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Introduction: Chronic obstructive pulmonary disease (COPD) is a multi-component disease characterized by not fully reversible airflow limitation and inflammatory response in the airways. Recently, local changes of T regulatory cell populations (Tregs) in the lung of COPD patients were evidenced and some data suggested a beneficial influence of glucocorticoids on Tregs function. Aims: Better understanding the role of Treg cells in systemic inflammation in COPD and their possible modulation by budesonide and formoterol might be of interest. Methods: Peripheral blood mononuclear cells (PBMCs) were obtained from 28 moderate COPD patients, 10 healthy volunteers smokers (HVS) and 10 never smokers (HVNS). PBMCs were isolated and analyzed by flow cytometry before treatments and Tregs were assessed as CD4 + CD25 ++ CD127 - cells. Fresh PBMCs were cultured for 48 hours in the absence or presence of budesonide (Bud) and/or formoterol (For) (Bud1 and 0,01microM, For30 and 0,3nM and Bud1microM+For30nM and Bud0,01microM+For0,3nM). Cells were evaluated by flow cytometry after treatments. Results: Our data show that CD4 + CD25 ++ CD127 - Treg cells percentage in patients is significantly reduced with respect to volunteers, both smokers and non-smokers. Statistical analysis shows that Treg cells in COPD patients have a reduced response to drugs with respect to healthy volunteers. Interestingly, Treg cells in healthy donors and in patients had a different behavior depending on smoke. Conclusions: Our results show that COPD patients have a different basal expression of Tregs with respect to healthy volunteers. All together these experiments highlight the role of tobacco smoke in making the cells unsusceptible to the action of drugs.

SUMMARYAccumulating data show that fibroblasts are important regulators in the development and maintenance of allergic airway inflammation. However, most studies so far have used individual recombinant cytokines in high concentrations, unlikely to be found in vivo. We aimed to investigate how cytokines produced by peripheral blood mononuclear cells (PBMC) affect fibroblast functions. Primary airway fibroblasts where incubated with allergen-stimulated or non-stimulated PBMC supernatants from allergic patients. The levels of cytokines in PBMC supernatants were measured and the expression of CD54, CD40 and CD106 as well as the production of eotaxin, interleukin (IL)-6 and IL-8 were assessed in fibroblasts. Although the levels of single cytokines measured in PBMC supernatants were low, a significant up-regulation of the surface molecules as well as of IL-6 and IL-8 production was found in fibroblasts cultured with allergen-stimulated PBMC supernatants as compared to non-stimulated, while the increase in eotaxin production was not significant. The evaluation of correlations between cytokines produced by PBMC and effects seen on fibroblasts did not indicate a crucial role for any single cytokine. Furthermore, the addition of comparably low concentrations of recombinant interferon (rIFN)-γ or recombinant tumour necrosis factor (rTNF)-α did not induce the same effects as PBMC supernatants, the only exception being TNF-α as a direct inducer of CD54 expression. Our results show that synergistic mechanisms has a more important role than single mediators, highlighting important differences between in vitro experiments, where effects of individual mediators are studied, versus the actual situation in vivo.

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.

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.

Limonium tataricum (Lt) is a plant belonging to the family of Plumbaginaceae. The role of this family and in particular, that of dried flowers (but not of the pollen) in occupational allergy has already been described. We have observed a farmer with asthma occurring in the presence of fresh flowers. Standard methacoline test demonstrated that the patient was a true asthmatic. The allergenicity of Lt pollen was thus investigated Skin prick tests (SPT) were carried out using both standard allergens and the Lt extract and the patient's mucosal reactivity was evaluated by nasal provocation test with the pollen extract. In vitro studies were also performed on the patient's serum by evaluating routine specific anti-allergen IgE on raw extracts and on Microarray Allergen Chip (ISAC). Finally, the raw extract of the fresh Lt pollen was also used in ELISA inhibition test, immunoblotting and Basophil Activation Test (BAT). The specific sensitization was demonstrated by Skin Prick test and nasal provocation test. The sensitization was also confirmed by specific IgE and by in vitro activation of basophils in the presence of the pollen. By using RAST inhibition test, the presence of cross-reactivity with other pollens was ruled out. According to our results, Lt extracts contain an allergenic activity not only as dried flowers, but also as fresh pollen. For its role in occupational asthma, this allergen should be included in any allergy screening at least in farmers or in the flower industry employers.

