HIV coreceptor tropism in paired plasma, peripheral blood mononuclear cell, and cerebrospinal fluid isolates from antiretroviral-naïve subjects.

A survey of HIV coreceptor usage in cerebrospinal fluid (CSF) samples, peripheral blood mononuclear cells (PBMCs), and plasma samples from naïve seropositive patients was conducted. One hundred patients were enrolled in this study. Of the 100 patients, 36 had a primary or recent infection (P-RI), 31 had an early chronic infection (>350 CD4 cells) (ECI), and 33 had a late chronic infection (LCI). All 3 compartments were sampled in a subset of 33 participants, while the remaining 67 patients provided plasma samples and PBMCs only. Seventy-seven patients harbored the R5 virus in plasma samples and had a significantly higher median and percentage of CD4(+) T cells than patients with X4 virus (437 and 281 cells/µl, respectively; P = 0.0086; 20.6% and 18.6%, respectively). The X4 strain was detected more frequently in patients with LCI than in patients with P-RI or ECI (39.3%, 19.4%, and 9.6%, respectively; P = 0.0063). PBMC and plasma tropism was concordant in 90 patients, and 73 had the R5 strain. Among patients with discordant results, 4 had the R5 virus in their plasma and the X4 virus in PBMCs; 6 showed the opposite profile. Plasma, PBMC, and CSF tropism determinations were concordant in 26/33 patients (21 patients had R5, and 5 had X4). The tropism was discordant in 5/33 patients, with the X4 virus in plasma and R5 in CSF; the HIV tropism in PBMCs was X4 in 3 patients. The remaining 2/33 patients had the R5 virus in plasma and PBMCs and the X4 virus in CSF; one of these patients had a P-RI. The discordant tropism in CSF and blood may have implications for chemokine (C-C motif) receptor 5 (CCR5) antagonist use in patients with limited response to antiretroviral therapy (ART) or in responding patients evaluated for simplification of treatment.

The pan-DAC inhibitor LBH589 is a multi-functional agent in breast cancer cells: cytotoxic drug and inducer of sodium-iodide symporter (NIS).

New drugs with anti-tumor activity, also able to modify the expression of selected molecules, are under evaluation in breast cancer which is becoming resistant to conventional treatment, or in metastatic disease. The sodium-iodide symporter (NIS), which mediates iodide uptake into thyroid cells, and is the molecular basis of radioiodine imaging and therapy in thyroid cancer, is also expressed in a large portion of breast tumors. Since NIS expression in breast cancer is not sufficient for a significant iodide uptake, drugs able to induce its expression and correct function are under evaluation. In the present study, we report for the first time that the pan-deacetylase (DAC) inhibitor LBH589 (panobinostat) significantly induced NIS, both as mRNA and as protein, through the increase of NIS promoter activity, with the final consequence of obtaining a significant up-take of iodide in MCF7, T47D, and MDA-MB231 breast cancer cells. Moreover, we observed that LBH589 causes a significant reduction in cell viability of estrogen-sensitive and -insensitive breast cancer cells within nanomolar range. The anti-tumor effect of LBH589 is sustained by apoptosis induction and cell cycle arrest in G(2)/M. In conclusion, our data suggest that LBH589 might be a powerful tool in the management of breast cancer due to its multiple effects and support a potential application of LBH589 in the diagnosis and treatment of this disease.

Elevated thrombopoietin in plasma of burned patients without and with sepsis enhances platelet activation.

BACKGROUND: Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. OBJECTIVES: To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. METHODS: We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. RESULTS: Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. CONCLUSIONS: Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

Conhecimento das gestantes sobre alguns aspectos da saúde bucal de seus filhos / The knowledge of pregnant women about some aspects of the oral health of their children

