The activation of human endogenous retrovirus K (HERV-K) is implicated in melanoma cell malignant transformation.

Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.

Herpes simplex virus 2 causes apoptotic infection in monocytoid cells.

Increasing evidence indicates that apoptosis can be associated with several viral infections. Here we demonstrate, that infection of monocytoid cells by Herpes simplex virus 2 (HSV-2) resulted, in time- and dose-dependent induction of apoptosis as an exclusive cytopathic effect. The phenomenon was confirmed using four different techniques. Conversely, apoptosis was not observed in the Vero cell line. Virus yield in monocytoid cells was delayed and reduced, although well detectable, in comparison with that observed in Vero cells. Nevertheless, released virions exhibited full infecting capability. Apoptosis induced by HSV-2 was not inhibited by cycloheximide and only partially by an UV-treatment which completely abrogated infectivity. Virus-induced apoptosis was partly inhibited by indomethacin and was associated with a down-regulation of Bcl-2. A similar, but less pronounced, apoptosis-inducing effect in monocytoid cells was also observed with HSV-1 infection. Depending on the target cells, therefore, HSV could complete a cycle of infection which is characterized by apoptosis of infected cells.

Primary macrophages infected by human immunodeficiency virus trigger CD95-mediated apoptosis of uninfected astrocytes.

Infection of macrophages (M/M) by human immunodeficiency virus (HIV) is a main pathogenetic event leading to neuronal dysfunction and death in patients with AIDS dementia complex. Alteration of viability of neurons and astrocytes occurs in vivo even without their infection, thus it is conceivable that HIV-infected M/M may affect viability of such cells even without direct infection. To assess this hypothesis, we studied the effects of HIV-infected M/M on an astrocytic cell-line lacking CD4-receptor expression. Exposure to supernatants of HIV-infected M/M triggers complete disruption and apoptotic death of astrocytic cells. This effect is not related to HIV transmission from infected M/M, because HIV-DNA and p24 production in astrocytic cells remained negative. Apoptotic death of astrocytes is mainly mediated by Fas ligand released in supernatants of HIV-infected M/M (as demonstrated by complete reversal of such phenomenon by adding neutralizing antibodies against CD95 receptor). Treatment of astrocytic cells with recombinant (biologically active) Tat induces < 10% apoptosis, and gp120 was totally ineffective. Treatment of HIV-infected M/M with AZT completely reverses the proapoptotic effect of their supernatants on astrocytes, thus demonstrating that productive virus replication within M/M is required for the induction of astrocytic cell death. Taken together, data suggest that homeostasis of astrocytes may be affected by HIV-infected M/M in the absence of productive infection of target cells. This phenomenon may help to explain the cellular damage found in HIV-infected patients also in areas of the brain not strictly adjacent to HIV-infected M/M.

Spontaneous and anti-Fas-induced apoptosis in lymphocytes from HIV-infected patients undergoing highly active anti-retroviral therapy.

OBJECTIVE: The aim of this study was to investigate susceptibility to spontaneous or anti-Fas-induced apoptosis in peripheral blood mononuclear cells (PBMC) from HIV-positive patients before and during highly active anti-retroviral therapy (HAART). DESIGN: A longitudinal study was performed on 12 evaluable patients on HAART. This cohort was analysed prior to and at week 2, 4, 8, 16 and 24 after beginning HAART. Variations in CD4 and CD8 cells, viral load, susceptibility to spontaneous or anti-Fas-induced apoptosis in the presence of IL-2, IL-4 or IL-12 were studied. Expression of Fas and Bcl-2 were also assessed. METHODS: Levels of HIV RNA were determined by a quantitative reverse transcription-PCR assay. Apoptosis was evaluated by staining isolated nuclei with propidium iodide followed by multiparameter flow cytometry analysis. RESULTS: Spontaneous apoptosis of PBMC was promptly inhibited after the start of treatment. Similarly, anti-Fas-induced apoptosis diminished greatly during treatment. Expression of Fas decreased significantly, while that of Bcl-2 remained statistically unchanged during the first 24 weeks of therapy. Levels of apoptosis correlated inversely to CD4 cell counts and directly to viral load in a highly significant way. Expression of Fas was directly correlated to apoptosis. Interleukin (IL)-2, but not IL-4 or IL-12, protected PBMC of HIV-positive individuals from spontaneous or anti-Fas-induced apoptosis before and during HAART. CONCLUSION: These results suggest that regulation of apoptosis and of Fas expression are involved in immunoreconstitution during HAART.

