Inverse relationship between erythrocyte size and platelet reactivity in elderly.

Previous work indicates that erythrocytes (RBCs) accumulate ß-amyloid X-40 (Aß40) in individuals with Alzheimer disease (AD) and to a lesser extent in healthy elderly. The toxin damages RBCs and increases their mean corpuscular volume (MCV). Furthermore, AD platelets demonstrate lower reactivity. This study investigated interactions between RBCs and platelets. Older individuals with moderate hypertension (n = 57) were classified into two groups, depending on MCV in whole blood: The MCVhigh group comprised individuals with higher MCV (n = 27; 97 ± 3(SD) fl) and MCVlow group had relatively lower MCV (n = 30; 90 ± 3(SD) fl). Flow cytometry was used to determine platelet reactivity, i.e., the surface binding of fibrinogen after provocation. Adenosine diphosphate (ADP) and a thrombin receptor-activating protein (TRAP-6) were used as agonists. Subsequently, blood cells were divided according to density into 17 subfractions. Intra-RBC Aß40 content was analyzed and in all platelet populations surface-bound fibrinogen was determined to estimate platelet in vivo activity. We found Aß40 inside RBCs of approximately 50% of participants, but the toxin did not affect MCV and platelet reactivity. In contrast, MCV associated inversely with platelet reactivity as judged from surface-attached fibrinogen after ADP (1.7 µmol/L) (p < 0.05) and TRAP-6 provocation (57 µmol/L (p = 0.01) and 74 µmol/L (p < 0.05)). In several density fractions (nos. 3, 4, 8, 11-13 (p < 0.05) and nos. 5-7 (p < 0.01)) MCV linked inversely with platelet-attached fibrinogen. In our community-dwelling sample, enhanced MCV associated with decreased platelet reactivity and lower in vivo platelet activity. It resembles RBCs and platelet behavior in AD-type dementia.

PTEN, Akt, and GSK3beta signalling in rat primary cortical neuronal cultures following tumor necrosis factor-alpha and trans-4-hydroxy-2-nonenal treatments.

PTEN is a dual phosphatase that negatively regulates the phosphatidylinositol 3-kinase (PI3K)/Akt signalling pathway important for cell survival. We determined effects of the inflammation and oxidative stresses of tumor necrosis factor-alpha (TNFalpha) and trans-4-hydroxy-2-nonenal (HNE), respectively, on PTEN, Akt, and GSK3beta signalling in rat primary cortical neurons. The inhibitors bisperoxovanadium [bpV(Pic)] and LY294002 were also used to determine PTEN and PI3K involvement in TNFalpha and HNE modulation of neuronal cell death. PTEN inhibition with bpV(Pic) alone did not affect Ser(473)Akt or Ser(9)GSK3beta phosphorylation. Instead, effects of this inhibitor were manifest when it was used together with TNFalpha and to a lesser extent with HNE. TNFalpha together with PTEN inhibition increased phosphorylation of Ser(473)Akt and Ser(9)GSK3beta. TNFalpha and HNE both gave decreased numbers of viable and increased numbers of early apoptotic neurons. PTEN inhibition partially reversed the toxic effect of TNFalpha as shown by an increased number of viable and a decreased number of early apoptotic neurons. All effects were reversed by PI3K inhibition. HNE together with inhibition of PTEN gave increased Ser(473)Akt but not Ser(9)GSK3beta phosphorylation and no effects on the number of viable or early apoptotic cells. In conclusion, PTEN inhibition gives a mild reversal of TNFalpha- but not HNE-induced cell death via the PI3K pathway.

Flow cytometry as a method for studying effects of stressors on primary rat neurons.

The mechanisms associated with cell death have been an important focus for neurobiology research. In the present study, the methodology of flow cytometry was used to optimize quantification of the toxic effects of tumor necrosis factor-alpha (TNF-alpha), trans-4-hydroxy-2-nonenal (4-HNE), and aged amyloid-beta (Abeta1-42) on rat primary cortical neurons. The fluorescent dyes annexin V-FITC and propidium iodide (PI) were used to identify populations of viable, early apoptotic, necrotic and late apoptotic cells by flow cytometry. Prior to exposure, the primary cultures showed 83% cell viability. Flow cytometry following labeling of cells with a specific neuronal marker, TUJ-1, revealed 82% pure neuronal populations, whereas approximately 7% were astrocytic as shown by glial fibrillary acidic protein positivity. Exposure of primary cultures to TNF-alpha, 4-HNE, and aged Abeta1-42 gave an increased number of early apoptotic cells. We show that flow cytometry is a suitable method for quantifying effects of different stressors on neurons in primary cultures. This technique could be useful for screening and testing of pharmacological compounds relevant to neurodegenerative disorders.

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.

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

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-gamma], interleukin [IL]-2, IL-4, IL-10, IL-1alpha, and IL-1beta) 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-gamma 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-1alpha and IL-1beta was reduced by week 4 but IL-1alpha 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.

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.

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.

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 x 10(6)/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)-1 alpha, IL-12, IL-2 and interferon (IFN)-gamma 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)-alpha, 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.

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.