Γδ T Cell-Based Immunotherapy in Melanoma: State of the Art.

Metastatic melanoma is still associated with a poor prognosis, and there is increasing interest in immunotherapy alone or in combination with other adjuvant therapies. Γδ T lymphocytes play a pivot role in the immune response against cancer, but while γδ-based immunotherapy is already a clinical reality for several solid tumors, data on melanoma are still limited and fragmented. This systematic review presents preclinical and clinical evidence for a role of γδ T lymphocytes in immunotherapeutic strategies for advanced melanoma and discusses research state of the art and future perspectives. Current strategies focus on in vivo stimulation, and ex vivo adoptive therapy and vaccination; results are promising, but further studies are needed to better investigate the interactions in tumoral microenvironment and to improve clinical efficacy of immunotherapeutic protocols.

Distinctive features of tumor-infiltrating γδ T lymphocytes in human colorectal cancer.

γδ T cells usually infiltrate many different types of cancer, but it is unclear whether they inhibit or promote tumor progression. Moreover, properties of tumor-infiltrating γδ T cells and those in the corresponding normal tissue remain largely unknown. Here we have studied features of γδ T cells in colorectal cancer, normal colon tissue and peripheral blood, and correlated their levels with clinicopathologic hallmarks. Flow cytometry and transcriptome analyses showed that the tumor comprised a highly variable rate of TILs (5-90%) and 4% γδ T cells on average, with the majority expressing Vδ1. Most Vδ1 and Vδ2 T cells showed a predominant effector memory phenotype and had reduced production of IFN- Î³ which was likely due to yet unidentified inhibitory molecules present in cancer stem cell secretome. Transcriptome analyses revealed that patients containing abundant γδ T cells had significantly longer 5-year disease free survival rate, suggesting their efficacy in controlling tumor at very early stage.

Short term respiratory effects of acute exposure to chlorine due to a swimming pool accident.

OBJECTIVE: Acute exposure to chlorine causes lung damage, and recovery may proceed slowly for several weeks. The short term respiratory effects of acute chlorine inhalation during a swimming pool accident were examined. METHODS: A total of 282 subjects (134 children, aged <14 years) inhaled hydrogen chloride and sodium hypochlorite during an accident caused by a malfunction of the water chlorinating system in a community pool in Rome in 1998. Most people received bronchodilators and cortisone at the emergency room; five children were admitted to hospital. A total of 260 subjects (92.2%) were interviewed about duration of exposure (<3, 3--5, >5 minutes), intensity of exposure (not at all or a little, a moderate amount, a lot), and respiratory symptoms. Lung function was measured in 184 people (82 children) after 15--30 days. The effects of exposure to chlorine were analysed through multiple linear regression, separately in adults and in children. RESULTS: Acute respiratory symptoms occurred among 66.7% of adults and 71.6% of children. The incidences were highest among those who had chronic respiratory disease and had a longer duration of exposure. In about 30% of the subjects, respiratory symptoms persisted for 15--30 days after the accident. Lung function levels were lower in those who reported a high intensity of exposure than in those who reported low exposure, both in children and in adults (mean (95% confidence interval (95% CI)) differences in forced expiratory volume in 1 second (FEV(1,)) were -109 (-310 to 93) ml, and -275 (-510 to -40) ml, respectively). CONCLUSION: Persistent symptoms and lung function impairment were found up to 1 month after the incident. Although community pool accidents happen rarely, the medical community needs to be alerted to the possible clinical and physiological sequelae, especially among susceptible people.

[Cause of death among workers of a refinery in Rome]. / Mortalità per causa dei lavoratori della raffineria di Roma.

We evaluated mortality among subjects employed in an oil refinery plant in Rome, Italy. We studied two subgroups of refinery employees: blue collar and white collar workers. A total of 682 men (505 blue collars, 148 white collars, 29 unknown) employed between 1965 and 1992, were followed up for mortality since employment in the plant to July 1999. Standardized Mortality Ratios (SMR), and their 90% Confidence Intervals (90% CI), comparing mortality rates of the cohort members with those of the general population of the Lazio region. For blue collar workers, we performed analyses by latency since first employment and by duration of employment for selected cancer sites. We observed 94 total deaths (100.8 expected) (SMR = 0.93; 90% CI = 0.78-1.11) among blue collar workers and 16 total deaths (31.7 expected) (SMR = 0.50; 90% CI = 0.32-0.77) among white collar workers, and a large deficit of deaths from non-neoplastic cardiovascular diseases (respectively SMR = 0.60; 90% CI = 0.41-0.86 and SMR = 0.18; 90% CI = 0.03-0.56). All cancer mortality was slightly increased only in blue collars (SMR = 1.27 CI = 0.97-1.65). There was an excess risk from cancer of the lung (20 obs SMR = 1.80, 90% CI = 1.19-2.62), bladder (5 obs SMR = 3.19, 90% CI = 1.26-6.72), and benign/unspecified cancer of the brain (4 obs SMR = 4.11, 90% CI = 1.12-10.6). The lower mortality from cardiovascular disease indicates the presence of a strong healthy worker effect. The findings of elevated mortality from cancer of the lung and bladder in blue collar workers are in agreement with those of other studies. Confounding factors from cigarette smoking might have played only a marginal role in influencing the results. Exposure to polynuclear aromatic hydrocarbons (PAHs) is a plausible explanation for the excesses found.

