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Biomedical subjects

G Melioli

Publications and source records attributed to G Melioli.

At least 55 records · Page 3Linked to original sources
PubMed1994

Release of peripheral blood progenitor cells during standard dose cyclophosphamide, epidoxorubicin, 5-fluorouracil regimen plus granulocyte colony stimulating factor for breast cancer therapy.

BACKGROUND: High dose chemotherapy with the support of peripheral blood progenitor cells (PBPC) is increasingly used in the treatment of solid tumors. Although the best method of PBPC mobilization is still under investigation, it should be optimized for different tumor types to obtain antitumor effect and mobilizing activity. The authors report these results in terms of the number of PBPC released and the time of maximum mobilization induced by standard dose cyclophosphamide, epidoxorubicin, 5-fluorouracil (CEF) (cyclophosphamide 600 mg/m2, epidoxorubicin 60 mg/m2, 5-fluorouracil 600 mg/m2) plus granulocyte colony stimulating factor (G-CSF) in patients with breast cancer. METHODS: Peripheral blood progenitor cells were studied by clonogenic assay of granulocyte macrophage colony-forming units (CFU-GM), megakaryocyte colony-forming unit (CFU-Meg) and erythrocyte burst-forming unit (BFU-E) and by flow cytometric analysis of CD34+ cells in 12 patients with early breast cancer throughout three cycles of CEF chemotherapy plus G-CSF. RESULTS: Colony assays and CD34+ cell determination were performed on 111 and 151 blood samples, respectively. The peak of CFU-GM and CD34+ cells occurred consistently at day 11 throughout all three cycles. At day 11 of the first cycle, the median peak values were 2223 CFU-GM/mL and 256 CD34+ cells/microL. A progressive decrease in peak value from the first to the third cycle was observed. CONCLUSIONS: Standard dose CEF chemotherapy plus G-CSF is a disease specific regimen allowing PBPC mobilization without any relevant toxicity. Maximum mobilization was recorded at day 11 of the first cycle.

Adult↗
PubMed1994

Isolation and in vitro expansion of lymphocytes infiltrating non-small cell lung carcinoma: functional and molecular characterisation for their use in adoptive immunotherapy.

Tumour infiltrating lymphocytes (TIL) have the capability of recognising and lysing autologous cancer cells, both in vitro and in vivo. Advanced non-small cell lung carcinoma (NSCLC) is partially insensitive to chemo radiotherapy and has a poor prognosis: thus, for this, an immunotherapeutic approach could be attempted. We expanded in vitro 46 out of 70 samples of TIL derived from NSCLC. From proliferating TILS, a number varying from 10 to 50 x 10(9) cells was obtained. These lymphocytes belonged to the T cell lineage, had the capability of growing for 45-60 days and lysed autologous better than allogeneic cancer cells. In addition, analysis of the restriction maps of T cell receptor (TRC)-beta, demonstrated that an oligoclonal population of T cells was preselected in vivo, near the tumour site, and might be expanded in vivo, using phytohaemagglutin and interleukin 2 while maintaining the same characteristics of the original population. These results give a clear rationale for the use of in vitro expanded TIL from NSCLC in protocols of adoptive immunotherapy in patients with residual disease following surgery.

Adult↗
PubMed1994

Proliferative, phenotypic and functional and molecular characteristics of tumour-infiltrating lymphocytes obtained from unselected patients with malignant melanomas and expanded in vitro in the presence of recombinant interleukin-2.