Introdução: A gravidez é uma fase ideal para o estabelecimento de bons hábitos, pois a gestante mostra-se psicologicamente receptiva em adquirir novos conhecimentos e a mudar padrões que provavelmente terá influência no desenvolvimento da saúde do bebê. É nesse período que a mulher pode ser incentivada pelo profissional dentista a modificações de hábitos bucais, visando ao bem-estar da sua própria saúde bucal e da de seu futuro bebê. Objetivo: Avaliar os conhecimentos das gestantes em relação a alguns aspectos da saúde bucal de seus filhos, como transmissibilidade da doença cárie, cárie de mamadeira, entre outros, e alguns cuidados que se deve ter para manter a saúde bucal dos filhos. Material e métodos: O estudo foi realizado com 100 gestantes que freqüentam as Unidades Básicas de Saúde (UBSs) da cidade de Varginha-Mg. Resultados: Entre as participantes, 54% receberam orientações sobre higiene bucal. Essas informações foram dadas por 28% dos médicos, 9% dos enfermeiros e 17% foram obtidas por meio de leitura. Entre as participantes, 98% pretendem amamentar seus filhos, 62% acreditam que a cárie é uma doença e 48% a consideram como doença transmissível. Conclusão: A maioria das gestantes levaria seus filhos ao cirurgião-dentista em uma idade precoce.

Pregnancy is an ideal stage for the establishment of good habits because it is a psychologically receptive stage; it can be used to acquire new kno-wledge and to change patterns that will probably influence the development of the baby´s health. This is the period of life that a woman may be encouraged by her dentist to change her oral habits, aimed at her own oral health and her future baby as well. Objectives: Furthermore, the present study has the purpose to evaluate the knowledge of pregnant women in relation to some aspects of the oral health of their children; these include the contagious aspect of the dental Caries, Caries of bottle; also, the care women must have to keep a good oral health of their children. Methods: Moreover, this study was conducted with 100 pregnant women who attended the USBs (basic Health Unity) in the city of varginha. Results: Among the participants, 54% received orientation about oral hygiene; the information was given by 28% of the physicians, 9% of the nurses, 17% through reading. Finally, among the participants, 98% planed to breastfeed their babies and 62% believed that dental caries was a disease and that 48% thought it was contagious.

High energy shock waves activate 5'-aminolevulinic Acid and increase permeability to Paclitaxel: antitumor effects of a new combined treatment on anaplastic thyroid cancer cells.

OBJECTIVE: Multimodal treatments do not meaningfully improve survival of anaplastic thyroid cancer. Consequently, new effective therapeutic modalities are needed. The use of paclitaxel is under clinical investigation; it shows about a 50% response rate, but it is not able to alter the fatal outcome for patients with anaplastic carcinoma. High energy shock waves (HESW) have been shown to cause a transient increase in the permeability of cell membranes thus allowing higher intracellular drug concentrations. 5-Aminolevulinic acid (ALA) is used in the photodynamic therapy (PDT) of cancer, and HESW are under evaluation for their use as an activator in ALA-PDT. DESIGN: We investigated the effect of HESW produced by a piezoelectric generator on the sensitivity to paclitaxel and ALA treatments of two different anaplastic thyroid cancer cell lines (ARO and CAL-62). Cells, treated sequentially with ALA and paclitaxel were exposed to HESW; thereafter, cell viability and apoptosis induction were evaluated. MAIN OUTCOME: Combined exposure to ALA, paclitaxel, and shock waves resulted in a significant enhancement of cytotoxicity and induction of apoptosis in thyroid cancer cells with respect to cells treated with paclitaxel alone. CONCLUSIONS: These preliminary data suggest the possibility of using HESW and ALA in combination with paclitaxel as a promising new therapy in the treatment of anaplastic thyroid cancer.

Cytotoxicity of anticancer drugs incorporated in solid lipid nanoparticles on HT-29 colorectal cancer cell line.

Solid lipid nanoparticles (SLN) carrying cholesteryl butyrate (chol-but), doxorubicin and paclitaxel had previously been developed, and the antiproliferative effect of SLN formulations versus conventional drug formulations was here evaluated on HT-29 cells. The 50% inhibitory concentration (IC(50) values were interpolated from growth curves obtained by trypan blue exclusion assay. In vitro cytotoxicity of SLN carrying chol-but (IC(50 72 h) 0.3 +/- 0.03 mM vs >0.6 mM) and doxorubicin (IC(50 72 h) 81.87 +/- 4.11 vs 126.57 +/- 0.72 nM) was higher than that of conventional drug formulations. Intracellular doxorubicin was double after 24 h exposure to loaded SLN versus the conventional drug formulation, at the highest concentration evaluated by flow cytometry. In vitro cytotoxicities of paclitaxel-loaded SLN and conventional drug formulation (IC(50 72 h) 37.36 +/- 6.41 vs 33.43 +/-1.17 nM) were similar. Moreover, the combination of low concentrations of chol-but SLN (0.1-0.2 mM) and doxorubicin (1.72 nM) or paclitaxel (1.17 nM) exerted a greater-than-additive antiproliferative effect at 24 h exposure, while the combination of Na-but and doxorubicin or paclitaxel did not. These preliminary in vitro results suggest that SLN could be proposed as alternative drug delivery system.

