Carmelo Mavilia

Tissue engineered human tracheas for in vivo implantation

Two years ago we performed the first clinical successful transplantation of a fully tissue engineered trachea. Despite the clinically positive outcome, the graft production took almost 3 months, a not feasible period of time for patients with the need of an urgent transplantation. We have then improved decellularization process and herein, for the first time, we completely describe and characterize the obtainment of human tracheal bioactive supports. Histological and molecular biology analysis demonstrated that all cellular components and nuclear material were removed and quantitative PCR confirmed it. SEM analysis revealed that the decellularized matrices retained the hierarchical structures of native trachea, and biomechanical tests showed that decellularization approach did not led to any influence on tracheal morphological and mechanical properties. Moreover immunohistological staining showed the preservation of angiogenic factors and angiogenic assays demonstrated that acellular human tracheal scaffolds exert an in vitro chemo-active action and induce strong in vivo angiogenic response (CAM analysis). We are now able to obtained, in a short and clinically useful time (approximately 3 weeks), a bioengineered trachea that is structurally and mechanically similar to native trachea, which exert chemotactive and pro-angiogenic properties and which could be successfully used for clinical tissue engineered airway clinical replacements.

In vivo CD30 expression in human diseases with predominant activation of Th2-like T cells.

CD3O is a member of the tumor necrosis factor (TNF) receptor family, originally described as a marker for Hodgkin and Reed‐Sternberg cells in Hodgkins disease, which has been found to be preferentially expressed by T cells producing Th2‐type cytokines. The presence of CD3O expression was assessed by both immunohistochemistry and reverse transcriptase‐polymerase chain reaction in the target organs of patients with Th1‐ or Th2‐dominated disorders. CD3O expression was found in neither the gut of patients with Crohns disease nor in the gastric antrum of Helicobacter pylori‐infected patients, where there was high interferon‐γ (IFN‐γ) expression. In contrast, high CD3O expression in the apparent absence of IFN‐γ expression was observed in the skin of patients with systemic sclerosis or chronic graft versus host disease (GVHD), which can be considered Th2‐dominated disorders. Moreover, high levels of soluble CD3O were found in the serum of both systemic sclerosis and GVHD patients but not in the serum of patients suffering from multiple sclerosis, a Th1‐dominated disorder. Thus, CD3O expression appears to be preferentially associated with Th2‐type responses not only in vitro but also in vivo. J. Leukoc. Biol. 61: 539–544; 1997.

Two Novel Mutations at Exon 8 of the Sequestosome 1 (SQSTM1) Gene in an Italian Series of Patients Affected by Paget's Disease of Bone (PDB)†

PDB is genetically heterogeneous. Mutations of the sequestosome1 gene have been reported in sporadic and familial forms of Pagets in patients of French Canadian and British descent. Mutational analyses in different ethnic groups are needed to accurately investigate hereditary diseases. We describe two novel mutations of sequestosome1 in 62 Italian sporadic patients, confirming the role of the encoded protein in this disorder.

Macrophage-Derived Chemokine and EBI1-Ligand Chemokine Attract Human Thymocytes in Different Stage of Development and Are Produced by Distinct Subsets of Medullary Epithelial Cells: Possible Implications for Negative Selection

