Stable changes in CD4+ T lymphocyte miRNA expression after exposure to HIV-1.

MicroRNAs (miRNAs) inhibit HIV-1 expression by either modulating host innate immunity or by directly interfering with viral mRNAs. We evaluated the expression of 377 miRNAs in CD4(+) T cells from HIV-1 élite long-term nonprogressors (éLTNPs), naive patients, and multiply exposed uninfected (MEU) patients, and we observed that the éLTNP patients clustered with naive patients, whereas all MEU subjects grouped together. The discriminatory power of miRNAs showed that 21 miRNAs significantly differentiated éLTNP from MEU patients and 23 miRNAs distinguished naive from MEU patients, whereas only 1 miRNA (miR-155) discriminated éLTNP from naive patients. We proposed that miRNA expression may discriminate between HIV-1-infected and -exposed but negative patients. Analysis of miRNAs expression after exposure of healthy CD4(+) T cells to gp120 in vitro confirmed our hypothesis that a miRNA profile could be the result not only of a productive infection but also of the exposure to HIV-1 products that leave a signature in immune cells. The comparison of normalized Dicer and Drosha expression in ex vivo and in vitro condition revealed that these enzymes did not affect the change of miRNA profiles, supporting the existence of a Dicer-independent biogenesis pathway.

The monocytic population in chronic lymphocytic leukemia shows altered composition and deregulation of genes involved in phagocytosis and inflammation.

Macrophages reside in tissues infiltrated by chronic lymphocytic leukemia B cells and the extent of infiltration is associated with adverse prognostic factors. We studied blood monocyte population by flow cytometry and whole-genome microarrays. A mixed lymphocyte reaction was performed to evaluate proliferation of T cells in contact with monocytes from patients and normal donors. Migration and gene modulation in normal monocytes cultured with CLL cells were also evaluated. The absolute number of monocytes increased in chronic lymphocytic leukemia patients compared to the number in normal controls (792 ± 86 cells/µL versus 485 ± 46 cells/µL, P=0.003). Higher numbers of non-classical CD14(+)CD16(++) and Tie-2-expressing monocytes were also detected in patients. Furthermore, we performed a gene expression analysis of monocytes in chronic lymphocytic leukemia patients, showing up-regulation of RAP1GAP and down-regulation of tubulins and CDC42EP3, which would be expected to result in impairment of phagocytosis. We also detected gene alterations such as down-regulation of PTGR2, a reductase able to inactivate prostaglandin E2, indicating immunosuppressive activity. Accordingly, the proliferation of T cells in contact with monocytes from patients was inhibited compared to that of cells in contact with monocytes from normal controls. Finally, normal monocytes in vitro increased migration and up-regulated CD16, RAP1GAP, IL-10, IL-8, MMP9 and down-regulated PTGR2 in response to leukemic cells or conditioned media. In conclusion, altered composition and deregulation of genes involved in phagocytosis and inflammation were found in blood monocytes obtained from chronic lymphocytic leukemia patients, suggesting that leukemia-mediated "education" of immune elements may also include the establishment of a skewed phenotype in the monocyte/macrophage population.

Immunological advantages of everolimus versus cyclosporin A in liver-transplanted recipients, as revealed by polychromatic flow cytometry.

Several immunosuppressive drugs with different mechanisms of action are available to inhibit organ rejection after transplant. We analyzed different phenotypic and functional immunological parameters in liver-transplanted patients who received cyclosporin A (CsA) or Everolimus (Evr). In peripheral blood mononuclear cells (PBMC) from 29 subjects receiving a liver transplant and treated with two different immunosuppressive regimens, we analyzed T cell activation and differentiation, regulatory T cells (Tregs) and Tregs expressing homing receptors such as the chemokine receptor CXCR3. T cell polyfunctionality was studied by stimulating cells with the superantigen staphylococcal enterotoxin B (SEB), and measuring the simultaneous production of interleukin (IL)-2 and interferon (IFN)-γ, along with the expression of a marker of cytotoxicity such as CD107a. The analyses were performed by polychromatic flow cytometry before transplantation, and at different time points, up to 220 days after transplant. Patients taking Evr had a higher percentage of total CD4⁺ and naïve CD4⁺ T cells than those treated with CsA; the percentage of CD8⁺ T cells was lower, but the frequency of naïve CD8⁺ T cells higher. Patients taking Evr showed a significantly higher percentage of Tregs, and Tregs expressing CXCR3. After stimulation with SEB, CD8⁺ T cells from Evr-treated patients displayed a lower total response, and less IFN-γ producing cells. The effects on the immune system, such as the preservation of the naïve T cell pool and the expansion of Tregs (that are extremely useful in inhibiting organ rejection), along with the higher tolerability of Evr, suggest that this drug can be safely used after liver transplantation, and likely offers immunological advantages.