Tumour-infiltrating lymphocytes (TIL) derived from several histotypes, including melanoma, can be expanded in vitro in the presence of recombinant interleukin-2 (rIL-2) and infused as part of experimental adoptive immunotherapy protocols. Several authors have described the isolation and the expansion of TIL, but little is known about the actual proportion of unselected melanoma biopsies that give rise to 'positive' TIL cultures. We evaluated the proliferative, phenotypic, functional and molecular characteristics of cultured TIL obtained from 26 patients with metastatic melanomas, eligible for inclusion in a protocol of adoptive immunotherapy. Sixteen proliferating cultures were obtained. All TIL belonged to the T cell lineage and were characterized by a wide range of cytolytic activity against autologous and, to a lesser extent, allogeneic melanoma cells. Molecular analysis of the constant region of T cell receptor-beta (TCR-beta) showed an oligoclonal population of TIL in the majority of expanded samples, indicating that a selected subset of lymphocytes present in the tumour site could be expanded in vitro. These features, together with the high number of proliferating samples, make these populations of TIL suitable for infusion into patients with advanced disease.

Adult↗
PubMed1993

Lymphocytes isolated from the peritoneal fluid of women with advanced ovarian carcinoma differ significantly from autologous peripheral blood lymphocytes.

The phenotypic and functional analysis of ovarian carcinoma-associated lymphocytes (OCAL), obtained from a group of patients with neoplastic carcinosis, reveals major differences between OCAL and peripheral blood lymphocytes (PBL). Virtually all of the OCAL belong to the T cell lineage, whereas both B and natural killer (NK) cells are virtually absent. NK activity is also significantly lower than that exerted by PBL. Following treatment with rIL-2, lymphokine-activated killer cell activity is poorly inducible in OCAL. In addition, in vitro stimulated OCAL produce low amounts of lymphokines. Clonal analyses reveal a low clonal efficiency, which indicates a severe proliferative defect. T cell clones obtained show peculiar features: the percentage of CD4 and CD8 clones varies greatly in the different samples. Both CD8 and a significant proportion of proliferating CD4+ clones are cytotoxic in a lectin-dependent cytolytic assay. These findings, together with the results obtained from comparative analyses of the peripheral blood of the same group of patients, demonstrate the unique characteristics of OCAL and should be considered in the preparation of protocols for the locoregional immunotherapy of ovarian carcinoma.

Adenocarcinoma↗
PubMed1993

Recombinant interleukin-2 corrects in vitro the immunological defect of endometriosis.

PROBLEM: It has been shown recently that women with endometriosis have several immunological defects. In particular, these patients have a defect in peripheral blood natural killer cell activity and have lost their capacity of recognizing and lysing autologous endometrial cells. METHOD: We evaluated the effect of recombinant interleukin-2 (rIL-2) on peripheral blood lymphocytes obtained from both healthy donors and endometriosis patients. The generation of a strong cytolytic activity against autologous endometrial cells was obtained from both normal donors and endometriosis patients. RESULTS: Interestingly, these cytolytic cells belong to the T-cell lineage and do not recognize both autologous keratinocytes and allogeneic endometrial cells, thus suggesting a mechanism of specific recognition of autologous cells. CONCLUSION: The capability of restoring cytolytic activity using rIL-2 suggests a new immunotherapeutic approach for the treatment of severe endometriosis.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1992

"HepG2/erythroid/brain" type glucose transporter (GLUT1) is highly expressed in human epidermis: keratinocyte differentiation affects GLUT1 levels in reconstituted epidermis.

In mature animals, the "HepG2/erythroid/brain" glucose transporter isoform (GLUT1) appears to be expressed at the highest levels at blood tissue barriers; however, these levels may still be lower than the levels of expression seen in fetal tissues. Also, glucose transporters might serve as water channels. Therefore, we decided to investigate GLUT1 expression in human epidermis, a very active tissue, in terms of metabolism, even if not directly vascularized. We found GLUT1 transcripts in human skin and demonstrated, by immunohistochemistry, that GLUT1 protein is highly expressed in the basal layer and, to a lower extent, in the immediately suprabasal layer of the epidermis. This distribution pattern suggested that GLUT1 expression is affected by keratinocyte differentiation. To investigate this possibility, we used human epidermis reconstituted in culture. Our culture system allows the reconstruction of a stratified squamous epithelium which has been successfully grafted onto patients presenting large skin defects. Human keratinocytes have been cultured under conditions which allow a modulation of cellular differentiation and stratification. We observed that (i) GLUT1 expression is 4-6-fold higher in "stem-like" basal cells than in large, differentiated keratinocytes; (ii) culture conditions causing cell differentiation reduce GLUT1 expression, while conditions which minimize either differentiation or stratification of keratinocytes enhance GLUT1 expression. Finally, we found that IGF-1 and insulin, probably acting through the IGF-1 receptor, increase GLUT1 expression and stimulate glucose transport activity in epidermis reconstituted in culture. In conclusion, our data demonstrate that GLUT1 is highly expressed in the basal layers of human epidermis and that its expression is modulated by keratinocyte differentiation.

