Different sensitivity to apoptosis in cells of monocytic or lymphocytic origin chronically infected with human immunodeficiency virus type-1

Apoptotic death of CD4+ T lymphocytes is a major cause of the immunodeficiency caused by human immunodeficiency virus (HIV), but it is still unclear how this process precisely occurs. To characterize a potentially useful cellular model, we have analyzed the tendency of chronically HIV-infected CD4+ human cell lines of different origin to undergo apoptosis. We studied ACH-2 and U1 lines, derived from the CD4+ T-cell A301 and the promonocytic U937 cell lines, respectively, and induced apoptosis via several stimuli that trigger different pathways. Their capacity to regulate plasma membrane CD95 expression and to produce soluble CD95 was also analyzed. Using staurosporine, TNF-alpha plus cycloheximide, and gamma-radiations, we observed that ACH-2 were more sensitive to programmed cell death than A301, while U1 were less sensitive than U937. Both infected cell types had a lower sensitivity to CD95-induced apoptosis; the analysis of changes in mitochondrial membrane potential corroborated these observations. Plasma membrane CD95 was similarly regulated in all cell types, which, however, presented a different capacity to produce soluble CD95 molecules. Our in vitro results may offer a new perspective for developing further studies on the pathogenesis of HIV infection. A chronically infected cell line of lymphocytic origin is more susceptible to apoptosis than its parental cell type, while infected monocytic cells are less sensitive than their uninfected counterpart. Thus, it is possible to hypothesize that one of the reasons by which circulating monocytes survive and represent a viral reservoir is the capacity of HIV to decrease the sensitivity to apoptosis of this cell type. However, further studies on ex-vivo collected fresh cells, as well as on other cell lines, are urgently needed to confirm such hypothesis.     

The reduction in electroporation voltages by the addition of a surfactant to planar lipid bilayers.

The effects of a nonionic surfactant, octaethyleneglycol mono n-dodecyl ether (C12E8), on the electroporation of planar bilayer lipid membranes made of the synthetic lipid 1-pamitoyl 2-oleoyl phosphatidylcholine (POPC), was studied. High-amplitude ( approximately 100-450 mV) rectangular voltage pulses were used to electroporate the bilayers, followed by a prolonged, low-amplitude ( approximately 65 mV) voltage clamp to monitor the ensuing changes in transmembrane conductance. The electroporation thresholds of the membranes were found for rectangular voltage pulses of given durations. The strength-duration relationship was determined over a range from 10 micros to 10 s. The addition of C12E8 at concentrations of 0.1, 1, and 10 microM to the bath surrounding the membranes decreased the electroporation threshold monotonically with concentration for all durations (p < 0.0001). The decrease from control values ranged from 10% to 40%, depending on surfactant concentration and pulse duration. For a 10-micros pulse, the transmembrane conductance 150 micros after electroporation (G150) increased monotonically with the surfactant concentration (p = 0.007 for 10 microM C12E8). These findings suggest that C12E8 incorporates into POPC bilayers, allowing electroporation at lower intensities and/or shorter durations, and demonstrate that surfactants can be used to manipulate the electroporation threshold of lipid bilayers.     

Mitochondrial Membrane Potential and DNA Stainability in Human Sperm Cells: A Flow Cytometry Analysis with Implications for Male Infertility

Sperm cells from control donors of proven fertility and men from barren couples were studied by conventional procedures, i.e., light microscopy as well as flow cytometry. Light microscopy analysis of semen included the measurement of spermatozoa concentration, morphology, and motility. All the men from barren couples were asthenozoospermic at the conventional analysis of semen samples. Flow cytometry was applied to study two important parameters of sperm cells: mitochondrial membrane potential (MMP) assessed by the cationic dye JC-1 and DNA stainability with propidium iodide (PI). JC-1 staining was more reliable than the classical procedure used for this purpose, i.e., rhodamine 123 (Rh123) staining, and allowed us to show a positive correlation between MMP and spermatozoa motility. Regarding DNA analysis, a higher relative percentage of immature spermatozoa, showing a high accessibility of DNA to the intercalating PI fluorochrome, was found in men from barren couples compared to donors of proven fertility. The relative percentage of immature spermatozoa was significantly higher in semen from oligoasthenozoospermic subjects. Moreover, a positive correlation was found between immature spermatozoa, as evaluated by PI staining, and cells with depolarized mitochondria, as evaluated by JC-1 staining, suggesting that spermatozoa defective for nuclear maturity could be functionally defective cells. No correlation between immature spermatozoa determined by FCM and immature spermatozoa determined by light microscopy was found, suggesting that these two techniques assess sperm cell maturity at different levels.     

Fine Exon–Intron Structure of the Fanconi Anemia Group A (FAA) Gene and Characterization of Two Genomic Deletions

Fanconi anemia (FA) is a genetically heterogeneous disease with at least eight genes on the basis of complementation groups (FAAtoFAH). The analysis of theFAAgene in patients suggested the existence of deletions, none of which have thus far been characterized at the genomic level. A detailed restriction map of theFAAgene with the fine localization of its 43 exons is reported in this paper. We also describe the first two genomic deletions, one of 5.0 kb and another of at least 120 kb. The former was likely the result of a recombination between relatedAlusequences. Since these interspersed repeats could generate deletions and insertions by mispairing, rearrangements of this gene are a possibility in those FA families in whichFAAmutations have not been identified.     

