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Biomedical subjects

R Zambello

Publications and source records attributed to R Zambello.

At least 91 records · Page 5Linked to original sources
PubMed1992

Spontaneous production of interleukin-6 by alveolar macrophages from human immunodeficiency virus type 1-infected patients.

To test the hypothesis that the lung represents a source of interleukin (IL)-6 in human immunodeficiency virus type 1 (HIV-1)-positive subjects, alveolar macrophages (AM) obtained from the bronchoalveolar lavage (BAL) fluid of 10 HIV-1-positive patients were investigated for the expression of IL-6 mRNA and the ability to release IL-6. The presence of IL-6 in BAL fluid was also investigated. It has been demonstrated that freshly recovered AM from HIV-1-positive patients show a strong IL-6 mRNA signal. The message for IL-6 increases following culture with LPS. Supernatants obtained from AM cultured in medium alone contain high amounts of IL-6; the values are three to four times higher following culture with LPS. IL-6 has also been detected in the BAL fluid from 5 of 8 HIV-1-positive patients. Results of immunoblotting analysis were consistent with those given above. These findings suggest that the lung represents a source of IL-6 production in HIV-1-infected subjects with lung disorders.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Clonally expanded CD3+, CD4-, CD8- cells bearing the alpha/beta or the gamma/delta T-cell receptor in patients with the lymphoproliferative disease of granular lymphocytes.

Among 60 retrospectively assessed patients with the lymphoproliferative disease of granular lymphocytes (LDGL), lymphocytes from only 2 patients had the CD3+, CD4-, CD8- phenotype, rarely observed in normal peripheral blood lymphocytes (about 3%). In this paper we report a detailed study of lymphocytes isolated from these two patients. The cells from patients 1 had the CD3+, CD4-, CD8-, WT31-, beta F1-, TCR delta 1+, Ti gamma A-, BB3+, CD7+, CD16-, CD57+ phenotype, while cells from patient 2 had a phenotype even more rarely observed on normal lymphocytes: CD3+, CD4-, CD8-, WT31+, beta F1+, TCR delta 1-, CD7+, CD16-, CD57+. Thus, in only the first case the cells expressed the gamma/delta T-cell receptor (TCR) on the membrane, while the cells from the second case had the alpha/beta TCR. Genetic studies showed that in case 1 the TCR gamma gene was rearranged and the beta chain gene configuration was germline; the TCR mRNA was of normal size for the gamma chain, while that of the beta chain was truncated. Case 2 had the beta and the gamma genes of the TCR rearranged, but only the alpha and beta mRNA were expressed. In agreement with these findings, the delta chain gene of the TCR was rearranged in case 1 and was deleted in case 2. Cytotoxic activity was absent in cells from case 1 and low in case 2; in the latter, the lytic activity could be up-regulated following incubation with IL-2 or an anti-CD3 monoclonal antibody. Our study indicates that CD3+, CD4-, CD8- lymphocytes are rarely expanded in patients with LDGL. The detection of a lymphoproliferative disease of a CD3+, CD4-, CD8-, alpha/beta + cell may contribute to a better characterization of this novel lymphocytic subpopulation.

Adult↗
PubMed1991

Hepatitis B virus binds to peripheral blood mononuclear cells via the pre S1 protein.

The hepatitis B virus has been documented in hepatic and extrahepatic compartments, including bone marrow and peripheral blood cells. The viral protein involved in the attachment to human hepatocytes has been identified within the N-terminus of the pre S1 envelope protein. Using recombinant particles containing the pre S1, pre S2 and S encoded sequences, we studied which virus envelope protein is involved in binding to peripheral blood cells. Mononuclear cells of 20 healthy subjects bound 125I-labelled particles, with a S/N ratio higher than 2.5 (range 2.69-7.77). Binding was abolished by trypsinization. B lymphocytes and monocytes were found to bind viral particles much more efficiently compared to T cells and granulocytes. A monoclonal antibody (MA 18/7), recognizing the (27-49) pre S1 sequence, completely inhibited viral particle attachment to PBMC, while anti-pre S2 (Q 19/10) and anti-S (20/2) monoclonal antibodies had no effect. We conclude that the pre S1 sequence is involved in HBV attachment to PBMC and to hepatocytes. The nature of the cellular attachment site is unknown, but it might be a receptor for physiologic ligands, as occurs with other viruses.

