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CD48 is a glycosyl phosphatidylinositol anchor protein known to be virtually expressed by all human leukocytes. Its ligand, 2B4, is a signaling lymphocyte activation molecule-related receptor involved in NK cell activation. Because dendritic cells (DCs) are strong inducers of NK cell functions, we analyzed the expression of CD48 in different human DC subsets. We observed that monocytes differentiating in DCs promptly down-regulate CD48. Similarly, DCs isolated from inflamed lymph nodes generally do not express CD48. Plasmocytoid DCs do not express CD48 either, whereas myeloid DCs harbored in blood, bone marrow, and thymus express it. In addition, we showed that CD48 expression in DCs affects NK cell functions during NK/DC cross-talk, because NK cells obtained from normal donors and from X-linked lymphoproliferative disease patients are, respectively, triggered or inhibited by DCs expressing surface CD48. Remarkably, IFN-gamma production by lymph node NK cells, in contrast to blood NK cells, can be negatively modulated by 2B4/CD48 interactions, indicating a 2B4 inhibitory pathway in lymph node NK cells. Therefore, the CD48 deficiency of DCs harbored in inflamed lymph nodes that we report in this study might be relevant to successfully activate lymph node NK cells in the early phase of the immune response. Our results show that distinct subsets of human DCs, differently from all other mononuclear hemopoietic cells, specifically do not express CD48. Moreover, the expression of CD48 depends on the anatomic location of DCs and might be related to the tissue-specific 2B4 function (activating or inhibitory) of the NK cells with which they interact.

The treatment of EBV-associated post-transplant lymphoproliferative disease (PTLD) poses a considerable challenge. Efforts have been made to define regimens based on combination of the available therapeutic agents, chosen and tailored on a patient-by-patient basis, with the aim of augmenting event-free patient and graft survival. Recently, autologous EBV-specific cytotoxic T-lymphocytes (CTL) have proved effective in enhancing EBV-specific immune responses and reducing viral load in organ transplant recipients with active infection. We investigated the use of a tailored combined approach including autologous EBV-specific CTL for the treatment of EBV-related PTLD developing after pediatric kidney transplantation. Five patients with disseminated monoclonal (n = 3) or localized polyclonal (n = 2) PTLD unresponsive to reduction of immunosuppression were enrolled. The patients with disseminated PTLD received 4-5 courses of reduced-dosage polychemotherapy, accompanied by rituximab on the first day of each course, while localized disease was removed surgically. At treatment completion, autologous EBV-specific CTL were infused. All patients showed a complete response to treatment, without therapy-related toxicity or rejection, and persist in remission with good renal function at a median follow-up of 31 months. These preliminary results suggest that a combined chemoimmunotherapy regimen including virus-specific T-cells is well tolerated and potentially effective as first-line treatment of EBV-related PTLD.

Indoleamine 2,3-dioxygenase (IDO), a catabolizing enzyme of tryptophan, is supposed to play a role in tumor immune escape. Its expression in solid tumors has not yet been well elucidated: IDO can be expressed by the tumor cells themselves, or by ill-defined infiltrating cells, possibly depending on tumor type. We have investigated IDO expression in 25 cases of non-small cell lung cancer (NSCLC). Using histochemistry and immunohistochemistry, we found that IDO was expressed not by tumor cells, but by normal cells infiltrating the peritumoral stroma. These cells were neither macrophages nor dendritic cells, and were identified as eosinophil granulocytes. The amount of IDO-positive eosinophils varied in different cases, ranging from a few cells to more than 50 per field at x200 magnification. IDO protein in NSCLC was enzymatically active. Therefore, at least in NSCLC cases displaying a large amount of these cells in the inflammatory infiltrate, IDO-positive eosinophils could exert an effective immunosuppressive action. On analyzing the 17 patients with adequate follow-up, a significant relationship was found between the amount of IDO-positive infiltrate and overall survival. This finding suggests that the degree of IDO-positive infiltrate could be a prognostic marker in NSCLC.

The ProteomeLab PF2D protein fractionation system is a rapid, semi-automated, 2 D-HPLC instrument that uses two different methods to separate plasma serum proteins: ion-exchange chromatography using a wide pH range in the first dimension, and non-porous reverse-phase chromatography in the second dimension. Because this methodology has only very recently been introduced in proteomic laboratories, little is known about the characteristics of PF2D fractionation of human serum proteins. To evaluate the system's application in a clinical laboratory setting, the characteristics of the ion-exchange chromatography-based separation were analyzed. Following fractionation of human serum proteins on a linear pH gradient (ranging from 8.0 to 4.0), each fraction was collected in a cool module of the instrument. Different fractions obtained from the first dimension were then pooled together and loaded on classic 2D gel electrophoresis instrumentation. The different spots obtained were then checked against the Swiss-Prot Data-base. A total of 36 human serum proteins were identified in different PF2D-generated fractions. Some important features of the separation system were observed. Different eluted fractions contained different proteins, thus demonstrating the reliability of the fractionation system. The proteins were also fractionated according to the theoretical isoelectric point (pI). This was consistent with the evidence that the vast majority of immunoglobulins, characterized by an alkaline pI, were not retained by the column and were eluted in the unbound fraction. This outcome also underlines a practical advantage: fractions eluted from pH 8 to pH 4 contained virtually immunoglobulin-depleted serum proteins. This finding supports an immediate use of the PF2D system in a clinical setting, where abundant proteins should be clearly identified to enable evaluation of other less abundant, but potentially relevant, species.

Neuroblastoma is the most common extracranial solid tumor in children. Abnormal secretion of catecholamines in tissues and body fluids allows for the differential diagnosis of neuroblastoma from other neoplasms and its distinction from non-neoplastic inflammatory diseases. This is achieved by assaying homovanillic acid and 4-hydroxy-3-methoxymandelic acid, the catabolites of catecholamine metabolism. In the course of an evaluation of children with suspected neuroblastoma, homovanillic acid and 4-hydroxy-3-methoxymandelic acid were analyzed in urine samples by capillary gas chromatography with flame ionization detection after extraction and derivatization of these compounds as trimethylsilyl derivatives. In three urine samples a significant increase in biogenic amines was observed, but these results were not confirmed by thin-layer chromatography. Patient history revealed that these children had been treated with ibuprofen, an analgesic and anti-inflammatory drug. To verify how ibuprofen or its metabolites may have interfered with capillary gas chromatography with flame ionization detection, we analyzed the same samples by capillary gas chromatography-mass spectrometry. In urine samples from patients on the drug, the presence of a peak identified as the trimethylsilyl ester of hydroxyibuprofen, which had the same retention time as 4-hydroxy-3-methoxymandelic acid, was found to interfere with the capillary gas chromatography with flame ionization detection analysis of the metabolite. This interference must be taken into account during the laboratory diagnosis of neuroblastoma.