NK-cell phenotype at interruption underlies widely divergent duration of CD4+-guided antiretroviral treatment interruption.

Long-term side effects may represent a relevant burden of antiretroviral treatment (ART) in HIV-infected patients with good CD4 immune reconstitution over extended time spans. CD4-guided treatment interruption (TI) has been evaluated to address this point and may result in a wide spectrum of time off ART in different patient cohorts. We studied whether differences in innate immune responses, in particular NK cells, are associated to patterns of longer (LoTI) or a shorter (ShTI) TI. Clinical cohort parameters were analyzed on a group of patients widely diverging for TI duration (<9 versus >18 months) on samples before TI, including NK-cell analysis and function by natural cytotoxicity receptor (NCR)-triggered γ-IFN production. Although persistently reduced NCR expression (NKp30) and function were observed in both LoTI and ShTI patients on ART compared with healthy donors, relevant differences were observed at baseline TI in those patients who subsequently developed LoTI course. Lower expression of NKG2D and NKp46 on NK cells. This also translates in reduced γ-IFN production in redirected functional assays. Thus, differences in innate immune balance exist during ART, may be associated to differential control of HIV infection and their understanding could explain clinical differences in individual patients that are not reflected by CD4(+) cell counts alone.

Quercetin and cancer chemoprevention.

Several molecules present in the diet, including flavonoids, can inhibit the growth of cancer cells with an ability to act as "chemopreventers". Their cancer-preventive effects have been attributed to various mechanisms, including the induction of cell-cycle arrest and/or apoptosis as well as the antioxidant functions. The antioxidant activity of chemopreventers has recently received a great interest, essentially because oxidative stress participates in the initiation and progression of different pathological conditions, including cancer. Since antioxidants are capable of preventing oxidative damage, the wide use of natural food-derived antioxidants is receiving greater attention as potential anti-carcinogens. Among flavonoids, quercetin (Qu) is considered an excellent free-radical scavenging antioxidant, even if such an activity strongly depends on the intracellular availability of reduced glutathione. Apart from antioxidant activity, Qu also exerts a direct, pro-apoptotic effect in tumor cells, and can indeed block the growth of several human cancer cell lines at different phases of the cell cycle. Both these effects have been documented in a wide variety of cellular models as well as in animal models. The high toxicity exerted by Qu on cancer cells perfectly matches with the almost total absence of any damages for normal, non-transformed cells. In this review we discuss the molecular mechanisms that are based on the biological effects of Qu, and their relevance for human health.

Quercetin inhibits lymphocyte activation and proliferation without inducing apoptosis in peripheral mononuclear cells.

Toxicity of chemotherapeutic drugs towards normal cells is a serious side effect of cancer treatment. Thus, finding of molecules with low toxicity for normal cells is crucial. Several natural compounds, such as flavonoid quercertin, are receiving a growing attention as "chemopreventers". Quercetin kills tumour-derived cell lines, but little is known about its effects on normal cells. Here we show that although quercetin exerts a higher apoptotic potential on leukemic cell lines than on peripheral blood mononuclear cells (PBMCs) and does not sensitize PBMCs to CD95-induced apoptosis, it is able to inhibit normal immune functions such as T cell proliferation and activation. Quercetin sensitivity is independent on cell cycle progression since it was not abrogated in serum-starved U937 cells, nor proliferating PBMCs underwent apoptosis after quercetin treatment. However, quercetin prevented PHA-induced PBMC proliferation and SEB-induced upregulation of activation markers. Our data suggest that quercetin, while incapable of inducing apoptosis in normal cells under several conditions, could interfere with effector T cell function.