B-Lymphocytes↗
PubMed1991

Shedding of the soluble form of the CD8 complex by CD8+/HLA-DR+ cells in HIV-1-infected patients.

High levels of the soluble form of the CD8 molecule (sCD8) are detectable in the serum of HIV-1-infected patients. To investigate the mechanisms accountable for the release of this molecule we evaluated the presence of sCD8 in the supernatants obtained from in vitro cultures of highly purified CD8 cells isolated from 20 HIV-1-infected patients. At resting conditions cultured CD8 cells from HIV-1-infected patients released low amounts of sCD8; no statistically significant differences were observed between unstimulated cultures from HIV-1-seropositive patients and from HIV-1-seronegative subjects at risk for HIV-1 infection or normal healthy controls. Following in vitro activation of highly purified CD8 cells with a series of stimulatory agents, including phorbol myristate acetate, phytohemagglutinin (PHA) and recombinant interleukin-2, CD8 cells of HIV-1-infected patients significantly increased the shedding of sCD8. By expressing the results of activation-related release index (ARRI = sCD8 levels detected in the cultures with stimulatory agent/sCD8 levels detected in the unstimulated cultures), significantly higher values were observed upon PHA stimulation in HIV-1-infected patients than in control subjects. In order to identify the cell subset responsible for the enhanced release of sCD8 by PHA-stimulated cultures, we correlated the amounts of sCD8 detected in the supernatants with the phenotypic profile of CD8+ cells recovered from the cultures. A significant relationship was demonstrated between the percentage of CD8+/HLA-DR+ lymphocytes and sCD8 levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1991

Alveolar macrophages from patients with AIDS and AIDS-related complex constitutively synthesize and release tumor necrosis factor alpha.

To verify the hypothesis that alveolar macrophages (AMs) from patients infected with HIV-1 could synthesize and release TNF alpha, AMs recovered from the BAL fluid of 11 patients with seropositive HIV-1 (six with AIDS and five with ARC) were tested in vitro for their ability to destroy TNF alpha-susceptible targets. Furthermore, the presence of TNF alpha was assessed in AM-conditioned supernatants on the basis of their cytotoxic activity and by using an immunoenzymatic test and immunoblotting. Transcription of the TNF alpha gene in AMs was also studied by means of the Northern blot analysis. AMs freshly recovered from patients infected with HIV-1 exhibited high levels of cell-mediated cytotoxicity against U937 targets, and the addition of a polyclonal anti-TNF alpha antibody resulted in a significant inhibition of the target lysis. Cell-free supernatants conditioned by unstimulated AMs exerted high levels of cytotoxic activity against TNF alpha-sensitive targets, whereas duplicate, neutralization experiments performed in the presence of an anti-TNF alpha antibody proved that the observed cytotoxic activity was mostly mediated by TNF alpha. The presence of high amounts of TNF alpha in the conditioned media was confirmed by the immunoenzymatic test. In addition, the immunoblot analysis showed that the TNF alpha released by AMs has a Mr 17,000 band, identical to a standard preparation of recombinant TNF alpha. The Northern blot demonstrated that unstimulated AMs express detectable levels of mRNA transcripts for TNF alpha. Taken together, our data support the concept that AMs from patients with HIV-1 infection constitutively release TNF alpha.(ABSTRACT TRUNCATED AT 250 WORDS)

AIDS-Related Complex↗
PubMed1991

Prognostic significance of the evaluation of bronchoalveolar lavage cell populations in patients with HIV-1 infection and pulmonary involvement.

