Infection of CD8+CD45RO+ Memory T-Cells by HIV-1 and Their Proliferative Response

CD8+ T-cells are involved in controlling HIV-1 infection by eliminating infected cells and secreting soluble factors that inhibit viral replication. To investigate the mechanism and significance of infection of CD8+ T-cells by HIV-1 in vitro, we examined the susceptibility of these cells and their subsets to infection. CD8+ T-cells supported greater levels of replication with T-cell tropic strains of HIV-1, though viral production was lower than that observed in CD4+ T-cells. CD8+ T-cell infection was found to be productive through ELISA, RT-PCR and flow cytometric analyses. In addition, the CD8+CD45RO+ memory T-cell population supported higher levels of HIV-1 replication than CD8+CD45RA+ naïve T-cells. However, infection of CD8+CD45RO+ T-cells did not affect their proliferative response to the majority of mitogens tested. We conclude, with numerous lines of evidence detecting and measuring infection of CD8+ T-cells and their subsets, that this cellular target and potential reservoir may be central to HIV-1 pathogenesis

Low-bone-mass phenotype of deficient mice for the cluster of differentiation 36 (CD36).

Bone tissue is continuously remodeled by bone cells and maintenance of its mass relies on the balance between the processes of resorption and formation. We have reported the expression of numerous scavenger receptors, namely scavenger receptor (SR) class B type I and II (SR-BI and SR-BII), and CD36, in bone-forming osteoblasts but their physiological roles in bone metabolism are still unknown. To unravel the role of CD36 in bone metabolism, we determined the bone phenotype of CD36 knockout (CD36KO) mice and characterized the cell functions of osteoblasts lacking CD36. Weights of CD36KO mice were significantly lower than corresponding wild-type (WT) mice, yet no significant difference was found in femoral nor tibial length between CD36KO and WT mice. Analysis of bone architecture by micro-computed tomography revealed a low bone mass phenotype in CD36KO mice of both genders. Femoral trabecular bone from 1 to 6 month-old CD36KO mice showed lower bone volume, higher trabecular separation and reduced trabeculae number compared to WT mice; similar alterations were noticed for lumbar vertebrae. Plasma levels of osteocalcin (OCN) and N-terminal propeptide of type I procollagen (PINP), two known markers of bone formation, were significantly lower in CD36KO mice than in WT mice, whereas plasma levels of bone resorption markers were similar. Accordingly, histology highlighted lower osteoblast perimeter and reduced bone formation rate. In vitro functional characterization of bone marrow stromal cells and osteoblasts isolated from CD36KO mice showed reduced cell culture expansion and survival, lower gene expression of osteoblastic Runt-related transcription factor 2 (Runx2) and osterix (Osx), as well as bone sialoprotein (BSP) and osteocalcin (OCN). Our results indicate that CD36 is mandatory for adequate bone metabolism, playing a role in osteoblast functions ensuring adequate bone formation

Preconditioning with Hemin Decreases <em>Plasmodium chabaudi adami</em> Parasitemia and Inhibits Erythropoiesis in BALB/c Mice

<div><p>Increased susceptibility to bacterial and viral infections and dysfunctional erythropoiesis are characteristic of malaria and other hemolytic hemoglobinopathies. High concentrations of free heme are common in these conditions but little is known about the effect of heme on adaptive immunity and erythropoiesis. Herein, we investigated the impact of heme (hemin) administration on immune parameters and steady state erythropoiesis in BALB/c mice, and on parasitemia and anemia during <em>Plasmodium chabaudi adami</em> infection. Intra-peritoneal injection of hemin (5 mg/Kg body weight) over three consecutive days decreased the numbers of splenic and bone marrow macrophages, IFN-γ responses to CD3 stimulation and T_h1 differentiation. Our results show that the numbers of erythroid progenitors decreased in the bone marrow and spleen of mice treated with hemin, which correlated with reduced numbers of circulating reticulocytes, without affecting hemoglobin concentrations. Although blunted IFN-γ responses were measured in hemin-preconditioned mice, the mice developed lower parasitemia following <em>P.c.adami</em> infection. Importantly, anemia was exacerbated in hemin-preconditioned mice with malaria despite the reduced parasitemia. Altogether, our data indicate that free heme has dual effects on malaria pathology.</p> </div

Impact of heme on the clearance of <i>Plasmodium</i> infection.