Characteristics of nonsmoking women exposed to spouses who smoke: epidemiologic study on environment and health in women from four Italian areas.

The aim of this study was to evaluate whether risk factors associated with cardiovascular or respiratory diseases and lung cancer occur differently among nonsmoking women in Italy with and without exposure to environmental tobacco smoke (ETS) from husbands that smoke. We performed a cross-sectional study of 1,938 nonsmoking women in four areas of Italy. Data on respiratory and cardiovascular risk factors and on diet were collected using self-administered questionnaires. Medical examinations and blood tests were administered; urine cotinine levels were measured. Nonsmoking women ever exposed to husbands' smoking were compared with unexposed women for several factors: education, husband's education, household crowding, number of children, current or past occupation, exposure to toxic substances at work, parental diseases, self-perceived health status, physician-diagnosed hypertension, hypercholesterol, diabetes, osteoporosis, chronic respiratory diseases, blood pressure medications, lifestyle and preventive behaviors, dietary variables, systolic and diastolic blood pressure, body mass index, waist-hip ratio, triceps skin folds, plasma antioxidant (pro-) vitamins (- and ss-carotene, retinol, l-ascorbic acid, -tocopherol, lycopene), serum total and HDL cholesterol, and triglycerides. Women married to smokers were more likely to be less educated, to be married to a less educated husband, and to live in more crowded dwellings than women married to nonsmokers. Women married to smokers were significantly less likely to eat cooked [odds ratio (OR) = 0.72; 95% confidence interval (CI), 0.55-0.93] or fresh vegetables (OR = 0.63; CI, 0.49-0.82) more than once a day than women not exposed to ETS. Exposed women had significantly higher urinary cotinine than unexposed subjects (difference: 2.94 ng/mg creatinine). All the other variables were not more prevalent among exposed compared to unexposed subjects. The results regarding demographic factors are easily explained by the social class distribution of smoking in Italy. A lower intake of vegetables among exposed women in our study is consistent with the available literature. Overall, our results do not support previous claims of more frequent risk factors for cardiovascular and pulmonary diseases among ETS-exposed subjects. In Italy, as elsewhere in Europe and North America, women who have never smoked but are married to smokers are likely to be of lower social class than those married to never-smokers. However, once socioeconomic differences are considered, the possibility of confounding in studies on the health effects of ETS is minimal.

[Malignant mesothelioma in the industrial area of Colleferro]. / Mesotelioma maligno nel comprensorio industriale di Colleferro.

The study describes the occurrence of pleural and peritoneal malignant mesothelioma in the Colleferro industrial area (Province of Rome, 9 municipalities, population 63,000, period 1993-98) which is the site of a large chemical plant (BPD) producing organic chemicals, acid mixtures, insecticides, explosives and dynamite, and was involved in manufacturing/maintenance of railroad rolling stock. Asbestos was extensively used in these plants in the past. Mesothelioma cases were actively searched from data in files of pathology archives, hospital admission and discharge (records), and death certificates recorded at local health authority register. 23 potential cases were identified for whom clinical charts and pathological slides were reviewed. A multidisciplinary evaluation of all collected information confirmed 18 cases of cyto-histologically proven malignant mesothelioma (pleural/peritoneal ratio of 2.75:1) among residents and/or workers at BPD. The remaining 5 cases were defined as not mesothelioma; however, two were cases of lung cancer (both occupationally exposed to asbestos). All subjects with malignant mesothelioma had been occupationally exposed to asbestos (14 males and 3 females), except one (1 female with domestic exposure). No mesothelioma case was attributable to environmental exposure. Of the 17 cases with occupational asbestos exposure, 15 occurred in BPD workers employed in manufacturing/maintenance of railroad rolling stock (3 cases), general maintenance services (5 cases), or in the armaments sector (7 cases) and 2 in residents but not BPD workers (1 baker, 1 pipefitter). The incidence rate in residents of the 9 municipalities was 5.5 in males and 1.3 in females (standardized on the Italian population x100,000, census 1981). For Colleferro municipality only, the incidence was 10.1 in males and 4.1 in females, which are the highest rates reported so far in Italy. Besides confirming the risk of mesothelioma risk in railroad rolling stock manufacturing and asbestos-insulated pipe maintenance workers, this study identifies a cluster of malignant mesothelioma in explosives production workers.