Streptococcus pneumoniae is one of the most important causative agent of pneumonia, meningitis, bacteremia, sinusitis and otitis media. The gold standard diagnostic method is still culture even if bacteriological diagnosis is making progress in molecular biology and in proteomics areas.

The resistance to antibiotics, appeared in the 50s, at the same time of the introduction of these drugs, has become today a public health emergency of international importance. This problem is extremely complex because it is based on many factors, including inappropriate use of antibiotics and their massive presence in the feed of farm animals. Today this problem has become a real public health priority not only for the clinical implications (increased morbidity, mortality and duration of disease, the possibility of developing complications and infections), but also for the economic impact of infections by bacteria resistant, due to the additional cost for more expensive drugs and procedures and for stretching the hospitalization. The European Centre for Disease Prevention and Control (ECDPC) in Stockholm has recently estimated that the bacteria with multiple resistances are responsible for about half of thirty-seven thousand deaths per year in the twenty member countries of the European Union. The importance of this phenomenon has prompted several surveillance systems based on the collection of laboratory data to local or national level. To standardize and interpret the data collected and to facilitate comparison between different realities, in 1998 the EU has decided to finance a European surveillance network (EARSS: European Antimicrobial Resistance Surveillance System), which involves several national surveillance networks. Despite the increase of antimicrobial resistance in clinical isolates of both gram positive and gram negative, there are very few new antimicrobial agents in development. Only a multidisciplinary approach, combining various skills in medicine, can help us cope with this emergency and to use the resources available.

The micro-array techniques for the detection of specific IgE has improved the diagnostic procedures for allergic diseases. This method also allows to define sensitisation profiles from an epidemiological point of view. We studied the sensitisation pattern in a population of polysensitized patients with respiratory allergy, living in a restricted geographical area in the north-west Italy.Consecutive patients with asthma/rhinitis, living in the province of Cuneo, and having at least two positive skin prick test for non related aeroallergens were studied by a microarray (Phadia, Milan Italy) which allowed to detect specific IgE against 103 different allergen components.The 70 patients included had specific IgE towards a mean of 4.3 allergens/patient (range 2-12 allergens). Concerning pollens, 63 (90%) had specific IgE to at least one genuine grass pollen allergen, 32 (45.7%) had Ole e 1 specific IgE antibodies, although olive tree is not present in the area. A relevant percentage of sensitisation to mite was found (47,1%). True co-sensitisation to grass-pollen allergens/Bet v 1/Ole e 1 was observed in 15 individuals (21.4%). Prup 1, resulted to be a sensitising allergen in 23 patients (32.85%), 4 of whom were co-sensitised to Prup 3 and/or Art v 3.A detailed knowledge of the sensitisation pattern may have relevant implications for the prescription of specific immunotherapy. Moreover, sensitisation to PR-10 (or profilin), frequently associated to oral allergy syndrome, in some cases could hide the sensitisation to LTPs which are clinically more relevant.

Background Allergic sensitization and diseases have been reported to have a very high and increasing prevalence in elite athletes. Over 80% of allergic athletes are poly-sensitized. Objective This study aims at evaluating the potential diagnostic added value of a microarray technology (ImmunoCAP ISAC, Phadia AB [at present Thermo Fisher Scientific] Uppsala, Sweden which detects IgE antibodies to specific or cross-reacting allergen components. Methods Seventy-two poly-sensitized athletes according to skin prick test (SPT) with different allergic phenotypes (asthma n = 19; rhino-conjunctivitis n = 20; food allergy and/or oral allergy syndrome n = 13; no clinical symptoms n = 20) and two different control populations (20 poly-sensitized sedentary subjects with respiratory allergy and 20 healthy athletes with negative SPT) were studied for detecting specific IgE (sIgE) both to allergen extracts (ImmunoCAPsIgE) and to allergen components (ImmunoCAP ISAC). Results ImmunoCAP ISAC detected the presence of sIgE in 90% of poly-sensitized athletes – in 96% with symptoms and in 75% without symptoms – and in 100% of allergic controls. The pattern of positivity towards the 103 components tested differed from subject to subject, even in those with the same sensitization to allergen extract SPT or sIgE. Based on the ISAC results, poly-sensitized athletes were classified into the following prototypical patterns, differently represented in the clinical phenotypes studied (P = 0.03): (1) One single predominant specific allergen positivity; (2) sIgE to two or more non-cross-reacting allergens; (3) sIgE to cross-reacting allergens; and (4) sIgE to components potentially responsible for severe allergic reactions. Conclusions The ImmunoCAP ISAC represents a useful additional tool for diagnosis and management of poly-sensitized athletes.