CD40/CD40 ligand interaction is an important pathway for B and T cell cooperation and function; functional CD40 molecules have recently been found on nonhematopoietic cells. We detected CD40 in vivo on normal human respiratory epithelial cells and showed that its expression is increased on inflamed airway epithelium. Subsequently, we analyzed its expression and function on primary cultures of human airway epithelial cells. Our data show that CD40 is up-regulated by IFN-beta and IFN-gamma, its ligation increases the surface expression of CD54 and CD106 and it may stimulate the release of IL-6 and IL-8. The use of Janus kinase 3 (JAK3) and NF-kappaB inhibitors suggests that both basal and CD40-induced release of the two cytokines is JAK3-dependent. Using colocalization techniques, we revealed the existence of CD40/JAK3 and CD40/TNFR-associated factor 2 interplay. The extent of these interactions may be partial (2-40% of the cells) or massive (80-90% of the cells) in cultured cells. Stimulation via CD40 causes a significant increase in the number of cells expressing colocalization only in the cultures displaying low frequency of initial colocalization. Thus, airway epithelial cells, activated by CD40, may behave as effector cells of the inflammation process and should be considered priority targets for anti-inflammatory therapy. This work identifies CD40 and the correlated JAK3 signaling molecule as potential molecular targets to block the inflammatory functions of epithelial cells.

Treatment of melanoma cell lines with IFN-gamma induces the switch from proteasome (PS) to immunoproteasome (iPS). This finding has profound implications for the immunobiology of melanoma cells since certain peptides (such as Melan-A(mart1)(27-35)) are cleaved differently by iPS, thus implying a different ability to be presented by HLA class I molecules. IFN-alpha is a cytokine not only produced during infectious diseases, but also used in the treatment of certain cancers. Nevertheless, the effects of IFN-alpha on the switch of PS to iPS are largely unknown. A comparison of the effect of both IFN-alpha and IFN-gamma was thus carried out on melanoma cell lines. RT-PCR showed that mRNA for iPS subunits (i.e. LMP-2, LMP-7 and MECL-1) was detectable both in untreated and IFN-treated melanoma cells. Immunoblotting analysis revealed that while IFN-gamma was able to consistently induce the switch from PS to iPS, IFN-alpha treatment did not, possibly due to post-transcriptional event(s) blocking the expression of iPS-specific subunits. Finally, Melan-A(mart1)(27-35) peptide was found only in the HPLC-MS spectra from both untreated and IFN-alpha-treated cells, but not upon IFN-gamma treatment. Altogether, these data demonstrate that IFN-alpha does not induce the switch from PS to iPS.

Natural killer (NK) cells have long been considered as "primitive" and "non-specific" effector cells. However, the past 10 years have witnessed dramatic progress in our understanding of how NK cells function and their role in innate defenses. Thanks to specialized inhibitory receptors specific for MHC-class I molecules, they can sense the decrease or loss of these molecules, a typical condition of potentially dangerous cells such as tumor or virally-infected cells. NK cell triggering and lysis of these cells is mediated by several activating receptors and co-receptors that have recently been identified and cloned. While normal cells are usually resistant to the NK-mediated attack, a remarkable exception is represented by dendritic cells (DC). In their immature form (iDC), they are susceptible to NK-mediated lysis because of the expression of low levels of surface MHC-class I molecules. Since the process of DC maturation (mDC) is characterized by the surface expression of high levels of MHC-class I molecules, mDC become resistant to NK cells. Exposure to live bacteria induces rapid DC maturation and, thus, resistance to NK cells. The cross-talk between DC and NK cells is more complex and involves also a DC-dependent NK cell activation and proliferation. Thus, two important players of the innate immunity may be involved in a coordinated regulation of critical events occurring at the interface between innate and adaptive immunity.

Background: In a group of patients with head and neck cancers (H&NC), the expansion of the population of CD3-,CD16+ natural killer (NK) cells in the peripheral blood was studied., Methods: Cytofluorimetric analysis of the expression of killer Ig-like receptors (KIR, namely p58.1, p58.2, p58.3, p70, and p140) and CD94-NKG2a was performed. Cytolytic activities were studied using 51Cr release assay. T and NK cell cloning was performed using limiting dilution culture conditions. Cytokine production was analyzed using commercial enzyme immunoassays., Results: Phenotypic analysis showed that the expanded populations were heterogeneous. Even in the presence of a large number of circulating NK cells, "nonspecific" cytolytic capacities were heavily reduced, whereas cytolytic capacity related to T cells was virtually normal. Unlike NK cell clones derived from healthy donors, most NK cells derived from H&NC patients expressed surface "activating" NK cell receptors (KAR) for HLA, detected by use of a redirected cytolytic assay. Analysis of the CD4+ subpopulation at the clonal level demonstrated that they had a severe proliferative defect., Conclusion: These experimental data indicated that H&NC patients have a polyclonal expansion of functionally deficient NK cells expressing KAR. In addition, the proliferative capacity of patients' "helper" cells was strongly inhibited, thus accounting for a severe impairment of cytolytic activity of the expanded NK cells., (Copyright 2003 Wiley Periodicals, Inc.)

Lyophilized bacterial lysates, which actively stimulate the immune response, are widely used as vaccines or 'biological response modifiers' in subjects with recurrent bacterial respiratory infections. Since vaccines are indicated in the absence or in the presence of a weak constitutive immune response activity, a better knowledge on the 'naturally' occurring antibacterial immune response at the oropharingeal level should be helpful. A study was, therefore, designed to quantify the presence of salivary IgA directed against surface antigens bacteria frequently involved in the pathogenesis of upper respiratory tract infections: Klebsiella pneumoniae (KP), Staphylococcus aureus (SA), Streptococcus pyogenes (SPy), Morraxella catarrhalis (MC), Haemophylus influenzae (HI), and Streptococcus pnumoniae (SPn). In 34 volunteers (21 adults and 13 children), salivary fluid was collected and the presence of microorganism-specific IgA antibodies evaluated by a novel enzyme immuno-assay. In the whole population only 29 and 24% of subjects had IgA directed, respectively, to KP and SA, while the immune-response against other microbes was detectable in a small population ranging from 12 to 15% of all subjects studied. We found higher proportions of individuals with strain specific salivary IgA in the adult than in the pediatric population for all the microorganism evaluated. In addition, in children, the only strain inducing a significant production of specific IgA at oropharingeal level was KP. Interestingly, only ten out of 21 adults and two out 13 children have at least one significantly high antibody titer against one of the bacteria evaluated. Nevertheless, when a group of healthy donors was treated with a polyvalent mechanical bacterial lysate (Ismigen t.), the large majority developed a specific immune-response in the salivary fluid. These results are thus consistent with the good features of the novel enzyme-immunoassay and with a poor frequency of naturally induced specific anti-microbe antibodies in children and in adults despite the presence on recurrent respiratory infections in their clinical history.

