-1 is also able to up-regulate the expression of vascular endothelial growth factor (VEGF)-C, VEGF receptor-3, and VEGF-A, and to stimulate hypoxia-inducible factor (HIF)-1alpha expression similarly to hypoxia. Moreover, HIF-1alpha silencing by siRNA desensitizes VEGF-C and VEGF-A production in response to ET-1 or hypoxia, implicating HIF-1alpha/VEGF as downstream signaling molecules of ET-1 axis. Double immunofluorescence analysis of human lymph nodes reveals that lymphatic vessels express ET(B)R together with the lymphatic marker podoplanin. Furthermore, a Matrigel plug assay shows that ET-1 promotes the outgrowth of lymphatic vessels in vivo. ET(B)R blockade with the specific antagonist, BQ788, inhibits in vitro and in vivo ET-1-induced effects, demonstrating that ET-1 through ET(B)R directly regulates lymphatic vessel formation and by interacting with the HIF-1alpha-dependent machinery can amplify the VEGF-mediated lymphatic vascularization. Our results suggest that ET-1 axis is indeed a new player in lymphangiogenesis and that targeting pharmacologically ET(B)R and related signaling cascade may be therapeutically exploited in a variety of diseases including cancer.

HIF-1α/GPER signaling mediates the expression of VEGF induced by hypoxia in breast cancer associated fibroblasts (CAFs)

IntroductionCarcinoma-associated fibroblasts (CAFs) play a pivotal role in cancer progression by contributing to invasion, metastasis and angiogenesis. Solid tumors possess a unique microenvironment characterized by local hypoxia, which induces gene expression changes and biological features leading to poor outcomes. Hypoxia Inducible Factor 1 (HIF-1) is the main transcription factor that mediates the cell response to hypoxia through different mechanisms that include the regulation of genes strongly associated with cancer aggressiveness. Among the HIF-1 target genes, the G-protein estrogen receptor (GPER) exerts a stimulatory role in diverse types of cancer cells and in CAFs.MethodsWe evaluated the regulation and function of the key angiogenic mediator vascular endothelial growth factor (VEGF) in CAFs exposed to hypoxia. Gene expression studies, Western blotting analysis and immunofluorescence experiments were performed in CAFs and breast cancer cells in the presence of cobalt chloride (CoCl2) or cultured under low oxygen tension (2% O2), in order to analyze the involvement of the HIF-1α/GPER signaling in the biological responses to hypoxia. We also explored the role of the HIF-1α/GPER transduction pathway in functional assays like tube formation in human umbilical vein endothelial cells (HUVECs) and cell migration in CAFs.ResultsWe first determined that hypoxia induces the expression of HIF-1α and GPER in CAFs, then we ascertained that the HIF-1α/GPER signaling is involved in the regulation of VEGF expression in breast cancer cells and in CAFs exposed to hypoxia. We also assessed by ChIP assay that HIF-1α and GPER are both recruited to the VEGF promoter sequence and required for VEGF promoter stimulation upon hypoxic condition. As a biological counterpart of these findings, conditioned medium from hypoxic CAFs promoted tube formation in HUVECs in a HIF-1α/GPER dependent manner. The functional cooperation between HIF-1α and GPER in CAFs was also evidenced in the hypoxia-induced cell migration, which involved a further target of the HIF-1α/GPER signaling like connective tissue growth factor (CTGF).ConclusionsThe present results provide novel insight into the role elicited by the HIF-1α/GPER transduction pathway in CAFs towards the hypoxia-dependent tumor angiogenesis. Our findings further extend the molecular mechanisms through which the tumor microenvironment may contribute to cancer progression.

HIV-1 matrix protein p17 increases the production of proinflammatory cytokines and counteracts IL-4 activity by binding to a cellular receptor

Purified recombinant HIV-1 p17 matrix protein significantly increased HIV-1 replication in preactivated peripheral blood mononuclear cell cultures obtained from healthy donors. Because HIV-1 infection and replication is related to cell activation and differentiation status, in the present study, we investigated the role played by p17 during the process of T cell stimulation. Using freshly isolated peripheral blood mononuclear cells, we demonstrate that p17 was able to enhance levels of tumor necrosis factor α and IFN-γ released from cells stimulated by IL-2. IL-4 was found to down-regulate IFN-γ and tumor necrosis factor α, and p17 restored the ability of cells to produce both cytokines. The property of p17 to increase production of proinflammatory cytokines could be a mechanism exploited by the virus to create a more suitable environment for HIV-1 infection and replication. Our data show that p17 exerts its biological activity after binding to a specific cellular receptor expressed on activated T lymphocytes. The functional p17 epitope involved in receptor binding was found to be located at the NH2-terminal region of viral protein. Immunization of BALB/c mice with a 14-aa synthetic peptide representative of the HIV-1 p17 functional region (SGGELDRWEKIRLR) resulted in the development of p17 neutralizing antibodies capable of blocking the interaction between p17 and its cellular receptor. Our results define a role for p17 in HIV-1 pathogenesis and contribute to our understanding of the molecular mechanism of HIV-1 infection and the development of additional antiviral therapeutic strategies.