Efficacy of 3'-azido 3'deoxythymidine (AZT) in preventing HTLV-1 transmission to human cord blood mononuclear cells.

The present study investigated the effect of 3'-azido 3'deoxythymidine (AZT) treatment on in vitro infection of human cord blood mononuclear cells (CBMCs) exposed to HTLV-1 by cocultivation with the MT-2 cell line. Cultures of CBMCs were grown in IL-2 and were either left untreated or were treated with concentrations of AZT ranging from 0.0078 to 32 microM. HTLV-1-infected cultures were monitored at different times of culture by evaluating proliferation activity, cell growth and the presence and expression of HTLV-1 genes. Results showed that untreated cultures infected with HTLV-1 were able to grow for several weeks, while those treated with AZT at 0.03 microM or higher concentrations were limited in their growth capacity. Moreover, the addition of AZT at the moment of infection significantly inhibited cell proliferation in a dose-dependent fashion. In the presence of AZT, detection of proviral DNA and, more remarkably, viral RNA expression were clearly reduced. In addition, treatment with AZT resulted in a noticeable decrease in Tax protein expression. Using treatment with relatively low doses of AZT, effective in exerting an antiviral action, cytotoxicity on CBMCs was not observed, whereas higher doses induced apoptosis in uninfected CBMCs. These data show that CBMCs are protected by AZT against HTLV-1 transmission even at low, non-toxic doses.

Leucocyte rheological properties are altered in patients with diffuse atherosclerosis.

To evaluate whether atherosclerosis may be associated with altered leucocyte rheology, we assessed leucocyte count (by Coulter counter), aggregation (by means of the leukergy test) and expression of adhesion molecules integrin LFA-1 and CD 44 (by means of immunofluorescence staining and flow cytometry) in 9 patients with carotid plus lower limb artery atherosclerosis (group A), 14 patients with carotid atherosclerosis only (group B) and 23 controls without atherosclerosis (group C). The level of LFA-1 (calculated as mean fluorescence channels-MFCs) on neutrophils, lymphocytes and monocytes was significantly higher (p < 0.05) in group A and B patients than in controls (group A-mean +/- SE: 383.77 +/- 9.42 vs 295.45 +/- 5.76; 474.22 +/- 8.86 vs 388.35 +/- 7.84; 457.66 +/- 12.03 vs 396.25 +/- 4.37. Group B: 322.42 +/- 6.36 vs 295.45 +/- 5.76; 421.42 +/- 7.21 vs 388.35 +/- 7.84; 415.71 +/- 7.73 vs 396.25 +/- 4.37, respectively); furthermore, the MFC of LFA-1 on neutrophils was significantly different (p < 0.05) between group A and B patients. The percentage of aggregated leucocytes was significantly higher (p < 0.05) in group A patients (4.46 +/- 1.07) than those in groups B (1.75 +/- 0.38) and C (1.43 +/- 0.25), whereas no significant difference was detected between groups B and C. Leucocyte number and expression of CD44 were not significantly different among the 3 groups. In conclusion, changes in leucocyte rheology are present in patients with atherosclerosis and may contribute to chronic ischaemia.

Umbilical cord blood: the role of apoptosis in the control of CD34+ cell counts.