Brain↗
PubMed1992

Perilymphatic injections of recombinant interleukin-2 (rIL-2) partially correct the immunologic defects in patients with advanced head and neck squamous cell carcinoma.

Patients with advanced head and neck squamous cell carcinoma (HNSCC) are severely immunocompromised. In virtually all such patients who have been studied, reduced numbers of circulating CD3+ T-cell-receptor (TCR)alpha/beta+ T lymphocytes, a reduction of natural killer (NK) activity, and a poor induction of lymphokine-activated killer (LAK) cell activity (following in vitro treatment with recombinant interleukin-2 [rIL-2]) have been detected. Recently, however, it has been demonstrated that perilymphatic injections of low doses of rIL-2 may induce a local reduction of tumor masses in these patients. The present study, a cooperative pilot effort on the clinical effects of this route of administration, showed an activation of the lytic machinery in lymphocytes belonging to the T-cell lineage, as well as a potentiation of NK activity in the peripheral blood. These findings demonstrated that the severe immunodeficiency of HNSCC patients may be at least partially corrected by in vivo administration of rIL-2.

Aged↗
PubMed1991

High expression levels of the "erythroid/brain" type glucose transporter (GLUT1) in the basal cells of human eye conjunctiva and oral mucosa reconstituted in culture.

The expression of the "erythroid/brain" type glucose transporter (GLUT1) seems to be a feature of "barrier" tissues, at least in humans. Recently, we reported that GLUT1 is highly expressed in the basal layers of either "authentic" human epidermis or human epidermis reconstituted in culture and that its expression seems to be related to keratinocyte differentiation. In this paper we demonstrate that GLUT1 is selectively expressed in the basal layers of either eye conjunctiva epithelia or oral mucosa, reconstituted in culture starting from 1-2 mm2 bioptic specimens of normal human tissue. GLUT1 mRNA and protein levels are very high in conjunctiva and oral mucosa, 2-3 times higher than in epidermis reconstituted in culture. Taking into account its localization at the border of tissues not directly vascularized, but metabolically active, GLUT1 could play an important role in controlling the entry of glucose into these firmly guarded tissues.

Blotting, Northern↗
PubMed1991

Induction of functional activities of human lymphocytes by monoclonal antibodies.

Monoclonal antibodies (MoAbs) specific for surface molecules involved in lymphocyte activation, represent an useful tool for the analysis of the activation pathways of different effector lymphocytes. We have analyzed the ability of MoAbs directed against "triggering" surface molecules, such as CD3/TCR, CD2 and CD16 to induce activation of the lytic machinery in T and NK lymphocytes, using a redirected killing assay. In addition, we have constructed bispecific monoclonal antibodies (BiMoAbs) directed against both the CD3/TCR complex (or the CD16 molecule) and a tumor associated antigen expressed on the surface of ovarian cancer cells. BiMoAbs were constructed by two different approaches: a) conjugation of two distinct MoAbs by a chemical heterobifunctional agent; b) selection of hybrid-hybridomas secreting BiMoAbs. BiMoAbs were able to focus appropriate lymphoid effector cells on tumor cells expressing the relevant antigen, and to induce tumor cell lysis. Therefore, these reagents were able to confer a new specificity for a given antigen to effector lymphocytes, independently from the original one. For these properties, BiMoAbs may represent a suitable tool for new strategies of adoptive immunotherapy, based on the use of effector cells that have been specifically armed by BiMoAbs against the tumor.

