Unbalanced expression of HLA-A and -B antigens: a specific feature of cutaneous melanoma and other non-hemopoietic malignancies reverted by IFN-gamma.

Conflicting evidences suggested that levels of HLA-A and -B antigens expressed on normal and neoplastic cells of given individuals are genetically predetermined, or, on the other hand, regulated by molecular mechanisms generating the down-regulated expression of HLA-B antigens frequently observed on melanoma cells. In our study, we quantitated, both at the protein and mRNA level, the amounts of HLA-A and -B antigens constitutively expressed on 23 primary cultures of metastatic melanomas and on autologous peripheral blood mononuclear cells (PBMC). Flow cytometric analyses identified a significantly (p < 0.01) lower expression of HLA-B antigens on melanoma cell cultures but not on autologous PBMC. Consistently, lower amounts of HLA-B antigens mRNA were detected by RNase protection assay exclusively in neoplastic cells. This unbalanced expression of HLA-A and -B antigens was readily reverted by interferon (IFN)-gamma but not by the DNA hypomethylating agent 5-aza-2'-deoxycytidine in 4 melanoma cell cultures investigated. Significantly (p < 0.05) lower levels of HLA-B antigens were also detected on cells from solid malignancies of different histotypes but not on neoplastic cells from hemopoietic neoplasms; levels of HLA-B antigens were rapidly up-regulated by IFN-gamma exclusively on non-hemopoietic transformed cells. Together, these data strongly argue against a genetic predetermination of the amounts of HLA-A and -B antigens expressed on normal and neoplastic cells of distinct melanoma patients and suggest that constitutively low levels of HLA-B antigens are a specific feature of non-hemopoietic transformed cells that is controlled by common regulatory mechanism(s) and that is possibly shared by non-hemopoietic normal cells.

In vitro analysis of the melanoma/endothelium interaction increasing the release of soluble intercellular adhesion molecule 1 by endothelial cells.

Melanoma cells constitutively release intercellular adhesion molecule 1 (ICAM-1) as soluble ICAM-1 (sICAM-1), and its levels are elevated in melanoma patients and correlate with disease progression. However, this correlation is not absolute, suggesting that specific characteristics of neoplastic cells and/or ICAM-1-positive non-neoplastic cells may influence the amounts of circulating sICAM-1. In this study, we found a weak correlation (r = 0.55; r2 = 0.3) between sICAM-1 release by 40 metastatic melanomas (36 primary cultures and 4 cell lines), and ICAM-1 expression on neoplastic cells. In addition, melanoma-secreted interleukin-1alpha (IL-1alpha) (1/40) but not vascular endothelial growth factor (VEGF) (29/40), significantly (P < 0.05) up-regulated the shedding of sICAM-1 by human umbilical vein endothelial cells (HUVEC). This was completely abolished by IL-1alpha/beta neutralizing antibodies both at the protein and mRNA level. Altogether, our results suggest that (i) the extent of sICAM-1 release is distinctive for individual melanomas and can be independent of ICAM-1 expression; (ii) tumor endothelia may sustain levels of sICAM-1 in selected melanomas; (iii) melanoma-released VEGF does not affect ICAM-1 expression and sICAM-1 release by HUVEC. Melanoma-derived sICAM-1 inhibits cell-mediated cytotoxicity of melanoma cells; therefore, constitutive levels of sICAM-1 release and IL-1alpha secretion by individual melanomas can differentially influence tumor progression and the clinical effectiveness of cytotoxic-cell-based vaccines.

Prolonged upregulation of the expression of HLA class I antigens and costimulatory molecules on melanoma cells treated with 5-aza-2'-deoxycytidine (5-AZA-CdR).

The immunogenic potential of melanoma cells and their recognition by the host's cytotoxic cells depends on the presence and on the level of expression of human leukocyte antigen (HLA) class I antigens, costimulatory molecules and melanoma-associated antigens (MAA), on neoplastic cells. In this study, we demonstrate that the DNA hypomethylating agent 5-aza-2'-deoxycytidine (5-AZA-CdR), significantly (p < 0.05) enhanced the constitutive expression of HLA class I antigens, HLA-A1 and -A2 alleles, and of the costimulatory molecules intercellular adhesion molecule-1 and lymphocyte function-associated antigen-3, on a panel of 12 melanoma cells. This upregulation peaked at day 4, slowly decreased thereafter, and returned to baseline levels 32 days after the end of treatment. In addition, treatment with 5-AZA-CdR induced a persistent expression of MAGE-1 in Mel 275 melanoma cells; this was still detectable, by reverse transcriptase polymerase chain reaction, 60 days after the end of treatment. In contrast, 5-AZA-CdR did not affect the constitutive expression of the high molecular weight-MAA by the melanoma cells investigated. These observations, together with data obtained comparing the effect of 5-AZA-CdR with that of interferon-gamma, strongly suggest that 5-AZA-CdR may have prospective therapeutic implications in active and/or passive specific immunotherapy for human melanoma.