NK and DC reciprocal interactions have only recently been investigated. In this study, we focused on the interplay between NK cells and DC in two models of bacterial infection. Immature monocyte-derived DC were cultured in the presence of live Escherichia coli or bacillus Calmette-Guérin. Upon exposure to either extracellular or intracellular bacteria, DC underwent maturation as assessed by the increased levels of expression of CD80,CD86, and HLA molecules and the de novo expression of CD83 and CCR7. Significant amounts of TNF-alpha and IL-12 were released by DC upon infection, whereas IL-2 and IL-15 were barely detectable in culture supernatants. Both infected and uninfected DC were capable of inducing in fresh autologous NK cells the expression of CD69 and HLA-DR and of inducing cell proliferation. Remarkably, however, infected DC were much stronger inducers of NK cell activation and proliferation than uninfected DC. Thus, after just 24 h of NK/DC coculture, only those NK cells that had been exposed to bacteria-infected DC had acquired the ability to lyse autologous immature DC. In addition, infected DC were more resistant to NK-mediated lysis as a consequence of the up-regulation of HLA class I molecule expression on their surface. This study suggests a regulatory circuit involving NK cells and DC in which DC-induced NK cell activation is effectively enhanced by the presence of pathogens. Activated NK cells, by limiting the supply of immature DC, may then exert a control on subsequent innate and adaptive immune responses.

Dendritic cells (DC) play a pivotal role in the initiation, maintenance and regulation of the immune response. Here we obtained the first evidence that DC, in the absence of any foreign antigens, induce the expression of B7 family costimulatory molecules, such as CD80, CD86, B7-H1, PD-L2, B7-H3, and B7RP-1, on autologous T lymphocytes. Cell-to-cell contact between DC and T cells was needed in order to obtain this expression on T cells. De novo expressed B7 molecules on T cells were functional since B7+ T cells were able to costimulate the proliferation of highly purified T cells. While both autologous and allogeneic DC were able to induce similar levels of costimulatory molecule expression, the chemokine receptor repertoire on B7+ T cells after interaction with DC varied depending on the presence of allo-antigens during the interaction (CCR7-, CCR5+) or the absence of antigens (CCR7+, CCR5-). In accordance with this different pattern of chemokine receptors in the two conditions, we propose that, after the encounter with DC in lymphoid organs, this peculiar T cell population should reside in the T cell areas of the lymph nodes or migrate to peripheral sites of inflammation, providing a second signal for activating or switching off, respectively, naive or peripheral effector T cells.

Bacillus Calmette-Guerin (BCG) is currently employed in the treatment of superficial bladder cancer but, despite its recognized effectiveness in preventing recurrences and progression, the immune mechanisms behind its antitumor activity remain to be delineated. In this study we provide evidence that a prolonged increase in the plasma levels of IL-2, but not IL-1beta, IL-4, IL-10, IL-2R or TNF-alpha occured in patients affected by bladder cancer following effective BCG treatment. Conversely, a drop in circulating IL-2 was consistently associated with tumor relapse. The level of IL-2 was elevated even further 15 days after the last BCG administration in patients who did not experience tumor recurrence, suggesting a prolonged T cell-mediated response against antigens other than BCG. Our results indicate that a specific type 1 immune response plays a major role in the anti-cancer activity of BCG. In addition, monitoring IL-2 plasma levels may offer a useful tool for predicting tumor recurrences.

During the innate response to many inflammatory and infectious stimuli, dendritic cells (DCs) undergo a differentiation process termed maturation. Mature DCs activate antigen-specific naive T cells. Here we show that both immature and mature DCs activate resting human natural killer (NK) cells. Within 1 wk the NK cells increase two-- to fourfold in numbers, start secreting interferon (IFN)-gamma, and acquire cytolytic activity against the classical NK target LCL721.221. The DC-activated NK cells then kill immature DCs efficiently, even though the latter express substantial levels of major histocompatibility complex (MHC) class I. Similar results are seen with interleukin (IL)-2--activated NK cell lines and clones, i.e., these NK cells kill and secrete IFN-gamma in response to immature DCs. Mature DCs are protected from activated NK lysis, but lysis takes place if the NK inhibitory signal is blocked by a human histocompatibility leukocyte antigen (HLA)-A,B,C--specific antibody. The NK activating signal mainly involves the NKp30 natural cytotoxicity receptor, and not the NKp46 or NKp44 receptor. However, both immature and mature DCs seem to use a NKp30 independent mechanism to act as potent stimulators for resting NK cells. We suggest that DCs are able to control directly the expansion of NK cells and that the lysis of immature DCs can regulate the afferent limb of innate and adaptive immunity.

In many types of cells, ligation of human leukocyte antigens (HLA) Class I molecules with specific mAbs results in the transduction of signals that trigger different cell functions. We have investigated the effects of Class I ligation in human neutrophils. After several hours in culture, neutrophils split spontaneously into two subpopulations, one with normal and the other with reduced levels of Class I. The latter subpopulation displayed high binding capacity for Annexin V, showed a hypodiploid peak, electrophoretic DNA fragmentation, and morphological features of apoptotic cells. The addition of drugs known to delay apoptosis (GM-CSF or cAMP) resulted in a reduction of Class I modulation. Furthermore, ligation of surface Class I with F(ab')2 fragments of the anti-Class I mAb W6/32 resulted in a delay in the progression of apoptosis. These data indicate that this surface Class I molecule is a marker of age-related apoptosis, and the ligation of these molecules results in the transduction of a signal that inhibits apoptosis. Thus, the downregulation of HLA Class I molecules in aging neutrophils prevents their halting the apoptotic process.

Natural killer (NK) cells have the capability of lysing targets that have down-regulated the expression of HLA class I molecules. Herpes simplex virus (HSV) infection results in a profound reduction of HLA class I molecules on the surface of infected cells. For this reason, NK cell populations kill efficiently HSV-infected cells. The recent availability of a panel of monoclonal antibodies directed to NK receptors for HLA class I (CD158a, CD158b, anti-p70, anti-p140, and CD94) allowed an accurate dissection of the NK cell subpopulations. Using this approach, the relationship between the expression of NK cell receptors and the capability of lysing HSV-infected cell targets was analyzed at the clonal level. NK cell clones were derived from healthy donors, and cytolytic properties were assayed against HSV-infected autologous fibroblasts. NK cell clones, classified according to the expression of natural killer-cell receptors on their surface, displayed a great heterogeneity of cytolytic properties against HSV-infected cells. Nevertheless, a more accurate functional analysis demonstrated not only that HSV infection downregulated the expression of HLA-A and HLA-B and did not modify the expression of HLA-C, but also that NK cell clones expressing the "activating" form of the anti HLA-C NK cell receptor were more cytolytic than other clones. This finding suggests that two different and clonally distributed mechanisms of NK cell activation may be employed by NK cells to kill HSV-infected autologous target cells.

