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Biomedical subjects

C Castelli

Publications and source records attributed to C Castelli.

At least 19 recordsLinked to original sources
PubMed2006

Opposite immune functions of GM-CSF administered as vaccine adjuvant in cancer patients.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) has been and is still widely used as an adjuvant in clinical trials of vaccination with autologous tumor cells, peptides and/or dendritic cells in a variety of human neoplasms. This cytokine was administered either as product of gene-transduced tumor cells or as recombinant protein together with the vaccine given subcutaneously or intradermally. Results of these trials were heterogeneous in terms of induction of vaccine-specific immune response and of clinical response. Though in some of these studies GM-CSF appeared to help in generating an immune response, in others no effect or even a suppressive effect was reported. Here, we review the literature dealing with the immune adjuvant activity of GM-CSF both in animal models and clinical trials. As a consequence of such analysis, we conclude that GM-CSF may increase the vaccine-induced immune response when administered repeatedly at relatively low doses (range 40-80 microg for 1-5 days) whereas an opposite effect was often reported at dosages of 100-500 microg. The potential mechanisms of the GM-CSF-mediated immune suppression are discussed at the light of studies describing the activation and expansion of myeloid suppressor cells by endogenous tumor-derived or exogenous GM-CSF.

Adjuvants, Immunologic↗
PubMed2005

Photosensitizing properties of 6-methoxy-2-naphthylacetic acid, the major metabolite of the phototoxic non-steroidal anti-inflammatory and analgesic drug nabumetone.

The photobiological properties of 6-methoxy-2-naphthylacetic acid (6-MNAA) were studied using a variety of in vitro phototoxicity assays: photohemolysis, photoperoxidation of linoleic acid, photosensitized degradation of histidine and thymine and the Candida phototoxicity test. 6-MNAA was phototoxic in vitro. 6-MNAA reduced nitro blue tetrazolium (NBT) when irradiated with lambda > or = 300 nm in deoxygenated aqueous buffer solution (pH 7.4). NBT can be reduced by reaction with the excited state of 6-MNAA subject to interference with molecular oxygen. The photohemolysis rate was inhibited by the presence of 1,4-diazabicyclo[2.2.2]octane (DABCO), sodium azide (NaN3) and reduced glutathione (GSH). Photoperoxidation of linoleic acid and photosensitized degradation of histidine and thymine were significantly inhibited by sodium azide and reduced glutathione. 6-MNAA was phototoxic to C. albicans, C. lipolytica and C. tropicalis. A mechanism involving singlet oxygen, radicals, and electron transfer reactions is suggested for the observed phototoxicity.

Aerobiosis↗
PubMed2004

Levocloperastine in the treatment of chronic nonproductive cough: comparative efficacy versus standard antitussive agents.

The medical and social impact of cough is substantial. Current antitussive agents at effective doses have adverse events such as drowsiness, nausea and constipation that limit their use. There is also recent evidence that standard antitussive agents, such as codeine, may not reduce cough during upper respiratory infections. Therefore, there is a need for more effective and better-tolerated agents. The efficacy of levocloperastine, a novel antitussive, which acts both centrally on the cough center and on peripheral receptors in the tracheobronchial tree in treating chronic cough, was compared with that of other standard antitussive agents (codeine, levodropropizine and DL-cloperastine) in six open clinical trials. The studies enrolled patients of all ages with cough associated with various respiratory disorders including bronchitis, asthma, pneumonia and chronic obstructive pulmonary disease. Levocloperastine significantly improved cough symptoms (intensity and frequency of cough) in all trials, and improvements were observed after the first day of treatment. In children, levocloperastine reduced nighttime awakenings and irritability, and in adults it was effective in treating cough induced by angiotensin-converting enzyme inhibitors. When compared with other antitussive agents, levocloperastine had improved or comparable efficacy, with a more rapid onset of action. Importantly, no evidence of central adverse events was recorded with levocloperastine, whereas drowsiness was reported by a significant number of patients receiving codeine. Levocloperastine is an effective antitussive agent for the treatment of cough in patients of all ages. It has a more rapid onset of action than standard agents with an improved tolerability profile.

Adolescent↗
PubMed2003

Vaccination of patients with solid tumours.