3-Acetyl-5-acylpyridin-2(1H)-ones and 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones: synthesis, cardiotonic activity and computational studies.

A series of milrinone analogues, namely 6-substituted 3-acetyl-5-acylpyridin-2(1H)-ones 4a-c, e, f and 7-substituted or unsubstituted 3-acetyl-7,8-dihydro-2,5(1H,6H)-quinolinediones 4g-j, in which the cyano group was replaced by the acetyl function, was prepared. In a preliminary pharmacological investigation on spontaneously beating atria from reserpine-treated guinea-pigs, all new compounds did not induce any inotropic effect equivalent or higher than that of the milrinone chosen as the reference compound. In order to rationalise how the structure modifications influence the activity and the selectivity of the title compounds, a computational study has been performed. The important role of the substituents in positions-3 and -6 on the pyridone nucleus has been highlighted.

Synthesis and biological evaluation of [alpha-(1,5-disubstituted 1H-pyrazol-4-yl)benzyl]azoles, analogues of bifonazole.

A series of pyrazole analogues of bifonazole, an antifungal drug used in clinical practice, 2a-h and 4a-h were synthesized and tested in vitro against Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, with no significant results. Imidazoles 2a-h were also tested in vivo for antiarrhythmic and antihypertensive activities; two of these compounds showed moderate activity against ventricular fibrillation caused by aconitine in rats. The above compounds were prepared by reaction of phenyl-[5 substituted 1-phenyl (or 1-methyl)-1H-pyrazol-4-yl]methanols with N,N'-carbonyldiimidazole (2a-h) or of the respective chloro derivatives with 1H-1,2,4-triazole (4a-h).

Synthesis of angelicin heteroanalogues: preliminary photobiological and pharmacological studies.

A series of angelicin heteroanalogues, in which the furan was replaced by thiophene or a 1-substituted pyrazole moiety, was synthesised in order to obtain potential therapeutic agents with antiproliferative and/or other biological activities. In general, the antiproliferative activity of the new thioangelicin, tested in different biological substrates, appeared to be higher than that of the angelicin, the natural parent compound, but lower than that of 8-MOP, the furocoumarin ordinarily used in PUVA therapy and photopheresis. Thioangelicin 6 induced strong inhibition of T2 bacteriophage infectivity and was able to significantly repress the DNA synthesis in Ehrlich ascites cells and the clonal growth in HeLa cells. The pyrazolocoumarins did not show any noticeable effect upon UVA irradiation in all the biological systems considered. All the new angelicin heteroanalogues appeared to be free of the known phototoxicity of furocoumarins on the skin. The pyrazolocoumarins have also been tested as anti-inflammatory, analgesic, antipyretic, local anaesthetic, anti-arrhythmic and platelet anti-aggregating agents by standard procedures. In this class of derivatives, 10a showed good anti-inflammatory and antipyretic properties, while 9a and 11a showed significant local anaesthetic activity.

Inotropic agents. Synthesis and structure-activity relationships of new milrinone related cAMP PDE III inhibitors.

The synthesis of 6-substituted 5-acyl-1,2-dihydro-2-oxo-3-pyridinecarbonitriles 1b,c, 1,2,5,6,7,8-hexahydro-2,5-dioxo-3-quinolinecarbonitriles 1d-g and esters of 5-cyano-1,6-dihydro-2-methyl-6-oxo-3-pyridinecarboxylic acid 2b-e is described. In the case of 1e and 1f, a careful elucidation of the reaction mechanism is discussed. As milrinone analogues, the above compounds were tested on contractile activity and frequency rate of spontaneously beating atria from reserpine-treated guinea pigs. The methyl and the benzyl esters 2b and 2e showed an appreciable positive inotropic activity when compared to milrinone. A fitting study with the DISCO (Distance Comparison) model has been carried out on 2e. This modeling approach allowed for the improvement of the pharmacophoric requirements for a better interaction with the cAMP-specific PDE (PDE III), thought to be the final biological target of these cardiotonic agents.

Use of pooled residual laboratory sera to assess human immunodeficiency virus prevalence among patients in Italy. The Italian Study Group on Occupational Risk of HIV infection.