Transforming Growth Factor-β1 and CD105 Promote the Migration of Hepatocellular Carcinoma–Derived Endothelium

Hepatocellular carcinoma (HCC) is one of most malignant and aggressive human tumors. Transforming growth factor-beta1 (TGF-beta1) and its coreceptor CD105 have been shown to contribute to HCC malignant progression. TGF-beta1 and CD105 have also been implicated in angiogenesis, but their role in the vascularization of HCC has not been investigated. To fill this gap, we studied the effect of TGF-beta1 and CD105 on HCC-derived endothelium. By using immunomagnetic beads, we isolated and cultured endothelial cells (ECs) from HCC (HCC-EC) and adjacent nonneoplastic tissue (nNL-ECs) obtained from 24 liver biopsies. HCC and nNL biopsies were also analyzed by immunohistochemistry for the expression of CD105, TGF-beta1, Ve-cadherin (Ve-cad), CD44, beta-catenin, and E-cadherin. Compared with nNL-ECs, HCC-ECs had higher expression of CD105, enhanced spontaneous motility, and greater capacity to migrate in response to TGF-beta1 (5 ng/mL), particularly in the presence of a fibronectin matrix. The chemotactic effect of TGF-beta1 was blocked by anti-CD105 antibodies and correlated with the grade of HCC malignancy. Histologic examination of HCC biopsies showed that HCCs with the worse malignant features had the highest expression of TGF-beta1, CD105, and angiogenic markers (Ve-cad and CD44). Because CD105 was highly expressed in microvessels at the tumor periphery and TGF-beta1 staining was only found in neoplastic hepatocytes, we conclude that HCC-derived TGF-beta1 may act as a chemoattractant for CD105-expressing ECs and as a promoter of tumor angiogenesis. Thus, drugs that selectively target the TGF-beta1/CD105 axis may interfere with HCC-related angiogenesis and HCC progression.

Inhibition of neuroblastoma‐induced angiogenesis by fenretinide

Retinoids are a class of natural or synthetic compounds that participate in the control of cell proliferation, differentiation and fetal development. The synthetic retinoid fenretinide (HPR) inhibits carcinogenesis in various animal models. Retinoids have also been suggested to be effective inhibitors of angiogenesis. The effects of HPR on certain endothelial cell functions were investigated in vitro, and its effects on angiogenesis was studied in vivo, by using the chorioallantoic membrane (CAM) assay. HPR inhibited vascular endothelial growth factor‐ (VEGF‐) and fibroblast growth factor‐2‐ (FGF‐2)‐induced endothelial cell proliferation without affecting endothelial motility; moreover, HPR inhibited growth factor‐induced angiogenesis in the CAM assay. Furthermore, a significant antiangiogenic potential of HPR has also been observed in neuroblastoma (NB) biopsy‐induced angiogenesis in vivo. We previously demonstrated that supernatants derived from NB cell lines stimulated endothelial cell proliferation. In the present study, we found that this effect was abolished when NB cells were incubated in the presence of HPR. VEGF‐ and FGF‐2‐specific ELISA assays, performed on both NB cells derived from conditioned medium and cellular extracts, indicated no consistent effect of HPR on the level of these angiogenic cytokines. Moreover, RT‐PCR analysis of VEGF and FGF‐2 gene expression confirmed the above lack of effect. HPR was also able to significantly repress the spontaneous growth of endothelial cells, requiring at least 48–72 hr of treatment with HPR, followed by a progressive accumulation of cells in G1 at subsequent time points. Finally, immunohistochemistry experiments performed in the CAM assay demonstrated that endothelial staining of both VEGF receptor 2 and FGF‐2 receptor‐2 was reduced after implantation of HPR‐loaded sponges, as compared to control CAMs. These data suggest that HPR exerts its antiangiogenic activity through both a direct effect on endothelial cell proliferative activity and an inhibitory effect on the responsivity of the endothelial cells to the proliferative stimuli mediated by angiogenic growth factors.