T cell activation but not polyfunctionality after primary HIV infection predicts control of viral load and length of the time without therapy.

OBJECTIVE: Immune changes occurring after primary HIV infection (PHI) have a pivotal relevance. Our objective was to characterize the polyfunctionality of immune response triggered by PHI, and to characterize immune activation and regulatory T cells, correlating such features to disease progression. PATIENTS AND METHODS: We followed 11 patients experiencing PHI for 4 years. By polychromatic flow cytometry, we studied every month, for the first 6 months, T lymphocyte polyfunctionality after cell stimulation with peptides derived from HIV-1 gag and nef. Tregs were identified by flow cytometry, and T cell activation studied by CD38 and HLA-DR expression. RESULTS: An increase of anti-gag and anti-nef CD8+ specific T cells was observed 3 months after PHI; however, truly polyfunctional T cells, also able to produce IL-2, were never found. No gross changes in Tregs were present. T lymphocyte activation was maximal 1 and 2 months after PHI, and significantly decreased in the following period. The level of activation two months after PHI was strictly correlated to the plasma viral load 1 year after infection, and significantly influenced the length of period without therapy. Indeed, 80% of patients with less than the median value of activated CD8+ (15.5%) or CD4+ (0.9%) T cells remained free of therapy for >46 months, while all patients over the median value had to start treatment within 26 months. CONCLUSIONS: T cell activation after PHI, more than T cell polyfunctionality or Tregs, is a predictive marker for the control of viral load and for the time required to start treatment.

CD4+ T-cell differentiation, regulatory T cells and gag-specific T lymphocytes are unaffected by CD4-guided treatment interruption and therapy resumption.

OBJECTIVES: Despite limiting exposure to antiretroviral drugs, structured treatment interruptions can influence multiple aspects of T-cell immunity, particularly those regarding CD4(+) T lymphocytes. We evaluated the impact of CD4-guided treatment interruption (CD4-GTI) and treatment resumption on regulatory T cells (Tregs), T-lymphocyte activation, differentiation and polyfunctional gag-specific response. METHODS: Patients were analyzed just prior to treatment interruption, at 2 and 6 months after treatment interruption, just prior to treatment resumption and at 2 and 6 months after treatment resumption. Thawed peripheral blood mononuclear cells were stained immediately for phenotype analysis or stimulated with HIV-gag peptides and analyzed by polychromatic flow cytometry. RESULTS: Treatment interruption resulted in a CD4(+) cell count decrease and plasma viral load (pVL) increase, but did not preclude a good immune reconstitution and a complete suppression of pVL after treatment resumption. Treatment interruption did not influence CD4(+) T-cell differentiation and Treg subsets. During treatment interruption, gag-specific CD4(+) T cells were not lost, although the frequency of HIV-specific CD8(+) cells increased. Most gag-specific CD4(+) T cells were potentially cytotoxic (CD107a(+)) and were not influenced by pVL or by HAART. Most helper (CD154(+)) gag-specific CD4(+) T lymphocytes did not produce interferon-γ or interleukin-2. CONCLUSION: CD4-GTI did not cause depletion of memory cells, Tregs or HIV-specific CD4(+) cells and, on the contrary, could induce HIV-specific responses. If guided by CD4(+) T-cell count, treatment interruption does not provoke irreversible immune damages.

Increased plasma levels of extracellular mitochondrial DNA during HIV infection: a new role for mitochondrial damage-associated molecular patterns during inflammation.