An anonymous unlinked seroprevalence study of human immunodeficiency virus (HIV) infection was performed by testing pools of ten sera remaining from specimens submitted consecutively to clinical pathology laboratories at 18 Italian public hospitals during four consecutive days in April 1991. Sera from positive pools were retested individually by three different enzyme immunoassays (EIAs) and considered positive if reactive by all three assays. Only the sera with discordant EIA results were retested by Western blot. Of a total of 22,590 sera, 278 were HIV positive (1.2%). The highest rates were seen in hospitals located in metropolitan areas (1.5%), in infectious disease departments (28%) and in drug addiction treatment units (28%); among men aged 21-30 (4.6%) and 31-40 years (4%); and among women aged 21-30 years (1.6%). The distribution of seropositive patients by gender and age group suggests an increasing role of heterosexual transmissions of the infection. The presence of anti-HIV antibodies in sera from patients of both sexes, in all age groups, and from all clinical settings reinforces the need for health care workers to adhere to universal precautions issued to prevent occupational bloodborne infections.

Virus-free survival and down-regulation of CD4 in C8166 cells infected with human immunodeficiency virus type 1 at low density.

Compared with other T cell lines, C8166 lymphocytes are particularly susceptible to human immunodeficiency virus (HIV) infection and the outcome is invariably cell death. The results reported in this study demonstrate that the virus-induced cytolysis is strongly dependent on the initial cell density of C8166 cultures. Cultures diluted to 50 to 500 cells/ml almost completely maintained their cell duplication rate and released infectious virus into the medium. HIV infection of diluted C8166 cells is a simple and easily reproducible procedure for obtaining persistently infected cultures. These cultures contained genomic and extragenomic HIV DNA, the latter being assayed by PCR for two-long terminal repeat circular forms. The status of persistent infection disappeared within 2 months. The recovery is due to the replacement of CD4 down-regulated infected cells by overgrowing uninfected cell variants, which are transcriptionally inactive for CD4. The mechanisms underlying the emergence of these variants in persistently infected cultures are considered.

Tumour necrosis factor-alpha increases the sensitivity of human immunodeficiency virus (HIV)-infected monocytic U937 cells to the complement-dependent cytotoxicity of sera from HIV type 1-infected individuals; role of the gp120 protein.

Sera of 40 intravenous drug addicts [25 seropositive and 15 seronegative for human immunodeficiency virus (HIV)] were tested for the presence of cytotoxic antibodies against uninfected and HIV-infected monocytic U937 cells. Six of the 25 seropositive samples proved to be cytotoxic for HIV-infected target cells in the presence of complement. The pretreatment of HIV-infected U937 cells with tumour necrosis factor (TNF)-alpha (which enhances virus production in these cells) increased the detection of serum cytotoxicity and 60% of these sera became cytotoxic. The percentage lysis was also increased after the TNF-alpha treatment of the target cells (from 16.2 +/- 4.5 to 71.2 +/- 4.9). The complement-dependent cytotoxic activity of these sera was significantly reduced by pretreatment with recombinant HIV gp120 antigen. This reduction was dose-dependent in the majority of cases. Immunofluorescence studies suggested that the cytotoxic sera mainly interacted with the viral antigens localized on the membrane of HIV-infected TNF-treated U937 cells. Moreover, comparative Western blot analyses using cellular extracts from untreated and TNF-treated U937 cells showed that there was a positive correlation between the cytotoxic phenotype and the capacity of sera to recognize the gp120 protein in extracts from TNF-treated HIV-infected cells. These results suggest that in some circumstances endogenous TNF-alpha can be a protective factor because it can render persistently infected cells highly sensitive to complement-dependent serum cytotoxicity as a result of increased expression of the relevant viral antigen (gp120) on the cell membrane.

[The seroprevalence of HIV, HBV and HCV infections in patients coming to the departments of general surgery of a public hospital (S. Camillo, Rome)]. / Sieroprevalenza di infezione da HIV, HBV e HCV in pazienti afferenti ai reparti di chirurgia generale di un ospedale pubblico (S. Camillo, Roma).

An anonymous unlinked seroprevalence study of HIV, HBV and HCV infections has been conducted on 485 sera consecutively submitted to the virology laboratory to be tested for HBsAg; sera came from patients attending general surgery at San Camillo Hospital in Rome. Carriers of HBsAg were 12 (2.5%); antibodies against HIV have been identified in 4 sera (0.8%) and against HCV in 35 (7.2) by ELISA (first generation assay); 25/35 of anti HCV ELISA positive sera were reactive also by RIBA (first generation assay). The observed prevalences of potentially infectious patients represent a risk of occupational transmission for health care workers. Vaccination against HBV, adherence with universal precautions and development of safer surgical devices and techniques are needed to prevent the risk of exposure and, consequently, of occupational acquired infections.