The VH4 genes expressed by both resting and in vivo-activated subepithelial (SE) B cells from human tonsils were studied. Resting SE B cells were subdivided according to the presence (IgDlow) or absence (IgM-only) of surface IgD. CD27 was abundant on activated SE B cells and low on resting IgM-only B cells. Resting IgDlow SE B cells could be subdivided into CD27low and CD27high cell fractions. Resting IgDlow SE B cells displayed VH4 genes with a substantial number of mutations (13/29 of the molecular clones were mutated), whereas 25/26 of the clones from resting IgM-only SE B cells were unmutated. Moreover, mutated VH4 genes were detected mainly within the CD27high cell fraction of the IgDlow SE B cells. Several identical unmutated VH4DJH sequences (11/32) were found in different molecular clones from resting IgM-only SE B cells, suggesting local cellular expansion. Both unmutated (14/25) and mutated (11/25) sequences were found in mu transcripts of activated SE B cells. Extensive mutation was observed in the gamma transcripts of activated SE B cells. Therefore, SE B cells are heterogeneous, being comprised of B cells with mutated Ig VH4 genes, that are Ag-experienced B cells, and a subset of B cells with unmutated VH4 genes that are either virgin cells or cells driven by Ags that did not induce or select for V gene mutations.

Interferons upregulate the expression of HLA class I antigens on cancer cells. Nevertheless, little is known about the panel of HLA class I antigen-associated peptides presented by recombinant alpha-interferon (r(alpha)-IFN)-treated cells. For this reason, peptides were eluted from five cancer cell lines (four melanoma and one non-small cell lung cancer) following treatment with r(alpha)-IFN. High-performance liquid chromatography (HPLC) profiles of the peptide fractions were compared with those obtained from untreated cells. No significant differences in peptide characteristics (detectable on the basis of retention times) were observed, but significant differences in terms of peptide quantities were observed. Mass spectrometry performed on HPLC peaks allowed not only the detection of three different peptides (two derived from the MAGE family of genes and one from the mart-1) both in untreated and in treated cells, but also gave an indication of the number of peptides within one HPLC peak. This data demonstrates that r(alpha)-IFN-treated cells express a similar peptide pattern as untreated cells, with significant quantitative differences. Interestingly, this finding also explains the higher susceptibility to lysis (mediated by specific cytolytic lymphocytes, which recognize cancer cells in an HLA-restricted fashion) of r(alpha)-IFN-treated cells.

An in vitro flow cytometric model has been developed to evaluate the effects of antiallergic drugs such as cetirizine (CTZ) on the expression of surface molecules on primary cultured normal cells. Quantitative analysis demonstrated that HLA class I and ICAM-1/CD54 molecules are present on both epithelial and stromal cells, and that their expression is strongly enhanced by treatment with interferon-gamma (IFN-gamma). Nevertheless, the IFN-gamma-mediated upregulation of ICAM-1/CD54 was inhibited by treatment with CTZ, demonstrating a direct effect on both cell types. This finding is particularly interesting because ICAM-1/CD54 is the main rhinovirus receptor, and rhinoviruses are the principal cause of asthma exacerbation in children. Thus, according to data derived from this in vitro model, CTZ should have an important role in the reduction of infectious exacerbation of asthma in atopic patients.

Although CD33 represents an important marker of myeloid cell differentiation, its function remains poorly defined. In view of its homology with p75/AIRM1, a recently identified surface molecule which exerts a potent inhibition on NK cell function, we re-evaluated the effect of CD33 engagement in defined myeloid cell functions. Addition of anti-CD33 mAb to cultures of CD14+ monocytes supplemented with granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-4 and TNF-alpha, prevented the generation of dendritic cells. In these cultured cells, engagement of CD33 resulted in an increased surface binding of annexin-V, followed by cell death. Mature dendritic cells were resistant to the CD33-mediated effect. Also in CD34+ precursors, cultured in the presence of flt3-ligand, c-Kit-ligand, GM-CSF, IL-4 and TNF-alpha, addition of anti-CD33 mAb prevented the recovery of mature dendritic cells. These data suggest a regulatory role of CD33 in the myeloid cell maturation and may offer a tool to interfere with the monocyte/macrophage cell function as well as with the development of dendritic cells.

An enzyme-immunoassay was developed to evaluate the presence of anti-erythropoietin antibodies in plasma samples obtained from renal failure patients treated with recombinant human erythropoietin (rh-EPO). The assay was specific and reproducible. Normal donors had no antibodies to EPO, while 67% of treated patients were positive to the assay. While the specificity of anti-EPO IgG antibodies was high, their affinity for the antigen was low. This finding can be explained by the very small differences in the structure of rh-EPO compared to that of natural EPO. The assay described could be useful in evaluating the long-term effects of rh-EPO treatment on the control of anaemia in renal failure patients.

The association of adoptive immunotherapy (AI) and radiotherapy has been shown to be effective in the control of residual intrathoracic disease, while having no systemic advantages, in patients operated on for locally advanced non-small-cell lung cancer (NSCLC). The potential synergy of coupling immunotherapy and chemotherapy has been emphasized in several tumors including NSCLC. The aim of this work was to determine the feasibility and activity of a combined therapeutic program, including AI, chemotherapy, and radiotherapy in patients who had undergone incomplete resections for NSCLC. In a phase II trial, 13 patients received the combined treatment. AI was given from week 4 after surgery until week 8. Concurrent chemo-(cisplatin and etoposide)-radiotherapy (60 Gy) was given from week 9 to week 14. Twenty eligible patients received chemoradiotherapy only and were used as a non-randomized concomitant group for merely descriptive purposes. At 9-month follow-up, 10 of the 13 patients had progression of disease and the study was stopped. Progression-free survival and survival were similar to those of the chemoradiotherapy group. The present study showed that the sequence of immunotherapy followed by chemotherapy is not effective as adjuvant treatment in patients operated on for stage III NSCLC, at least when used according to the adopted schedule.

