The Toxoplasma gondii Peptide AS15 Elicits CD4 T Cells That Can Control Parasite Burden

The apicomplexan parasite Toxoplasma gondii can cause severe disease in immunocompromised individuals. Previous studies in mice have focused largely on CD8(+) T cells, and the role of CD4 T cells is relatively unexplored. Here, we show that immunization of the C57BL/6 strain of mice, in which the immunodominant CD8 T cell response to the parasite dense-granule protein GRA6 cannot be generated, leads to a prominent CD4 T cell response. To identify the CD4 T cell-stimulating antigens, we generated a T. gondii-specific, lacZ-inducible, CD4 T cell hybridoma and used it as a probe to screen a T. gondii cDNA library. We isolated a cDNA encoding a protein of unknown function that we call CD4Ag28m and identified the minimal peptide, AS15, which was presented by major histocompatibility complex (MHC) class II molecules to the CD4 T cells. Immunization of mice with the AS15 peptide provided significant protection against subsequent parasite challenge, resulting in a lower parasite burden in the brain. Our findings identify the first CD4 T cell-stimulating peptide that can confer protection against toxoplasmosis and provide an important tool for the study of CD4 T cell responses and the design of effective vaccines against the parasite.     

Thymic microenvironments for T cell differentiation and selection

The adult thymus provides a variety of specialized microenvironments that support and direct T cell differentiation and selection. In this review, we summarize recent advances in the understanding of the function of microenvironments in shaping a diverse T cell repertoire. In particular, we focus on how thymocytes move in and out of these specialized thymic compartments in response to homing signals, differential chemokine gradients and other factors that regulate T cell migration. In addition, we discuss the diverse developmental signals provided by these microenvironments that contribute to the generation of divergent T cell lineages.     

In vivo formation of bone and haematopoietic territories by transplanted human bone marrow stromal cells generated in medium with and without osteogenic supplements

Autologous transplantation of human bone marrow stromal cells (BMSCs) has been successfully used for bone reconstruction. However, in order to advance this approach into the mainstream of bone tissue engineering, the conditions for BMSC cultivation and transplantation must be optimized. In a recent report, cultivation with dexamethasone (Dex) significantly increased bone formation by human BMSCs in vivo. Based on this important conclusion, we analysed the data accumulated by our laboratory, where human BMSCs have been routinely generated using media both with and without a combination of two osteogenic supplements: Dex at 10^–8 m and ascorbic acid phosphate (AscP) at 10^–4 m . Our data demonstrate that for 22/24 donors, BMSC strains propagated with and without Dex/AscP formed similar amounts of bone in vivo. Thus, human BMSCs do not appear to need to be induced to osteogenic differentiation ex vivo prior to transplantation. Similarly, for 12/14 donors, BMSC strains cultured with and without Dex/AscP formed haematopoietic territories to a comparable extent. While Dex/AscP did not increase bone formation, they significantly stimulated BMSC in vitro proliferation without affecting the number of BMSC colonies formed by the colony‐forming units–fibroblasts. We conclude that for the substantial majority of donors, Dex/AscP have no effect on the ability of BMSCs to form bone and myelosupportive stroma in vivo. However, due to increased BMSC proliferation, the total osteogenic population obtained from a single marrow sample is larger after cultivation with Dex/AscP than without them. Secondary to increased BMSC proliferation, Dex/AscP may stimulate bone formation if BMSCs and/or the transplantation system are less than optimal. Published 2011. This article is a U.S. Government work and is in the public domain in the USA     

In vitro model of bromodeoxyuridine or iron oxide nanoparticle uptake by activated macrophages from labeled stem cells: implications for cellular therapy

There is increasing interest in using exogenous labels such as bromodeoxyuridine (BrdU) or superparamagnetic iron oxide nanoparticles (SPION) to label cells to identify transplanted cells and monitor their migration by fluorescent microscopy or in vivo magnetic resonance imaging (MRI), respectively. Direct implantation of cells into target tissue can result in >80% cell death due to trauma or apoptosis. Bystander uptake of labeled cells by activated macrophages (AM) can confound the interpretation of results. This study investigated the frequency of BrdU or SPION uptake by AM using the Boyden chamber model of inflammation. SPION/BrdU-labeled bone marrow stromal cells or HeLa cells, AM, and mouse fibroblasts (MF) or human fibroblasts (HF) were mixed in various ratios in Matrigel in the upper chamber and incubated for up to 96 hours. The AM were chemotactically induced to migrate to the lower chamber. Fluorescence-activated cell sorting analysis of AM from lower and upper chambers, in the presence of either MF or HF using anti-CD68, anti-BrdU, anti-dextran antibodies, revealed 10%-20% dextran-positive or 10% BrdU-positive AM after 96 hours of incubation. Transfer of iron to AM accounted for <10% of the total iron in labeled cells. The uptake of BrdU and SPION was dependent on the ratio of labeled cells to inflammatory cells and microenvironmental conditions. Direct implantation of BrdU/SPION-labeled cells into target tissue can result in uptake of label by AM; therefore, care should be taken to validate by histology transplanted cells for bystander cell markers and correlation with MRI results.     

