Homira Behbahani

The amyloid β-peptide is imported into mitochondria via the TOM import machinery and localized to mitochondrial cristae

The amyloid β-peptide (Aβ) has been suggested to exert its toxicity intracellularly. Mitochondrial functions can be negatively affected by Aβ and accumulation of Aβ has been detected in mitochondria. Because Aβ is not likely to be produced locally in mitochondria, we decided to investigate the mechanisms for mitochondrial Aβ uptake. Our results from rat mitochondria show that Aβ is transported into mitochondria via the translocase of the outer membrane (TOM) machinery. The import was insensitive to valinomycin, indicating that it is independent of the mitochondrial membrane potential. Subfractionation studies following the import experiments revealed Aβ association with the inner membrane fraction, and immunoelectron microscopy after import showed localization of Aβ to mitochondrial cristae. A similar distribution pattern of Aβ in mitochondria was shown by immunoelectron microscopy in human cortical brain biopsies obtained from living subjects with normal pressure hydrocephalus. Thus, we present a unique import mechanism for Aβ in mitochondria and demonstrate both in vitro and in vivo that Aβ is located to the mitochondrial cristae. Importantly, we also show that extracellulary applied Aβ can be internalized by human neuroblastoma cells and can colocalize with mitochondrial markers. Together, these results provide further insight into the mitochondrial uptake of Aβ, a peptide considered to be of major significance in Alzheimers disease.

Repertoire of Chemokine Receptor Expression in the Female Genital Tract : Implications for Human Immunodeficiency Virus Transmission

Sexually transmitted diseases, genital ulcer disease, and progesterone therapy increase susceptibility to lentivirus transmission. Infection of cells by human immunodeficiency virus (HIV) is dependent on expression of specific chemokine receptors known to function as HIV co-receptors. Quantitative kinetic reverse transcription-polymerase chain reaction was developed to determine the in vivo expression levels of CCR5, CXCR4, CCR3, CCR2b, and the cytomegalovirus-encoded US28 in peripheral blood mononuclear cells and cervical biopsies from 12 women with and without sexually transmitted diseases, genital ulcer disease, and progesterone-predominant conditions. Our data indicate that CCR5 is the major HIV co-receptor expressed in the female genital tract, and CXCR4 is the predominantly expressed HIV co-receptor in peripheral blood. CCR5 mRNA expression in the ectocervix was 10-fold greater than CXCR4, 20-fold greater than CCR2b, and 100-fold greater than CCR3. In peripheral blood, CXCR4 expression was 1.5-fold greater than CCR5, 10-fold greater than CCR2b, and 15-fold greater than CCR3. US28 was not expressed in cervical tissue despite expression in peripheral blood mononuclear cells from five individuals. CCR5 was significantly increased (p < 0.02) in biopsies from women with sexually transmitted diseases and others who were progesterone predominant. In vitro studies demonstrate that progesterone increases CCR5, CXCR4, and CCR3 expression and decreases CCR2b expression in lymphocytes and monocytes/macrophages. Characterization of chemokine receptors at the tissue level provides important information in identifying host determinants of HIV-1 transmission.

Early reduction of immune activation in lymphoid tissue following highly active HIV therapy.

Objective:To evaluate immune reconstitution within HIV-infected lymphoid tissue during highly active antiretroviral therapy (HAART). Design and methods:In situ cellular responses were studied in sequential tonsillar biopsies in three asymptomatic HIV-infected (CD4 cells greater than 400 × 106/l) antiretroviral treatment-naive volunteers enrolled in a clinical trial to determine the early effect of HAART. Computerized image analysis was used to study immunohistochemically stained sequential tonsil sections for the patterns of local cytokine production, chemokine receptor expression and cellular distribution. Replicate quantitative assessments of samples before and after 4 weeks of therapy were used for the evaluation of drug effects and compared with four uninfected controls. Tonsillar HIV proviral-DNA was determined by fluorescent in situ 5′-nuclease assay. Results:HIV-infected tonsil tissue was characterized by extensive pro-inflammatory and type 1 cytokine expression. A five- to 15-fold elevation of interleukin (IL)-1α, IL-12, IL-2 and interferon (IFN)-γ protein expression was found compared with controls, and each encompassed a mean of at least 4.5% of the tissue compartment. This was reduced by 20–90% in all individuals after 4 weeks of HAART. In contrast, type 2 cytokine expression (IL-4, IL-10), plus tumour necrosis factor (TNF)-α, remained low throughout the study. HAART reduced, by 40%, the expression of HIV co-receptors, CCR5 and CXCR4, which initially were elevated four to six times over the control values. In addition, the myelomonocytic inflammatory proteins, CD68 and calprotectin, diminished by 26–83% after therapy. The HIV RNA was reduced to undetectable levels in plasma by HAART. However, a large pool of tonsil cells (2–7%), remained HIV DNA positive after 4 weeks of therapy. Conclusions:Although immune activation may be the direct consequence of HIV replication, HAART-associated reconstitution begins with a reduction in inflammatory cytokine production which precedes the elimination of local proviral reservoirs.