The molecular characterisation of human tumour antigens recognised by T cells has provided new impetus for immunisation of patients bearing tumours expressing well-defined antigens. After evaluating the immunogenicity of the new, molecularly characterised antigens in vitro, several clinical studies were conducted to assess the in vivo immunogenicity and the clinical efficacy of vaccines including these antigens. The findings generated by trials based on the administration of peptides or DNA-encoding antigens are discussed to highlight the limits of this therapeutic approach; however, this approach has resulted in some complete and durable regressions, although still in a unsatisfactory small number of cases (5-25%). The recent use of dendritic cells loaded ex vivo with tumour antigens suggests that a high frequency of tumour-specific immune responses can be achieved. Possible means of overcoming the clinical limits and improving the outcome of previous studies are also discussed.

Animals↗
PubMed2001

Tumor necrosis factor-alpha induces coordinated changes in major histocompatibility class I presentation pathway, resulting in increased stability of class I complexes at the cell surface.

It is demonstrated that similar to interferon gamma (IFN-gamma), tumor necrosis factor-alpha (TNF-alpha) induces coordinated changes at different steps of the major histocompatibility complex (MHC) class I processing and presentation pathway in nonprofessional antigen-presenting cells (APCs). TNF-alpha up-regulates the expression of 3 catalytic immunoproteasome subunits--LMP2, LMP7, and MECL-1--the immunomodulatory proteasome activator PA28 alpha, the TAP1/TAP2 heterodimer, and the total pool of MHC class I heavy chain. It was also found that in TNF-alpha--treated cells, MHC class I molecules reconstitute more rapidly and have an increased average half-life at the cell surface. Biochemical changes induced by TNF-alpha in the MHC class I pathway were translated into increased sensitivity of TNF-alpha--treated targets to lysis by CD8(+) cytotoxic T cells, demonstrating improved presentation of at least certain endogenously processed MHC class I--restricted peptide epitopes. Significantly, it was demonstrated that the effects of TNF-alpha observed in this experimental system were not mediated through the induction of IFN-gamma. It appears to be likely that TNF-alpha--mediated effects on MHC class I processing and presentation do not involve any intermediate messengers. Collectively, these data demonstrate the existence of yet another biologic activity exerted by TNF-alpha, namely its capacity to act as a coordinated multi-step modulator of the MHC class I pathway of antigen processing and presentation. These results suggest that TNF-alpha may be useful when a concerted up-regulation of the MHC class I presentation machinery is required but cannot be achieved by IFN-gamma. (Blood. 2001;98:1108-1115)

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed2001

Human heat shock protein 70 peptide complexes specifically activate antimelanoma T cells.

Members of the heat shock protein 70 (HSP70) family display a broad cellular localization and thus bind a repertoire of chaperoned peptides potentially derived from proteins of different cellular compartments. In this report, we show that HSP70 purified from human melanoma can activate T cells recognizing melanoma differentiation antigens in an antigen- and HLA class I-dependent fashion. HLA class I-restricted anti-melanoma T cells were susceptible to MHC-restricted, HSP70-dependent stimulation, indicating that HSP70 complexed peptides were able to gain access to the class I HLA presentation pathway. In addition, MHC matching between the melanoma cells used as a source of HSP and the responding T cells were not required, indicating that HSP70 activation may occur across MHC barriers. Besides the MHC-restricted and peptide-dependent activation pathway, HSP70 with no endogenous complexed peptides or HSP70 purified from antigen-negative cells was also able to induce IFN-gamma release by antimelanoma T cells by a MHC-independent mechanism. In this case, however, higher doses of HSP70 were required. The capacity to activate class I-restricted, antitumor T cells as well as antigen-presenting cells, together with the finding that the HSP70 chaperoned peptide repertoire includes melanoma-shared epitopes, holds promise for a HSP70-based cancer vaccine.

Antigen-Presenting Cells↗
PubMed2001

Simultaneous transduction of B7-1 and IL-2 genes into human melanoma cells to be used as vaccine: enhancement of stimulatory activity for autologous and allogeneic lymphocytes.