BACKGROUND AND OBJECTIVE: Cord blood obtained at delivery can be used for hematopoietic precursor cells (HPC) transplantation. The major limit for its success is represented by the low cellular yield of the stem cell population. The objective of this study was to determine the role played by apoptosis in the numerical control of CD34+ cell counts. DESIGN AND METHODS: Umbilical cord blood samples were collected from 15 women at the time of the delivery and cord blood units processed. Cells, collected following 24h and 48h of incubation, were analysed by flow cytometry using the gating strategy. RESULTS: Remarkable levels of apoptosis were detected in the stem cell population and a significant difference between apoptosis mean values at 24h and 48h within CD34+ cells were found. The difference between the percentage of apoptosis in CD34+ cells and that in the remaining population was significant both at 24h and at 48h. CONCLUSIONS: CD34+ cells have a higher likelihood to undergo apoptosis in comparison to the remaining ones present in umbilical cord blood. This process of cellular death plays a major role in the control of CD34+ cell counts in placental blood and influence, for this reason, the possibility of success of a cord blood transplantation.

Impaired apoptosis in mitogen-stimulated lymphocytes of patients with multiple sclerosis.

We investigated the sensitivity to cell death of peripheral blood mononuclear cells (PBMCs) from patients with multiple sclerosis (MS). PBMCs from MS patients, following PHA stimulation, were less sensitive to cell death than those from healthy donors (mean +/- s.e.m., 22.5 +/- 1.9 in MS patients vs 36.5 +/- 2.8 in healthy controls; p = 0.0003). However, when Fas-agonist antibody was added, the increase in respect to apoptosis induced by mitogen alone was even higher in MS patients than in controls. In addition, PHA-activated PBMCs from MS patients showed higher surface expression of Fas than controls, while Bcl-2 expression was decreased. This finding raised the question of whether an impaired generation of apoptotic signals may be contributing to the immune component of MS.

Defective Fas ligand production in lymphocytes from MS patients.

In the present transectional study, Fas ligand (Fas-L) levels, either in membrane or in soluble form, in cells from multiple sclerosis (MS) patients were investigated. Expression of Fas was evaluated after PHA stimulation of peripheral blood mononuclear cells from MS patients with relapsing-remitting or secondary-progressive disease, and in healthy donors. There was statistically significant decreased expression (p = 0.001), as well as release of Fas-L, (p = 0.045) in lymphocytes from MS patients, in comparison with healthy donors. Moreover, levels of Fas-L production were inversely correlated with the EDSS scores of patients in an highly significant way. Impairment of Fas-L release in stimulated PBMC from MS patients might influence the ability to eliminate autoreactive clones in vivo.

Recombinant interferon alpha 2a, thymopentin and low doses of cytosine arabinoside for the treatment of myelodysplastic syndromes: a pilot study.

Eighteen patients (pts) with myelodysplastic syndrome (MDS) were treated with thymopentin (TP) (50 mg subcutaneously for 5 days) and recombinant interferon alpha 2a (rIFN alpha 2a) (3 MU/m2 subcutaneously on the sixth day); the courses were delivered every week. Moreover those pts with > or = 10% blasts in the bone marrow were additionally treated with low dose cytosine arabinoside (LDARAc) (20 mg standard dose, subcutaneously, twice a day for seven days every four weeks). Sixteen pts were finally assessable for response. Seven pts (44%) were classified as good responders, 5 (31%) had a PR; the overall response rate (GR+PR) was 75%. Two pts (12.5%) showed stable disease and the 2 remaining (12.5%) had progressive disease. Six pts with an initial moderate anemia never required supportive care before and during the therapy; in contrast to 10 pts who were transfusion-dependent. After six months of therapy 2 pts decreased their transfusional needs by 50% (1 of them did not receive any transfusion over the following six months of therapy); 2 pts needed no packed red cell infusions and 1 pt decreased his transfusional support by 75%. Five pts kept an unchanged supportive care load. The overall median survival was 12.5 months. Therapy was generally well tolerated with acceptable compliance; the most frequently recorded side effects were neutropenia and thrombocytopenia grade 2-3 among the group receiving LDARAc. However no life-threatening infectious episodes or bleeding were observed. TP, rIFN alpha 2a and LDARAc can be safely administered on an outpatient basis to MDS pts and appears to have significant activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunological and endocrinological disturbances in patients after prolonged coma following head injury.