Although conventional cytology represents the most widely performed cytometric analysis of bladder cancer cells, DNA flow cytometry has, over the past decade, been increasingly used to evaluate cell proliferation and DNA ploidy in cells from bladder washings. We have investigated whether DNA flow cytometry and conventional cytology of epithelial cells obtained from bladder washings provide reliable surrogate endpoint biomarkers in clinical chemoprevention trials. We used cytometric and clinical data from a chemoprevention trial of the synthetic retinoid Fenretinide on 99 patients with superficial bladder cancer. A total of 642 bladder washing specimens obtained from the patients at 4 month intervals was analyzed. Intra-individual agreement and correlation of flow cytometric DNA ploidy (diploid vs. aneuploid), DNA Index, Hyper-Diploid-Fraction (proportion of cells with DNA content higher than 2C), and conventional cytologic examination, as assessed by kappa statistics and Spearman's correlation test, were poor from baseline through 24 months. Moreover, no correlation was found between DNA ploidy and cytology at each time point. The same results were obtained when the analyses were stratified by treatment group. In addition, the association between the results of bladder washing (by either DNA flow cytometry or cytology) and concomitant tumor recurrence was significant only for abnormal cytology, while neither biomarker was predictive of tumor recurrence at the subsequent visit. During the time of this study only four patients progressed to muscle-invasive bladder cancer, indicating the "low-risk" features of the patient population. We conclude that DNA flow cytometry and conventional cytology on epithelial cells obtained from bladder washings do not appear to provide suitable surrogate endpoint biomarkers during the early stages of bladder carcinogenesis., (Copyright 1999 Wiley-Liss, Inc.)

This study shows that human postthymic T cells express CD10 when undergoing apoptosis, irrespective of the signal responsible for initiating the apoptotic process. Cells from continuous T-cell lines did not normally express CD10, but became CD10(+) when induced into apoptosis by human immunodeficiency virus (HIV) infection and exposure to CD95 monoclonal antibody, etoposide, or staurosporin. Inhibitors of caspases blocked apoptosis and CD10 expression. Both CD4(+) and CD8(+) T cells purified from normal peripheral blood expressed CD10 on apoptotic induction. CD10 was newly synthesized by the apoptosing cells because its expression was inhibited by exposure to cycloheximide and CD10 mRNA became detectable by reverse transcription-polymerase chain reaction in T cells cultured under conditions favoring apoptosis. To show CD10 on T cells apoptosing in vivo, lymph node and peripheral blood T cells from HIV(+) subjects were used. These suspensions were composed of a substantial, although variable, proportion of apoptosing T cells that consistently expressed CD10. In contrast, CD10(+) as well as spontaneously apoptosing T cells were virtually absent in peripheral blood from normal individuals. Collectively, these observations indicate that CD10 may represent a reliable marker for identifying and isolating apoptosing T cells in vitro and ex vivo and possibly suggest novel functions for surface CD10 in the apoptotic process of lymphoid cells.

Background: Adoptive immunotherapy (AI) of cancer, based upon the injection of in vitro manipulated autologous lymphocytes is still in an experimental phase. Our group started different clinical trials of AI in early 1990, and, at present, some specific targets for this approach seem to have been identified., Patients and Methods: 296 patients with solid tumors (melanoma, kidney carcinoma, non-small-cell lung cancer, mesothelioma, neoplastic pleural effusion, and liver cancer) were treated with either locoregional or systemic adoptive immunotherapy (AI) using both LAK and TIL cells in combination with s.c. rIL-2., Results: The surgery/AI combination resulted in good clinical results, characterized by enhanced survival and long lasting disease free periods in a significant number of patients., Conclusions: AI seems to be efficacious in the treatment of melanoma, lung and hepatic cancers. Further studies will expand the application of the treatment to other malignancies.

In this report, a subset of CD4(+) cells which kills autologous HLA class I positive and HLA class II negative non-small-cell lung cancer (NSCLC) cells is described. Killing was performed both by direct cytolytic mechanisms and by apoptosis. The peculiar characteristic of these effectors was their capability of lysing only interferon (IFN)-gamma-treated NSCLC target cells, expressing high numbers of HLA class I molecules. Analysis at the clonal level confirmed that cytolytic capability was distributed clonotypically. The addition of anti-HLA class I monoclonal IgM abrogated the susceptibility to lysis. Notably, the CD4-mediated cytolytic effect on IFN-gamma-treated targets was incomplete. Thus cytofluorimetric DNA and HLA class I expression analyses of target cells were performed: "resistant" targets consisted of large, aneuploid cells expressing a low number of HLA class I molecules. These findings suggested a direct role of HLA class I expression in the recognition of autologous cancer cells mediated by cytolytic CD4(+) cells., (Copyright 1999 Academic Press.)

Background: On the basis of our previous experience, we designed this study to determine the activity and toxicity of outpatient treatment with autologous tumor-infiltrating lymphocytes (TIL) together with intermediate-dose recombinant interleukin-2 (rIL-2) and low-dose recombinant interferon alfa-2a (rIFN-alpha2a), for patients with metastatic melanoma., Methods: Between April 1992 and October 1994, we processed 38 melanoma samples derived from 36 patients with metastases. Proliferative cultures of expanded lymphocytes (TIL) were infused only once into patients with metastatic melanoma. rIL-2 was administered subcutaneously for 1 month, starting on the day of TIL infusion, at an escalating dose of 6-18 x 10(6) IU/m2/day for the first week and at the maximum-tolerated dose for the subsequent 3 weeks and then, after a 15-day interval, for 1 week/month for 3 months. rIFN-alpha2a was administered subcutaneously at 3 X 10(6) IU three times each week until progression., Results: Of 38 melanoma samples, 19 (50%) resulted in proliferative cultures and were infused. The median number of expanded lymphocytes was 18 x 10(9) (range, 1-43 x 10(9)), and the median period of culture was 52 days (range, 45-60). rIL-2 was administered at doses ranging between 6 and 18 x 10(6) IU/m2/day. Toxicity was mild or moderate, and no life-threatening side effects were encountered. Two of 19 treated patients experienced complete responses of their metastatic sites (soft tissue), 10 had stable disease, and 7 showed progressive disease. The response rate was 11% (95% confidence interval, 2-35%)., Conclusions: Outpatient treatment with TIL plus rIL-2 and rIFN-alpha2a is feasible, although, within the context of the small sample size, the activity of the combination was no different from the reported activity of any of the components used alone.