HIV infection is characterized by a chronic inflammatory state. Recently, it has been shown that mitochondrial DNA (mtDNA) released from damaged or dead cells can bind Toll like receptor-9 (TLR9), an intracellular receptor that responds to bacterial or viral DNA molecules. The activation of TLR9 present within monocytes or neutrophils results in a potent inflammatory reaction, with the production of proinflammatory cytokines. We measured plasma levels of mtDNA in different groups of HIV(+) patients, i.e., those experiencing an acute HIV infection (AHI), long term non progressors (LTNP), late presenters (LP) taking antiretroviral therapy for the first time, and healthy controls. We found that in AHI and LP mtDNA plasma levels were significantly higher than in healthy individuals or in LTNP. Plasma mtDNA levels were not correlated to peripheral blood CD4(+) T cell count, nor to markers of immune activation, but had a significant correlation with plasma viral load, revealing a possible role for mtDNA in inflammation, or as a biomarker of virus-induced damage.

Interfering with ROS Metabolism in Cancer Cells: The Potential Role of Quercetin.

A main feature of cancer cells, when compared to normal ones, is a persistent pro-oxidative state that leads to an intrinsic oxidative stress. Cancer cells have higher levels of reactive oxygen species (ROS) than normal cells, and ROS are, in turn, responsible for the maintenance of the cancer phenotype. Persistent ROS stress may induce adaptive stress responses, enabling cancer cells to survive with high levels of ROS and maintain cellular viability. However, excessive ROS levels render cancer cells highly susceptible to quercetin, one of the main dietary flavonoids. Quercetin depletes intracellular glutathione and increases intracellular ROS to a level that can cause cell death.

Cytotoxic granule release dominates gag-specific CD4+ T-cell response in different phases of HIV infection.

BACKGROUND: The activity of virus-specific T lymphocytes, among which those capable of a polyfunctional response against the viral protein gag, is crucial to control HIV infection. OBJECTIVE: The objective of this study is to investigate the polyfunctionality of gag-specific T cells in different phases of HIV infection, analyzing markers related to T-helper cell 1 (Th1) and degranulation/cytotoxicity, and the production of Th1 cytokines in peripheral blood lymphocytes from patients experiencing an acute primary infection, long-term nonprogressors, patients naive for antiretroviral drugs, and patients taking HAART. MATERIALS AND METHODS: Cells were stimulated with a pool of gag-derived peptides or with a superantigen (staphylococcal enterotoxin B). Using eight-color polychromatic flow cytometry, we analyzed the expression of interleukin-2, interferon-gamma, CD154, and CD107a by CD4 and CD8 T cells. RESULTS: The main finding was that in all HIV-positive patients, about half gag-specific CD4 T cells were CD107a, that is, able to degranulate. CD4CD154 cells unable to produce Th1 cytokines were the second most represented population. Truly polyfunctional CD4 T cells were very rare and present only in a few long-term nonprogressors. Superantigen stimulation showed that CD4 T lymphocytes from all patients displayed a typical Th response, including interleukin-2 and interferon-gamma production, lacking CD107a expression. CONCLUSION: In all the aforementioned phases of HIV infection, the large majority of gag-specific CD4 T lymphocytes cannot be identified by the sole expression of interleukin-2 and interferon-gamma, which is early impaired. Degranulation and helper functions other than Th1 cytokine production are the predominant features of HIV-specific CD4 lymphocytes.

Simultaneous analysis of reactive oxygen species and reduced glutathione content in living cells by polychromatic flow cytometry.

Reactive oxygen species (ROS) are continuously produced in the cell as a consequence of aerobic metabolism, and are controlled by several antioxidant mechanisms. An accurate measurement of ROS is essential to evaluate the redox status of the cell, or the effects of molecules with the pro-oxidant or antioxidant activity. Here we report a cytofluorimetric technique for measuring simultaneously, at the single-cell level, hydrogen peroxide and superoxide anion, reduced glutathione (a main intracellular antioxidant) and cell viability. The staining is performed with the fluorescent dyes 2',7'-dichlorodihydrofluorescein diacetate (H2DCFH-DA), hydroethidine (HE), monobromobimane (MBB) and TO-PRO-3. This analysis is possible with new-generation flow cytometers equipped with several light sources (in our case, four lasers and an UV lamp), which excite different fluorochromes. This approach is extremely useful to study the balance between ROS content and antioxidants in cells receiving different stimuli, and to analyze the relationship between oxidative stress and cell death.