The chemoattractant activity of macrophage-derived chemokine (MDC), EBI1-ligand chemokine (ELC), and secondary lymphoid tissue chemokine (SLC) on human thymocytes was analyzed. Both ELC and SLC caused the accumulation of CD4+CD8− or CD4−CD8+ CD45RA+ thymocytes showing high CD3 expression. By contrast, a remarkable proportion of MDC-responsive thymocytes were CD4+CD8+ cells exhibiting reduced levels of CD8 or CD4+CD8− cells showing CD3 and CD45R0, but not CD45RA. MDC-responsive thymocyte suspensions were enriched in cells expressing the MDC receptor, CCR4, selectively localized to the medulla, and in CD30+ cells, whereas ELC-responsive thymocytes never expressed CD30. Reactivity to both MDC and ELC was localized to cells of the medullary areas, but never in the cortex. Double immunostaining showed no reactivity for either MDC or ELC by T cells, macrophages, or mature dendritic cells, whereas many medullary epithelial cells were reactive to MDC or ELC. However, MDC reactivity was consistently localized to the outer wall of Hassal’s corpuscles, whereas ELC reactivity was often found in cells surrounding medullary vessels, but not in Hassal’s corpuscles. Moreover, while most MDC-producing cells also stained positive for CD30L, this molecule was never found on ELC-producing cells. We suggest therefore that CD30L-expressing MDC-producing medullary epithelial cells attract CCR4-expressing thymocytes, thus favoring the CD30/CD30L interaction, and therefore the apoptosis, of cells that are induced to express CD30 by autoantigen activation. By contrast, ELC production by CD30L-lacking medullary epithelial cells may induce the migration into periphery of mature thymocytes that have survived the process of negative selection.

Expression and release of LAG-3-encoded protein by human CD4+ T cells are associated with IFN-gamma production .

The lymphocyte activation gene (LAG) ‐3 is a member of the immunoglobulin superfamily that is selectively transcribed in human activated T and NK cells. In this work, the possibility that LAG‐3 expression by human CD4+ T cells was preferentially related to one or another phenotype of cytokine secretion was investigated. Surface LAG‐3 expression correlated with IFN‐γ, but not IL‐4, production in antigen‐stimulated T cells and it was up‐ regulated by IL‐12. Most activated CD4+ T cell clones with established Thl or Th0 profiles of cytokine secretion expressed LAG‐3 on their surface, whereas the great majority of Th2 clones showed 1 neither surface LAG‐3 nor LAG‐3 mRNA expression. After activation, the majority of CD4+ T cell clones also released soluble LAG‐3‐related peptides, and such a release correlated positively with the production of IFN‐γ and inversely with the production of IL‐4. Thus, LAG‐3 expression by activated CD4+ human T cells appear to be preferentially associated with the differentiation/activation pathway leading to the production of IFN‐γ.—Annunziato, F., Manetti R., Tomase vie, L., Guidizi, M.‐G., Biagiotti, R., Giannö, V., Germano, P., Mavilia, C., Maggi, E., Romagnani, S. Expression and release of LAG‐3‐encoded protein by human CD4+ T cells are associated with IFN‐γ production. FASEB J. 10, 769‐775 (1996)

Enhanced HIV expression during Th2‐oriented responses explained by the opposite regulatory effect of IL‐4 and IFN‐γ on fusin/CXCR4

The human α‐chemokine receptor fusin/CXCR4 is an important cofactor for entry of T lymphocyte‐tropic HIV‐1 strains. We investigated the possible regulatory role of T cell cytokine patterns on CXCR4 as well as HIV expression by using in vitro models of both secondary and primary immune responses. Antigen‐specific memory CD4+ T cells infected with a T‐tropic HIV‐1 strain showed significantly higher CXCR4 and HIV‐1 expression in Th0/2‐oriented responses in comparison with Th1‐oriented responses. Similarly, in naive CD4+ T cells activated in the presence of IL‐4 or IL‐12 and infected with the same T‐tropic strain, IL‐4 up‐regulated whereas IL‐12 down‐regulated both CXCR4 and HIV‐1 expression. The down‐regulatory effect of IL‐12 on CXCR4 expression was found to be dependent on its capacity to induce IFN‐γ production. These observations can account for the higher risk of progression in HIV‐1‐infected individuals undergoing Th0/2‐oriented immune responses.