Meta-analysis of BACTEC MGIT 960 and BACTEC 460 TB, with or without solid media, for detection of mycobacteria.

In a meta-analysis of 10 studies, the BACTEC 960/MGIT and BACTEC 460 systems showed a sensitivity and specificity in detecting mycobacteria (1,381 strains from 14,745 clinical specimens) of 81.5 and 99.6% and 85.8 and 99.9%, respectively. Combined with solid media, the sensitivity of the two systems increased to 87.7 and 89.7%, respectively.

Determinants of health-related quality of life in HIV-infected patients.

In the era of new antiretroviral treatments that have dramatically reduced both morbidity and mortality, a primary goal is to maximize function and wellbeing in the everyday life of HIV-infected patients. To be able to do so, it would be important for clinicians and policy makers to identify factors that influence health-related quality of life (HRQoL). The objective of this multicentre prospective cohort study was to identify determinants of HRQoL in a cohort of Italian HIV-infected patients, the majority of whom were taking highly active antiretroviral therapy (HAART). A total of 809 patients were enrolled. The MOS-HIV Health Survey (summarized using two scores, physical health (PHS) and mental health (MHS)), and an HIV-related symptom scale were administered at enrolment and six months later. At baseline, low CD4+ cell count, hospitalization during the three months before the enrollment and symptoms were independently related to poor PHS; hospitalization during the three months before the enrollment, symptoms and poor satisfaction with information from providers were independently related to MHS. Predictors of PHS at six months included the stage of HIV infection, baseline CD4+ cells count, PHS and symptom score; while age, baseline MHS, symptom score and education predicted six-month MHS. Among these factors, symptoms, recent hospitalization and satisfaction with information are most amenable to clinical intervention.

Meta-analysis of diagnostic procedures for Pneumocystis carinii pneumonia in HIV-1-infected patients.

Sputum induction is a simple and noninvasive procedure for Pneumocystis carinii pneumonia (PCP) diagnosis in human immunodeficiency virus-1-positive patients, although less sensitive than bronchoalveolar lavage (BAL). In order to obtain an overview of the diagnostic accuracy of sputum induction, a systematic review and meta-analysis of studies reporting the comparative sensitivity and specificity of BAL (the "gold standard") and sputum induction was performed. The odds ratio and related 95% confidence interval were calculated using summary receiving operating characteristic curves as well as fixed-effect and random-effect models. Based on pooled data, the negative and positive predictive values were calculated for a range of PCP prevalence using a Bayesian approach. Seven prospective studies assessed the comparative accuracy of BAL and sputum induction. On the whole, sputum induction demonstrated 55.5% sensitivity and 98.6% specificity. The sensitivity of sputum induction was significantly higher with immunofluorescence than with cytochemical staining (67.1 versus 43.1%). In settings of 25-60% prevalence of PCP, the positive and negative predictive values ranged 86-96.7 and 66.2-89.8, respectively, with immunofluorescence, and 79-94.4 and 53-83.5% with cytochemical staining. In conclusion, in a setting of low prevalence of Pneumocystis carinii pneumonia, sputum induction, particularly with immunostaining, appears to be adequate for clinical decision-making.

The impact of human immunodeficiency virus type 1 on infectiousness of tuberculosis: a meta-analysis.