Animals↗
PubMed1991

A phase I study of recombinant interleukin-2 intraperitoneal infusion in patients with neoplastic ascites: toxic effects and immunologic results.

A phase I study to evaluate the use of i.p. infusion of recombinant interleukin-2 (rIL-2) was planned. The following dose levels were calculated: 0.1, 0.3, 1.0, 3.0 and 10 mg/m2/day for 14 days, but only the second levels were reached. In this trial the acute toxic effects at this dosage included cardiac ischemia, transient liver impairment and septic peritonitis. The maximum tolerated dose (MTD) was 0.3 mg/m2/day for 14 days. In addition, two patients developed peritoneal fibrosis. No objective responses were observed. Therefore, in order to explore the biological activity of low (nontoxic) doses, three patients (one untreated and two previously treated with rIL-2) were infused with 0.01 and 0.03 mg/m2/day for 7 days. Potentiation of cytolytic activities in peritoneal lymphocytes and activation of a lymphokine cascade in the ascitic fluid were observed at doses ranging from 0.03 mg/m2/day to 0.3 mg/m2/day. These findings in association with the toxic effects observed at the MTD suggest the use of the minimum effective dose for future locoregional immunotherapeutic protocols.

Adult↗
PubMed1989

CD16 surface molecules regulate the cytolytic function of CD3CD16+ human natural killer cells.

Monoclonal (IgG) antibodies (MAbs) directed to CD16 molecules efficiently induced lysis of the IgG-binding P815 target cells. A similar effect was observed with selected anti-CD2 MAbs. While combinations of 2 appropriate anti-CD2 MAbs were required for induction of T lymphocyte activation, single stimulatory anti-CD2 MAbs were sufficient for inducing cytolytic function in CD3- CD16+ lymphocytes. In order to study possible regulatory mechanisms existing in the process of activation and induction of the cytolytic machinery of CD3- CD16+ effector cells, we utilized the anti-CD16 OKNK MAb. Being of IgM isotype, the OKNK MAb does not allow cross-linking between CD3- D16+ lymphocytes and target cells. Pre-treatment of effector cells with OKNK MAb sharply inhibited the target cell lysis induced by either anti-CD16 (IgG) MAbs or stimulatory anti-CD2 MAb. Moreover, a strong inhibitory activity of PHA-induced target cell lysis and even of "spontaneous" lysis (at high effector:target ratio) was observed. In contrast, in CD3+ CD16+ clones, OKNK MAb selectively inhibited the cell triggering induced by anti-CD16 MAbs (but not by anti-CD3, anti-CD2 MAbs or PHA). Our data indicate that CD16 receptor molecules expressed by CD3- CD16+ lymphocytes down-regulate cell responses to anti-CD2 MAbs or PHA, and then exert a regulatory role in the cytolytic function of these cells.

Antibodies, Monoclonal↗
PubMed1989

Microplate selection technique (MPST). A new method for selecting mouse transfectants expressing human gene products.

This paper examines the analytical power of fluorescence activated cell sorting and immunorosetting technique as compared with the newly devised microplate selection technique in identifying transfected murine L cells expressing human surface molecules. The microplate selection technique relies on the mechanical transfer of transfected cells to a terasaki microplate, where an indirect immunofluorescence assay is carried out. It is a simple procedure not requiring costly equipment and with a detection capacity equivalent to that of the fluorescence activated cell sorter. The microplate selection technique proved to be sensitive enough to detect all the transfectants produced during the present study.

Animals↗
PubMed1989

Phenotypic and functional characteristics of tumor-associated lymphocytes in patients with malignant ascites receiving intraperitoneal infusions of recombinant interleukin-2 (rIL-2).