In order to construct an immunogenic cellular vaccine, we transduced three HLA-A*0201 human melanoma lines, selected for expression of classes I and II HLA, adhesion molecules and the T cell-defined melanoma antigens Melan/MART-1, gp100 and tyrosinase, with both interleukin-2 (IL-2) and B7-1 genes by the use of a polycistronic retroviral vector. The lines were selected to share only the HLA-A*0201 allele to avoid generation of strong alloreactivity in case of their multiple in vivo use in HLA-A*0201 + patients. Phenotypic and functional analysis of B7-1-IL2 transduced melanoma lines in comparison with B7-1 transduced and/or parental untransduced counterparts were then carried out. Tumor cells expressing either B7-1 or both genes did not change their original antigenic profile. From a functional point of view, expression of both genes in melanoma lines: (1) improved the response of anti-melanoma cytotoxic T lymphocytes (CTL) over singly transduced or untransduced melanoma cells when subthreshold levels of MHC-peptide complexes were expressed by melanoma cells; (2) conferred a distinct advantage in the ability to stimulate cytotoxicity and interferon-gamma release by autologous and/or HLA-A*0201-compatible allogeneic lymphocytes; (3) allowed the generation of a high number of specific CTL by in vitro stimulation of lymphocytes of HLA-A*0201-melanoma patients. Thus, B7-IL2 gene-transduced melanoma lines appear to display a high immunogenicity and could be used as vaccine in melanoma patients.

B7-1 Antigen↗
PubMed2001

Ultrastructural complexity of nuclear components during early apoptotic phases in breast cancer cells.

Fractal morphometry was used to investigate the ultrastructural features of the plasma membrane, perinuclear membrane and nuclear chromatin in SK-BR-3 human breast cancer cells undergoing apoptosis. Cells were incubated with 1 microM calcimycin (A23187) for 24 h. Cells in the early stage of apoptosis had fractal dimension (FD) values indicating that their plasma membranes were less rough (lower FD) than those of control cells, while their perinuclear membranes were unaffected. Changes of the chromatin texture within the entire nucleus and in selected nuclear domains were more pronounced in treated cells. This confirms that the morphological reorganization imputable to a loss of structural complexity (reduced FD) occurs in the early stage of apoptosis, is accompanied by the inhibition of distinct enzymatic events and precedes the onset of conventional cellular markers, which can only be detected during the active phases of the apoptotic process.

Annexin A5↗
PubMed2000

T cell responses in colorectal cancer patients: evidence for class II HLA-restricted recognition of shared tumor-associated antigens.

Few cases of anti-colon cancer specific T lymphocytes have been described so far. Moreover, the majority of these effectors were generated in vitro by stimulating PBMC from patients or healthy donors with peptides that were derived from proteins expressed and/or secreted by colon cancer tissue such as CEA, Mucin or Her-2/neu. The aim of our study was to evaluate the immunogenicity of colorectal carcinomas in an autologous setting. We exploited the antigen processing and presentation capacity of dendritic cells (DC) to establish an in vitro autologous system that can bypass the need of obtaining cultured tumor cells. DC were generated from the adherent monocyte fraction of PBMC taken from stage II/III colorectal cancer patients. A single cell suspension was prepared by mechanical and enzymatic disruption of the surgical specimens immediately after resection. DC were loaded with autologous tumor lysate, obtained by repeated freezing and thawing, before being used as stimulators for autologous PBL. HLA-class II restricted T cells that recognize the autologous tumor could be generated in a proportion of patients. The fine specificity of the anti-tumor T cells indicates that differentiation as well as tumor restricted antigens are expressed in colon cancer and that these antigens can evoke a class II HLA-restricted response in an autologous setting. Altogether these findings may open a new perspective for a DC based vaccination of colon cancer patients.

Antigens, Neoplasm↗
PubMed2000

T-cell recognition of melanoma-associated antigens.

In this review, we summarize the significant progress that has been made in the identification of melanoma-associated antigens (MAA) recognized by cytotoxic T-lymphocytes (CTL). These antigens belong to three main groups: tumor-associated testis-specific antigens (e.g. , MAGE, BAGE, and GAGE); melanocyte differentiation antigens (e.g., tyrosinase, Melan-A/MART-1); and mutated or aberrantly expressed molecules (e.g, CDK4, MUM-1, beta-catenin). Although strong CTL activity may be induced ex vivo against most of these antigens, often in the presence of excess cytokines and antigen, a clear understanding of the functional status of CTL in vivo and their impact on tumor growth, is still lacking. Several mechanisms are described that potentially contribute to tumor cell evasion of the immune response, suggesting that any antitumor efficacy achieved by immune effectors may be offset by factors that result ultimately in tumor progression. Nevertheless, most of these MAA are currently being investigated as immunizing agents in clinical studies, the conflicting results of which are reviewed. Indeed, the therapeutic potential of MAA has still to be fully exploited and new strategies have to be found in order to achieve an effective and long-lasting in vivo immune control of melanoma growth and progression.

Antigens, Neoplasm↗
PubMed1999

Cytotoxic T lymphocytes define multiple peptide isoforms derived from the melanoma-associated antigen MART-1/Melan-A.