Structure, distribution, and functional role of protectin (CD59) in complement-susceptibility and in immunotherapy of human malignancies (Review).

Protectin (CD59) is a glycosyl-phosphatidylinositol-anchored cell membrane glycoprotein, ubiquitously expressed, though to a different extent, on benign and malignant cells. CD59 inhibits complement (C)-mediated lysis of target cells by preventing the formation of the membrane attack complex, in the terminal step of C-activation. Recent experimental evidence demonstrates that CD59 is the main restriction factor of C-mediated lysis of malignant cells of different histotype. Additionally, a soluble form of CD59, that retains its anchoring ability and functional properties, has been most recently identified in body fluids and in culture supernatants of different malignant cells. In view of its functional role, CD59 may protect neoplastic cells from C-mediated lysis, contributing to their escape from innate C-control and to tumor progression; additionally, the expression of CD59 by neoplastic cells may contribute to impair the therapeutic efficacy of C-activating monoclonal antibodies (mAb) directed to tumor-associated antigens. In the light of the functional role of CD59, this review focuses on the structural features, tissue distribution and regulation of the expression of CD59 in malignant tissues, and on the foreseeable application(s) of CD59 to improve the therapeutic efficacy of clinical approaches of humoral immunotherapy with C-activating mAb in human malignancies.

Melanoma cells constitutively release an anchor-positive soluble form of protectin (sCD59) that retains functional activities in homologous complement-mediated cytotoxicity.

Protectin (CD59), a glycosylphosphatidylinositol-anchored cell membrane glycoprotein, is differentially expressed on melanocytic cells and represents the main restriction factor of C-mediated lysis of melanoma cells. In this study, we report that CD59-positive melanoma cells constitutively release a soluble form of CD59 (sCD59), and that its levels directly correlate (r = 0.926; P < 0.05) with the amount of membrane-bound CD59. SDS-PAGE analysis showed that the molecular components of sCD59 are similar to those of cellular CD59 expressed by melanoma cells. Melanoma-released sCD59 is anchor positive since it inserts into cell membranes of homologous cells that transiently increase their expression of CD59. Moreover, sCD59 is functional: it blocks the binding of the anti-CD59 mAb YTH53.1 to melanoma cells and reverses its effects on C-mediated lysis. In fact, preincubation of mAb YTH53.1 with scalar doses of conditioned media of CD59-positive but not of CD59-negative melanoma cells reduced significantly (P < 0.05), and in a dose-dependent fashion, the enhancement of C-mediated lysis of anti-GD3-sensitized melanoma cells induced by the masking of cellular CD59 by mAb YTH53.1. Altogether, these data demonstrate that CD59-positive human melanoma cells release a soluble form of CD59 that is structurally similar to cellular CD59, retains its anchoring ability, is functional, and may impair the effectiveness of clinical approaches to humoral immunotherapy for human melanoma.

Tumour-derived interleukin 1alpha (IL-1alpha) up-regulates the release of soluble intercellular adhesion molecule-1 (sICAM-1) by endothelial cells.

Levels of circulating soluble intercellular adhesion molecule-1 (sICAM-1) are elevated in patients affected by solid malignancies; however, the cellular sources generating high levels of sICAM-1 remain to be characterized. Using conditioned media (CM) from seven ICAM-1-positive or -negative neoplastic cells, we demonstrate that tumour-derived interleukin 1alpha (IL-1alpha) significantly (P < 0.05) up-regulates the release of sICAM-1 by human umbilical vein endothelial cells. The intensity of the effect correlated with the amounts of IL-1alpha detectable in CM. Levels of ICAM-1 mRNA were also up-regulated by tumour-secreted IL-1alpha. The up-regulation of the shedding of sICAM-1 and of its expression at protein and mRNA level were completely reversed by the addition of anti-IL-1alpha neutralizing antibodies. Consistent with the in vitro data, tumour endothelia were strongly stained for ICAM-1 compared with autologous normal tissue endothelia. Taken altogether, our observations reveal an IL-1alpha-mediated tumour-endothelium relationship sustaining the shedding of sICAM-1 by endothelial cells. This is a general phenomenon in solid malignancies that correlates with the ability of neoplastic cells to secrete IL-1alpha rather than with their expression of ICAM-1 and/or histological origin. sICAM-1 has been previously shown to inhibit LFA-1/ICAM-1-mediated cell-cell interactions; therefore, the ability of neoplastic cells to secrete IL-1alpha is likely to represent a mechanism for their escape from immune interaction.