In the course of a phase I trial, in which recombinant IL-2 (rIL-2) was infused intraperitoneally (i.p.) in patients with peritoneal carcinomatosis, we evaluated the effect on "tumor-associated lymphocytes" (TAL) isolated from the ascitic fluid. No major changes in the percentages of cells expressing the CD3, CD4, CD8, Leu-7, OKM1 and WT-31 antigens were detected either in TAL or in peripheral blood lymphocytes (PBL) after 7 days of rIL-2 infusion. In contrast the percentages of TAL (but not PBL) expressing surface IL-2 receptor (Tac), or LAK-1 antigen were sharply increased. Analysis of cytolytic functions showed a potentiation of the lytic activity against natural-killer (NK) sensitive K562 target cells and the de novo appearance of lytic activity against fresh melanoma cells. In one patient IFN-gamma was detected in the ascitic fluid following rIL-2 infusion. T-cell clones derived from the patient were analyzed for the IFN-gamma production. While only approximately 40% of PB-derived control clones produced medium to low amounts of IFN-gamma, all of the TAL-derived clones produced medium to high amounts of the lymphokine.

Ascites↗
PubMed1989

Human T lymphocytes expressing gamma/delta T cell antigen receptor.

The majority of mature T lymphocytes express CD3-associated antigen receptor molecules (TCR) formed by alpha and beta chains. Recently, a minor subset has been identified that expresses a different CD3-associated heterodimer composed of gamma and delta chains. The TCR gamma/delta+ cell subset differs from conventional T cells for a number of phenotypic and functional characteristics. The simultaneous lack of both CD4 and CD8 antigens allows to greatly enrich TCR gamma/delta+ cells (by monoclonal antibodies and complement). Cloning of CD4-8- peripheral blood lymphocytes, under limiting dilution conditions, revealed that they are homogeneously composed of cytolytic cells which, in most instances, lyse tumor target cells. The formal proof has been provided that TCR gamma/delta+ cells are able to recognize antigens. Indeed they proliferated in response to allogeneic cells in mixed lymphocyte culture (MLC) and MLC-derived TCR gamma/delta+ cells specifically lysed PHA-induced blast cells bearing the stimulating alloantigens. The use of different monoclonal antibodies specific for TCR gamma/delta molecules allowed to identify two distinct subsets which bound BB3 and delta-TCS-1 mAbs, respectively. The BB3-reactive TCR molecules were represented by C gamma 1-encoded disulfide-linked heterodimers, whereas delta-TCS-1 reacted with C gamma 2-encoded nondisulfide-linked molecules. Both BB3 and delta-TCS-1 mAb induced activation of cloned cells expressing the corresponding antigenic determinants (as assessed by measurements of intracellular Ca2+ and lymphokine production or cytolytic activity). Analysis of the unfrequent delta-TCS-1+ clones which express surface CD8 molecules revealed that the "heavy" 55 kDa form of (C gamma 2-encoded) gamma chain is selectively expressed by this cell type. Analysis of the distribution of subsets expressing different TCR gamma/delta isotypes showed that the C gamma 1-encoded, BB3-reactive form is prevalent in the peripheral blood, but virtually absent in the thymus. In contrast, cells expressing the C gamma 2-encoded, delta-TCS-1 reactive form are relatively unfrequent in peripheral blood, but represent the majority of TCR gamma/delta+ thymocytes. In addition, upon culture in rIL-2, approximately half of the delta-TCS-1+ thymocytes expressed CD8 antigen, thus providing further evidence that major differences exist in the distribution of TCR gamma/delta+ subsets in thymus and in peripheral blood.

Antibodies, Monoclonal↗
PubMed1989

Surface molecules involved in the activation and regulation of T or natural killer lymphocytes in humans.