Peptides derived from the melanoma-associated MART-1/Melan-A antigen are currently implemented in immunotherapy for inducing or augmenting T-cell responses directed against peptides expressed by autologous tumor cells in HLA-A2+ patients with melanoma. Here, we describe the specificity of the T-cell clone SK29-FFM1.1, which secretes GM-CSF in response to a panel of synthetic MART-1/Melan-A-derived peptides, including the naturally presented ILTVILGVL(32-40), but exhibits cytotoxicity and IFN-gamma secretion exclusively to the MART-1/Melan-A derived peptide AAGIGILTV(27-35). In addition, cytotoxic T-lymphocyte (CTL) clone SK29-FFM1.1 recognizes 3 different naturally processed and presented peptides on HLA-A2+ MART-1/Melan-A+ melanoma cells, as defined by cytotoxicity and IFN-gamma and GM-CSF secretion. Processing and presentation of MART-1/Melan-A peptides appears to be different in cells of non-melanocytic origin, as shown by the characterization of naturally presented peptides displayed by HLA-A2+ colorectal cancer cells transduced with a MART-1/Melan-A gene-containing retrovirus. Our data suggest that multiple epitopes, including ILTVILGVL and different isoforms of AAGIGILTV derived from MART-1/Melan-A may be naturally presented by melanoma cells to the immune system.

Amino Acid Sequence↗
PubMed1999

Novel HLA-Cw8-restricted T cell epitopes derived from tyrosinase-related protein-2 and gp100 melanoma antigens.

The identification of T cell epitopes presented by alternative HLA-B and -C alleles may provide a means to counteract the tumor escape mechanism based on the selection of tumor cells no longer susceptible to HLA-A-restricted T cell recognition. Several T cell clones and lines were obtained from T lymphocytes purified from melanoma-infiltrated or noninfiltrated lymph nodes of a patient who remained disease free 8 yr after surgery. Selected T cells recognized the autologous melanoma as evaluated by direct cytolysis and production of cytokines. These effectors were directed against the tyrosinase-related protein-2 (TRP-2) and gp100 melanoma epitopes restricted by HLA-Cw8. The nonamer and decamer peptides containing the sequence ANDPIFVVL (residues 387-395) of TRP-2 and the octamer, nonamer, and decamer peptides containing the sequence SNDGPTLI (residues 71-78) of gp100 reconstituted the epitope for TRP-2- and gp100-specific T cell lines and clones, respectively. However, only the nonameric form of TRP-2 and the nonameric and octameric forms of gp100 were able to induce peptide-specific T cells recognizing the autologous tumor in an HLA-class I-restricted fashion from PBMC of the melanoma patient studied. Together these data indicate that HLA-Cw8 can restrict the recognition of gp100 and TRP-2 epitopes by CTL, and that such peptides could stimulate a patient's PBL, suggesting that these Ags could have contributed to a systemic immunity against melanoma.

Alleles↗
PubMed1999

A superagonist variant of peptide MART1/Melan A27-35 elicits anti-melanoma CD8+ T cells with enhanced functional characteristics: implication for more effective immunotherapy.

In the present study, we show that a singly substituted peptide derived from the epitope MART1(27-35) and containing a Leu in position 1 (LAGIGILTV; 1L) behaves as a superagonist by in vitro inducing specific T cells with enhanced immunological functions. 1L-specific CTLs can be raised from peripheral blood of HLA-A2+ melanoma patients more efficiently than T cells specific for the cognate peptide. These T cells show a greater sensitivity to native MART1(27-35) when compared with CTL variable raised to parental peptide from the same patients. More importantly, anti-1L but not anti-native T cells display high levels of interferon gamma production at early time points, and readily secreted interleukin-2 in response to native epitope endogenously presented by melanoma cells. Additionally, anti-1L T cells are insensitive to the inhibitory effects of MART1(27-35) natural analogues that antagonize the lytic response of CTLs raised to the cognate peptide. Analysis of T-cell receptor variable beta usage suggests that the native and 1L peptides stimulate different components of the MART1(27-35)-reactive T cell population. These data provide rationale to the use of superagonist analogues of tumor antigens for inducing in vivo immunization potentially able to overcome tumor immune escape and mediate a more significant control of tumor growth.

CD8-Positive T-Lymphocytes↗
PubMed1999

Gas exchange response to exercise in patients with chronic heart failure.