<p><i>P. chabaudi adami</i> DK (10⁵ parasitized RBCs) were inoculated by the intravenous route in PBS (Ctrl)- and hemin (HE)-preconditioned mice (5 mg/kg, for 3 consecutive days) 24 h after the last injection. Parasitemia was followed daily from tail-tip blood smears for estimation of the kinetics of infection (A) and cumulative (B) and peak parasitemia (C). Values represent the mean ± SEM from two independent experiments (n = 8) and were compared using a non-parametric Student <i>t</i> test. **<i>p</i><0.01; ***<i>p</i><0.001.</p

Immune parameters in control and heme-preconditioned BALB/c mice 10 days after <i>P. c. adami</i> infection.

<p>PBS (Ctrl) and hemin (HE)-treated mice were euthanized and spleen was recovered to assess the number of macrophages (F4/80⁺ cells) (A) and CD4 T cells (B). Spleen cells were stimulated with anti-CD3 monoclonal antibody for 48 h to assess IFN-γ (C) and IL-4 (D) production by ELISA. Purified CD4 T cells were stimulated with anti-CD3 and anti-CD28 monoclonal antibodies to estimate the production of IFN-γ (E) and IL-4 (F). Data represent the mean ± SEM from two independent experiments (n = 7–12) and values were compared using a non-parametric Student <i>t</i> test.</p

Bone marrow and spleen erythroid parameters.

<p>Control (Ctrl) and hemin (HE)-treated mice were euthanized 24 h after the last injection with PBS or HE. Populations of erythroid cells were analyzed in the bone marrow (A) and in the spleen (B) by staining cells with anti-CD71-FITC and anti-Ter119-PE antibodies. The percentage of reticulocytes (CD71⁺ cells) (C) and hemoglobin levels were measured in the blood (D). Macrophages (F4-80⁺ cells) were quantified in femoral bone marrow cell suspensions (E) by staining with anti-F4/80-PE antibody. Erythropoietin (EPO) was measured in the plasma by ELISA (F). Data are mean ± SEM from two independent experiments (n = 4–11 mice per group) and values were compared using a non-parametric Student <i>t</i> test. <i>*p<</i>0.05; **<i>p</i><0.01; ***P<0.001.</p

Body weight and bone length of 1 to 6 month-old WT and CD36KO mice.

<p>A) Body weight of WT and CD36KO male and female mice from 1 to 6 months of age. Data are expressed as mean ± SEM from 10–37 mice per group of age. Bonferroni post-test: ***P<0.001 compared to WT male; ^£P<0.05, ^££P<0.01,^£££P<0.001 compared to WT female. B) Femur and tibia lengths for WT and CD36KO mice of 1–3 months old. Data are expressed as mean ± SEM from 11–35 mice per group of age.</p

Cell Survival of CD36-deficient osteoblasts.

<p>MTT assays and protein measurements were performed on osteoblasts isolated from bone marrow (A) and long bone (B) of WT and CD36KO mice after 0 and 14 of culture in differentiation medium. Data represent mean ± SD of two independent experiments performed in triplicates.</p

Expression of osteoblastic genes by cells from WT and CD36KO mice.

<p>Total RNA was isolated from MSC and cultured for 7 days. The levels of transcripts were determined by semi-quantitative RT-PCR using specific primers for (A) type I collagen (Col1α1), bone sialoprotein (BSP) and osteocalcin (OCN) or (B) osteoblastic transcription factors Runx2 and Osx as described in Material and methods. Expression levels were normalized against expression of reference gene. Data are mean ± SEM of 3–8 independent cell preparations. Student T test: *P<0.05, **P<0.01.</p