Plasmodium vivax: a cause of malnutrition in young children

We studied the aetiology of malnutrition in a cohort of 1511 children < 10 years old in Espiritu Santo, Vanuatu. Malnutrition was categorized using standard anthropometric criteria as: underweight [weight-for-age (WA) Z score < -2], wasting [weight-for-height (WH) Z < -2], or stunting [height-for-age (HA) Z < -2]. On multiple logistic regression analysis, the only factors significantly associated with wasting were age < 5 years [OR (95% CI) 1.8 (1.2-2.9), p = 0.01] and having suffered one or more episodes of clinical P. vivax malaria in the 6 months preceding nutritional assessment [OR 2.4 (1.3-4.4), p = 0.006]. The incidence of P. vivax infection was significantly higher during the 6 months preceding assessment in underweight vs. non-underweight children [incidence rate ratio (IRR) 2.6 (1.5-4.4), p < or = 0.0001). These groups had similar incidences of clinical P. falciparum infection during the same period [IRR 1.1 (0.57-2.1) p = 0.8] and of either species during the 6 months following assessment [IRR P. vivax 1.3 (0.9- 2.0) p = 0.2; IRR P. falciparum 1.3 (0.9-1.9) p = 0.2]. In these children, P. vivax malaria was a major predictor of acute malnutrition; P. falciparum was not. Wasting neither predisposed to nor protected against malaria of either species. Although P. vivax malaria is generally regarded as benign, it may produce considerable global mortality through malnutrition.      

Differential effect of phorbol esters and interleukin-3 on protein kinase C isoform content and kinase activity in the FDC-P1 cell line

FD/PMA is a subclone of the interleukin-3 (IL-3)-dependent, FDC-P1 cell line, which proliferates in response to either 12-O- tetradecanoylphorbol-13 acetate (PMA) or IL-3. While several endogenous substrates were phosphorylated in response to protein kinase C (PKC) activation in FDC-P1, phospholipid-dependent phosphorylation in the FD/PMA grown in PMA was not observed. Basal, phosphatidylserine- independent, and diolein-independent phosphorylation of cytosolic substrates with molecular weights of 17, 52, 57, and 105 Kd were enhanced in FD/PMA cells grown in PMA as compared with FDC-P1 cells cultured in IL-3. Phosphorylation of a 105-Kd substrate was enhanced in the particulate fraction of FD/PMA cells maintained in PMA. The 17-Kd substrate in FD/PMA cells comigrated with a substrate phosphorylated in a PKC-dependent manner in FDC-P1 cells. Phosphorylation of the 52- and 57-Kd substrates, but not of the 17-Kd substrate, was inhibited by H-7 and staurosporine. A portion of the PMA-induced cytosolic kinase activity coeluted with PKC on diethyl aminoethyl chromatography. While FD/PMA cells cultured in PMA contained negligible PKC-dependent phosphorylation of endogenous substrates or histone, alpha and epsilon PKC isoforms were detected by Western blot analysis. PKC phosphotransferase activity was observed in FD/PMA cells grown in PMA when peptides corresponding to residues 720 to 737 of PKC-epsilon or residues 4 to 14 of myelin basic protein were used as substrates. These data indicate that maintenance of FD/PMA cells in PMA stimulates proliferation and markedly alters PKC substrate specificity. Generation of at least two phospholipid-independent kinases occurs in PMA-treated cells.     

Detection of minimal residual disease in acute lymphoblastic leukemia by in vitro amplification of rearranged T-cell receptor delta chain sequences

Human T-cell receptor (TCR) delta-chain diversity mainly originates from high junctional variability, since only a limited number of germline elements is available. This extraordinary diversity at the V.J junction, due to the use of two D delta elements and extensive incorporation of N nucleotides, constitutes a specific clonal marker for cell populations exhibiting rearranged TCR delta genes. To this end we amplified in vitro by polymerase chain reaction (PCR) the TCR delta junctional region of five acute lymphoblastic leukemias (ALL), isolated respective DNA fragments, and used them directly as clonospecific probes. The combination of PCR technology and hybridization to clonospecific probes permitted the detection of leukemia DNA at dilution of 1:100,000 in all five cases. Moreover, we were able to investigate one of the ALL patients 11 months after achieving continuous complete remission. Conventional Southern blot analysis failed to detect rearranged TCR genes at this stage. However, residual leukemic cells could readily be detected by PCR technique. We conclude that the strategy proposed here is a very sensitive tool to detect minimal residual disease in a significant proportion of human lymphoid neoplasias.