Natural killer (NK) cells and NK-like T cells have been described as efficiently lysing neoplastic cells derived from hematopoietic tumors. By modulating the expression of HLA class I surface molecules on normal epithelial cells, we also observed that nonneoplastic cells can efficiently be lysed by "nonspecific" effectors. Clonal analysis clearly demonstrates that the presence of HLA-specific inhibitory NK receptors, such as CD94, CD158a, and CD158b, described on NK cells, is responsible for the inhibitory signal. Thus, NK cells, as well as NK-like T cells, in the absence of HLA surface molecules on normal target cells, efficiently lyse epithelial cells., (Copyright 1998 Academic Press.)

It has recently been shown that the infusion of tumor infiltrating lymphocytes (TIL) and recombinant interleukin-2 (rIL-2) in patients operated on for advanced non-small-cell lung cancer (NSCLC), has significant effects in terms of the survival time and control of local relapses. Despite the evident clinical effects, the treatment is unavailable for patients in which TIL have lost their proliferative potential. In an attempt to identify new sources of effector cells using mixed lymphocyte/tumor cultures (MLTC), populations of peripheral blood (PB) lymphoid cells, which have the capability of lysing autologous NSCLC, were studied at the clonal level. Specific cytolytic lymphocytes were detected at very low frequencies in two out of four patients, whereas non-MHC restricted cytolytic T cells were frequently detected. Cytolytic CD4+ belonged to the Th0 or Th2 subsets and were characterized by cytokine secretion patterns suggestive of a lymphokine cascade that could be involved in cancer control. The identification of different efficient effector mechanisms at the clonal level strongly supports the use of in vitro activated lymphocytes, derived from PB, in protocols of adoptive immunotherapy in patients with advanced NSCLC in which TIL are unavailable.

A pilot study was set up in order to evaluate the feasibility and safety of infusing in vitro expanded tumour-infiltrating lymphocytes (TILs) and recombinant interleukin-2 (rIL-2) in a group of patients with advanced melanoma after radical resection of lymph node metastases. Twenty-four patients were eligible for the study and proliferating TILs were collected in 16. These patients were infused with TILs and then treated with rIL-2 and alpha-interferon. Short-term toxic effects (such as fever) were in general controlled by symptomatic drugs, whereas chronic toxicities were absent. The median follow-up period was 19 months; at present, 13 patients are alive and disease free, one patient is in progression and two patients have died. The approach was feasible and safe and the clinical results observed are comparable to those obtained by long-term treatment with other biological response modifiers.

The number of HLA class I molecules and the susceptibility to lysis mediated by natural killer (NK) cells were evaluated on cell targets obtained from confluent and sparsely plated cultures of both normal and tumor cell lines. Sparsely plated proliferating cells expressed high amounts of HLA class I molecules and were more resistant to NK cell-mediated lysis than confluent cells, which expressed low amounts of HLA class I antigens. This characteristic could be involved in the control of cancer progression and could also explain the wide variability of assays of lymphocyte-mediated cytotoxic activity.

Purpose: Although erythropoietin (EPO) is known to be useful in treating chemotherapy-induced anemia, few data are available on its potential preventive role. The aim of this study was to evaluate the ability of EPO in preventing the development of clinically significant anemia in patients treated with chemotherapy., Patients and Methods: Sixty-two early-stage breast cancer patients undergoing accelerated adjuvant chemotherapy were randomized to receive EPO 150 U/kg three times a week or no additional treatment. Chemotherapy consisted of six cycles of cyclophosphamide 600 mg/m2, epirubicin 60 mg/m2, and fluorouracil 600 mg/m2 (CEF) intravenously on day 1, every 2 weeks with the support of granulocyte colony-stimulating factor (G-CSF), 5 microg/kg subcutaneously from day 4 to day 11., Results: Throughout the six cycles of chemotherapy, EPO-treated patients maintained stable values of hemoglobin, whereas control patients developed a progressive anemia. At the end of chemotherapy, the mean (+/- SD) hemoglobin decrease in the control group was 3.05 g/dL (+/- 1.0; 95% confidence interval [CI], 2.6 to 3.5), whereas in the EPO group it was 0.8 (+/- 1.4; 95% CI, 0.3 to 1.4). Clinically significant anemia (hemoglobin < or = 10 g/dL) occurred in 16 patients (52%; 95% CI, 33 to 69) in the control arm and in no patient (0%; 95% CI, 0 to 14) in the EPO arm (P = .00001)., Conclusion: EPO prevents anemia in patients undergoing chemotherapy. Further trials are required to identify subsets of patients in which the preventive use of this drug could be cost-effective.

Primary and metastatic liver cancers have a poor prognosis. At present, sonographically guided alcohol injection results in a partial reduction of cancer masses even if severe toxic effects (including pain and bleeding) are always present. For these reasons, a pilot study was started to evaluate the feasibility of an intralesional adoptive immunotherapeutic approach, using lymphokine-activated killer (LAK) cells and recombinant interleukin-2 (rIL-2). Nine patients (one with primary hepatocarcinoma and eight with liver metastases) entered the study. Four cycles of weekly injections of LAK cells (ranging from 2 to 9 x 10(8)) and 10(6) IU rIL-2 were performed percutaneously under ultrasonic guidance. In the same period, 3 x 10(6) IU rIL-2/day, for 24 days, was injected subcutaneously. All patients but one completed the therapy. Side effects were limited to grade 1-2 fever and were mostly related to rIL-2 subcutaneous injections. No patients complained of having pain during intralesional therapy. Two complete responses were detected. One partial response, four stable diseases, and one progressive disease were observed. One patient was not evaluable. These preliminary results suggest that sonographically guided intralesional adoptive immunotherapy of liver tumors is feasible, safe, and could offer promising therapeutic advantages in cancers for which conventional treatment is generally unsatisfactory.

Adoptive immunotherapy with in vitro expanded tumor infiltrating lymphocytes (TIL) and recombinant interleukin-2 (rIL-2) is a recent option in the treatment of advanced melanoma resistant to conventional chemotherapy. In the course of a protocol of treatment of advanced melanoma with in vitro expanded TIL and rIL-2, we obtained 38 samples from 27 different patients. Lymphocytes derived were cultured in the presence of rIL-2 in vitro for a 4-6 week period and 23 resulted in proliferative cultures. Eighteen samples were infused in the course of the clinical trial. The median number of lymphocytes obtained was 18x10(9) (range 1-43x10(9)) cells. Phenotypic analysis showed that all samples contained a pure population of T cells. These data confirm that lymphocytes can be expanded from advanced metastatic sites, have peculiar characteristics and are suitable to be infused in vivo.