To investigate the prognostic utility of the morphologic and immunologic evaluation of BAL cell populations in determining mortality risk, we analyzed BAL data obtained from 115 patients infected with HIV-1. Forty fatal outcomes occurred within 73 patients with OI. The OI patients who died showed a significant increase in neutrophils with respect to surviving patients. Furthermore, the finding of a BAL neutrophilia in HIV-1-infected patients with OI strongly correlated with a high risk of death. Among 42 cases without OI, 11 patients died. Patients without OI who had a fatal outcome showed an increase in CD3+ and CD8+ BAL lymphocytes with respect to the survivors. The presence of a lymphocytic alveolitis was associated with a significant increase in the mortality rate. Taken together our data suggest that the evaluation of the BAL cell populations might be useful in predicting the risk of fatal outcome in patients with HIV-1 infection.

Acquired Immunodeficiency Syndrome↗
PubMed1991

Tumor necrosis factor-alpha and B-cell growth factor induce leukemic hairy cells to proliferate in vitro.

To clarify the mechanisms accounting for the hairy cell proliferation, in five patients with hairy cell leukemia (HCL), we evaluated the ability of neoplastic cells to proliferate in vitro in response to tumor necrosis factor-alpha (TNF-alpha), B-cell growth factor (BCGF), and interleukin 4 (IL-4). In addition, supernatants recovered from cultures of unstimulated hairy cells were tested for the capability to induce the proliferation of allogeneic hairy cells. We demonstrated that TNF-alpha and BCGF were able to induce the in vitro growth of hairy cells (stimulation index (SI) ranging, at different concentrations, from 1.6 to 10.7 and from 1.6 to 25.1 for TNF-alpha and BCGF, respectively). IL-4 did not show any proliferative effect on hairy cells. When unstimulated hairy cell supernatants were tested for the proliferative activity on allogeneic hairy cells, we demonstrated a low proliferative effect that was not inhibited by an anti-TNF-alpha antibody. Our findings demonstrate that unstimulated hairy cells proliferate in vitro in response to TNF-alpha and BCGF, thus suggesting that these cytokines could be involved in an autocrine model of cell proliferation.

Cell Division↗
PubMed1991

Different mechanisms of activation of proliferating CD3+ cells in patients with lymphoproliferative disease of granular lymphocytes.

To investigate the role of p55 and p75 chains of interleukin-2 receptor (IL-2R) on the activation of granular lymphocytes (GL) in patients with lymphoproliferative disease of granular lymphocytes (LDGL), the cells obtained from 11 LDGL patients (belonging to the CD3+ group) were studied for (a) the surface expression and (b) mRNA transcripts of the p55 and p75 IL-2R after activation with anti-CD3 monoclonal antibody (mAb) or interleukin-2 (IL-2). The effects of mAbs specifically blocking the p55 and p75 IL-2R on the generation of proliferative and cytotoxic functions were studied following anti-CD3 mAb stimulation. A significant difference was observed in the expression of p55 and p75 antigens on LDGL cells under resting conditions: a low number of p55 IL-2R+ (mean 1.2 +/- 0.4%) and high values of p75 IL-2R+ cells (54.9 +/- 7.4%). Accordingly, a barely detectable message for the p55 IL-2R and a strong signal for the p75 IL-2R mRNA were demonstrated. Following activation with anti-CD3 or IL-2, different patterns of IL-2R expression were observed. Anti-CD3 mAb induced an increase in the expression of the p55 IL-2R both at the mRNA and antigen level, whereas the p75 values remained consistently raised. In contrast, IL-2 induced the expression of p55 IL-2R mRNA associated with only a slight expression of this antigen. This finding was associated with a decrease in the cell expression of the p75 IL-2R, whereas the amount of p75 mRNA was unchanged. Both anti-CD3 mAb and IL-2 induced cell proliferation and cytotoxicity against the K-562 target cells. Anti-p55 IL-2R mAb did not affect the cytotoxic activity mediated by anti-CD3, but it markedly inhibited cell proliferation. Anti-p75 mAb did not inhibit either lytic function or cell proliferation mediated by anti-CD3 mAb, suggesting that only the high affinity IL-2R (p55 plus p75) is involved in anti-CD3 mediated cell activation in LDGL patients. This mechanism is different from that responsible for the IL-2 activation of CD3+ GL in LDGL patients, which is achieved through the p75 IL-2R alone. These results provide new insights into the pathophysiology of proliferating GL in LDGL patients and may also contribute to further characterization of the normal CD3+ GL population.