The surface molecules that mediate activation of different subsets of T or NK cells have been reviewed. A suitable approach to the study of different lymphocyte activation pathways is provided by mAbs specific for these molecules. MAbs directed to the CD3 surface molecules mediate a polyclonal T-cell activation, whereas mAbs to "clonotypic" structures of TCR only trigger cells bearing the corresponding clonotypic determinant (thus mimicking the effect of antigen/MHC). MAbs directed to appropriate epitopes of CD2 molecules or to CD28 molecules mediate polyclonal T-cell activation, leading to triggering of the functional program of the cell (i.e. proliferation, lymphokine production or activation of the cytolytic machinery). Interaction of specific mAbs with CD3/TCR molecules leads to surface modulation of these molecules which lasts for 48-72 h. During this interval the cell is refractory to any further activation stimulus. No such refractoriness occurs following mAb-induced modulation of CD2 or CD28 surface molecules. The mechanisms by which CD3/TCR modulation results in the inactivation of T-cell function appears to involve the early metabolic steps of T-cell activation, as neither Ca++ mobilization nor IP3 formation could be further induced by any stimulus. The surface molecules and mechanisms involved in the activation of TCR gamma/delta cells are similar to those of TCR alpha/beta + cells. TCR gamma/delta molecules are heterogeneous in size and charge mobility. MAbs directed to one or another form of TCR gamma/delta trigger the functional program of the cell (primarily cytolytic function). However, a receptor form composed of a heavy form (55 kD) of the gamma chain appears to be relatively inefficient in signal transduction upon binding with anti-TCR mAbs. Evidence has also been provided that TCR gamma/delta + cells are capable of (allo)antigen responses and that polymorphic determinants of class I can be recognized (specific lysis of P815 cells transfected with HLA-A24 allele). Although the mechanisms and the surface receptor molecules involved in (CD3-, CD16+) NK cell activation are still poorly understood, several surface molecules have been identified that mediate NK-cell triggering. These include CD2 and CD16 and the novel GL183 molecule which is selectively expressed by a fraction of NK cells and thus identifies a well-defined NK subsets. Under appropriate conditions, mAbs to CD16 or GL183 mediate an inhibitory effect on the NK cell activation. These data suggest that also NK cells are characterized by surface molecules capable of initiating distinct pathways of cell activation and that, similarly to T lymphocytes, mechanisms exist which regulate NK cell function.

Antibodies, Monoclonal↗
PubMed1989

Intraperitoneal infusion of recombinant interleukin-2 in malignant ascites in patients with gastrointestinal and ovarian cancer.

In a phase-I-study recombinant interleukin-2 (rIL-2) in a dose from 0.01 to 0.3 mg/m2/day for 7 to 14 days was infused intraperitoneally. Side effects were fever and fluid retention. 2 patients showed a bacterial peritonitis after paracentesis. The investigations showed that the intraperitoneal (i.p.) administration of interleukin-2 activates the whole lymphokine cascade.

Catheters, Indwelling↗
PubMed1988

Specific lysis of allogeneic cells after activation of CD3- lymphocytes in mixed lymphocyte culture.

Human CD3- lymphocyte populations were obtained by treating peripheral blood lymphocytes with mAbs directed to CD3, CD4, and CD8 surface antigens. The resulting populations were cultured with irradiated allogeneic cells; at day 4, 100 U/ml IL-2 were added and cultures continued for an additional 10 d. The resulting populations were CD3-CD2+CD7+ and displayed cytolytic activity against PHA-induced blast cells bearing the stimulating alloantigens but not against autologous or unrelated allogeneic blast cells. When CD3- populations were cultured with irradiated autologous cells, no cytolytic activity could be detected either against autologous or allogeneic blast cells. On the other hand, K562 target cells were lysed by both MLC-derived CD3- cell populations regardless of the origin (autologous or allogeneic) of the stimulating cells. CD3- clones were further derived from MLC-stimulated CD3- populations. These clones displayed a cytolytic pattern similar to the original MLC populations as only specific PHA blasts could be lysed. These clones did not express detectable surface TCR-alpha/beta or -gamma/delta molecules and lacked productive mRNA for TCR alpha and beta chains, while small amounts of TCR-gamma mRNA were detectable in one of four clones tested. Also mRNA for CD3 gamma and delta chains were undetectable in all clones, however, CD3 epsilon mRNA was consistently present.

Antigens, Differentiation, T-Lymphocyte↗