Fourteen patients with advanced soft tissue sarcomas (STS), all pre-treated with one or more chemotherapy (CT) lines, entered an outpatient phase 11 study in which subcutaneous recombinant Interleukin-2 (rIL-2) and intramuscular recombinant alpha-2a-interferon (r-alphaIFN) were concomitantly administered. Both the cytokines were given for 5 days/week for 3 consecutive weeks followed by a 2 weeks period during which only r-alphaIFN was administered. r-alphaIFN was provided at a dose of 3 x 10(6) International Units (IU), while rIL-2 was given at a dosage of 6 x 10(6)/m2/day IU (in 2 subcutaneous injections), starting from 2 x 10(6)/m2/day IU in the first week and progressively increasing to 4 and 6 x 10(6)/m2/day IU in the second and third weeks; in 4 patients the dose of 8 x 10(6)/m2/day IU was reached. Toxicity was moderate and correlated with rIL-2 dose; main side effects included changes in liver functionality tests (14/14), fever (13/14), fatigue (13/14), nausea and vomiting (9/14). In all 11 patients evaluable for response, stable disease (SD) was observed (duration 4-43 weeks; median 9 weeks); the median survival from the starting treatment was 18 weeks (range 10-52). In all treated patients, an immunological monitoring was performed: an increase in percentage (from 10 to 74%) and in absolute number (from 400 to 4.500 cells/mm3) of CD16+ lymphocytes (NK cells) was observed in the majority of cases. Our data indicate that this regimen can be administered in pre-treated and severely immunocompromised patients with minimal to moderate toxicity on ambulatory and home bases, with acceptable clinical results.

We evaluated the rates of gastroenteritis admissions to the emergency department and of rotavirus-related hospitalisations in children ≤5 years of age in 2006 at an Italian paediatric hospital. We calculated the number of rotavirus cases avoidable through the universal vaccination of children. Epidemiological data were extracted from the Data Elaboration Centre. To calculate the hospitalisation rate due to rotavirus, the virus was sought in the faeces of children hospitalised for acute gastroenteritis by means of rapid immunochromatographic assay. Emergency department admissions due to gastroenteritis numbered 2,396 (11.58% of the total admissions). Of these, 276 children (11.52%) were examined and then sent home, 1,286 (53.67%) were kept in short observation and 776 (32.38%) were hospitalised. In 27.83% of hospitalised cases, the rotavirus test proved positive. The rotavirus hospitalisation rate was 55 per 10,000 children ≤5 years of age in Genoa in 2006. In 85.6% of hospitalised patients with community-acquired rotavirus infection, the disease was severe. The number of avoidable cases confirmed that the vaccination of children ≤1 year of age could reduce the burden of rotavirus infection, especially with regard to hospitalisation (45 per 10,000 children ≤5 years of age) and admissions to short observation (85 per 10,000), generating benefits for the Italian healthcare system.

An HIV-positive woman receiving antiretroviral therapy developed an opportunistic toxoplasma infection, detected by morphological examination of bone marrow aspirate in the absence of serological positivity. The intracellular presence of Toxoplasma gondii was confirmed by indirect immunofluorescence microscopy, using a polyclonal antitoxoplasma antiserum on marrow smears. This case report confirms the utility of morphological bone marrow examination for the diagnosis of parasitic infections in patients with impaired host defenses.

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.

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.

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.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.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.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.

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.

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.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.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%).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.

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.

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.

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.

A previous pilot study from our group suggested that: (1) adoptive immunotherapy (A1) with tumor-infiltrating lymphocytes (TIL) and recombinant interleukin-2 (rIL-2) may be applied with safety to more than 80% of the patients who had surgery for Stage III nonsmall cell lung carcinoma (NSCLC); and (2) AI could be useful in patients with locally advanced disease. The present randomized study was planned to assess the efficacy of AI in the postoperative treatment of Stage II, IIIa, or IIIb NSCLC:TIL were expanded in vitro from tissue samples obtained from the surgically removed specimens of 131 patients. Eighteen cultures yielded no growth of TIL. The remaining 113 patients were stratified according to disease stage and randomized to receive AI or standard chemoradiotherapy. TIL were infused intravenously 6 to 8 weeks after surgery, rIL-2 was administered subcutaneously at escalating doses for 2 weeks, and then at reduced doses for 2 weeks and then for 2 to 3 months.Three-year survival was significantly better (P0.05) for patients who underwent AI than for controls. AI was of no benefit to patients with Stage II NSCLC, potentially useful to patients with Stage IIIa NSCLC (P = 0.06), and significantly advantageous to patients with Stage IIIb (T4) NSCLC (P0.01). For patients with Stage III NSCLC, local relapse (but not distant relapse) was significantly reduced following AI (P0.05).AI should be considered when designing future adjuvant therapy protocols for the treatment of 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-Guerin (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.