Adult↗
PubMed1990

Cell membrane expression and functional role of the p75 subunit of interleukin-2 receptor in lymphoproliferative disease of granular lymphocytes.

The cell membrane expression and functional role of the interleukin-2 receptor (IL-2R) was analyzed in nine patients with lymphoproliferative disease of granular lymphocytes (LDGL) using monoclonal antibodies (MoAbs) specific for the p75 (TU27) and the p55 (anti-Tac) subunits of IL-2R. Four patients were characterized by the proliferation of CD3+CD8+ granular lymphocytes (GL) expressing the alpha/beta T-cell receptor (T alpha beta) and one case by the proliferation of CD3+CD4-CD8- GL expressing the gamma/delta T-cell receptor (T gamma delta); in four additional cases proliferating cells were CD3 negative GL. Consistent with data observed on normal GL, phenotypic analysis demonstrated that patients' GL lack the expression of the p55 IL-2R, whereas the p75 subunit is constitutionally expressed by expanding GL of both T-cell (either T alpha beta and T gamma delta) and natural killer (NK) origin in variable proportions (11% to 94% of cells). The analysis of the cytotoxic and proliferative activity demonstrated that the anti-p55 MoAb failed to inhibit IL-2-mediated activation, whereas a marked inhibition of both cytotoxicity and proliferation were obtained using the anti-p75 chain specific MoAb. These data indicate that the p75 chain of IL-2R is responsible for IL-2 signal transduction in both CD3+ and CD3- LDGL patients' GL.

Adult↗
PubMed1990

Mechanisms accounting for lymphocytic alveolitis in hypersensitivity pneumonitis.

Hypersensitivity pneumonitis (HP) is a lung disorder characterized by an exaggerated accumulation of CD8+ T lymphocytes in the pulmonary parenchyma. To investigate the mechanisms accounting for the T cell alveolitis taking place in the lung of HP patients and their pattern of growth, cells recovered from the bronchoalveolar lavage (BAL) of seven patients were evaluated for: 1) the expression of activation markers, including IL-2R (p55 and p75 subunits), HLA-DR and VLA-1 Ag; 2) the ability of IL-2 and IL-4 to induce in vitro proliferation; 3) the capability to synthesize and release IL-2 by determining the levels of IL-2 in BAL cell-free supernatants and by evaluating the presence of mRNA transcripts for IL-2; and 4) the molecular configuration of the beta- and gamma-genes of the TCR. This study demonstrates that a high number of BAL lymphocytes recovered from the lungs of HP patients express activation markers including the p75 chain of IL-2R, VLA-1, and HLA-DR Ag. These cells express the CD3+,CD8+,CD16-,CD56+ phenotype and proliferate in vitro in the presence of IL-2 but do not release this cytokine. Furthermore, IL-2 transcripts could not be detected in BAL resting T lymphocytes. No proliferation was observed in the presence of IL-4. The analysis of the configuration of the TCR beta- and gamma-genes showed a polyclonal pattern, with the exception of one case in which extra bands were observed following digestion with BamHI and EcoRI restriction enzymes. Taken together, our data suggest that the IL-2 system may play a central role in the mechanisms accounting for lymphocytic alveolitis in HP patients. Although the pattern of growth is usually polyclonal, such polyclonal recruitment seems to be biased toward cells that have rearranged and possibly expressed particular V beta or V gamma genes, thus leading to the hypothesis that the events that take place in the lung of these patients may occasionally elicit an oligoclonal expansion of the cells proliferating in lung parenchyma.