Stage IIIb non-small-cell lung cancer (NSCLC) has a poor prognosis. The median survival is approximately 6 months, and only 30% of patients are alive 1 year after diagnosis. The need for effective treatment is evident. The aim of this study was to evaluate whether the infusion of tumor-infiltrating lymphocytes (TILs), isolated from resected tumor, expanded in vitro and injected together with recombinant Interleukin-2, is feasible and may at least partially modify the poor prognosis in these patients. The infusion of TILs, derived from surgically resected NSCLC and expanded in vitro, together with subcutaneous (s.c.) injections of recombinant interleukin-2 (rIL-2) was attempted in a group of 11 patients. Treated patients were infused i.v. with in vitro expanded TILs (from 4 to 70 x 10(9) cells), and rIL-2 was injected s.c. at doses varying from 61 to 378 x 10(6) IU. Toxic side effects (fever and, in some cases, hypotension) were observed and limited the dose of rIL-2 infused. Follow-up was continued for 40 months. The mean survival time was 13.8 months. Three of five TIL-treated patients with residual disease have no evident disease after 1 year, and two of them are still alive and have no evidence of disease after 40 months. This pilot study suggests that the infusion of in vitro expanded TILs, derived from surgical samples, is feasible and seems to prolong overall survival and to control the residual disease in patients with advanced NSCLC.

We have recently reported the results of a phase I study on the intravesical perfusion of recombinant interleukin-2 in patients with superficial bladder cancer. The treatment feasible with mild toxic effects, especially compared with the treatment using TUR and instillations of Bacillus Calmette-Guerin. The follow-up of these phase I study patients was continued for another twelve months. During this period, cytoscopy and cytological examination of cells washed from bladder were performed every four months. The results showed that three out of 9 patients relapsed, over a period ranging from 6 to 20 months after treatment. All these data clearly confirm that the intravesical perfusion of rIL-2 is feasible, safe and should be an effective and nontoxic treatment of patients with superficial bladder cancer.

Bladder-washing-derived lymphocytes (BWDL) from 67 patients with bladder cancer were studied. The large majority of samples contained a pure population of T lymphocytes, whereas B and NK cells were absent. A comparative analysis of bladder lymphocytes and peripheral blood lymphocytes (PBL), collected in parallel, showed that BWDL significantly differed from PBL. In vitro cultures of bladder lymphocytes were attempted on 21 samples but in vitro expansion was only possible on six patients treated with bacillus Calmette-Guérin (BCG). This finding indicates that BWDL are characterized by a severe proliferative defect. Nevertheless, the addition of BCG on bladder lymphocytes expanded in vitro enhanced their proliferation, suggesting that this population is sensitized against BCG antigen(s). The analysis of T cell receptor restriction patterns showed that bladder lymphocytes from patients under BCG treatment were oligoclonal. A possible explanation for the efficiency of the immune response and good clinical outcome in patients treated with BCG could be found in the high homology between some BCG antigens and human heat-shock proteins, which are overexpressed in transformed cells.

Objective: To evaluate whether the expression of human leukocyte antigen (HLA) class I on eutopic and ectopic endometrial cells modify the susceptibility to lysis mediated by lymphocytes., Design: Evaluation of T lymphocyte cytotoxic activity and HLA class I expression on endometrial cells., Setting: Subjects were recruited at laparoscopy., Patients: Patients with endometriosis (n = 7). Healthy women as controls (n = 10)., Main Outcome Measures: Human leukocyte antigen class I molecule analysis of endometrial cells was carried out by immunofluorescence and flow cytometry. Phenotyping of T lymphocytes was performed to analyze T-cell subsets. Cytotoxicity was performed to determine cytolytic activity against endometrial cells., Results: In vitro culture of endometrial cells down-regulates the expression of HLA class I molecules and enhances the susceptibility to lysis mediated by natural killer (NK)-like T lymphocytes. Cytolytic T-cell clones, expressing the CD94 antigen, are inhibited by the HLA-B7 allele on endometrial cells. Ectopic endometrial cells modulate the expression of HLA class I molecules., Conclusions: The resistance to lysis of endometrial cells is related to expression of surface HLA class I molecules, which send a negative signal for lysis mediated by NK-like T lymphocytes. The HLA-B7 allele inhibits the cytotoxic activity, suggesting that the growth of ectopic endometrial cells might be under a genetic control.

This study assesses the feasibility and toxicity of adoptive immunotherapy with tumor infiltrating lymphocytes and recombinant interleukin-2 in 29 patients who underwent resection for stage III non-small-cell lung cancer. In five patients cultures yielded no growth of tumor infiltrating lymphocytes. In the remaining 24 patients (stage IIIa, 14 cases; stage IIIb, 10 cases) tumor infiltrating lymphocytes were in vitro expanded from surgically obtained tissue samples, including samples from both the tumor and surrounding lung. A number of tumor infiltrating lymphocytes, ranging from 4 to 70 billion cells, were reinfused intravenously 4 to 6 weeks after operation. Interleukin-2 was administered subcutaneously at escalating does for 2 weeks and then at reduced doses for 2 to 3 months. Median survival was 14 months, and the 2-year survival was 40%. Three patients remain alive and disease-free at more than 2 years after operation. Two of these patients did not have complete resection at thoracotomy. Multivariate analysis showed no correlation between the factor of incomplete resection and survival. Intrathoracic recurrence without concomitant distant failure was documented in two patients only and none of the patients with incomplete resection (12 cases) had relapse within the thorax. The present experience demonstrates that adoptive immunotherapy may be applied with safety in patients operated on for stage III non-small-cell lung cancer and suggests that it can be useful, notably in patients with locally advanced disease.

Despite the combined use of surgery and chemoradiotherapy, the poor prognosis of advanced non-small-cell lung cancer (NSCLC) requires the definition of new therapeutic approaches. The presence of T lymphocytes, with peculiar phenotypic, functional and molecular characteristics within the tumour, suggested the possible use of these cells, expanded in vitro, in protocols of adoptive immunotherapy. We have described how a population of oligoclonal T lymphocytes, derived from advanced NSCLC, can be expanded in vitro and has the capability of lysing autologous cancer cells. What is more important, we observed that patients with advanced NSCLC, treated with TIL expanded in vitro and recombinant interleukin-2, seemed to have a disease-free period longer than that of patients treated with conventional chemoradiotherapy. In an attempt to find new sources of specific lymphocytes for immunotherapy, we describe the analysis of the phenotypic, functional and molecular characteristics of T lymphocytes, derived from lymph nodes draining advanced NSCLC. In this paper we show that these cells, have restriction patterns of T cell receptor beta chain similar to those detectable in the population of infiltrating T lymphocytes. This finding suggests that T cells derived from draining lymph nodes of advanced NSCLC have peculiar characteristics and can be a suitable source of effector cells for protocols of adoptive immunotherapy in lung cancer treatment.