To assess if the relative infectiousness of patients with tuberculosis is enhanced by coinfection with human immunodeficiency virus type 1 (HIV-1), data from 6 studies of 1240 health care workers who had contact with tuberculosis patients were analyzed. Overall rates of tuberculin skin test conversion were similar regardless of HIV-1 positivity of tuberculosis patients (odds ratio [OR], 1.04; 95% confidence interval [CI], 0.23-1.84). However, when only 3 studies during nosocomial outbreaks of multidrug-resistant Mycobacterium tuberculosis were analyzed, rates of skin test conversion were higher among contacts of HIV-1-positive index cases (OR, 2.85; 95% CI, 1.85-3.85; P=.0002). A second meta-analysis included data from 11 studies of 10,714 household contacts of tuberculosis patients. Prevalence of both skin test positivity (OR, 0.45; 95% CI, 0.20-1.03) and active disease (OR, 1.17; 95% CI, 0.78-1.56) were similar regardless of HIV-1 positivity of index cases. These data suggest that tuberculosis patients with HIV-1 infection are not intrinsically more infectious to their contacts than are HIV-1-negative tuberculosis patients.

Resistance to thromboembolism in PI3Kgamma-deficient mice.

Platelet aggregation and subsequent thrombosis are the major cause of ischemic diseases such as heart attack and stroke. ADP, acting via G protein-coupled receptors (GPCRs), is an important signal in thrombus formation and involves activation of phosphoinositide 3-kinases (PI3K). When platelets from mice lacking the G protein-activated PI3Kgamma isoform were stimulated with ADP, aggregation was impaired. Collagen or thrombin, however, evoked a normal response. ADP stimulation of PI3Kgamma-deficient platelets resulted in decreased PKB/Akt phosphorylation and alpha(IIb)beta(3) fibrinogen receptor activation. These effects did not influence bleeding time but protected PI3Kgamma-null mice from death caused by ADP-induced platelet-dependent thromboembolic vascular occlusion. This result demonstrates an unsuspected, well-defined role for PI3Kgamma downstream of ADP and suggests that pharmacological targeting of PI3Kgamma has a potential use as antithrombotic therapy.

Aberrant activation of c-kit protects colon carcinoma cells against apoptosis and enhances their invasive potential.

Multiple genetic aberrations contribute to the development of biologically aggressive, clinically malignant colorectal carcinomas (CRCs). Some of these have been linked to inappropriate signaling through the tyrosine kinase moieties of growth factor receptors. We have described previously (G. Bellone et al., J. Cell. Physiol., 172: 1-11, 1997) that human CRCs overexpress both the receptor tyrosine kinase c-kit and its ligand, stem cell factor (SCF), relative to normal mucosa cells, thus establishing an autocrine c-kit-mediated loop. In addition, we noted that exogenous SCF contributes to anchorage-independent growth of HT-29 colon carcinoma cells in semisolid medium. Here, we investigated possible roles of the c-kit/SCF autocrine/paracrine system in survival and invasive capacity of DLD-1 colon carcinoma cells. We report that SCF was required for migration and invasion of DLD-1 cells through reconstituted basement membranes (Matrigel) and up-regulated gelatinase (matrix metalloproteinase-9) activity in DLD-1 cells. Furthermore, we describe that SCF supported survival of DLD-1 cells in growth factor-deprived conditions. These results suggest multiple roles of c-kit activation in support of the malignant phenotype of DLD-1 cells related to growth, survival, migration, and invasive potential.

The induction of distinct cytokine cascades correlates with different effects of granulocyte-colony stimulating factor and granulocyte/macrophage-colony-stimulating factor on the lymphocyte compartment in the course of high-dose chemotherapy for breast cancer.

The availability of the myeloid hemopoietic growth factors (HGF) granulocyte- and granulocyte/macrophage-colony stimulating factor (G-CSF and GM-CSF) has enhanced the therapeutic index of high-dose chemotherapeutic antitumoral regimens (HDCT), as well as the rate of severe damage to immune competence. We investigated some immune functions before, during and after one course of HDCT for poor-risk breast cancer and compared the effects of G-CSF and GM-CSF on the immune recovery. They exerted different influences on the functions we examined and showed distinctive patterns of both qualitative and quantitative in vivo activities on the immune system. The main findings were that (a) granulocyte and lymphocyte recovery rates were faster in the patients receiving G-CSF; (b) looking at the lymphocyte compartment, this difference was restricted to the CD3(+)/CD8(+) and CD56(+) lymphocyte subsets; (c) the reconstitution rate of CD19(+) lymphocytes was slow in both groups; (d) at the end of follow-up HLA-DR expression by CD3(+) lymphocytes was higher in the GM-CSF group; (e) the lymphocyte proliferative capacity was restored at a faster rate in the GM-CSF group, whereas cytotoxic activities recovered better in the G-CSF group; (f) the early repopulating phase was characterized by higher interleukin-6 serum levels in the GM-CSF group. Overall, GM-CSF seemed to exert an earlier effect on all T lymphocyte subsets, preventing them from a complete drop during the long-lasting "nadir" of the cell count, whereas G-CSF appeared to boost them strongly, though a few days later, hastening their final recovery. The distinct pattern of the cytokine cascade induced by each factor, consistent with the different functional changes, seemed to account for the peculiarities of their immune modulations.