Adult↗
PubMed1990

Mechanisms accounting for the defective natural killer activity in patients with hairy cell leukemia.

Natural killer (NK) cell activity is severely impaired in untreated patients with hairy cell leukemia (HCL). In an attempt to investigate whether this impairment is related to a defect at the target cell binding and/or at the post target cell binding level, we evaluated the peripheral blood mononuclear cells (PBMC) of HCL patients for their ability to: (1) bind and kill K-562 NK-sensitive targets at the single cell binding level; (2) release the NK cytotoxic factor (NKCF) under different in vitro stimuli, including K-562 and phytohemoagglutinin; and (3) kill K-562 targets in a lectin-dependent cellular cytoxicity (LDCC) assay. This study demonstrates that untreated HCL patients' PBMC show a low ability to form conjugates with K-562 targets at the single cell binding level (5.7% +/- 1.0%) with respect to patients studied after treatment (9.3% +/- 1.3%) and controls (15.0% +/- 4.0%); P less than .05 and P less than .001, respectively. A decreased ability to kill the bound target was demonstrated in untreated cases (1.2% +/- 1.1%) versus patients studied after treatment and controls (12.3% +/- 1.6%, 17.0% +/- 3.1% respectively); P less than .001 in both conditions. After activation of effector cells with interleukin-2 (IL-2) in vitro, an increase in the ability of PBMC to form conjugates with K-562 targets and kill the bound target was demonstrated in each group of patients. Moreover, IL-2 was able to increase the cytotoxicity against NK-sensitive targets in all patients tested. Evaluation of NKCF production showed that untreated patients release low levels of NKCF when PBMC were incubated in the presence of K-562 stimulators (1.8% +/- 0.7%) with respect to patients after interferon-alpha (IFN-alpha) therapy (7.6% +/- 2.1%) and controls (12.9% +/- 2.2%); P less than .02 and P less than .001, respectively. When the recognition mechanisms were bypassed by triggering the cells with lectins in an LDCC assay, we demonstrated an increase of the lytic activity in both groups of patients with respect to the baseline values. However, the cytotoxic capacity observed in untreated patients was significantly lower than that observed in subjects after IFN-alpha therapy and controls (P less than .001). These findings suggest that the impaired NK activity observed in patients with HCL is related to defects both at the target and posttarget cell binding levels.

Adult↗
PubMed1990

Susceptibility to lysis of pulmonary alveolar macrophages by human lymphokine-activated killer cells.

In this study we addressed the question of whether lymphokine-activated killer (LAK) cells, besides killing neoplastic cells, may exert a certain degree of lysis on the normal counterpart; in particular we took into consideration the toxicity against pulmonary alveolar macrophages (PAM). We demonstrated that human LAK cells generated in vitro following incubation of peripheral blood mononuclear cells with recombinant interleukin 2 for 4 days were able to lyse normal PAM in a 4-h 51Cr release assay. Similarly, PAM recovered from patients suffering from nonneoplastic interstitial lung disorders, i.e., sarcoidosis and hypersensitivity pneumonitis, were shown to be susceptible to the cytotoxic function provided by LAK cells. Both autologous and allogeneic PAM were lysed by LAK cells, thus suggesting that the phenomenon we observed does not require a major histocompatibility complex restriction. Preincubation of PAM under study with gamma-interferon did not affect their susceptibility to the lysis mediated by LAK cells. Furthermore, cold target inhibition assay demonstrated that normal PAM could efficiently compete with both NK-sensitive and NK-resistant target lines for the binding sites on LAK cells, thus indicating that the putative receptor(s), or at least the mechanism of target recognition, is shared by PAM and these different target cell lines. The evidence herein provided that LAK cells are cytotoxic to normal, nontransformed PAM points out that the pathogenetic mechanisms involving this self-addressed lytic activity could account for some adverse reactions related to LAK/interleukin 2 immunotherapy.

Cell Line↗