It has been previously reported that following severe brain damage, a deficit of cellular immunity could be detected in the early phase after the occurence of the lesion. We report here the results of a cross-sectional study on long term effects of severe brain damage on immunological and neuro-endocrine changes in patients who recovered from prolonged coma caused by head injury. Results obtained from post-comatose (PC) patients were compared with those obtained from two control groups made up of spinal-cord injury (SCI) patients and healthy subjects, respectively. The following parameters were studied: lymphomonocyte subsets; interleukin 2 (IL-2) production; natural killer (NK) activity and serum levels of adrenocorticotrophic hormone (ACTH), cortisol, follicle-stimulating hormone (FSH), luteinizing hormone (LH), prolactin, tri-iodothyronine (T3) and thyroxine (T4). With respect to healthy controls the PC1 subgroup, i.e. patients examined 3-6 months after injury, showed a statistically significant decrease in IL-2 production, NK activity and CD25+ lymphocytes. Similar immunological disturbances were observed in SCI but not in the PC2 subgroup, i.e. patients examined later than 6 months after injury. The same sub-group of PC1 patients showed high serum levels of cortisol and PRL. These results could be related to the immunological status and may be interpreted as a transient but prolonged condition of chronic stress or "chronic alarm reaction". Copyright Rapid Science Ltd

Mechanism of production of interferon-gamma: role of arachidonic acid metabolites.

The effects of arachidonic acid metabolites on mitogen-induced interferon (IFN)-gamma production by human peripheral blood mononuclear cells (PBMC) were examined. Both prostaglandins E2 (PGE2) and leukotrienes B4 (LTB4) were produced after macrophage activation stimulated by galactose oxidase (GO) and Staphylococcal enterotoxin B (SEB), two well known inducers of IFN-gamma. To test the involvement of PGE2 and LTB4 in IFN-gamma production, GO- and SEB-activated PBMC were treated with two inhibitors of cyclooxygenase (aspirin and indomethacin) and with an inhibitor of lipoxygenase [nordihydroguaiaretic acid (NDGA)]. The results of these experiments showed that aspirin and indomethacin cause a marked increase of IFN-gamma production by GO- and SEB-activated PBMC. On the contrary, NDGA treatment reduced IFN-gamma production induced by the same agents. Moreover, whereas the addition of exogenous PGE2 reduces IFN gamma production, the addition of exogenous LTB4 does not affect IFN-gamma production. Taken together these findings indicate that arachidonic acid metabolites, produced during mitogenic activation, are involved in the regulation of IFN-gamma production and suggest that, in our system, LTB4 exerts a positive modulating signal while PGE2 represents a negative signal.

Susceptibility to influenza A virus infection in mice immunosuppressed with cyclophosphamide.

It is known that immunocompromised hosts show an enhanced susceptibility to microbial infections. Among these, viral infections represent a particular problem because of the lack of really efficient antiviral drugs. In the present report we have studied the effect of pharmacological immunosuppression with cyclophosphamide (CY) in virus infection in vivo, using Balb/c mice infected with influenza A virus (PR8). At the dose of 0.1 hemagglutinating units of viral inoculum, intranasal administration of PR8 virus caused the death of 50 to 60% of the animals within a period of 3-10 days. A single injection of CY (200 mg/kg) significantly increased mouse mortality to 90%, when PR8 virus challenge was performed 4 days after chemotherapy pretreatment. When the PR8 virus infection was performed at different times after CY-treatment, a similar appreciable effect was not observed. Severe alterations of some immunological parameters such as NK activity and analysis of lymphoid spleen cell subsets were related to the increased susceptibility to virus infection.