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.

In the past two decades interferons have undergone extensive phase I and II evaluations in various types of cancers, including breast cancer. This article reviews the experience, obtained from preclinical and clinical studies, about the clinical rationale efficacy and toxicity of interferons in cancer treatment. In particular, we examine the preclinical experience in which antineoplastic activity of interferon against breast cancer has been demonstrated. Finally we discuss clinical data accumulated using interferon-alpha, interferon-beta and lymphoblastoid interferon alone or in combination in metastatic breast cancer patients. The use of the interferons in the treatment of breast cancer remains investigational and the optimal scheduling undetermined. New methods of administration may maximize the antitumor effects in order to better understand the role of interferon in clinical oncology.

Human alpha- and gamma-interferons (IFNs) have the capability of enhancing the expression of major histocompatibility complex-class I and class II (MHC-I and MHC-II) as well as other surface tumor associated antigens (TAA) on cancer cell lines. This capability, associated to the anti-proliferative effect observed both in vitro and in vivo, was the basis of the extended use of IFN in cancer patients. Despite the great expectations, several solid neoplasms resulted resistant to treatment with IFNs. In order to analyze the actual effects of IFNs on cancer, we treated primary cultures of different histotypes of cancer cell, which are well known to differ significantly from established cell lines, with escalating doses of alpha-IFN. In this paper, we demonstrate that a variable proportion of culture was able to potentiate the expression of MCH-I and MCH-II molecules on the surface. This finding suggests that a clear functional heterogeneity was present in primary cultures of cancer cells, that strictly reflected the in vivo situation. In addition, it is of note that in some histotypes, "natural" lymphoblastoid alpha-IFN was more effective than recombinant human alpha-IFN in the induction of this capability.

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.

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.

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.

Several observations indicate that a retrovirus might be involved in the pathogenesis of multiple sclerosis (MS). We report that the sera from 40 Italian MS patients did not contain antibodies to the human T-lymphotropic type I (HTLV-I) and type III viruses (HTLV-III) at levels detectable by commercial ELISA kits. Nevertheless, it cannot be ruled out that a distinct retrovirus is the etiologic factor of MS.

T derived colony inhibitory activity (Td/CIA) was obtained from unstimulated T cells from aplastic anemia patients (SAA), or from PWM primed normal T cells. Td/CIA suppressed CFUC growth of normal allogeneic marrow to less than 30% of expected growth. Td/CIA was then added to normal peripheral blood T and B cells, primed with PWM, to test whether it would interfere with in vitro immunoglobulin (Ig) production. When Td/CIA from normal T cells was added to cultures of T + B cells + PWM there was a 2-2.1-fold increase in Ig production. Similarly the addition of Td/CIA from SAA patients also resulted in a 1.4 up to 166-fold increase in Ig production. These results indicate that either (a) the targets for Td/CIA are expressed on hemopoietic but not on T and B cells, or (b) that Td/CIA inactivates an accessory cell which is essential for CFUC growth but not for the PWM driven in vitro B cell differentiation system.

Human peripheral blood null cells were conjugated in vitro with K-562 cells and expanded into continuous cell lines using IL-2 containing medium (CM) and periodical restimulation with phytohaemagglutinin (PHA). Most of these lines were made up of granular blasts expressing high natural killer (NK) activity. When analysed for different surface markers, the large majority of the blasts were E rosette+, T3+, Tac+, DR+, Leu7+ with a variable proportion of cells expressing T8 and M1 antigens (range: 20-80%). In contrast, T4 antigen was expressed by the majority of cells of the control cell lines originated in the absence of K-562 cells. Twenty-nine clones were obtained from one of the above lines using the limiting dilution technique and subsequently maintained in CM for 4 months or more. The majority of these clones maintained their cytotoxic potential and were able to produce different interferon (IFN) types (IFN-alpha, IFN-gamma or both) when growing in CM. In addition in a number of selected clones, simultaneous stimulation with PHA and K-562 cells was able to induce or support the production of both IFN types.

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.