Dexamethasone modulates interleukin-12 production by inducing monocyte chemoattractant protein-1 in human dendritic cells

Glucocorticoids have long been used as first-line immunosuppressants, although their precise mechanism of action has not been fully elucidated yet. This study evaluated the gene and protein expression of monocyte chemoattractant protein-1 (MCP-1), and its relationship with interleukin-12 and interleukin-10 synthesis, in human monocyte-derived dendritic cells exposed to dexamethasone. Dendritic cells were differentiated in the presence or in the absence of dexamethasone and then activated by IFN-gamma+soluble CD40 ligand; the gene and protein expression of target cytokines was measured by real-time PCR and ELISA, respectively. Our results showed that dexamethasone-primed mature dendritic cells expressed low levels of interleukin-12, and, at the opposite, high levels of interleukin-10 and MCP-1. Transfection experiments confirmed the ability of dexamethasone to activate MCP-1 gene promoter. Dexamethasone increased also MCP-2, but not MCP-3 synthesis, and the gene expression of CC chemokine receptor-2 by mature dendritic cells. The addition of anti-MCP-1 blocking antibody depressed MCP-1 release, and increased interleukin-12 production in dexamethasone-treated dendritic cells, thus demonstrating that interleukin-12 downregulation is largely dependent on MCP-1 overexpression. Our findings suggest that the induction of MCP expression in human dendritic cells by dexamethasone, and the amplification of cell response via the upregulation of the chemokine cognate receptor, may be critical to inhibit type 1 T-helper-biased immune response and, possibly, to favor type 2 T-helper-skewed response.     

Analysis of the Dynamics of a Bacillus subtilis Spore Germination Protein Complex during Spore Germination and Outgrowth

Germination of Bacillus subtilis spores is normally initiated when nutrients from the environment interact with germinant receptors (GRs) in the spores'' inner membrane (IM), in which most of the lipids are immobile. GRs and another germination protein, GerD, colocalize in the IM of dormant spores in a small focus termed the “germinosome,” and this colocalization or focus formation is dependent upon GerD, which is also essential for rapid GR-dependent spore germination. To determine the fate of the germinosome and germination proteins during spore germination and outgrowth, we employed differential interference microscopy and epifluorescence microscopy to track germinating spores with fluorescent fusions to germination proteins and used Western blot analyses to measure germination protein levels. We found that after initiation of spore germination, the germinosome foci ultimately changed into larger disperse patterns, with ≥75% of spore populations displaying this pattern in spores germinated for 1 h, although >80% of spores germinated for 30 min retained the germinosome foci. Western blot analysis revealed that levels of GR proteins and the SpoVA proteins essential for dipicolinic acid release changed minimally during this period, although GerD levels decreased ∼50% within 15 min in germinated spores. Since the dispersion of the germinosome during germination was slower than the decrease in GerD levels, either germinosome stability is not compromised by ∼2-fold decreases in GerD levels or other factors, such as restoration of rapid IM lipid mobility, are also significant in germinosome dispersion as spore germination proceeds.     

Genetic polymorphisms of Fas (CD95) and Fas ligand (CD178) influence the rise in CD4+ T cell count after antiretroviral therapy in drug-naïve HIV-positive patients

Fas and Fas ligand (FasL) are the main genes that control cell death in the immune system. Indeed, they are crucial for the regulation of T lymphocyte homeostasis because they can influence cell proliferation. A strong debate exists on the importance of Fas/FasL system during HIV infection, which is characterized by the loss of CD4+ T cells directly, or indirectly, caused by the virus. To investigate whether the genetic background of the host plays a role in the immunoreconstitution, we studied the influence of different Fas and FasL polymorphisms on CD4+ T lymphocyte count and plasma viral load following initiation of highly active antiretroviral therapy (HAART) in drug-naïve HIV+ patients. We studied 131 individuals, who were compared to 136 healthy donors. Statistical analysis was performed by using X ² test, Fischer's Exact Test, and analysis for repeated measurements. The group of HIV+ patients had an unexpected lower frequency of FasLnt169 polymorphism (delT allele) than healthy controls (p=0.039). We then observed no significant differences in the immune reconstitution, in terms of CD4+ T cell increase, when the influence of single alleles of the gene Fas or FasL was considered. However, the combination of some polymorphisms of Fas or FasL significantly influenced CD4+ T cell production and viral load decrease, showing that these genes can play a role in the immunoreconstitution triggered by antiretroviral therapy.     

Essential requirement of reduced glutathione (GSH) for the anti-oxidant effect of the flavonoid quercetin

We have analyzed the anti- or pro-oxidant effects of the flavonoid quercetin (QU) by evaluating, in U937 cell line, hydrogen peroxide (H(2)O(2)), superoxide anion reduced glutathione (GSH) content, mitochondrial membrane potential, DNA content, phosphatidylserine exposure on the outer face of the plasma membrane and cell viability. Polychromatic flow cytometry was used to evaluate in the same cells several functional parameters. For short periods of treatment QU exerted an anti-oxidant effect (decrease in H(2)O(2) levels), whereas for long periods it showed a pro-oxidant activity (increase in ). In these conditions, GSH content was reduced, and this correlated with a lack of anti-oxidant activity of QU, which in turn could be correlated with proapoptotic activity of this molecule. Thus, QU can exert different effects (anti-/prooxidant) depending on exposure times and oxidative balance, and in particular on stores of GSH.