Interaction between thymic hormones and other immunomodulatory agents.

We have investigated the effects of a combination in vivo treatment with thymosin alpha 1 (TA1) and murine alpha/beta interferon (IFN) on natural killer (NK) activity and on tumor growth in B-16 melanoma tumor-bearing mice. The results indicated that treatment with a single injection of IFN (3 x 10(4] 24 h before testing, enhanced NK activity in tumor-bearing mice if the test was performed 10 days after tumor inoculation, when the animals have normal NK responsiveness. On the other hand, the same treatment led to lower or no improvement of NK responses when the assay was performed 14 or 18 days after tumor inoculation, at a time when tumor growth caused NK-suppression. However, combination treatment with TA1 (200 micrograms/kg) for 4 days, followed by IFN was found to restore normal NK cell activity. On the other hand primary tumor growth was unaffected by combination therapy, while the same treatment with TA1 and IFN was able to significantly prolong survival time of B-16 tumor-bearing mice, when administered starting on day 6 after tumor inoculation. The last evidence, together with results on NK activity stimulation, indicates that combination therapy with TA1 and IFN could be an interesting approach to cancer immunotherapy.

D(-)lentiginosine-induced apoptosis involves the intrinsic pathway and is p53-independent.

We have recently found that D(-)lentiginosine, a synthetic iminosugar exerting glucosidase inhibitory activity, but not its natural enantiomer lentiginosine, is endowed with an unexpected, pro-apoptotic activity. Here, we investigated mechanisms involved in apoptosis induced by D(-)lentiginosine in MOLT-3, HT-29 and SH-SY5Y tumour cell lines. The results showed that D(-)lentiginosine increased caspase 9 expression at 18 h in all the cell lines from 1.5-3.1 folds. Cytochrome c in the cytoplasm was found to be increased from 2.3-2.6 folds in treated cells with respect to control cells. These effects were accompanied by a remarkable collapse of the mitochondrial membrane potential and by the downregulation of anti-apoptotic genes, as well as the upregulation of pro-apoptotic genes of the Bcl-2 family. U937Bcl-2 transfectants, highly expressing Bcl-2, were reluctant to undergo apoptosis even following treatment with 500 µM D(-)lentiginosine, whereas apoptosis by D(-)lentiginosine was induced also in U937 cells, naturally deficient in P53. Thus, our study establishes that the enantiomer of a natural iminosugar is endowed with a possible anti-tumorigenic effect that might be ascribed not only to their capacity to inhibit glycosidases but also to other unknown mechanisms. These data encourage further investigation on similar compounds to make them an interesting platform for the generation of new anticancer drugs.

Inhibition of NF-κB activation sensitizes U937 cells to 3'-azido-3'-deoxythymidine induced apoptosis.

In this study, we investigated molecular mechanisms underlying low susceptibility to apoptosis induced by the nucleoside analog azidothymidine (AZT) and the role of nuclear factor-κB (NF-κB) activation in these phenomena. A preliminary screening in different cell lines indicated U937 monocytic cell line as suitable to this purpose. Treatment of U937 cells even with suprapharmacological concentrations of AZT induced only moderate levels of apoptosis. Surprisingly, SuperArray analysis showed that AZT induced the transcriptional activity of both pro- and anti-apoptotic genes. Interestingly, moreover, several genes upregulated by AZT were NF-κB related. In fact, AZT, after an initial inhibition of NF-κB activation with respect to control, induced a transient, but consistent, increase in NF-κB-binding activity. Inhibition of NF-κB activation in U937 cells, stably transfected with a dominant-negative IκBα or by pharmacological treatment, sensitized them to apoptosis induced by AZT and impaired the upregulation of anti-apoptotic genes in response to AZT treatment, with respect to control cells. These results indicate that NF-κB activation by AZT has a role in protecting target cells from apoptotic cell death, improving our understanding of the toxicology and the therapeutic usage of this drug.