Intravenous drug use, relationship with providers, and stage of HIV disease influence the prescription rates of protease inhibitors.

OBJECTIVE: To assess rates of prescriptions of protease inhibitors (PI) and determinants of not being prescribed PIs in a cohort of HIV-infected people eligible (according to published guidelines) for highly active antiretroviral therapy (HAART). DESIGN: Cross-sectional survey. METHODS: A total of 684 patients with CD4+ counts <500 cells/microl were enrolled from seven Italian HIV treatment centers from October 1997 to April 1998. A questionnaire on health-related quality of life (MOS-HIV) and patient ratings of the quality of care was administered. Sociodemographic variables, HIV disease-related factors, and prescribed antiretroviral therapy were also recorded. RESULTS: 61% of those enrolled were prescribed PI (median, 7.5 months). In addition, 75% of patients had previously received antiretroviral therapy. Fewer than 1% were prescribed nonnucleoside reverse transcriptase inhibitors (NNRTIs). Using multivariable logistic regression considering those with CD4+ counts <500 cells/microl, patients reporting the least information received (odds ratio [OR], 1.78; 95% confidence interval [CI], 1.23-2.58), injecting drug users (IDUs; OR, 1.73; 95% CI, 1.18-2.54), people with CD4+ counts >200 cells/microl (OR, 1.76; 95% CI, 1.19-2.61), and patients with early stage disease (OR, 2.24; 95% CI, 1.73-2.90) were less likely to have be prescribed PIs. CONCLUSIONS: Of patients eligible for HAART, only 61% were prescribed PIs. People who wanted more information, IDUs, and patients in earlier disease stages are significantly less likely to be prescribed PIs. Access to HAART remains a critical issue in the management of HIV disease.

Decreased surface antigen density on lymphocytes of elderly humans.

The aim of our study was to analyze some poorly investigated and controversial aspects of senescent lymphocyte phenotype and functions. We examined 100 healthy aging individuals, divided into 4 age groups, and 30 young controls, correlating lymphocyte responsiveness to mitogenic stimulation with membrane phenotypic pattern and surface molecular densities of the main functional lymphoid markers. Stability of values in the period of study was established. No age-related differences in the parameters evaluated were detected among aging subjects. Phytohemagglutinin-induced lymphocyte proliferation was found severely impaired (about halved) in all elderly individuals with respect to controls. There was no significant difference between elderly group and controls in CD2, CD3, CD4, CD8, CD19, CD25 and HLA-DR antigen distribution. CD56 positive cell percentages were slightly decreased in the elderly groups. In apparent correlation with reduced lymphocyte responsiveness, CD2, CD3, CD4 and CD8 molecular densities, to different extents, were found relevantly (about 1 to 3-fold) lower in all aging groups than in controls. We could not ascertain if those antigens were poorly synthesized, defectively transported to membrane or shed in excess. However, we suggest that decreased surface molecular densities of antigens involved in functional processes of immune responses may be responsible for an abnormal costimulatory pattern during lymphocyte activation, leading to apoptotic rather than proliferative signals in a greater proportion of cells than normal.

Idiopathic chronic fatigue and chronic fatigue syndrome: a comparison of two case-definitions.

The aim of the study was to compare the signs and symptoms of individuals meeting two different definitions of chronic fatigue syndrome (CFS). Ninety-four patients fitting the eligibility criteria for idiopathic fatigue were enrolled into the study. Of the 94 patients, 48 met the 1988 definition of CFS, 20 the 1994 (but not the 1988) definition of CFS, and 26 met neither definition. The 1994 defined cases were more likely than 1988 defined cases, and non-syndromal individuals to be male, married, and high school educated. The 1994 cases were less likely than 1988 cases to present acute onset, self reported sore throat, mild fever lymphadenopathy, pharyngitis. In conclusion, the 1994 criteria increased the number of patients classified as CFS; however, those who fit only the 1994 criteria were less likely to have an acute symptomatic onset and signs and symptoms suggestive of an infectious process.

