Simultaneous expression and detection of multiple retroviral constructs in haematopoietic cells after bone marrow transplantation

Due to the complexity and redundancy of molecular processes governing the development and function of haematopoietic cells, experimental procedures allowing simultaneous alteration in gene expression of multiple genes in vivo are needed. Here, we describe a protocol allowing for simultaneous transduction of haematopoietic stem cells (HSC) with two different replication incompetent retroviral expression vectors followed by transplantation of lethally irradiated recipient mice. These bicistronic retroviral vectors carried genes for the enhanced green and yellow florescent proteins (EGFP and EYFP) respectively. Spleen cells from reconstituted animals were stained for common lymphocyte and myeloid markers, then analysed on a two-laser, 488 and 635 nm, flow cytometer equipped with a 510/20-nm bandpass filter for FL1, a 550/30-nm bandpass filter for FL2 and a 530-nm short-pass dichroic mirror. It was demonstrated that cells expressing EGFP, EYFP or combinations thereof could be distinguished and analysed for staining with PerCP- and APC-conjugated reagents. We found that a sizable proportion of cells (70%) from reconstituted animals expressed EGFP and/or EYFP and that expression of these genes did not affect lymphoid or myeloid development. We also demonstrated that the alternative optical configuration allowed for conventional multiparameter flow cytometric analyses.     

Nutritional iron status in children with alpha+ thalassemia and the sickle cell trait in a malaria endemic area on the coast of Kenya

Although hemoglobinopathies such as alpha+ thalassemia and the sickle cell trait might contribute to anemia in African children, we hypothesized that they might also enhance iron absorption under circumstances of critical availability, and that this could attenuate their hematologic effects. We found no support for this hypothesis in a cohort of children living on the coast of Kenya.     

Malaria and nutritional status in children living on the coast of Kenya

BACKGROUND: The relation between malnutrition and malaria is controversial. On the one hand, malaria may cause malnutrition, whereas on the other hand, malnutrition itself may modulate susceptibility to the disease. OBJECTIVE: The objective was to investigate the association between Plasmodium falciparum malaria and malnutrition in a cohort of Kenyan children. DESIGN: The study involved the longitudinal follow-up of children aged 0-95 [corrected] mo for clinical malaria episodes and anthropometric measurements through 4 cross-sectional surveys. We used Poisson regression analysis to investigate the association between malaria and nutritional status. RESULTS: The crude incidence rate ratios (IRRs) for malaria during the 6-mo period before assessment in children defined as malnourished on the basis of low height-for-age or low weight-for-age z scores (<-2) were 1.17 (95% CI: 0.91, 1.50; P=0.21) and 0.94 (0.71, 1.25; P=0.67), respectively, which suggests no association between malaria and the subsequent development of protein-energy malnutrition. However, we found that age acted as an effect modifier in the association between malaria episodes and malnutrition on prospective follow-up. The IRR for malaria in children aged 0-2 y, who were subsequently characterized as underweight, was 1.65 (1.10, 2.20; P=0.01), and a significant overall relation between malaria and stunting was found on regression analysis after adjustment for the interaction with age (IRR: 1.91; 1.01, 3.58; P=0.04). CONCLUSION: Although children living on the coast of Kenya continue to experience clinical episodes of uncomplicated malaria throughout the first decade of life, the effect of malaria on nutritional status appears to be greatest during the first 2 y of life.     

Enhanced Induction of HIV-specific Cytotoxic T Lymphocytes by Dendritic Cell-targeted Delivery of SOCS-1 siRNA

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in the activation of T cells. RNA interference (RNAi)-mediated silencing of negative immunoregulatory molecules expressed by DCs may provide a strategy to enhance the potency of DC-based vaccines and immunotherapy. Ablation of suppressor of cytokine signaling-1 (SOCS-1) in antigen-presenting cells has been shown to enhance cellular immune response in mice. Here, we used a previously reported DC-targeting approach to deliver small interfering RNA (siRNA) against SOCS-1 to human myeloid-derived DCs (MDDCs). SOCS1-silencing in MDDCs resulted in enhanced cytokine responses to lipopolysaccharide (LPS) and a strong mixed-lymphocyte reaction. Moreover, only DCs treated with SOCS-1 siRNA, and not controls, elicited strong primary in vitro responses to well-characterized HLA-A*0201-restricted Melan-A/MART-1 and human immunodeficiency virus (HIV) Gag epitopes in naive CD8⁺ T cells from healthy donors. Finally, stimulation of CD8⁺ T cells from HIV-seropositive subjects with SOCS1-silenced DCs resulted in an augmented polyfunctional cytotoxic T-lymphocyte (CTL) response, suggesting that SOCS-1 silencing can restore functionally compromised T cells in HIV infection. Collectively, these results demonstrate the feasibility of DC3-9dR-mediated manipulation of DC function to enhance DC immunogenicity for potential vaccine or immunotherapeutic applications.     