Perforin is not co-expressed with granzyme A within cytotoxic granules in Cd8 T lymphocytes present in lymphoid tissue during chronic Hiv infection

BACKGROUND Residual HIV-1-infected cells are poorly eliminated from lymphoid tissue (LT) reservoirs by effector cytotoxic T lymphocytes (eCTL) despite antiretroviral therapy. Perforin and granzyme A (grA) constitute major effector molecules within eCTL granules that induce apoptosis and lysis of virally infected cells. OBJECTIVE Expression of perforin and grA was studied at the single cell level in LT and blood from 16 patients infected with HIV-1 (stage A1-C) who were not taking antiretroviral therapy. METHOD Immunohistochemical analysis by in situ imaging of cells from blood and LT. RESULTS Quantitative in situ imaging showed that perforin-expressing CD8 T cells comprised 0.3-1.5% of total cells within the LT from recent HIV-1 seroconverters, while grA was found in 2.1-7.2% of total cells. However, despite high-level grA upregulation (1.5-4.5% of total cells) compared with that in non-infected individuals (0.4-0.9%), perforin expression remained low (< 0.1% of total cells) (P < 0.02) in LT from patients with chronic HIV-1 infection (stage A2-C). This contrasted with findings in peripheral blood mononuclear cells (PBMC) from the same HIV-1 infected cohort where perforin was detected in 13-31% of all PBMC, which was 10- to 100-fold higher than in lymphoid tissue (P < 0.001); grA was found in 14-32% of total PBMC. Two-colour staining showed that granular expression of perforin and grA was restricted to CD8 T cells in over 90% of total cells in both LT and blood. CONCLUSIONS These findings indicate that cytotoxic perforin expression is impaired at local sites of HIV replication within lymphoid tissue. Since perforin is required together with grA for granule-mediated cytolysis, the low perforin expression in the LT may limit the ability of eCTL to eliminate HIV-1 infected cells in lymphoid tissue.

Active γ‐secretase is localized to detergent‐resistant membranes in human brain

Several lines of evidence suggest that polymerization of the amyloid β‐peptide (Aβ) into amyloid plaques is a pathogenic event in Alzheimer’s disease (AD). Aβ is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by β‐secretase and γ‐secretase, and it has been suggested that these enzymes could be targets for treatment of AD. γ‐Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that γ‐secretase is partially localized to lipid rafts, which are detergent‐resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied γ‐secretase in detergent‐resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in γ‐secretase components and activity. The DRM fraction was subjected to size‐exclusion chromatography in CHAPSO, and all of the γ‐secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co‐immunoprecipitation studies further supported the notion that the γ‐secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time‐dependent decline in γ‐secretase activity, suggesting that DRMs from fresh rat brain may be useful for γ‐secretase activity studies. Finally, confocal microscopy showed co‐localization of γ‐secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active γ‐secretase complex is localized to lipid rafts in human brain.

Leukemia inhibitory factor inhibits HIV-1 replication and is upregulated in placentae from nontransmitting women

The placenta may play a critical role in inhibiting vertical transmission of HIV-1. Here we demonstrate that leukemia inhibitory factor (LIF) is a potent endogenous HIV-1-suppressive factor produced locally in placentae. In vitro, LIF exerted a potent, gp130-LIFRbeta-dependent, HIV coreceptor-independent inhibition of HIV-1 replication with IC50 values between 0.1 pg/ml and 0.7 pg/ml, depending on the HIV-1 isolate. LIF also inhibited HIV-1 in placenta and thymus tissues grown in ex vivo organ culture. The level of LIF mRNA and the incidence of LIF protein-expressing cells were significantly greater in placentae from HIV-1-infected women who did not transmit HIV-1 to their fetuses compared with women who transmitted the infection, but they were not significantly different from placentae of uninfected mothers. These findings demonstrate a novel pathway for endogenous HIV suppression that may prove to be an effective immune therapy for HIV infection.