The cell interactions that take place between Toxoplasma gondii trophozoites and the human immune system have been investigated by using an in vitro model of infection. PBMC were co-cultured with live, appropriately attenuated, trophozoites. When cells from immune (seropositive) donors were used, a proliferative response was observed. At the same time, the proliferating T cells proved capable of controlling the growth of live trophozoites. By contrast, cells from seronegative donors failed to mount a proliferative response and intracellular overgrowth of trophozoites with subsequent cell injury occurred. Actively proliferating T cells were expanded in continuous cell lines with IL-2 and periodical restimulation with Ag in the presence of autologous irradiated mononuclear cells. From some of the lines obtained, clones were also derived. Ten clones were selected for further studies. They proliferated in response to trophozoites but not to unrelated Ag. Their response required the presence of autologous monocytes-macrophages isolated from peripheral blood on Percoll density gradients. B cells that were obtained from the same donors and immortalized by EBV infection proved inefficient as APC. These data suggest that live trophozoites have to be processed by macrophages in order to be presented to T cells. Upon appropriate antigen stimulation, all of the clones produced IL-2 and IFN-gamma, a finding that was consistent with both their CD4+ surface phenotype and their helper capacity on B cell proliferation and differentiation in vitro. The supernatants of all of the stimulated clones released a factor that activated macrophages to kill intracellular trophozoites as well as an unrelated pathogen, Listeria monocytogenes. This factor was identified as IFN-gamma because it was neutralized by specific anti-IFN-gamma antibodies. The present in vitro model of response to live protozoa may prove suitable to assess the role of both T lymphocytes and macrophages in intracellular parasite infections in man. Furthermore, this experimental system may be applied to detect specific lesions of cell mediated immunity in a number of immunodeficiency syndromes.

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.

The case of a patient with encephalomyelitis and laboratory signs of a central nervous system herpes zoster infection without cutaneous lesions is reported. The diagnosis was supported by the serological evidence of intrathecal synthesis of specific antibodies against Varicella-zoster virus (VZV).

An enzyme-linked immunosorbent assay (ELISA) for the evaluation of growth kinetics of Toxoplasma gondii in tissue cultures was developed. Tissue culture microplates (96 wells) were seeded with Vero cells, infected with a virulent strain of T. gondii, and incubated for different time periods. The ELISA was performed with anti-T. gondii antibodies on the infected cells. The method was simple, rapid, and accurate, and very good correlations between the ELISA results and the percentage of T. gondii rosettes in infected cells, the number of free T. gondii in the supernatant, and the amount of T. gondii antigen in the supernatant were observed.

Primary biliary cirrhosis (PBC) is a chronic cholestatic progressive liver disease frequently associated with various immunological abnormalities. We have studied the influence of normal and PBC adherent cells (AC) on peripheral blood lymphocytes (PBL) colony growth both in liquid and soft agar culture. Co-culture experiments with different combinations of AC and PBL of PBC patients and normal donors provided evidence that PBC adherent cells function abnormally. However, this impaired function is not the sole factor involved, since the number of colonies formed by PBC lymphocytes in the presence of normal AC was twice the normal value. The possibility that this increase is correlated with the state of activation of T cells is discussed in the light of the high number of DR-positive T cells found in the PBC patients studied. Finally, none of these immunological abnormalities was related to the stage of the disease.

Most mature T lymphocytes express CD3-associated antigen receptor molecules (TCR) formed by alpha and beta chains. Recently, a minor subset has been identified that express a different CD3-associated TCR composed of gamma and delta chains. The cell subset expressing TCR gamma/delta differs from conventional T cells in a number of phenotypic and functional characteristics. The simultaneous lack of both CD4 and CD8 antigens at the cell surface allows one to greatly enrich for TCR gamma/delta + cells (by monoclonal antibodies, mAbs and complement). Cloning of CD4-8- peripheral blood lymphocytes revealed that they are homogeneously composed of cytolytic cells which, in most instances, lyse tumor target cells. TCR gamma/delta + cells 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, thus providing the formal proof that they recognize (allo)antigens. The use of different mAbs specific for TCR gamma/delta molecules allowed us to identify two distinct subsets which bound BB3 and delta-TCS-1 mAbs, respectively. The BB3-reactive molecules in peripheral blood-derived TCR gamma/delta + cells were represented by C gamma 1-encoded disulphide-linked heterodimers, whereas delta-TCS-1 reacted with C gamma 2-encoded non disulphide-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 Ca++ and/or lymphokine production or cytolytic activity).(ABSTRACT TRUNCATED AT 250 WORDS)