Estrogen Receptor Expression in Cutaneous Melanoma A Real-Time Reverse Transcriptase-Polymerase Chain Reaction and Immunohistochemical Study

OBJECTIVE To evaluate estrogen receptor (ER) expression in human melanoma tissues and in the adjacent healthy skin with the aim of explaining whether the ERalpha:ERbeta expression ratio has a role in neoplastic progression. DESIGN Prospective study. SETTING Department of Dermatology, University of Florence, Florence, Italy. Patients Fourteen patients, 12 with cutaneous melanoma (6 women and 6 men) and 2 with melanocytic nevi (1 woman and 1 man). MAIN OUTCOME MEASURES Using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical analysis, we analyzed ERalpha and ERbeta messenger RNA (mRNA) and ERbeta protein expression in cutaneous melanoma and in the healthy skin surrounding the lesions. RESULTS All melanocytic lesions expressed detectable levels of ERalpha and ERbeta mRNA as well as ERbeta protein. Dividing melanoma cases into 2 groups according to Breslow thickness, we found lower ERalpha and ERbeta mRNA levels and lower ERbeta protein levels in thicker, more invasive tumors. CONCLUSIONS These observations suggest a role for ERs in the metastatic process of melanoma cells, pointing at the possibility of using ERbeta expression as a prognostic indicator of melanoma. The possibility of distinguishing proliferative melanomas, which are associated with dismal prognosis, from the so-called dormant melanomas opens up novel avenues in tailoring individual treatments, as already happens for other tumors.

Production of IL-4 and leukemia inhibitory factor by T cells of the cumulus oophorus: a favorable microenvironment for pre-implantation embryo development

The nature and the functional activity of immunocytes present in the cumulus oophorus, a mass of cells surrounding the oocyte, were examined here for the first time. The cumuli oophorus were obtained from women who had taken part in an in vitro fertilization program and were suffering from blocked fallopian tubes. Both macrophages and CD4+ T cellswere detected in all cumuli. CD4+ T cell clones, generated from T cells of these cumuli, showed higher potential to produce IL‐4 and leukemia inhibitory factor (LIF) thanCD4+ T cell clones generated from peripheral blood or ovary specimens from the same women. More importantly, IL‐4 and LIF, but not IFN‐γ mRNA was found to be constitutively expressed in vivo by cumulus oophorus cells. Progesterone is highly produced by the cumulus oophorus/oocyte complex. We recently showed that progesterone up‐regulates the production of LIF by T cells and that the progesterone‐induced LIF production is mediated by IL‐4. Progesterone produced by cumulus granulosa cells may favor IL‐4 production by T cells, which in turn can produce LIF. As the treatment with LIF enhances the in vitro growth and development of mammalian embryos, our data suggest that T cells present in the cumulus oophorus produce cytokines that may provide a microenvironment suitable for pre‐implantation development of the mammalian embryo.

Relaxin favors the development of activated human T cells into Th1-like effectors.

Differentiation of naive CD4+ helper T (Th) cells into Th1 or Th2 effectors, as characterized by their opposite pattern of cytokine production, can be influenced by several factors, including hormones. In this study, we demonstrate that porcine relaxin, at concentrations ranging from 10–10, to 10–6M, favors the in vitro development of human antigen‐specific T cells into Th1‐like effectors and enhances both IFN‐γ mRNA expression and IFN‐γ production by established human T cell clones. The promoting effect of relaxin on the development of IFN‐γ‐producing cells was not due to a relaxin‐induced release of IL‐12 and/or IFN‐α by antigen‐presenting cells. These results suggest that relaxin may contribute to the regulation of the immune homeostasis during pregnancy and may also play some role in counteracting Th2‐dominated disorders.

Azidothymidine Induces Apoptosis and Inhibits Cell Growth and Telomerase Activity of Human Parathyroid Cancer Cells in Culture

Telomerase activity has been correlated to parathyroid carcinoma. Because its role in acquisition of a malignant phenotype by parathyroid cells is unclear, we treated telomerase‐positive cultured human parathyroid cancer cells with the telomerase inhibitor AZT, evaluating cell telomerase activity, cytotoxic effects, growth, and morphological changes. In vitro exposure of these cells to AZT correlated with inhibition of cell proliferation.