Downregulation of HLA class I antigen expression in CD4+ T lymphocytes from HIV type 1-infected individuals.

The expression of HLA class I antigens is downregulated in CD4+ T cells following in vitro HIV-1 infection. We determined whether the expression of HLA class I antigens is downmodulated in peripheral blood lymphocytes (PBLs) of HIV-1-positive subjects and whether this defect correlates with disease progression. A cohort of 62 HIV-1-seropositive individuals in different stages of disease was studied. Among these, four subjects were evaluated at yearly intervals for 6 years. The expression of HLA class I, HLA class II, and CD38 antigens was analyzed in PBLs and in CD4+ and CD8+ T lymphocyte subpopulations. The percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in PBLs from HIV-1-positive individuals than in PBLs from HIV-negative controls, proportionally decreased with disease progression, and significantly correlated with the decrease in CD4+ T lymphocytes. Furthermore, the percentage of HLA class I-positive cells and the membrane density of HLA class I antigens were significantly lower in CD4+ T lymphocytes from AIDS patients with respect to CD4+ T lymphocytes from HIV-negative controls and to CD8+ T lymphocytes from HIV-negative controls and AIDS patients. By contrast, the expression of HLA class II and CD38 antigens was upregulated in CD4+ and CD8+ T lymphocytes from HIV-1-positive subjects. The defective expression of HLA class I antigens could impair the lysis of HIV-infected CD4+ cells by virus-specific HLA class I-restricted cytotoxic T lymphocytes and contribute to the progression of disease.

Prednisone increases apoptosis in in vitro activated human peripheral blood T lymphocytes.

Glucocorticoid hormones (GCH) regulate, through the apoptotic process, the negative selection of immature T cells in the thymus. Because apoptosis seems to occur also in the maintenance of peripheral tolerance, we have investigated whether GCH may induce apoptosis in human mature lymphocytes. Peripheral blood lymphocytes (PBL) or peripheral CD4(+) and CD8(+) T cell subsets were cultured in the presence of phytohaemaglutinin (PHA) or PHA and prednisone (PDN) at 10(-3)-10(-12)M concentrations for 72, 96 and 120h. Cell cycle and membrane antigen expression were evaluated by flow cytometry and DNA degradation was detected by agarose gel electrophoresis. PDN blocks PBL growth in the G1 phase of cell cycle and inhibits both IL-2 receptor (IL-2R) expression and IL-2 secretion. Apoptosis is clearly increased by PDN in PHA-activated human PBL, and the apoptotic effect of PDN is stronger on CD8(+) than on CD4(+) T lymphocytes. All these effects are dose- and time-dependent. The addition of exogenous IL-2 did not rescue lymphocytes from PDN-increased apoptosis. These results show that PDN increases apoptosis in mature activated human peripheral blood lymphocytes, suggesting a possible role of GCH in the maintenance of immune tolerance at post-thymic level.

CD4+ Th0 cell clones, isolated from a metastatic lymph node of a melanoma patient, possess cytolytic function.

In the present study T lymphocytes isolated from a metastatic lymph node (T-LNL) of a melanoma patient have been cloned. In the attempt to verify whether T-LNL may acquire in vitro functional activities in the absence of tumour-associated antigens, they were cloned utilizing allogenic lymphocytes as feeder cells. Nineteen clones generated from T-LNL proved to be CD4+ and, among these, five were able to kill autologous and allogeneic human melanoma cells in HLA-class-II-restricted way. On the basis of their cytokine production, these CD4+ cytolytic T-LNL clones were shown to belong to the Th0 subset and three of them expressed the V beta 17 chain of the T cell receptor. These results suggest the presence of melanoma-specific but functionally inactive lymphocytes with T cell receptor oligoclonality in the lymph node environment. These specific T cells may acquire in vitro the capacity to kill autologous and allogeneic tumours without any induction by autologous melanoma cells.