Natural killer (NK) cells express surface receptors for defined groups of HLA class I alleles. The specific interaction between these receptors and HLA class I molecules expressed on target cells results in inhibition of NK-mediated target cell lysis. In this report, we analyzed whether similar mechanisms were operating in cytolytic T lymphocytes (CTLs) capable of lysing NK-sensitive target cells. T cell clones were screened for their ability to lyse K562 target cells. The selected clones expressed either gamma delta or alpha beta TCR. The majority of these clones failed to lyse the HLA class I+ R8/15375 cell line; however, upon addition of the previously described A6-136 (IgM) or 6A4 F(ab')2 anti-HLA class I mAbs, target cells were efficiently lysed. Lysis of autologous phytohemagglutinin blasts in the presence of anti-HLA class I mAbs occurred primarily with TCR gamma delta+ CTLs. Recognition of HLA class I molecules on target cells implies the expression of NK-related specific receptors in CTL clones. Indeed, phenotypic analysis of > 300 CTL clones with NK-like activity revealed that 28% expressed p58 molecules (specific for HLA-C alleles) while 30% expressed CD94 molecules (specific for the Bw6 specificity). These receptor molecules were found to function as inhibitory receptors, as revealed by the effect of anti-p58 or anti-CD94 mAbs (of IgG isotype) on the lysis of the Fc gamma R+ K562 target cells.(ABSTRACT TRUNCATED AT 250 WORDS)

A Phase I study was started to evaluate the locoregional and/or systemic toxic effects of the continuous perfusion of recombinant interleukin-2 (rIL-2) in superficial bladder cancer. Three different dose levels were used: 3 x 10(6) IU/day (3 patients), 9 x 10(6) IU/day (3 patients) and 27 x 10(6) IU/day (3 patients). Two patients (one treated with 3 x 10(6) and another with 27 x 10(6) IU/day of rIL-2) had hematuria after the end of the treatment, one patient had fever (grade I) and 7 of 9 patients experienced hypotension (grade I-II). All effects were not dose related. Routine laboratory tests indicated that no significant variations of biochemical parameters occurred. A phenotypic analysis of white blood cells detectable in the bladder, showed an evident locoregional activation of lymphoid cells. In particular, T lymphocytes expressed activation antigens (such as CD25 and HLA-DR) following treatment with rIL-2. A 6- to 12-month clinical follow-up, showed that all patients but one (which recurred after 5 months) are alive and disease-free. This therefore indicates that the locoregional perfusion of rIL-2 is safe and gives clinical results similar to those obtained using Calmette-Guérin bacillus locoregional instillation, in patients who underwent a transurethral resection of superficial bladder cancer.

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.

cis-diamminechloro-[2-(diethylamino)ethyl 4-amino-benzoate, N4]-chlorideplatinum(II) monohydrochloride monohydrate (DPR) is a new platinum-triamine complex containing as ligand the local anesthetic procaine. In this study DPR was compared to the parent compound cisplatin (cis-DDP) in order to study the influence of both molecules on the cell cycle phases, and particularly on the induction of apoptosis. P388 murine leukemic cells were used as cellular model, and were exposed in vitro to either compound, continuously for 24 hours. At the end of the incubation, the thymidine uptake, the trypan blue dye exclusion assay, and the flow cytometry were assessed. Both the cytotoxic activity and the inhibition of DNA synthesis evaluated after 24 h incubation with DPR or cis-DDP were comparable. Moreover, cell cycle was modified in a comparable manner by both molecules. In particular the induction of the apoptotic effect was similarly induced by the same concentrations of the compounds and time exposure. In conclusion, DPR and cis-DDP seem to have a similar effect on the cell cycle of P388 leukemic cells and particularly on the induction of the programmed cell death.

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.

The clonal growth of cell lines derived from human ovarian tumours can be stimulated by GM-CSF in vitro. Among these cell lines one of the most responsive is the IGROV-1 cell line. This report describes the influence of GM-CSF on the in vivo growth of IGROV-1 cell xenografts in nude mice. Beginning one day after transplantation of the tumour, the cytokine was administered daily for 31 consecutive days as i.m. injections distant from the tumour lesion at doses of 0.1 microgram/kg and 1 microgram/kg. GM-CSF caused no significant effects on the growth modulation of the ovarian cancer cells in vivo.

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.

Background: Our recent findings in vitro in the human colon adenocarcinoma cell line HCT-8 suggest that resistance to fluorouracil (5-FU) in patients with advanced colorectal cancer might be overcome by use of a different treatment schedule., Purpose: We tested the hypothesis that HCT-8 cells resistant to short-term 5-FU exposure retain sensitivity to continuous exposure and studied interactions between the two schedules., Methods: HCT-8 cell lines resistant to short-term (pulse) treatment with 5-FU or to continuous exposure were obtained by six exposures to different concentrations of 5-FU for 4 hours or 7 days. We used a monolayer clonogenic assay to determine 5-FU-induced cell kill in resistant HCT-8 cells and sensitive parent cells. Parent cells were exposed to different concentrations of 5-FU for 1, 4, or 24 hours (short term), for 7 days (continuous exposure), or in a combination of both types of schedules. In a study of the mechanism of interaction between short-term and continuous exposure in parent cells, we performed flow cytometric DNA analysis to determine the percentage of cells in S phase and assays of thymidylate synthase inhibition in intact cells and of incorporation of [6-3H)]5-FU nucleotides into nucleic acids., Results: Sensitive HCT-8 cells became fully resistant to 5-FU within five or six treatments, and low-dose continuous exposure almost immediately produced resistant clones. HCT-8 cells resistant to 5-FU given every 4 hours retained full sensitivity to continuous exposure, suggesting lack of cross-resistance between the two schedules, but cells resistant to continuous exposure were cross-resistant to short-term treatment. Parent cells showed a statistically significant (synergistic) enhancement of the cytotoxic activity for 5-FU exposure for 1 hour (100, 300, or 500 microM) followed by continuous exposure (0.5, 1, or 2 microM) or 4 hours (10, 30, or 60 microM) followed by continuous exposure (1 or 2 microM). Short-term plus continuous exposure produced a marked increase in percentage of S-phase cells, compared with the percentage for each schedule alone. The combination of 1-hour exposure and continuous exposure (1000 and 2 microM, respectively) produced a marked accumulation of cells in S phase at 24 hours (59%), which lasted up to 96 hours (53%). The combination of the two schedules produced only additive enhancement of thymidylate synthase inhibition as well as incorporation of [6-3H]5-FU nucleotides into nucleic acids of HCT-8 cells., Conclusions: Our findings provide a rationale for the use of bolus 5-FU and continuous infusion 5-FU in sequence., Implication: We are conducting a clinical trial of bolus methotrexate followed by continuous-infusion 5-FU plus leucovorin.