Counselling and testing for HIV infection in Rome 1985-1990: analysis of client characteristics and seroprevalence.

To analyze the characteristics of persons seeking HIV testing, and to evaluate the potential effectiveness of a regional program of confidential counselling and testing for HIV infection, we reviewed data on 5127 clients seen from July 1985 to December 1990 in a major counselling and testing site of the city of Rome. During the study period, the number of clients attending the service increased without a parallel rise of the number of newly identified HIV seropositive subjects. Consequently, the seroprevalence rate decreased from 27.6% in the second half of 1985, to 4.3% in the second half of 1990 (p < 0.001). This decrease appears to be associated with the increasing attendance of low risk subjects and of those referred for testing by family practitioner. The study suggests the need for more efficient strategies to reach persons at higher risk for HIV infection.

Monocytotoxic antibodies in HIV-infected persons. TNF-alpha treatment of U937 cells increases the complement dependent cytotoxicity.

Sera of 40 intravenous drug addicts were tested for the presence of cytotoxic antibodies against uninfected and HIV-infected monocytic U937 cells. Twelve out of 31 seropositive samples proved to be cytotoxic for HIV-infected, untreated target cells in the presence of complement. The TNF-alpha treatment of HIV-infected U937 cells increased the detectability of cytotoxic effect of sera (21/31). The complement dependent cytotoxic activity of sera was reduced by pretreatment with recombinant HIV gp120. This reduction proved to be dose-dependent in the majority of cases. Immunofluorescence studies indicated that the cytotoxic sera interacted with antigens mostly localized on the cell membrane of HIV-infected TNF-alpha treated U937 cells. The specificity, the possible role and origin of monocytotoxic antibodies in HIV-infected persons is discussed.

Prevalence of hepatitis B and C viruses and human immunodeficiency virus infections in women of reproductive age.

OBJECTIVE: To determine the prevalence of hepatitis B and C viruses, and human immunodeficiency virus infections in women of reproductive age attending a health care system. DESIGN: Prospective cross-sectional study. SETTING: Public Obstetric Clinic and Service for Pre- and Perinatal Prevention of Infectious Diseases, Rome, Latium Region, Italy. SUBJECTS: 1142 women attending our centres consecutively for delivery, miscarriage, voluntary interruption of pregnancy or screening for pre- and perinatal prevention of infectious diseases. INTERVENTIONS: Serum samples, collected after informed consent over a period of 2 months, were tested for hepatitis B virus markers (anti-HBc and HBsAg) by enzyme linked immunosorbent assay (ELISA), for antibodies against hepatitis C virus (by ELISA and, if positive, by RIBA) and for human immunodeficiency virus antibodies (by ELISA and, if positive by Western blot). RESULTS: The seroprevalence of hepatitis B virus was 14.4% (95% CI Poisson distribution 12.2-16.5) for anti-HBc and 1.6% (95% CI, 0.9-2.5) for HBsAg. Antibodies against hepatitis C virus were detected by ELISA in 2.4% (CI 1.6-3.5) and by first generation RIBA in 0.9% (CI 0.4-1.6). Human immunodeficiency virus seroprevalence was 1.0% (CI 0.5-1.7). No significant differences were observed by age or by reason for attending. CONCLUSIONS: Women attending our centres have a higher prevalence of hepatitis B virus, hepatitis C virus and human immunodeficiency virus infection than those observed in our country in larger national surveys of newborn babies, in reproductive-aged women or in other selected low-risk groups such as blood donors. This could be due to the attendance of women at increased risk such as drug addicts. The information has the additional value of emphasizing the need for adherence by health care personnel, to the recommendations issued for the prevention of occupational infections.

DNA methylases separated through the HeLa cell cycle methodology show allosteric properties.

Two DNA methylases (DNAmets) can be separated through the cell cycle. The first appears as a minor peak in G1, the second as a major peak in S. Both enzymes protect from HpaII a plasmid (H31), constructed with the pBR322 vector (4.3 kbp) and the inverted A gamma fragment of the human globin gene (3.5 kbp), inserted at its HindIII site (the vector carries several HpaII sites, the insert only one HpaII site). DNAmets G1 and S show distinct Km values and different kinetics vs the ionic strength of the medium, while their Michaelis-Menten and Lineweaver-Burk plots are sigmoidal and hyperbolical curves, respectively. This is the first suggestion about the allosteric nature of the eukaryotic DNAmet system.