The objective of the study was investigate the pulmonary gas exchange response to exercise in 16 male patients with chronic heart failure (CHF) due to previous myocardial infarction and left ventricular dysfunction (ejection fraction < 45%). All patients underwent a symptom-limited exercise test during which cardiac frequency (fC), tidal volume (VT), respiratory frequency (fR), minute ventilation (V'E), oxygen consumption (V'O2) and carbon dioxide production (V'CO2) were measured on a breath-by-breath basis. Ventilatory equivalent for carbon dioxide (V'E/V'CO2) and lactate threshold (LT) were calculated. Arterial blood gas levels were measured at rest and at peak exercise. The dead space (VD) to tidal volume ratio (VD/VT) and alveolar-arterial oxygen gradient (PA-a,O2) were computed. Two subgroups of patients were identified according to peak V'O2 (V'O2,peak), group A (n = 7), V'O2,peak > 14 mL.kg-1.min-1 (17.2 +/- 2.5 SEM, range 14.5-20.8), and group B (n = 9), V'O2,peak < 14 mL.kg-1.min-1 (11.9 +/- 1.8, range 9.2-13.6). Arterial oxygen tension (Pa,O2) increased from rest to peak exercise in both groups (group A: 12.2 +/- 0.94 to 13.4 +/- 0.82 kPa (91.4 +/- 7.1 to 100.4 +/- 6.2 mmHg), p < 0.05; group B: 11.7 +/- 1.0 to 13.4 +/- 1.1 kPa (88.0 +/- 7.8 to 100.9 +/- 8.2 mmHg), p < 0.01), while a significant reduction in arterial carbon dioxide tension (Pa,CO2), from rest to peak exercise, was observed in group B only (4.64 +/- 0.39 to 4.08 +/- 0.36 kPa (34.9 +/- 2.8 to 30.7 +/- 2.7 mmHg), p < 0.005). Maximal V'E and maximal power (Powermax) were significantly lower in group B compared to group A (V'E 37.6 +/- 8.4 versus 52.1 +/- 13.8 L.min-1, p < 0.05; Powermax 64.4 +/- 12 versus 82.8 +/- 14.1 W, p < 0.01). fC was not significantly different at peak exercise, although the work load was significantly higher in group A. VD/VT failed to decrease significantly at maximal exercise in both groups. In group B, V'E/V'CO2 tended to be higher than in group A. In chronic heart failure patients, measurements of arterial blood gas levels during exercise might help to identify those subjects with a more pronounced depression of left ventricular function. At peak exercise, high ventilatory demand and respiratory alkalosis were observed in group B patients, suggesting an increased responsiveness of the respiratory centre that might be one major factor contributing to this excessive ventilatory response to exercise; vice versa, a combination of ventilation-perfusion mismatch, wasted ventilation and unpaired peripheral blood circulation seem to play only a minor role.

Aged↗
PubMed1998

Human melanoma-reactive CD4+ and CD8+ CTL clones resist Fas ligand-induced apoptosis and use Fas/Fas ligand-independent mechanisms for tumor killing.

Tumor cells have been shown recently to escape immune recognition by developing resistance to Fas-mediated apoptosis and acquiring expression of Fas ligand (FasL) molecule that they may use for eliminating activated Fas+ lymphocytes. In this study, we report that tumor-specific T lymphocytes isolated from tumor lesions by repeated in vitro TCR stimulation with relevant Ags (mostly represented by normal self proteins, such as MART-1/Melan A and gp100) can develop strategies for overcoming these escape mechanisms. Melanoma cells (and normal melanocytes) express heterogeneous levels of Fas molecule, but they result homogeneously resistant to Fas-induced apoptosis. However, CD4+ and CD8+ CTL clones kill melanoma cells through Fas/FasL-independent, granule-dependent lytic pathway. In these lymphocytes, Ag/MHC complex interaction with TCR does not lead to functional involvement of FasL, triggered, on the contrary, by T cell activation with nonspecific stimuli such as PMA/ionomycin. Additionally, melanoma cells express significant levels of FasL (detectable on the cell surface only after treatment with metalloprotease inhibitors), although to a lesser extent than professional immune cells such as Thl clones. Nevertheless, antimelanoma CTL clones resist apoptosis mediated by FasL either in soluble form or expressed by Thl lymphocytes or FasL+ melanoma cells. These results demonstrate that CD4+ and CD8+ antimelanoma T cell clones can be protected against Fas-dependent apoptosis, and thus be useful reagents of immunotherapeutic strategies aimed to potentiate tumor-specific T cell responses.

Apoptosis↗