Expression of protectin (CD59) in human melanoma and its functional role in cell- and complement-mediated cytotoxicity.

Immunohistochemical and/or indirect immunofluorescence analysis with monoclonal antibody (MAb) H19 demonstrated the expression of protectin (CD59) in 54 surgically removed metastatic melanoma lesions and on 8 out of 12 melanoma cell lines. CD59 expression had a low degree of intra- and intertumor heterogeneity. SDS-PAGE analysis showed that the molecular weight of CD59 expressed on melanoma cells is about 20 kDa. Treatment of melanoma cells with 5U/ml of phosphatidylinositol-specific phospholipase C completely abolished cell-surface expression of CD59. Interferon-gamma and/or tumor necrosis factor-alpha or phorbol 12-myristate 13-acetate neither modulated the expression of CD59 by melanoma cells nor influenced the amounts of CD59-specific mRNA. F(ab')2 fragments of anti-CD59 MAb YTH53. I did not inhibit the lysis of melanoma cells by allogeneic natural killer (NK) cells or lymphokine-activated killer (LAK) cells. In contrast, the whole Ig molecule of MAb HI9 or YTH53.I significantly (p < 0.05) enhanced NK-cell-mediated lysis of melanoma cells, suggesting the induction of antibody-dependent cell-mediated cytotoxicity. Lastly, masking of CD59 by MAb YTH53.I or its F(ab')2 fragments significantly (p < 0.05) enhanced, in a dose-dependent fashion, the lysis of anti-GD3-sensitized melanoma cells by homologous complement. These data demonstrate that CD59 expressed by human melanoma cells might regulate host-tumor interaction by protecting neoplastic cells from complement-mediated lysis.

Differential expression of cell adhesion molecules CD54/CD11a and CD58/CD2 by human melanoma cells and functional role in their interaction with cytotoxic cells.

Immunohistochemical staining with monoclonal antibodies showed a differential distribution of intercellular adhesion molecule 1 (ICAM-1/CD54) and lymphocyte function-associated antigen 3 (LFA-3/CD58) and their respective counterreceptors lymphocyte function-associated antigens 1 (LFA-1/CD11a) and 2 (LFA-2/CD2) on ten melanoma cell lines and in 46 surgically removed metastatic melanoma lesions. CD11a and CD2 were not detected on melanoma cells while CD54 and CD58 were coexpressed on the majority of the melanoma cell populations investigated. CD54 showed a higher degree of intra- and intertumor heterogeneity than CD58. gamma-Interferon and/or tumor necrosis factor alpha upregulated the expression of CD54 by melanoma cells, but neither modulated that of CD58 nor induced that of CD11a and CD2. Anti-CD54 and anti-CD58 monoclonal antibodies partially inhibited the lysis of melanoma cells by allogeneic natural killer cells, lymphokine-activated killer cells and, to a greater extent, by autologous tumor-infiltrating lymphocytes. Soluble CD54 (cCD54) purified from serum of patients with melanoma inhibited the lysis of melanoma cells F0-1 by natural killer cells in a dose-dependent fashion. These results suggest that membrane-bound CD54 and CD58 and cCD54 play a role in host-tumor interactions in patients with malignant melanoma and may account for the relationship between CD54 expression in primary lesions and the clinical course of disease.

[Values of plasma testosterone in infancy and adolescence]. / Valori del testosterone plasmatico nella infanzia ed adolescenza.

We have performed a research to establish the normal values of the plasmatic testosterone among 111 pupils, of both sexes, from 7 to 16 years attending our provincial schools. As we expected, the greatest increase of the plasmatic testosterone, in comparison with the previous ages, has been found in the group of the 13 years old females of the same age is more restrained, even if it's significative. By this way we could know the physiological values of the plasmatic testosterone in the children and adolescents of our population.