Low levels of perforin expression in CD8+ T lymphocyte granules in lymphoid tissue during acute human immunodeficiency virus type 1 infection

Human immunodeficiency virus (HIV)-specific cytotoxic T lymphocyte (CTL) responses are detectable shortly after the acute phase of HIV infection, but they cannot control viral replication and prevent development of chronic immune suppression. This article describes a defect in the coexpression of perforin in granzyme A-positive CD8(+) T cells in lymphoid tissue from patients with acute HIV infection and a reduction in the perforin-dependent nuclear translocation of granzyme A. Furthermore, intracellular levels of HIV DNA and RNA found in lymphoid tissue were higher (10-100 times) than those found in blood, and blood samples showed more-coordinated cellular perforin/granzyme A expression. This suggests that mechanisms inhibiting CTL-mediated cytotoxicity are operative in lymphoid tissue early in the course of HIV infection.

Synaptic and Endosomal Localization of Active γ-Secretase in Rat Brain

Background A key player in the development of Alzheimers disease (AD) is the γ-secretase complex consisting of at least four components: presenilin, nicastrin, Aph-1 and Pen-2. γ-Secretase is crucial for the generation of the neurotoxic amyloid β-peptide (Aβ) but also takes part in the processing of many other substrates. In cell lines, active γ-secretase has been found to localize primarily to the Golgi apparatus, endosomes and plasma membranes. However, no thorough studies have been performed to show the subcellular localization of the active γ-secretase in the affected organ of AD, namely the brain. Principal Findings We show by subcellular fractionation of rat brain that high γ-secretase activity, as assessed by production of Aβ40, is present in an endosome- and plasma membrane-enriched fraction of an iodixanol gradient. We also prepared crude synaptic vesicles as well as synaptic membranes and both fractions showed high Aβ40 production and contained high amounts of the γ-secretase components. Further purification of the synaptic vesicles verified the presence of the γ-secretase components in these compartments. The localization of an active γ-secretase in synapses and endosomes was confirmed in rat brain sections and neuronal cultures by using a biotinylated γ-secretase inhibitor together with confocal microscopy. Significance The information about the subcellular localization of γ-secretase in brain is important for the understanding of the molecular mechanisms of AD. Furthermore, the identified fractions can be used as sources for highly active γ-secretase.

Up-Regulation of CCR5 Expression in the Placenta Is Associated with Human Immunodeficiency Virus-1 Vertical Transmission

The role of placenta in vertical transmission is not yet fully understood. A protective role of the placenta during gestation is suggested by the finding that caesarian sections reduce the risk of transmission of human immunodeficiency virus (HIV)-1 from mother to child three- to fourfold. Here we investigated whether the immunological milieu of the placenta might be important in HIV-1 transmission. In situ imaging of immunohistochemically stained placenta sections and reverse transcriptase-polymerase chain reaction demonstrated a fourfold increase in CCR5:CXCR4 expression ratio in placentae from transmitting women compared to placentae from nontransmitting women. This chemokine receptor repertoire was consistent with an up-regulation of interleukin-4 and interleukin-10 expression in placentae from nontransmitting placentae compared to transmitting placentae. In situ imaging demonstrated that CCR5 and CXCR4 were expressed on placental macrophages and lymphocytes but not in trophoblasts. Simultaneous immunofluorescence/ultrasensitive in situ hybridization for HIV-1 gag-pol mRNA revealed that HIV-1 infects primarily CXCR4-expressing cells in placentae from nontransmitting women whereas predominantly CCR5-expressing cells were infected in placentae from transmitting women. These data are consistent with transmission of a homogeneous population of nonsyncytium-inducing HIV-1 isolates that use CCR5 as co-receptor.

Normalization of immune activation in lymphoid tissue following highly active antiretroviral therapy.

&NA;Although significant progress has been made in understanding immune reconstitution in peripheral blood following highly active antiretroviral therapy (HAART), less is known about immune changes in lymphoid tissue. Here, the expression of cytokine proteins (interferon gamma [IFN‐&ggr;], interleukin [IL]‐2, IL‐4, IL‐10, IL‐1&agr;, and IL‐1&bgr;) and surface antigens (CD4, CD8, CD1a, CD68) as well as cellular proviral HIV‐1 DNA were determined in sequential tonsil biopsies before and at 4, 12, and 48 to 56 weeks posttherapy by quantitative in situ image analysis and fluorescent in situ 5′‐nuclease assay (FISNA). Despite plasma virus suppression, a fraction of tonsil cells harbored pro‐viral DNA for up to 1 year. A fourfold to eightfold increase in CD8+ T cells in tissue compared with seronegative controls and an increased frequency of CD1a+ dendritic cells prior to HAART reached control levels at week 56. The frequency of IFN‐&ggr; expressing cells was 10‐ to 15‐fold higher than controls before therapy and was comparable with findings in seronegative controls by week 56. Elevated baseline expression of IL‐1&agr; and IL‐1&bgr; was reduced by week 4 but IL‐1&agr; levels remained elevated in 1 of 3 patients at week 56. These findings suggest that with effective viral suppression the immune system in tissue may return to a more resting state.