Iron deficiency and malaria among children living on the coast of Kenya

Both iron deficiency and malaria are common in much of sub-Saharan Africa, and the interaction between these conditions is complex. To investigate the association between nutritional iron status, immunoglobulins, and clinical Plasmodium falciparum malaria, we determined the incidence of malaria in a cohort of children between the ages of 8 months and 8 years who were living on the Kenyan coast. Biochemical iron status and malaria-specific immune responses were determined during 2 cross-sectional surveys. We found that the incidence of clinical malaria was significantly lower among iron-deficient children (incidence-rate ratio [IRR], 0.70; 95% confidence interval [CI], 0.51-0.99; P<.05), that the incidence of malaria was significantly associated with plasma ferritin concentration (IRR for log ferritin concentration, 1.48; 95% CI, 1.01-2.17; P<.05), and that iron status was strongly associated with a range of malaria-specific immunoglobulins. We conclude that iron deficiency was associated with protection from mild clinical malaria in our cohort of children in coastal Kenya and discuss possible mechanisms for this protection.     

Immunoglobulin E (IgE) containing complexes induce IL-4 production in human basophils: effect on Th1-Th2 balance in malaria

Human basophils are potent producers of IL-4 following cross-linking of the high affinity receptor for IgE (Fc epsilon R1). Elevated levels of both total- and malaria-specific IgE have been demonstrated in sera from people living in malaria-endemic regions. Whether or not these IgE antibodies are pathogenic is unclear. Serum containing high IgE levels obtained from malaria individuals was used to establish whether IgE-immune complexes could induce IL-4 production in human basophils. The basophils, obtained from healthy donors, were primed with 10 ng/ml of IL-3 before being transferred to wells containing goat anti-human IgE or human antimalarial IgE-immune complexes. IL-4 was induced upon stimulation of human basophils by plate bound IgE-containing immune complexes. Basophils treated similarly but with goat anti-IgG/human antimalarial- IgG-immune complexes did not secrete IL-4. Similarly mononuclear cells depleted of basophils in parallel culture did not secrete IL-4. Thus, human basophils may contribute to the polarization of T-helper type 2 in the (Th2) responses in malaria hosts via IgE-induced IL-4 production.     

Malaria and Nutritional Status in Children Living on the Coast of Kenya

The relationship between malnutrition and malaria is controversial. On one hand, malaria may cause malnutrition, while on the other, malnutrition itself may modulate susceptibility to the disease. We investigated the association between Plasmodium falciparum malaria and malnutrition in a cohort of children living on the coast of Kenya. The study involved longitudinal follow-up for clinical malaria episodes and anthropometric measurements at four cross-sectional surveys. We used Poisson regression analysis to investigate the association between malaria and nutritional status. Compared to baseline (children with a WAZ or HAZ score of ≥−2), the crude incidence rate ratios (IRRs) for malaria in children with low HAZ or WAZ scores (<−2) during the period prior to assessment were 1.17 (95% CI 0.91–1.50; 0 = 0.21) and 0.94 (0.71–1.25; 0.67), respectively, suggesting no association between malaria and the subsequent development of PEM. However, we found that age was acting as an effect modifier in the association between malaria and malnutrition. The IRR for malaria in children 0–2 years old who were subsequently characterized as wasted was 1.65 (1.10–2.20; P  = 0.01), and a significant overall relationship between malaria and low-HAZ was found on regression analysis when adjusting for the interaction with age (IRR 1.89; 1.01–3.53; P  < 0.05). Although children living on the coast of Kenya continue to suffer clinical episodes of uncomplicated malaria throughout their first decade, the association between malaria and malnutrition appears to be limited to the first 2 years of life.     

Highly active antiretroviral therapy in patients infected with human immunodeficiency virus increases CD40 ligand expression and IL-12 production in cells ex vivo

Highly active anti-retroviral therapy (HAART) restores CD4(+) T-cell numbers in the periphery; however, its efficacy in restoring functional immunity is not fully elucidated. Here we evaluated longitudinal changes in the expression of several key markers of T-cell activation, namely CD40 ligand (CD154), OX40 (CD134), or CD69, after anti-CD3/CD28 activation, as well as levels of IL-12 production after Staphylococcus aureus Cowan stimulation in 28 HIV-infected adult patients. Patients were followed up to 12 mo post-HAART initiation. Viral burdens and CD4 cell counts were measured at the same time points. A control group of 15 HIV-uninfected adult subjects was included for comparison. Significant increases in CD40L and OX40 expression, but not of CD69 expression, were observed over time in the overall patient population, and more particularly in patients with baseline CD4 counts lower than or equal to 200 cells/μL, or those with baseline viral loads lower than or equal to 10(5) RNA copies/mL. Similar increases were seen for IL-12 production. Viral loads were inversely associated with CD40L expression or IL-12 production in a mixed linear model analysis, while CD4 counts were directly associated. CD40L expression and IL-12 production were significantly correlated. In conclusion, HAART-mediated control of viral replication led to partial restoration of CD40L upregulation/expression, and to increased IL-12 production, but the magnitude of the response depended on the baseline characteristics of the treated patients.